ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
112 RPE/Retina Cell Biology and Degeneration, I
Sunday, May 04, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 352–383/C0123–C0154
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 352 Poster Board Number: C0123
Presentation Time: 8:30 AM–10:15 AM
Optimization of Rod Photoreceptor Culture and Rod Outer
Segment Isolation from a Single Canine Retina
Raghavi Sudharsan1, Natalia Dolgova1, Michael H. Elliott2, William
A. Beltran1. 1School of Vet Medicine, University of Pennsylvania,
Philadelphia, PA; 2Department of Ophthalmology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK.
Purpose: Rapid rod outer segment (ROS) disorganization followed
by acute cell death of both photoreceptors and retinal pigment
epithelium (RPE) occurs after brief exposure to light in the canine
model of autosomal dominant retinitis pigmentosa (ADRP) caused
by a naturally-occurring T4R mutation in the rhodopsin (RHO)
gene. Cell death is seen as early as 6 hours after light exposure and
peaks by 24-48 hours. The molecular pathways that are activated
immediately following light exposure and culminate in death of rods
and RPE are not well characterized. The purpose of this study is to
optimize rod photoreceptor culture and
rod outer segment isolation from single canine retinas that
could eventually be used to elucidate the mechanisms of acute
photoreceptor and RPE loss in the T4R RHO model of ADRP.
Methods: Rod photoreceptors were isolated under dim red
illumination from a single wild type (WT) canine retina and cultured
in dark. Various substrates (poly-d-lysine, wheat germ agglutinin,
laminin and matrigel) were evaluated for optimum cell attachment
and survival. Live cell imaging was used to study phenotypic
and viability changes in the photoreceptors on exposure to light.
Previously published sucrose step gradient protocol was scaled down
to prepare ROS from a single WT canine retina in dark. Cultured
ARPE19 cells were exposed to fluorescently-labeled or unlabeled
ROS and efficiency of ROS phagocytosis by ARPE19 cells was
evaluated by confocal microscopy.
Results: Canine rod photoreceptors were successfully cultured for a
period of 3 to 5 days when maintained in dark. Cell viability was not
compromised when observed by confocal microscopy using a 633
nm far-red laser. Approximately 10,000 intact ROS could be obtained
from a single canine retina. Phagocytosis of both fluorescentlylabeled and unlabeled ROS by ARPE19 cells was observed.
Conclusions: Optimization of the isolation of rods and ROS
from a single retina circumvents the limited access to abundant
material when working with rare and valuable retinal tissues from
animal models and/or humans. These techniques will be applied to
investigate the mechanism of extreme light sensitivity in the T4R
RHO canine retina and the pathways responsible for rod and RPE cell
death.
Commercial Relationships: Raghavi Sudharsan, None; Natalia
Dolgova, None; Michael H. Elliott, None; William A. Beltran,
None
Support: R24EY-022012; EY-06855, EY-17549, Foundation
Fighting Blindness.
Program Number: 353 Poster Board Number: C0124
Presentation Time: 8:30 AM–10:15 AM
Comprehensive characterization of retinal phenotypic changes in
methylene tetrahydrofolate reductase (MTHFR) deficient mice: a
model of hyperhomocysteinemia (HHcy)
Shanu Markand1, 2, Arul Shanmugam1, 2, Amany Tawfik1, 2, Penny
Roon1, Alan Saul1, 2, Rima Rozen3, Vadivel Ganapathy1, 2, Sylvia B.
Smith1, 2. 1Cellular Biology and Anatomy, Georgia Regents University,
Augusta, GA; 2The James and Jean Culver Vision Discovery Institute,
Georgia Regents University, Augusta, GA; 3Human Genetics and
Pediatrics, McGill University, Montreal, QC, Canada.
Purpose: MTHFR, a crucial enzyme in homocysteine metabolism,
is mutated in 44% (heterozygous) and 12% (homozygous) of
Americans. HHcy is implicated in CRVO, diabetic retinopathy,
glaucoma & AMD. MTHFR mutations are the most common genetic
cause of Hhcy, but there is discrepancy clinically regarding MTHFR
mutations & ocular problems. Mice deficient in mthfr provide an
excellent tool to address whether HHcy due to absence/lack of
MTHFR manifests as retinal disruption/dysfunction.
Methods: The retinal phenotype was characterized in mthfr +/+
(n=24),mthfr +/- (n=40), mthfr -/- (n=20) mice (8-52 wks) using
fundus photography, fluorescein angiography (FA), IOP, OCT, ERG,
flatmount immunostaining, morphometry, light/electron microscopy
(EM).
Results: Retinal phenotypic changes in mthfr -/- mice were observed
as early as 8 wks (~20%); by 12 wks ~60% were affected; by
24 wks 100% manifested fundoscopic evidence of disruption
including geographic atrophy; FA evidence of dilated, tortuous,
beaded, leaky blood vessels (BV), which was confirmed by OCT.
Morphometric analysis revealed ~20% decrease in RGCs by 8
wks; EM showed apoptotic cells in the INL and evidence of RPE
atrophy adjacent to areas of RPE hypertrophy. In mthfr +/- (mice
retinal phenotypic changes began at 24 wks (~50% were affected),
while 100% showed gross disruption by 1 yr. Fundoscopy showed
abnormal retinal deposits, FA revealed tortuous, leaky BV and these
changes progressively worsened. Neovascularization and gliosis
were observed in retinal flatmounts by isolectin -B4 and GFAP
immunostaining. Disruption was confirmed by OCT. Morphometry
indicated ~20% RGC loss observed by 24 wks, EM alterations were
substantial by 1 yr. ERG analysis showed marked decrease in a, b, c
wave amplitudes at 1 yr in ),mthfr+/- mice. IOP was similar in all three
mouse groups.
Conclusions: Absence of MTHFR manifests as marked retinal
neurovasculopathy, while moderate deficiency of MTHFR shows
a similar but later-onset retinal phenotype. The study provides
the first comprehensive, systematic analysis of retinal function
and architecture in MTHFR deficient mice. Future experiments
will explore the mechanisms involved in HHcy-induced retinal
neurovascular damage.
Commercial Relationships: Shanu Markand, None; Arul
Shanmugam, None; Amany Tawfik, None; Penny Roon, None;
Alan Saul, None; Rima Rozen, None; Vadivel Ganapathy, None;
Sylvia B. Smith, None
Support: R01 EY012830
Program Number: 354 Poster Board Number: C0125
Presentation Time: 8:30 AM–10:15 AM
Identification of (pro)renin receptor/Atp6ap2-binding proteins in
the retina of adult mice
Atsuhiro Kanda, Kousuke Noda, Susumu Ishida. Laboratory of Ocular
Cell Biology and Visual Science, Department of Ophthalmology,
Hokkaido University Graduate School of Medicine, Sapporo, Japan.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: (Pro)renin receptor [(P)RR] (also called Atp6ap2),
identified as a molecule existing in the major organs but not in the
circulation, has attracted growing attention due to its diverse roles in
tissue renin-angiotensin system (RAS) and in fundamental cellular
physiology related to vacuolar H+-ATPase (v-ATPase). The purpose
of this study is to determine retinal proteins bound to Atp6ap2/(P)RR
in adult mice.
Methods: We performed immunoprecipitation and mass
spectrometry (MS) experiments to identify candidate interacting
proteins of Atp6ap2/(P)RR. Pre-cleared C57BL/6J retina lysates
with protein G beads were immunoprecipitated with normal IgG
or anti-Atp6ap2 antibodies. The beads were washed, and protein
samples were then eluted by boiling in sample buffer and separated
by SDS-PAGE. Eluted samples observed by silver staining that were
identified as being unique to anti-Atp6ap2 antibody as compared to
normal IgG have been examined by MS.
Results: MS analysis results showed candidate proteins for possible
Atp6ap2/(P)RR binding partners include Ablim2, Atp6v0d1 and
Rpl6. We confirmed those protein interaction with co-transfection/
co-immunoprecipitation experiments and co-localizations in mouse
retina with immunofluorescence.
Conclusions: Our current findings may help elucidate various
physiological activities of Atp6ap2/(P)RR in the retina of adult mice.
Given that Atp6ap2/(P)RR functions as a cell polarity determinant
required for retinal lamination during embryonic development in
mice (Kanda A et al. J Neurosci. 2013), Atp6ap2/(P)RR is thought to
be involved in multiple physiological and pathological events in the
eye, besides the v-ATPase function and tissue RAS activation.
Commercial Relationships: Atsuhiro Kanda, None; Kousuke
Noda, None; Susumu Ishida, None
Support: Takeda Science Foundation, Mishima Saiichi
Memorial Ophthalmic Research Japan Foundation, and JSPS
KAKENHI-24791823.
Program Number: 355 Poster Board Number: C0126
Presentation Time: 8:30 AM–10:15 AM
TESK1/Cofilin pathway controls primary cilia assembly by
regulating actin dynamics and CP110 cap removal
Jongshin Kim, Joon Kim. Graduate School of Medical Science and
Engineering, KAIST, Daejeon, Republic of Korea.
Purpose: Primary cilia is a microtubule-based sensory organ and its
malfunction, known as ciliopathy, is associated with photoreceptor
degeneration. Previous studies have reported that drugs or proteins
that induce change in actin cytoskeleton affect ciliogenesis.
However the mechanism of the link between actin cytoskeleton and
ciliogenesis is not fully understood. The aim of this study is to find
actin-related key molecules that control ciliogenesis and to determine
the exact mechanism of cilia assembly.
Methods: A high-throughput assay using siRNA was performed
to evaluate the functional impact of protein kinases that regulate
actin dynamics in ciliogenesis. RPE1 cells stably expressing
Smo-EGFP were transfected with 10 nM siRNAs. For indirect
immunofluorescence, cells were fixed in methanol for 4 minutes at
−20°C. Primary and secondary antibodies were applied for 1 hour at
room temperature. Cytochalasin D (actin depolymerizing agent) was
applied to the cells at 200 nM.
Results: We found that TESK1, which inactivates cofilin that severs
actin filaments, is a potent negative regulator of ciliogenesis. TESK1
knockdown increased the number of ciliated cells compared with
the nonspecific siRNA control (33.8% vs 2.1%). Double knockdown
of TESK1 and cofilin did not induce ciliogenesis, which indicates
that cofilin is a downstream effector of TESK1 in cilia assembly.
When TESK1 was knockdowned, more ciliary vesicles that carry
Smo-EGFP accumulated around the centrosome than that of the
nonspecific siRNA control (90.2% vs 21.6%), suggesting that
up-regulated actin dynamics by TESK1 knockdown facilitates
the directional trafficking of ciliary vesicles to the centriolar
compartment. CEP164 is a molecule that is known to be essential
for ciliary vesicle docking to the basal body. In the condition of its
knockdown, an increased CP110 cap removal, an important step in
ciliogenesis initiation, was observed when treated with Cytochalasin
D versus without treatment (46.1% vs 13.3%). This suggests that
although ciliary vesicle docking does not occur, actin dynamics upregulation can remove the CP110 cap in a significant number of cells.
Conclusions: Up-regulation of actin dynamics through TESK1
knockdown facilitates ciliogenesis by directional ciliary vesicle
trafficking to the centrosome and CP110 cap removal. TESK1 could
therefore be exploited as a potential therapeutic target for retinal
ciliopathies.
Commercial Relationships: Jongshin Kim, None; Joon Kim, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Support: Outstanding Young Investigator Grant (No. 2012-0004114)
and Global Ph.D. Fellowship (No. 2011-002393) supported by
National Research Foundation of Korea.
Program Number: 356 Poster Board Number: C0127
Presentation Time: 8:30 AM–10:15 AM
Comparison of Basal Firing Patterns of rd1 mice Retinal
Ganglion Cell (RGC) Spikes in Freshly-isolated and Retinal
Explants
Yongsook Goo1, 2, Kun No Ahn1, 2, Joo Yeon Kim1, 2. 1Physiology,
Chungbuk National Univ Med School, Cheongju, Republic of Korea;
2
Nano Artificial Vision Research Center, Seoul National University
Hospital, Seoul, Republic of Korea.
Purpose: Retinal prosthesis is being developed for patients with
retinitis pigmentosa (RP) and age-related macular degeneration
(AMD), and this is regarded as the most feasible method to restore
vision. Extracting long-term safety margin for electrical stimulation
for the prosthesis is one of the most important elements for the
development of a viable retinal prosthesis. Here, we established
a retinal explant culture method for our safe long-term electrical
stimulation studies. We compared the firing pattern of retinal
ganglion cell (RGC) spikes during 0 to 4 days in culture with those
from freshly-isolated retinae. No electrical stimulation was applied to
both tissues in order to explore basal firing patterns.
Methods: The well-known animal model for RP, rd1 (Pde6brd1) mice
at postnatal 8 weeks were used (n=15). After isolation of retinae,
retinal explants (n=15) were cultured in serum-free media (2% B27/
1% N2) at 34 °C and 5% CO2 for up to 4 days. Each day, retinal
waveforms were recorded with 8 × 8 MEA. Each day, we compared
raster plot, mean frequency, inter-spike interval histogram (ISIH),
power spectral density (PSD), and cross-correlation function of RGC
spikes between explants and freshly-isolated tissues.
Results: In freshly-isolated retinae, RGC spikes as well as ~10 Hz
spontaneous oscillatory rhythm were observed while in explants, no
spontaneous oscillatory rhythm was found. In explants, the mean
frequency of RGC spikes were hypoactive compared with freshlyisolated retinae (ANOVA, p<0.05). There is no significant frequency
change amongst the explants at days 1 to 4. Furthermore, no 2nd peak
in ISIH and PSD were found in the explants. When the number of
cross-correlated RGCs was counted at each day, the size of crosscorrelated cell cluster becomes smaller with day.
Conclusions: We successfully established retinal explant tissue
culture system. This may serve as a good model for future electrical
stimulation studies regarding long-term safety margins.
Commercial Relationships: Yongsook Goo, None; Kun No Ahn,
None; Joo Yeon Kim, None
Support: grants of MEST (NRF-2010-0020852 and NRF2013R1A1A3009574) to Y.Goo
Program Number: 357 Poster Board Number: C0128
Presentation Time: 8:30 AM–10:15 AM
Distinct profiles of abnormal ganglion cell activity in two forms of
Leber’s congenital amaurosis (LCA): Implications for therapy
Steven F. Stasheff1, 5, Kelsey N. Spalding2, Frederick R. Blodi3,
Malini Shankar3, Sajag Bhattarai4, 5, Stewart Thompson4,
5
, Jeannette Bennicelli6, Jean Bennett6, Arlene V. Drack4, 5.
1
Pediatrics,Ophthalmology,Neurosci & BME, Univ of IowaChildren’s Hospital, Iowa City, IA; 2Program in Neuroscience,
University of Iowa, Iowa City, IA; 3Pediatrics, University of Iowa,
Iowa City, IA; 4Ophthalmology & Visual Science, University of Iowa,
Iowa City, IA; 5The Stephen A. Wynn Institute for Vision Research,
University of Iowa, Iowa City, IA; 6F.M. Kirby Center for Molecular
Ophthalmology/Scheie Eye Institute, University of Pennsylvania and
Children, Philadelphia, PA.
Purpose: To help improve therapy for Leber’s congenital
amaurosis (LCA), we sought to better understand differences in the
abnormalities of inner retinal neurophysiology particular to its varied
genetic forms. We studied mice with mutations in 1) Rpe65, the gene
affected in the only form currently treatable with gene therapy; and
2) Cep290, the most commonly affected gene. We also assessed the
effectiveness of gene therapy in reversing these abnormalities in
Rpe65-/- mice, at the level of single cell and circuit physiology.
Methods: Using in vitro multielectrode recording, we measured
retinal ganglion cell (RGC) activity in rd12 (Rpe65-/-), rd16
(Cep290-/-), and wild type (wt) mouse retinas. We compared
spontaneous and light-evoked activity (full field flashes or
pseudorandom checkerboard stimuli) from perinatal to adult ages.
Some rd12 RGCs were recorded 7-90 days following subretinal
injection of a viral vector to transfect photoreceptors with normal
RPE65 (AAV2/1-hRPE65).
Results: Before eye-opening, normal developmental waves of
correlated firing appeared in both rd12 and rd16 RGCs. Spontaneous
hyperactivity emerged shortly after eye opening in both strains, but
was more prominent in rd12. The frequency spectrum of spontaneous
activity in rd16 cells included several peaks, indicating a rhythmic
oscillatory component (similar to some other degenerate strains).
In contrast, rd12 RGCs lacked such rhythmicity and had a broad
frequency spectrum.
Untreated rd12 RGCs had no light responses at any age, while
rd16 cells had reduced responses at early ages but decayed slowly.
Gene therapy restored robust light-evoked responses in many rd12
RGCs, with multiple recognizable response types and some reliable
receptive field maps. However, treatment after first eye opening did
not prevent hyperactivity, which was associated with less precise
receptive fields. Only treatment prior to eye opening prevented
hyperactivity and preserved more normal receptive field architecture
in some cases.
Conclusions: Our results provide a high resolution view of distinct
abnormalities in RGC activity particular to Rpe65- or Cep290based LCA. They indicate that the ability of gene therapy to reverse
different abnormalities in visual processing varies, and depends on
optimizing the age of treatment administration. These principles may
guide further improvements in gene therapy and other treatments.
Commercial Relationships: Steven F. Stasheff, None; Kelsey N.
Spalding, None; Frederick R. Blodi, None; Malini Shankar, None;
Sajag Bhattarai, None; Stewart Thompson, None; Jeannette
Bennicelli, None; Jean Bennett, Avalanche Technologies (C),
Gensight Biologics (C), Spark Therapeutics (C); Arlene V. Drack,
None
Support: March of Dimes Research Award # 12-FY11-200, Stephen
A. Wynn Institute for Vision Research, & The Grousbeck Family
Foundation
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 358 Poster Board Number: C0129
Presentation Time: 8:30 AM–10:15 AM
Adenovirus-mediated overexpression of SOAT1 attenuates
7-Ketocholesterol-induced cytotoxicity and inflammation in
ARPE-19 cells
Jung W. Lee, Jiahn-Dar Huang, Juan Amaral, Ignacio R. Rodriguez.
Mechanisms of Retinal Diseases Section, LRCMB, National Eye
Institute/NIH, Bethesda, MD.
Purpose: We have found that the only significant metabolism
of 7-Ketocholesterol (7KCh) in ARPE-19 cells is via fatty acid
esterification catalyzed by Sterol O-acyltransferase (SOAT1/ACAT1). The object of this study was to investigate whether adenovirusmediated overexpression of SOAT1 can attenuate 7KCh-induced
cytotoxicity and inflammation in ARPE-19 cells.
Methods: ARPE-19 cells were infected with adenovirus encoding
SOAT1 prior to 7KCh treatment. Adenovirus-GFP was used as a
negative control. Overexpression of SOAT1 protein was confirmed
by immunoblot using a rabbit polyclonal affinity purified anti-SOAT1
antibody. Cells were collected and analyzed for 7KCh, Cholesterol
(Ch) and 7KCh-fatty acid esters (7KFAEs) 24 h after 7KCh
treatment. Cell viability was determined by celluar dehydrogenase
activity. Identification and quantification of 7KCh, Ch and 7KFAEs
were performed by HPLC-UV and LCMS. IL-6, IL-8 and VEGF
protein levels in conditioned media were measured by Milliplex
Cytokine Kit using the Luminex MAGPIX instrument.
Results: Adenovirus SOAT1 infection resulted in a high level of
SOAT1 overexpression in ARPE-19 cells. In normal ARPE-19 cells
(without SOAT1 overexpression) 4 main 7KFAEs are formed which
account for 20% of the internalized 7KCh. SOAT1 overexpression
increased 7KFAEs synthesis by 1.5-fold and reduced intracellular
7KCh levels by 40%. SOAT1 overexpression significantly decreased
IL-6 and IL-8 expression in conditioned media but had no effect on
VEGF levels. SOAT1 overexpression protected the cells from 7KChinduced cytotoxicity.
Conclusions: Overexpression of SOAT1 in ARPE-19 cells attenuates
7KCh-induced cell toxicity and inflammation. The protective effect
seems to be caused by the formation of non-toxic 7KFAEs. This
seems to be the only metabolic pathway available to extra-hepatic
tissues.
Commercial Relationships: Jung W. Lee, None; Jiahn-Dar
Huang, None; Juan Amaral, None; Ignacio R. Rodriguez, None
Support: NEI intramural research program
Program Number: 359 Poster Board Number: C0130
Presentation Time: 8:30 AM–10:15 AM
INSULIN LIKE GROWTH FACTOR 1 INDUCTION OF
HYPOXIA-INDUCIBLE FACTOR MEDIATED VASCULAR
ENDOTHELIAL GROWTH FACTOR SYNTHESIS IS
DEPENDENT ON MAP KINASE ACTIVITY
Piyush C. Kothary, Allen Lee, Eric Frontera, Priyanka Varma, Danna
Bismar, Monte A. Del Monte. Ophthalmology, Univ of MichiganKellogg Eye Ctr, Ann Arbor, MI.
Purpose: Human retinal pigment epithelium (hRPE) cells appear to
play a role in the pathogenesis of proliferative diabetic retinopathy
(PDR). Hypoxia-inducible factor (HIF) is a heterodimer made of
HIF1 alpha (HIF1a) and HIF1 beta (HIF1b). We have shown that
HIF1a mediates the angiogenic factor, vascular endothelial growth
factor (VEGF) synthesis in cultured hRPE cells. However, very
little is known about the role of HIF1b in the regulation of VEGF
synthesis in hRPE cells. Since IGF1 also plays an important role in
pathogenesis of diabetes and proliferative diabetic retinopathy (PDR),
we investigated the effect of IGF1 on HIF1a, HIF1b and VEGF
synthesis in hRPE cells.
Methods: hRPE cultures were established from normal human eyes.
Cell proliferation and viability were quantitated by 3H-thymidine
(3H-thy) incorporation and by the trypan blue exclusion (T) method.
14C-VEGF, 14C-HIF1 alpha and 14C-HIF1b production were
measured by immunoprecipitation and immunohistostochemistry.
PD98059 (PD), an inhibitor of MAPK was used to study the
mechanism. Statistical significance was determined by Student “t”
test.
Results: Increasing concentrations of fetal bovine serum and IGF1
stimulated hRPE proliferation in culture as determined by T and
3H-thy. IGF1 (0-50 ng/ml) also stimulated 14C-VEGF, 14C-HIFa
and 14C-HIFb synthesis in a dose-dependent manner. PD98059
inhibited IGF1 stimulated 14C-VEGF synthesis (646.73±165.61 vs.
1184.40±247.95, CPM±SEM, p<0.05, n=4), 14C-HIFa synthesis
(249.18±60.14 vs. 383.81±96.43, CPM±SEM, p<0.05, n=5) and
14C-HIF1b synthesis (87.78±18.36 vs. 166.49±25.96, CPM±SEM,
p<0.05, n=4). Immuno-histochemical studies confirmed that IGF1
increases VEGF, HIF1a and HIF1b protiens when compared to
controls. Linear curve analysis showed that IGF1 stimulation of
HIFa and HIFb were superimposable. VEGF synthesis was less than
HIF1a and HIF1b synthesis at a lower concentration of IGF1 but was
almost same as that of HIF1a and HIF1b when exposed to higher
concentrations of IGF1.
Conclusions: We conclude that IGF1 stimulated VEGF synthesis in
hRPE cells. In addition, IGF-1 induced HIF1a and HIF1b-mediated
VEGF expression is dependent on MAP kinase signaling in these
cells. Therefore an inhibitor of MAP kinase signaling may be of
therapeutic value in PDR or proliferative vitreoretinopathy.
Commercial Relationships: Piyush C. Kothary, None; Allen
Lee, None; Eric Frontera, None; Priyanka Varma, None; Danna
Bismar, None; Monte A. Del Monte, None
Support: This work was sponsored by the Skillman Foundation
Program Number: 360 Poster Board Number: C0131
Presentation Time: 8:30 AM–10:15 AM
Organotypic Culture System for the Adult Canine Retina
Hsiang-Rong Tsai1, 2, Ákos Lukáts3, Arnold Szabo3, Christine D.
Harman1, Andras M. Komaromy1. 1Small Animal Clinical Sciences,
Michigan State University, East Lansing, MI; 2Physiology, Michigan
State Unviversity, East Lansing, MI; 3Human Morphology and
Developmental Biology, Semmelweis University, Budapest, Hungary.
Purpose: Organotypic retinal culture systems have been established
for a number of species, including swine, rodents, rabbit, and
chicken. Because of our research focus on dog models of inherited
retinal diseases, the aim of this study was to develop such a culture
system for the adult canine retina.
Methods: Globes were collected from two young adult wt dogs
(ages: 29 and 22 wks). The neurosensory retinas were collected
within 3-4 hrs post enucleation, cut into smaller pieces (2-4 mm
diameter), transferred onto a semi-porous polycarbonate membrane,
and kept in culture for 21 days. Between 24 and 31 retinal pieces
were cultured per globe. The cell culture media contained a 1:1
mixture of Dulbecco’s Modified Eagle Medium and F-12 Nutrient
Mixture, supplemented with vitamins, amino acids, and hormones.
Every 2 days, retinal pieces were fixed and processed for frozen
sections and flat-mounts, followed by cell-specific immunolabeling
for cones (hCAR, L/M-, and S-opsin), rods (rhodopsin), bipolar
(G0alpha), and glial cells (glutamine synthetase and GFAP).
Results: The adult canine retina could be maintained with our
organotypic culture system for the entire 21-day study period with
all cell types evaluated remaining viable. Progressive degenerative
changes were observed within the photoreceptors, mostly at the 2and 3-wk time points. These included gradual loss of outer and inner
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
segments and mislocalization of opsins. Sustained upregulation of
GFAP expression with formation of new glial processes was seen in
both astrocytes and Müller cells starting within the first few days in
culture.
Conclusions: This is the first successful attempt of an organotypic
culture system for the adult canine retina. The integrity of the
photoreceptors was best preserved within the first 7 days. This
may represent a valuable tool for detailed examination of disease
mechanisms and preliminary evaluation of new therapies in mutant
retinas.
Commercial Relationships: Hsiang-Rong Tsai, None; Ákos
Lukáts, None; Arnold Szabo, None; Christine D. Harman, None;
Andras M. Komaromy, None
Support: NIH Grant EY019304, Republic of China (Taiwan)
Government Scholarship for Studying Abroad, Hungarian
Scientific Research Fund (OTKA #73000), TÁMOP4.2.1.B-09/1KMRB2010-0001, MSU Faculty Startup Funds, MSUCVM Visiting International Scholar Award
Program Number: 361 Poster Board Number: C0132
Presentation Time: 8:30 AM–10:15 AM
Opposite roles of MerTK ligands Gas6 and Protein S for RPE
phagocytosis regulation
Celia Parinot1, 2, Jonathan Chatagnon1, 2, Emeline F. Nandrot1, 2.
1
Therapeutics, INSERM, U968, UPMC Univ Paris 06, UMR_S 968,
Paris, France; 2CNRS, UMR_7210, Centre de Recherche Institut de
la Vision, Paris, France.
Purpose: Daily clearance of aged photoreceptor outer segment
(POS) tips by retinal pigment epithelial (RPE) cells is crucial for
retinal health and function. 2 main extracellular ligands for the
internalization receptor MerTK, Gas6 and Protein S, have been
shown to stimulate POS phagocytosis in vitro. In vivo, both ligands
are expressed and necessary for retinal survival, however their exact
role for MerTK regulation remains unclear. We showed previously
that Protein S expression follows a circadian rhythm peaking at
maximum phagocytosis time, whereas Gas6 expression is stable
overtime. Thus, we set out to analyze how stimulation of RPE cells
by ligand/s or ligand/s production by RPE cells themselves could
regulate POS phagocytosis. We also aimed at identifying MerTK
extracellular residues required for ligand binding and POS uptake.
Methods: We incubated RPE-J cells with various doses of ligands,
either alone or in combination. We inhibited ligand expression,
alone or in combination, using siRNA in RPE-J cells. We introduced
mutations in the 2 putative binding sites of mouse MerTK
extracellular domain by site-directed mutagenesis and constructs
were transfected into rat NRK-49F fibroblasts devoid of endogenous
MerTK and in RPE-J cells. We then analyzed the phagocytic
capabilities of the cells following these various treatments using in
vitro phagocytosis assays. Treatments and results were verified using
immunoblots.
Results: Exogenous recombinant Gas6 inhibits phagocytosis with
increasing doses while Protein S is stimulatory. When combined
to a 1:1 ratio, Gas6 has a dominant effect. Inhibiting endogenous
Protein S has not effect while Gas6 or Gas6/Protein S combined
down-regulates phagocytosis. Gas6 effect is more pronounced for the
binding step whereas Protein S seems to be more implicated in the
internalization step of phagocytosis. Mutagenesis of either MerTK
minor or major binding site appears to stimulate phagocytosis,
altough to various extents.
Conclusions: These results indicate an opposite role of Gas6 and
Protein S for the regulation of daily MerTK function, Gas6 inhibiting
and Protein S stimulating MerTK. Associated with our previous in
vivo data, Gas6 seems to sustain a continuous inhibitory role on
MerTK function, whereas Protein S appears to help stimulate MerTK
at the peak phagocytic time, thus participating in the fine-tuning of
this intricate function.
Commercial Relationships: Celia Parinot, None; Jonathan
Chatagnon, None; Emeline F. Nandrot, None
Support: This study is supported by a grant from Sanofi-Fovéa.
Program Number: 362 Poster Board Number: C0133
Presentation Time: 8:30 AM–10:15 AM
Regulation of Ocular Functions by Dopamine and Melatonin
Gianluca Tosini, Kenkichi Baba. Pharmacology, Morehouse School
of Medicine, Atlanta, GA.
Purpose: Previous studies have shown that the retina, the retinal
pigment epithelium (RPE) and the cornea contain a circadian clock
that controls the circadian rhythms in PER2::LUC bioluminescence.
Additional studies have indicated that only in the retina the
PER2::LUC rhythm can be phase-shifted by light, thus suggesting
that other signals are used by the retina to entrain the circadian
rhythms in other ocular structures. Melatonin (MLT) and dopamine
(DA) are known to be the key molecules to regulated retinal circadian
rhythms. In this study we investigated the role of DA and MLT to
and their associated receptors in the regulation of RPE and cornea
circadian rhythm.
Methods: Eyes were obtained from PER2::LUC mice, the eyecups were dissected, and the retina, RPE-choroid and cornea were
cultured at 37 oC with 199 medium containing 0.1mM D-Luciferin
K salt. The bioluminescence emitted by these tissues was measured
using a LumiCycle. After 3-4 days of culture, DA (100uM), MLT
(100nM) and DA or MLT receptor agonists were added to the culture
at different circadian times, and the recording was continued another
5 days. Activity of focal adhesion kinase (pFAK/FAK) in control
and D2R knock-out mice was measured by western blotting whereas
central corneal thickness (CCT) in mice lacking melatonin receptors
was measured using a ultrasound pachometer, Corneo-Gage Plus
1AS.
Results: Administration of DA, but not MLT, phase-shifted
PER2::LUC bioluminescence rhythm in the RPE-choroid.
Sumanirole (D2R agonist) induced a significant phase-shift during
the late night -early subjective day, whereas PD168077 (D4R
agonist) did not produce a significant phase-shift of the PER2::LUC
bioluminescence rhythm. Conversely, administration of MLT, but
not DA, phase-shifted PER2::LUC bioluminescence rhythm in the
cornea. Mice lacking D2R signaling showed a change in the timing of
the daily rhythm in FAK activation whereas removal MLT signaling
affected the daily rhythm in CCT.
Conclusions: Our data indicate that DA via D2 receptors can phaseshift the circadian rhythm in PER2::LUC bioluminescence rhythm in
the mouse RPE, whereas MLT can phase-shift the circadian rhythm
in PER2::LUC bioluminescence in the cornea. Thus indicating that
the retina uses DA and MLT to entrain other tissues within the eye.
Our data also indicate that activation of these signaling pathways is
the correct timing of circadian functions in these tissues.
Commercial Relationships: Gianluca Tosini, None; Kenkichi
Baba, None
Support: EY022216
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 363 Poster Board Number: C0134
Presentation Time: 8:30 AM–10:15 AM
Inhibition of αB Crystallin Induces Mesenchymal to Epithelial
Transition in RPE cells Through Down-regulation of Snail and
Slug
Keijiro Ishikawa1, 2, Ram Kannan1, Christine Spee2, Parameswaran
G. Sreekumar1, David R. Hinton2. 1Arnold and Mabel Beckman
Macular Research Center, Doheny Eye Institute, Los Angeles, CA;
2
Ophthalmology and Pathology, University of Southern California,
Los Angeles, CA.
Purpose: Epithelial-to-mesenchymal transition (EMT) of the
retinal pigment epithelium (RPE) is a hallmark of proliferative
vitreoretinopathy and age-related macular degeneration. The role
of small heat shock proteins (sHSPs) in EMT has not been studied.
The aim of this study was to investigate the role of αB crystallin, a
prominent member of sHSP family, in EMT of RPE cells.
Methods: All studies were conducted in cultured human primary
fetal RPE cells at two to four passages. We studied the expression
changes of αB crystallin after stimulation with TGFβ2 (5ng/ml) by
Western blotting and realtime RT-PCR analysis. To examine whether
modulation of αB crystallin expression can alter EMT markers, we
investigated the effects of αB crystallin silencing on the expression
of E-cadherin, α-SMA and the transcriptional factors such as Snail
and Slug with or without TGFβ2 by Western blotting and realtime
RT-PCR. To examine whether αB crystallin is involved in TGFβ/
SMAD signaling pathway, we also studied the effects of αB crystallin
knockdown on phosphorylation of SMAD2/3 induced by TGFβ2
stimulation.
Results: The levels of mRNA and protein of αB crystallin were
not significantly altered in RPE cells after TGFβ2 stimulation.
Suppression of αB crystallin by siRNA induced significant upregulation of E-cadherin and down-regulation of α-SMA, Snail
and Slug in mRNA and protein expressions (p<0.05 vs controls).
Knockdown of αB crystallin did not alter TGFβ2-induced
phosphorylation of SMAD2/3.
Conclusions: Our results show that αB crystallin plays a critical role
through modulation of Snail and Slug in EMT but not through TGFβ/
SMAD signaling pathway in this process.
Commercial Relationships: Keijiro Ishikawa, None; Ram
Kannan, None; Christine Spee, None; Parameswaran G.
Sreekumar, None; David R. Hinton, None
Support: EY03040, EY01545, Arnold and Mabel Beckman
Foundation and Research to Prevent Blindness
Program Number: 364 Poster Board Number: C0135
Presentation Time: 8:30 AM–10:15 AM
Valproic Acid Induced Inhibition of Fibroblast Growth Factor 2
Synthesis in Human Retinal Pigment Epithelial Cells
Andrew Kumar1, Piyush C. Kothary2, Benjamin Rossi2, Chintan
Mehta2, Monte A. Del Monte2. 1Wayne State University, Detroit, MI;
2
Department of Ophthalmology, University of Michigan, Ann Arbor,
MI.
Purpose: Human retinal pigment epithelial cells (hRPE) have been
implicated in the pathogenesis of age related macular degeneration.
Valproic acid (VPA), an epigenetic factor, reduces apoptosis in
immortalized ARPE19 cells that were subjected to oxidative injury.
In addition, VPA has also been shown to inhibit cancer cell growth
and angiogenesis in various animal models. Since very little is known
about the effect VPA on primary cultured hRPE cells, we examined
the effect of VPA on hRPE cell viability, as well as synthesis of the
angiogenic factor, fibroblast growth factor 2 (FGF2) and its receptor
(FGFR1) in hRPE cells.
Methods: Primary hRPE cell cultures were established from donor
non-pathologic human eyes obtained from the Michigan Eye Bank.
Cell viability was assessed by the trypan blue exclusion method
(T) and the cell proliferation was measured by 3H-thymidine
incorporation (3H-thy). 14C-methionine labeled FGF2 (14C-FGF2)
and 14C-FGFR1 synthesis were quantitated by immunoprecipitation
using FGF2, and FGFR1 specific antibody. Phase contrast
microscopy and nuclear staining studies were performed by DAPI.
Data were tabulated on Excel spreadsheets, mean ± SEM were
determined and statistical significance was established by student’s
t-test.
Results: Fetal Bovine Serum (FBS) and FGF2 stimulated hRPE cell
proliferation and viability in a dose-dependent manner as shown with
trypan blue exclusion and 3H-Thymidine incorporation. Valproic acid
(0.5 mM), in contrast, reduced the cell viability (1.75 ± 0.37 vs 3.25
± 0.87 cells x 10000 ± SEM, n=8, p<0.05). Increasing concentrations
of valproic acid decreased the production of immunoprecipitated
14C-FGF2, and 14C-FGFR1 in hRPE cells in a dose dependent
manner. Nuclear staining studies confirmed a reduction in hRPE cell
number when compared with controls.
Conclusions: Valproic acid reduces the synthesis of the angiogenic
factor FGF2, and its receptor FGFR1 in cultured hRPE cells. It
also reduces overall cultured hRPE cell proliferation and viability.
Since others have shown an anti-apoptotic effect of VPA in injured
ARPE19 cells and we show an inhibitory effect of VPA on primary
cultured normal hRPE cells, it appears that additional research into
the mechanism of VPA anti- apoptotic and anti-proliferative action
is required to support its use as a possible therapeutic agent in retinal
degenerative and proliferative diseases.
Commercial Relationships: Andrew Kumar, None; Piyush C.
Kothary, None; Benjamin Rossi, None; Chintan Mehta, None;
Monte A. Del Monte, None
Support: Skillman Foundation
Program Number: 365 Poster Board Number: C0136
Presentation Time: 8:30 AM–10:15 AM
Inflammasome-related cytokine secretion secondary to lysosomal
membrane permeabilization in retinal pigment epithelial cells
Susannah M. Spieker, Lena K. Mohr, Carolina Brandstetter, Andrea
V. Hoffmann, Frank G. Holz, Tim U. Krohne. Ophthalmology,
University of Bonn, Bonn, Germany.
Purpose: Photooxidative damage to the retinal pigment epithelium
(RPE) as well as chronic inflammatory processes in the sub-RPE
space are involved in the pathogenesis of age-related macular
degeneration (AMD). We have shown that lipofuscin phototoxicity
can activate the NLRP3 inflammasome in RPE cells by inducing
lysosomal membrane permeabilization (LMP). Here, we investigate
the effects of LMP-induced inflammasome activation on the secretion
of inflammatory and angiogenic cytokines related to the pathogenesis
of AMD.
Methods: LMP was induced in primary human RPE cells and
ARPE-19 cells either by Leu-Leu-OMe or by incubation with
4-hydroxnonenal-modified photoreceptor outer segments (HNE-POS)
to induce lipofuscinogenesis and subsequent irradiation with blue
light (0.8 mW/cm2) for up to 6 hours. LMP was quantified by flow
cytometry by means of acridine orange staining. Cytokine secretion
was measured using dot blot antibody arrays (RayBiotech) and
specific ELISAs (R&D Systems). Polarized cytokine secretion was
analyzed in RPE cells cultured on permeable membranes. Paracrine
cytokine effects were investigated by incubating human vascular
endothelial cells (HUVEC) with RPE-conditioned media.
Results: Secretion levels of 42 inflammation- and angiogenesisrelated cytokines were investigated in RPE cells following LMP
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
induction. LMP resulted in NLRP3 inflammasome activation
with increased secretion of the interleukins IL-1β and IL-18. In
contrast, secretion of vascular endothelial growth factor (VEGF)
was significantly decreased. Migration and proliferation of vascular
endothelial cells incubated with conditioned media of LMP-treated
RPE cells was reduced. Specific inhibition of caspase-1 or cathepsin
B prevented inflammasome-related cytokine secretion.
Conclusions: Activation of the NLRP3 inflammasome by lipofuscinmediated lysosomal membrane permeabilization in RPE cells results
in the release of pro-inflammatory interleukins with simultaneous
reduction of constitutive VEGF secretion. While these results support
a potential role of inflammasome activation in lipofuscin-related RPE
cell damage in early-stage and atrophic AMD, they argue against a
direct involvement of the NLRP3 inflammasome in the development
of neovascular AMD.
Commercial Relationships: Susannah M. Spieker, None; Lena K.
Mohr, None; Carolina Brandstetter, None; Andrea V. Hoffmann,
None; Frank G. Holz, Acucela (C), Acucela (F), Allergan (C),
Allergan (F), Bayer (C), Bayer (F), Boehringer Ingelheim (C),
Genentech (C), Genentech (F), Heidelberg Engineering (C),
Heidelberg Engineering (F), Merz (C), Novartis (C), Novartis (F),
Optos (F), Roche (C), Zeiss (F); Tim U. Krohne, Novartis (C)
Support: German Research Foundation (DFG, grant KR 2863/71); Pro Retina Research Foundation; German Ophthalmological
Society (DOG): University of Bonn BONFOR/SciMed Program; Dr.
Eberhard und Hilde Rüdiger Foundation
Program Number: 366 Poster Board Number: C0137
Presentation Time: 8:30 AM–10:15 AM
RETINAL DEGENERATION IN CCL2/DAF1 DOUBLEDEFICIENT MICE
Minzhong Yu1, Ping Bu2, Brent A. Bell1, Evgenii Boriushkin5, Feng
Lin3, James Qiao2, Gwen Sturgill-Short4, 1, Xiaoshan Yu1, Sarah X.
Zhang5, Neal S. Peachey1, 4. 1Cole Eye Institute, Cleveland Clinic
Foundation, Cleveland, OH; 2Department of Ophthalmology, Loyola
University Chicago, Maywood, IL; 3Department of Immunology,
Cleveland Clinic Lerner College of Medicine, Cleveland, OH;
4
Research Service, Cleveland Veterans Affairs Medical Center,
Cleveland, OH; 5Departments of Ophthalmology and Biochemistry,
SUNY-Buffalo and SUNY Eye Institute, Buffalo, OH.
Purpose: Chemoattractant chemokine ligand 2 (CCL2) can recruit
choroidal macrophages to clean up retinal debris, and reduce
the inflammation response related to complement activation.
Complement decay-accelerating factor (DAF) is a protein which
is encoded by the Daf1 gene. DAF is a membrane regulator of the
alternative pathway of complement that protects self cells from
autologous complement attack through blocking the assembly the C3convertase (C3bBb complex) of the alternative pathway, accelerating
the disassembly of existing C3-convertase, and finally preventing
the formation of the membrane attack complex. In view of the link
between complement genes and retinal disease, we evaluated the
retina of mice lacking Ccl2 and/or Daf1.
Methods: C57BL/6J, Ccl2−/−, Daf1−/− and Ccl2-/-/ Daf1-/- (DKO) mice
were studied. Electroretinography, histological analysis by light
microscopy, c3d staining in situ, GRP78 staining in situ, detection of
apoptotic cells in situ by TUNEL and fundus imaging by SLO were
performed.
Results: At 1 year of age, the a- and b-wave amplitudes of Ccl2/Daf1-/-, Ccl2-/- and Daf1-/- mice were reduced compared to those
of WT mice, and the reduction in DKO mice was more prominent
than in either single knockout line. TUNEL staining showed no
apoptotic cells in WT retina, but many in the ONL and INL in
the DKO retina, and a reduced density in these layers of Ccl2-/- or
Daf1-/- mice. H&E staining of retinal sections showed that cell loss
was more pronounced in the inner and outer retina of DKO than in
Ccl2-/- or Daf1-/- mice. Immunohistochemical analysis showed that
c3d deposition along the RPE is elevated in the DKO mice, and
to a lesser degree in Ccl2-/- retina, compared to WT mice. In Daf1/mice, c3d deposition is comparable to WT mice. SLO-AF488nm
fundus imaging showed increased number of autofluorescent foci,
suggestive of inflammatory cell infiltration, in the three mutant
lines with the greatest number in the DKO retina compared to WT.
Background autofluorescence emanating from lipofuscin in RPE of
DKO appears elevated relative to C57BL/6J, Ccl2-/-, and Daf1-/- mice.
Immunostaining showed reduced levels of GRP78 in the DKO retina
and modest decrease in the inner retina of ccl2-/- mice.
Conclusions: Mouse mutants for Ccl2-/-, and/or Daf1-/- mice have a
mild retinal degeneration while Ccl2-/-/Daf1-/- DKO mice have a more
severe loss of retinal cells. Cell loss involves both the inner and outer
nuclear layers.
Commercial Relationships: Minzhong Yu, None; Ping Bu, None;
Brent A. Bell, None; Evgenii Boriushkin, None; Feng Lin, None;
James Qiao, None; Gwen Sturgill-Short, None; Xiaoshan Yu,
None; Sarah X. Zhang, None; Neal S. Peachey, None
Support: American Health Assistance Foundation, Research
to Prevent Blindness, Foundation Fighting Blindness, VA and a
challenge grant from Research to Prevent Blindness.
Program Number: 367 Poster Board Number: C0138
Presentation Time: 8:30 AM–10:15 AM
Cell Death after Ocular Blast Trauma is Primarily Non-Apoptotic
Courtney Bricker-Anthony, Tonia S. Rex. Ophthalmology, Vanderbilt
University, Nashville, TN.
Purpose: To determine what cell death pathways are activated after
ocular blast trauma.
Methods: We exposed the left eyes of 3 month-old mice to a single,
26psi overpressure airwave. Mice were collected at 1, 3, 7, 14, and 28
days post-injury. All eyes were cryo-embedded, sectioned and labeled
with antibodies against caspase 1, RIP, RIP3, cleaved caspase-3,
calpain, cathepsin D, and TUNEL.
Results: The peak of cell death was identified by TUNEL and this
time point was then used as a reference for all cell death marker
analyses. No changes in calpain or cathepsin D were detected postblast. Rare photoreceptors were labeled with the apoptosis marker,
cleaved caspase 3 and some condensed nuclei were detected in resin
tissue sections. Labeling with the pyroptosis marker, caspase 1, was
present in the inner retina post-blast. RIP, a marker for necroptosis,
was localized to the Müller glia and inner plexiform layer in normal
and post-blast retina. RIP3 was detected in the inner plexiform layer
and in a small number of cells in the retinal ganglion cell layer and
inner nuclear layer in controls. After blast, RIP3 was also present in
the outer nuclear layer.
Conclusions: These results suggest that blast injury causes cell death
via multiple pathways. The majority of photoreceptor cell death
appears to be through necroptosis, although some apoptosis also
occurs. The inner retina cells may die via necroptosis and pyroptosis.
Commercial Relationships: Courtney Bricker-Anthony, None;
Tonia S. Rex, None
Support: DoD W81XWH-10-1-0528, RPE P30-EY008126
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 368 Poster Board Number: C0139
Presentation Time: 8:30 AM–10:15 AM
Profile of N-Glycans in Human Vitreous
Saori Takashina1, 2, Kousuke Noda1, 2, Maho Amano3, Tetsu Ohashi3,
Yoko Dong1, 2, Satoshi Kinoshita1, 2, Wataru Saito2, Atsuhiro Kanda1, 2,
Shin-Idhiro Nishimura3, Susumu Ishida1, 2. 1Laboratory of Ocular Cell
Biology and Visual Science, Hokkaido University Graduate School of
Medicine, Sapporo, Japan; 2Department of Ophthalmology, Hokkaido
University Graduate School of Medicine, Sapporo, Japan; 3Field
of Drug Discovery Research, Faculty of Advanced Life Science,
Graduate School of Life Sciences, Hokkaido University, Sapporo,
Japan.
Purpose: Glycans are biopolymers bearing biological information
and regarded as the third major class of cellular macromolecules,
following nucleic acids and proteins. Glycosylation is an important
process of post-translational modification that changes the functions
of proteins, and it has recently been reported that serum profile
of N-glycans is largely altered in patients with systemic diseases.
However, little is known about the profiles of glycans in ocular
tissues. In this study, we analyzed the profile of N-glycans in
human vitreous fluid obtained from patients with non-proliferative
vitreoretinal diseases.
Methods: A total of 20 vitreous samples and 20 plasma samples (10
males and 10 females, mean age 64.6±4.7 y/o) were collected from
patients with epiretinal membrane (ERM, n=9) or idiopathic macular
hole (MH, n=11) who underwent pars plana vitrectomy at Hokkaido
University Hospital from 2010 to 2013. Profiles of N-glycans in
the vitreous and plasma samples were analyzed using the novel
technique, “glycoblotting method”.
Results: Thirteen N-glycans were detected in all the vitreous
samples. All the glycans were included in 32 N-glycans detected in
plasma samples. The concentration of N-glycans in vitreous samples
(132.3±29.1pmol/100μg protein) was significantly lower compared
with those in plasma samples (505.7±16.9pmol/100μg protein,
P<0.001). Predominant N-glycan [(Hex)2 (HexNAc)2 (NeuAc)2
+ (Man)3(GlcNAc)2] was common in both groups, but the most
of the other N-glycans content ratios were different. There was no
significant difference in concentration and composition of N-glycans
in the vitreous between ERM and MH, or between males and females
either.
Conclusions: Our data for the first time provide the information on
profile of N-glycans in human vitreous humor.
Commercial Relationships: Saori Takashina, None; Kousuke
Noda, None; Maho Amano, None; Tetsu Ohashi, None; Yoko
Dong, None; Satoshi Kinoshita, None; Wataru Saito, None;
Atsuhiro Kanda, None; Shin-Idhiro Nishimura, None; Susumu
Ishida, None
Support: JSPS KAKENHI (Grant-in-Aid for Challenging
Exploratory Research, Grant Number 24659754, Charitable Trust
Fund for Ophthalmic Research in Commemoration of Santen
Pharmaceutical’s Founder (to A.K.),
Program Number: 369 Poster Board Number: C0140
Presentation Time: 8:30 AM–10:15 AM
A newly recognized molecule in the Retinal Pigment Epithelium
Karina E. Guziewicz1, Emily V. Dutrow1, Keiko Miyadera1, Jackie
Meyer2, Ruchira Singh2, 3, Kathleen Boesze-Battaglia5, David M.
Gamm2, 4, Gustavo D. Aguirre1. 1Clinical Studies-Philadelphia,
University of Pennsylvania, Philadelphia, PA; 2Waisman Center,
Madison, WI; 3McPherson Eye Research Institute, Madison, WI;
4
Department of Ophthalmology, University of Wisconsin, Madison,
WI; 5Department of Biochemistry, University of Pennsylvania,
Philadelphia, PA.
Purpose: Genotype-phenotype correlations in human BEST1associated disorders are considered complex. The pleiotropic effects
of BEST1, together with incomplete penetrance and multifaceted
clinical expression, strongly suggest that other factors modulate
the rate of progression and severity of the disease. Still, no genetic
components explaining this substantial clinical variability have
been identified. Canine multifocal retinopathy (cmr) caused by
mutations in the dog ortholog is a well-established model for human
bestrophinopathies that captures the full clinical picture of the
disease, including the variation in its clinical presentation. To expand
the spectrum of potential disease modifiers, we employed liquid
chromatography-tandem mass spectrometry (LC-MS/MS) to explore
the pool of molecules in a healthy and cmr-affected retinal pigment
epithelium (RPE).
Methods: LC-MS/MS technology (Thermo LTQ-Orbitrap XL)
performed on total canine and human RPE lysates was utilized in
these discovery studies. The findings were verified by western blot
and immunohistochemistry (mouse monoclonal EM-09) in vivo and
in vitro, and visualized using confocal microscopy (Leica TCS SP5).
Exon-intron boundaries of the canine gene were defined by direct
sequencing and verified against cDNA library.
Results: Mass spectrometry-based RPE proteome profiling revealed
a molecule previously uncharacterized in the eye, desmoyokin. Its
RPE-specific expression in the retina was confirmed by western blot
analysis and immunohistochemistry in both retinal cryosections
of dog and human and in the RPE-derived in vitro model systems
such as canine primary RPE, hfRPE, hiPSC-RPE and ARPE19
cells. Confocal microscopy defined desmoyokin as a plasma
membrane-associated protein localized at the intracellular face of the
plasmalemma, however, its subcellular localization depends on the
formation of cell-cell contacts. The canine gene contains two noncoding and three protein-coding exons (5549aa) and exhibits high
level of sequence conservation with its human ortholog (5890aa).
Conclusions: Here, we report a novel protein that is RPE-specific
in the retina and has not been previously characterized in the eye.
Our preliminary findings suggest its role in cell-cell adhesion and
cytoarchitecture of the plasma membrane; however, its exact function
in a healthy and BEST1-affected RPE is still under investigation.
Commercial Relationships: Karina E. Guziewicz, None; Emily
V. Dutrow, None; Keiko Miyadera, None; Jackie Meyer, None;
Ruchira Singh, None; Kathleen Boesze-Battaglia, None; David M.
Gamm, None; Gustavo D. Aguirre, None
Support: NEI/NIH EY-06855, -17549, -001583, -10420, Macula
Vision Research Foundation, Foundation Fighting Blindness, FFB
Wynn-Gund TRAP Award, Hope for Vision, Van Sloun Fund for
Canine Genetic Research;
Program Number: 370 Poster Board Number: C0141
Presentation Time: 8:30 AM–10:15 AM
Differential Composition of Docosahexaenoic Acid and Very
Long Chain Polyunsaturated Fatty Acids in Rod and Cone
Photoreceptor Membranes
Martin-Paul G. Agbaga1, 3, Dana K. Merriman4, Richard S. Brush1,
3
, Todd Lydic6, Shannon M. Conley2, Muna I. Naash2, Reid E.
Gavin7, Julia V. Busik5, Robert E. Anderson1, 2. 1Ophthalmology,
Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Cell Biology,
University of Oklahoma Health Sciences Center, Oklahoma City,
OK; 3Ophthalmology, Dean McGee Eye Institute, Oklahoma City,
OK; 4McPherson Eye Research Institute, University of Wisconsin,
Madison, WI; 5Physiology, Michigan State University, East Lansing,
MI; 6Biochemistry& Molecular Biology, Michigan State University,
East Lansing, MI; 7Chemistry, Michigan State University, East
Lansing, MI.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Polyunsaturated fatty acids (PUFA) 22:6n3 and 20:4n6
are highly enriched in the outer segment (OS) membranes of
vertebrate retina, where they are further elongated to very long
chain polyunsaturated fatty acids (C28-C40, VLC-PUFA). In
photoreceptors, these PUFA are found only in phosphatidylcholine
(PC). The cone-rich human macula has lower levels of 22:6n3
compared to rod-rich peripheral retina. To get a better understanding
of the role of C20-C40 PUFA in the retina, we performed
comprehensive lipidomic analyses of photoreceptor membrane
preparations from rod- and cone-dominant retinas.
Methods: Total lipids were extracted from whole retinas and
photoreceptor OS membrane preparations of rod-dominant
[C57BL/6J (WT), Nrl+/- mice, Wistar rats] and cone-dominant [Nrl/- mice, 13-lined ground squirrels (Ictidomys tridecemlineatus), Fox
squirrel (Sciurus niger) and Tree Shrew (Tupaia belangeril)] retinas.
Fatty acid methyl esters and whole lipids were analyzed by GC/MS
and ESI/MS/MS, respectively.
Results: While there were no significant differences between glycerol
phosphatidylcholine (GPC) lipids GPC-32:0 (16:0/16:0), GPC-34:01
(16:0/18:1), GPC-36:04 (16:0/20:4), GPC 36:01 (18:0/18.1) and
GPC-38:04 (18:0/20:4n6) in rod- and cone-dominant retinas, there
was a significant decrease in GPC-40:06 (18:0/22:6) and GPC-44:12
(22:6/22:6) in both whole retina and OS membranes isolated from
cone-dominant retinas compared to rod dominant retinas. VLCPUFA GPC-54:12 (22:6/32:6), GPC-56:12 (22:6/34:6), GPC-54:11
(22:6/32:5), GPC-54:10 (22:6/32:4), GPC-56:11 (22:6/34:5), and
GPC-58:12 (22:6/36:6) were highly enriched in rod-dominant retinas,
but were significantly reduced in the cone-dominant retina.
Conclusions: Compared to the well-characterized rod-dominant
retinas, cone-dominant retinas have significantly different lipid
composition in both whole retina and OS enriched membranes. Since
PUFA are necessary for optimal G-protein-coupled receptor signaling
in rods, these findings suggest that cones may not have the same lipid
requirements as rods. This may account for the reduced 22:6n3 and
VLC-PUFA levels found in the cone-rich macula compared to rodrich peripheral retina.
Commercial Relationships: Martin-Paul G. Agbaga, None; Dana
K. Merriman, None; Richard S. Brush, None; Todd Lydic, None;
Shannon M. Conley, None; Muna I. Naash, None; Reid E. Gavin,
None; Julia V. Busik, None; Robert E. Anderson, None
Support: NIH/NEI Grants EY04149, EY00871, and EY021725;
Research to Prevent Blindness, Inc.; the Foundation Fighting
Blindness to REA; BrightFocus Foundation grant to MPA, University
of Wisconsin Oshkosh 13-lined Ground Squirrels Captive Breeding
Program and Max Planck Florida Institute Animal Resource Center
NEI-7R01EY006821-27
Program Number: 371 Poster Board Number: C0142
Presentation Time: 8:30 AM–10:15 AM
Autophagy over the lifespan: using fetal, stem cell, and adult
RPE cultures to model the pathogenesis of AMD
Katherine J. Davis1, 2, Shabnam Pakneshan1, 2, Peter Y. Zhao1,
2
, Haben Kefella1, Ron A. Adelman1, Lawrence J. Rizzolo1, 2.
1
Ophthalmology and Visual Science, Yale School of Medicine, New
Haven, CT; 2Surgery, Yale School of Medicine, New Haven, CT.
Purpose: Dysfunctional autophagy in the retinal pigment epithelium
(RPE) has been implicated as a potential therapeutic target in agerelated macular degeneration (AMD). To explore how autophagy
changes over the lifespan and in response to photoreceptor outer
segments (POS), we compared induced pluripotent stem cell RPE
(iPS-RPE), human fetal RPE (hfRPE), and adult donor RPE (adRPE).
Methods: RPE was cultured from 16-week human fetuses and
cadaveric eyes. Stem cell-derived RPE was prepared from human
embryonic stem cells (hESC-RPE) and induced pluripotent stem
cells (iPS-RPE). LC3 conversion (immunoblotting) and changes in
autophagy-related gene expression (qRT-PCR) were used to monitor
autophagy. Relative maturity of RPE cultures was assessed using a
panel of signature and maturation genes (qRT-PCR). Autophagy was
manipulated with an inhibitor, Spautin-1, and inducer, Rapamycin.
iPS-RPE were challenged with porcine POS daily for up to 1 month,
and monitored with confocal-immunomicroscopy. The health of
RPE cultures was assessed by the transepithelial electrical resistance
(TER).
Results: Autophagic flux (LC3 conversion) increased from stem
cell to 53-year-old ad-RPE, but was reduced in 90-year-old RPE.
Rapamycin stimulated RPE autophagy, but Spautin-1 inhibited
autophagy only partially—the strongest effect was on 90-year-old adRPE. qRT-PCR revealed quantitative differences in the expression of
autophagy- and maturation-related genes. In iPS-RPE, the expression
level of most maturation genes was similar to hfRPE, but some
were at the level of less mature hESC-RPE. However, iPS-RPE and
ad-RPE exhibited substantially higher levels of autophagy-related
genes than hfRPE. In iPS-RPE, continuous feeding of POS over three
weeks lowered TER to physiologic levels. One-week exposure to
POS had little effect on iPS-RPE autophagy gene-expression, but did
result in the accumulation of autofluorescent granules.
Conclusions: The characteristics of autophagy depended on the
culture model: autophagy gene expression in iPS-RPE more closely
resembled ad-RPE than hfRPE. Pending the examination of more
donors, partial inhibition by Spautin-1 suggests that a non-canonical
autophagy pathway is replaced in old age by the canonical pathway.
The accumulation of lipofuscin-like granules induced by POS
indicates that complementary RPE cultures will be a valuable aid to
explore targets for therapeutic agents for AMD.
Commercial Relationships: Katherine J. Davis, None; Shabnam
Pakneshan, None; Peter Y. Zhao, None; Haben Kefella, None; Ron
A. Adelman, None; Lawrence J. Rizzolo, None
Support: James G. Hirsch, M.D. Yale University Medical Student
Research Fellowship (Davis), CT Stem Cell Research Fund 10SBC02
(Rizzolo), Leir Foundation (Adelman), Newman’s Own Foundation
(Adelman)
Program Number: 372 Poster Board Number: C0143
Presentation Time: 8:30 AM–10:15 AM
Protective Effect of Photoreceptor Outer Segments Phagocytosis
for Retinal Pigment Epithelial Cells by PGC-1α/SIRT1
Murilo F. Roggia, Yasuo Noda, Tomoyasu Shiraya, Takashi Ueta.
Ophthalmology, University of Tokyo, Tokyo, Japan.
Purpose: Aging process is accepted to present an association with
different diseases affecting the retinal pigment epithelial cells (RPE).
Both peroxisome proliferator-activated receptor gamma coactivator
1-alpha (PGC-1α) and sirtuin 1 (SIRT1) are considered as important
regulators of metabolism and physiological aging phenomenon. The
objective of this study was to evaluate the role of photoreceptor outer
segments (POS) phagocytosis in aging signaling in RPE cells.
Methods: Human RPE cells (ARPE-19) were treated with POS
isolated from porcine eyes for 3-9 hours. Different concentrations
of POS were used. Both PGC-1α and SIRT1 mRNA and protein
expression were measured before and after treatment with POS.
Expressions of antioxidant enzymes and senescence was analyzed.
Mitochondrial biogenesis was evaluated by measuring the
mitochondrial DNA replication. Knockdown of PGC-1α and SIRT1
in RPE cells was analyzed.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: First, we observed the knockdown of PGC-1α or SIRT1 by
small interfering RNAs resulted in decreased expression of antioxidant enzymes and increased expression of senescence markers
in RPE cells, confirming the involvement of these molecules in
aging signaling in RPE. When treated with POS, a dose-dependent
elevation in the expression of PGC-1α and SIRT1 was observed
in RPE cells while the treatment with latex beads did not change
the expression. Consistently, expression of anti-oxidant enzymes
was upregulated and expression of senescence markers was
downregulated in RPE cells phagocytosing POS. Furthermore,
mitochondrial biogenesis was induced by POS in RPE cells shown by
the increased mitochondrial DNA replication and the expression of
prohibitin, a mitochondrial marker protein.
Conclusions: Our data indicates that one of the physiological roles of
POS phagocytosis for RPE cells might be the regulation of PGC-1α/
SIRT1 pathway that leads to mitochondrial biogenesis and expression
of anti-oxidant enzymes, as well as the downregulation of senescence
markers.
Commercial Relationships: Murilo F. Roggia, None; Yasuo Noda,
None; Tomoyasu Shiraya, None; Takashi Ueta, None
Program Number: 373 Poster Board Number: C0144
Presentation Time: 8:30 AM–10:15 AM
Inflammatory and neovascular markers identified by deep
shotgun proteomic profiling of human retinal and choroidal
vascular endothelial cells
Binoy Appukuttan1, Phillip A. Wilmarth2, Yuzhen Pan3, Larry L.
David2, Justine Smith1. 1Clinical & Molecular Medicine, Flinders
Univ of South Australia - FU, Adelaide, SA, Australia; 2Biochemistry
& Molecular Biology, Oregon Health & Science University, Portland,
OR; 3Casey Eye Institute, Oregon Health & Science University,
Portland, OR.
Purpose: Endothelial cells of the retina (REC) and choroid (CEC)
have distinct protein phenotypes that may influence tissue-specific
diseases. Characterizing the proteomic profiles of REC and CEC may
allow identification of disease-associated cell-specific factors.
Methods: Cultures of REC and CEC were prepared, using Dynal
magnetic beads linked to an anti-CD31 antibody, from 5 human
cadaver donors. Cell proteins extracts (1mg) were digested and
peptides were fractionated on a polysulfoethyl A cation exchange
column. Fractions were analysed by reverse-phase LC-MS using an
Agilent 1100 series capillary LC system and LTQ linear ion trap.
BioWorks generated DTA files and SEQUEST (no enzyme, 2.5 Da
tolerance, C+57 static mod) was used to assign peptide sequences
to MS2 spectra with human Sprot target/decoy database. The PAW
pipeline was used to control peptide error rates, and count peptide
identifications and total protein spectral counts (SpC). Differential
protein expression analysis was performed using the edgeR package
in R. Differential expression candidates were selected based on
Benjamini-Hochberg FDR-corrected p-values: high significance
(P<0.01); medium (0.01≤P<0.05); low (0.05≤P<0.1); no difference
(P≥0.1). EdgeR candidates were verified using log-transformed
ratios of the average SpC re-normalized by a sliding window
Z-transformation. Gene ontology (GO) and GO Slim annotations
were compiled.
Results: PAW processing identified 1,112,384 peptides from
5,281,964 MS2 spectra, mapping to 3,200–4,000 proteins per sample.
Overall 4,942 proteins were identified in all REC and CEC donors.
2,521 (53%) proteins were observed in all 10 samples. Of the 280
proteins showing highly significant differential expression between
EC types, 176 were significantly more abundant in REC and 104
were significantly more abundant in CEC. GO for “angiogenesis”,
“inflammation” and “hypoxia” identified ANGPT2, CTGF,
ADAMTS-1, ICAM1 and VCAM1 as highly expressed proteins in
REC and NRP1, UACA and CAPN2 as more abundant in CEC.
Conclusions: Analysis of ocular EC-specific molecular phenotypes
may provide insights into the pathogenesis of disease. The impact of
differentially expressed angiogenic or inflammatory proteins will be
tested in disease-relevant functional assays. These proteins may be
targets for specific therapies of retinal or choroidal vasculopathies.
Commercial Relationships: Binoy Appukuttan, None; Phillip
A. Wilmarth, None; Yuzhen Pan, None; Larry L. David, None;
Justine Smith, None
Support: National Institutes of Health (R01 EY019875); Australian
Research Council (Future Fellowship to JRS); Ophthalmology Core
Facility: NIH grant P30 EY010572; NIH grant EY007755 NIH grant
P30 EY010572; NIH grant EY007755
Program Number: 374 Poster Board Number: C0145
Presentation Time: 8:30 AM–10:15 AM
Proteomic profiles of normal and gliotic human retinae and
comparison with Müller stem cells in vitro
Karen Eastlake1, Wendy Heywood2, Kevin Mills2, Philip J. Banerjee1,
David G. Charteris1, Peng Khaw1, G Astrid Limb1. 1National
Institute for Health Research (NIHR) Biomedical Research Centre at
Moorfields Eye Hospital NHS Foundation Trust and UCL Institute of
Ophthalmology, UCL, London, United Kingdom; 2Institute of Child
Health, UCL, London, United Kingdom.
Purpose: Müller glial stem cells are responsible for the retinal
regeneration observed in fish and amphibians after injury. The
human retina contains a population of Müller glia with stem cell
characteristics, but it is not known to undergo regeneration following
disease. It may be possible that gliotic retina may express factors that
prevent growth and differentiation of Müller glia in situ and this may
be important to investigate in order to explore potential therapies to
promote endogenous regeneration of the human retina. This study
aimed to examine differences between the proteomic profiles of
normal and gliotic human retinae and to compare these with the
proteomic profile of Müller stem cells.
Methods: Normal cadaveric and gliotic retina were obtained
according to guidelines from the Local Ethics Committee at
Moorfields Eye Hospital and the Institute of Ophthalmology.
Gliotic specimens were obtained upon written consent from patients
undergoing retinectomy at Moorfields Eye Hospital. Protein was
extracted from Müller stem cell lines established in our laboratory.
Differential protein profile expression was performed using
Label-free proteomics by LC-MS/MS. Protein identification and
bioinformatics analysis was performed using Protein Lynx Global
Server (PLGS), and Non-Linear dynamics Progenesis software.
Results: Proteomic analysis of human retinae identified a total of
478 proteins in the normal samples and 453 proteins in the gliotic
retina. 15 proteins were shown to be >2fold upregulated and 150
proteins to be >2fold downregulated in the gliotic retinas compared
to controls. Of those differentially expressed, synapsin, CD166,
TRAP1, RKIP, reticulon4 and SOD may be of interest to investigate
for their ability to modify Müller stem cell growth and differentiation
in vitro. Analysis of Müller glial stem cell lines identified a total of
477 proteins, which included common Müller cell markers including
Vimentin, glutamine synthetase, CRALBP, CD44 and GFAP, as well
as the factors identified above.
Conclusions: This proteomic-based study has identified proteins
and signalling pathways that are upregulated during retinal gliosis.
Investigation of these factors on the proliferation and neural
differentiation of human Müller stem cell functions in vitro may
provide important clues for the identification of novel approaches
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
to promote endogenous repair mechanisms in retinal degenerative
diseases.
Commercial Relationships: Karen Eastlake, None; Wendy
Heywood, None; Kevin Mills, None; Philip J. Banerjee, None;
David G. Charteris, None; Peng Khaw, None; G Astrid Limb,
None
Support: Special Trustees of Moorfields Eye Hospital. MRC (MR/
K008722/1)
Program Number: 375 Poster Board Number: C0146
Presentation Time: 8:30 AM–10:15 AM
ERK1/2 signaling pathway is activated by complement serum in
UV-POS pretreated ARPE-19 cells
Martin Busch1, Susanne Wasmuth1, Albrecht Lommatzsch2, Daniel
Pauleikhoff2. 1Ophtha-Lab, Department of Ophthalmology at
St. Franziskus Hospital, Muenster, Germany; 2Department of
Ophthalmology at St. Franziskus Hospital, Muenster, Germany.
Purpose: Retinal pigment epithelial (RPE) cells undergo functional
changes upon complement stimulation, which plays a role in the
pathogenesis of age-related macular degeneration. The aim of this
study was to investigate the effects of human complement serum
(HCS) treatment on p44/42 mitogen-activated protein kinase
(ERK1/2) activation in ARPE-19 cells pretreated with UV-irradiated
photoreceptor outer segments (UV-POS), which was demonstrated to
result in an accumulation of intracellular autofluorescent particles as
a possible sign of “aging cells”.
Methods: ARPE-19 cells were grown in DMEM/F12 medium
supplemented with 10 % fetal calf serum (FCS). For experiments,
ARPE-19 cells were cultured for one week in 1 % FCS containing
medium. Afterwards, cells were pretreated three times with 10 mg/
ml UV-POS. Subsequently, cells were thoroughly washed and were
stimulated with 5 % HCS or heat-inactivated HCS (HI-HCS) as a
control in FCS-free medium for 24 hours. Total protein was analyzed
for phosphorylated (activated) and non-phosphorylated ERK1/2 by
western blotting. Cell culture supernatants were examined for their
content of VEGF, IL-8, and MCP-1 via ELISA.
Results: The stimulation of UV-POS pretreated ARPE-19 cells with
HCS increased the amount of phosphorylated ERK1/2 (P-ERK1/2),
whereas UV-POS in combination with HI-HCS or HCS alone
(without UV-POS pretreatment) had no strong impact on the
frequency of P-ERK1/2 when compared to the HI-HCS control. In
contrast, the non-phosphorylated form did not differ between all
treatment groups. Also, homogeneous expression of the housekeeping
gene β-actin could be observed under the various conditions. The
ELISA data revealed the trend that the production of VEGF, IL-8,
and MCP-1 was increased in response to HCS. This HCS effect was
pronounced in UV-POS pretreated ARPE-19 cells.
Conclusions: ERK1/2 signaling pathway demonstrated an
increased phosphorylation and therefore activation in UV-POS
pretreated ARPE-19 cells in response to complement serum. This
was associated with an increased secretion of cytokines. Although
possibly superimposed by other signaling pathways, ERK1/2
activation may be involved in the proinflammatory and proangiogenic
changes of RPE-cells in response to complement and these effects
may be even more distinct, if RPE cells are under metabolic stress.
Commercial Relationships: Martin Busch, None; Susanne
Wasmuth, None; Albrecht Lommatzsch, None; Daniel
Pauleikhoff, None
Support: Voltmann Foundation
Program Number: 376 Poster Board Number: C0147
Presentation Time: 8:30 AM–10:15 AM
Effect of SPARC deletion on retinal neovascularization and
capillary drop out in mouse model of ischemic retinopathy
Doaa Sobeih1, 2, Khaled Hussein1, 2, Neveen Said3, Kouros Motamed4,
Mohamed Al-Shabrawey1, 2. 1Oral biology/Anatomy, Collage of dental
medicine, Augusta, GA; 2Department of Ophthalmology and Vision
Discovery Institute, Medical College of Georgia, Augusta, GA;
3
Department of Radiation Oncology, University of Virginia School of
Medicine, Charlottesville, VA; 4IGDRASOL, Irvine, CA.
Purpose: : The matricellular Secreted Protein Acidic and Rich in
Cysteine (SPARC/osteonectin) has been shown to play an important
role in pathological angiogenesis. Previous animal studies have also
supported the concept of SPARC as an angiogenesis regulator in
some retinal diseases such as choroidal neovascularization (CNV)
as well as proliferative diabetic retinopathy (PDR). However, the
exact role of SPARC in regulation of the angiogenesis process is still
unclear. The aim of the current study was to investigate the effect of
the matricellular SPARC deletion on pathological angiogenesis and
capillary drop out in the mouse model of oxygen induced retinopathy
(OIR).
Methods: Whole-mount retinas labeled with vascular marker
(Isolectin-B4) were examined in OIR wild type (n=7) and OIR
SPARC-deficient mice (n=7). Analysis was performed at postnatal
day 17 (P17) after they were exposed to 77% Oxygen for five days
(P7- P12), followed by exposing the animal to normal air for 5 days
until P17. Analysis of retinal vasculatures in retinal flat mounts
included the total area of new capillary tufts and area of capillary
dropout normalized to the total retinal area. Using Western Blot
(WB), SPARC expression was analyzed in retinal homogenates of
OIR wild type mice (P14 and P17) versus control mice at same time
points. Human retinal endothelial cells (HRECs) were exposed to
low oxygen treatment (1%) for 6 hrs before measuring the levels of
SPARC by WB.
Results: We noticed a modest increase in the new capillary tufts
in SPARC-deficient mice, compared to the wild type. However,
lack of SPARC was associated with significant increase in the
capillary dropout in comparison with the wild type (p value=0.03).
Meanwhile, a significant decrease in the SPARC protein levels were
detected by WB performed on the retinas of OIR wild type animal
model compared to the control animals at P14 (p value=0.01) while
no significant difference was noticed at P17. Moreover, hypoxia
significantly abrogated SPARC expression in HRECs.
Conclusions: SPARC deletion augments the severity of ischemic
retinopathy in the form of increasing capillary drop out areas
with modest increase in pathological neovascularization.
Therefore, modulation of SPARC expression in retina could
be a novel therapeutic approach to prevent pathological retinal
neovascularization
Commercial Relationships: Doaa Sobeih, None; Khaled Hussein,
None; Neveen Said, None; Kouros Motamed, None; Mohamed
Al-Shabrawey, None
Support: 1R01EY023315-01 and QNRF (NPRP4-1046-3-284)
Program Number: 377 Poster Board Number: C0148
Presentation Time: 8:30 AM–10:15 AM
ATF4 Deficiency Leads to Structural and Functional Preservation
of T17M RHO Retina
Yogesh Bhootada, Marina S. Gorbatyuk. Vision Science, University
of Alabama at Birmingham, Birmingham, AL.
Purpose: The T17M mutation within the rhodopsin gene (RHO)
causes protein misfolding, endoplasmic reticulum (ER) stress,
and activation of the unfolded protein response (UPR) leading to
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
autosomal dominant retinitis pigmentosa (ADRP). The activation of
the UPR in T17M RHO retina occurs through induction of the PERK
UPR signaling and leads to up-regulation of phosphorylated eIF2a,
ATF4, and pro-apoptotic CHOP. Our long term goal is to reverse
retinal degeneration in T17M RHO mice. Therefore, we hypothesize
that the modulation of the PERK signaling could prevent or slow
down the rate of retinal degeneration in T17M RHO mice.
Methods: T17M RHO ATF4+/-, T17M RHO ATF4+/+, ATF4+/- and
ATF4+/+ (C57Bl/6) mice were used in the study. All groups were
subjected to electroretinogram (ERG) and spectral domain optical
coherent tomography (SD-OCT) analysis at postnatal (P) day 30,
P60 and P90 of retinal degeneration. RNA and protein were extracted
from retina in all the four groups of mice at P30 to perform qRT-PCR
and western blot analysis.
Results: An analysis of the scotopic ERG response demonstrated
that the a- and b-wave amplitudes were dramatically increased in
ATF4-deficient T17M RHO retinas by 392% and 207%, respectively
at P30 compared to T17M RHO ATF4+/+and reached the level of
the ERG amplitudes measured in C57Bl6 mice. At P90 the increase
was only 172% and 125%, respectively compared to T17M RHO
ATF4+/+ and was lower compared to one measured in C57Bl6 retina
pointing a slow loss of physiological function. The thickness of the
outer nuclear layer (ONL) in the superior (S) and the inferior (I)
regions of the T17M RHO ATF4+/- retina was dramatically increased
by 141% and 145%, respectively compared to control ADRP mice
and was compatible with the length of the ONL in C57Bl6 retina.
However, at P90 we observed a 204% and 224% increase in the
thickness of the ONL for S and I, respectively compared to T17M
RHO ATF4+/+ mice pointing out a slow loss of photoreceptors in
ADRP retina. QPCR and protein analysis of P30 retinas revealed a
lack of activation of the UPR in the T17M RHO retina deficient in
ATF4 suggesting the presence of a link between the activated UPR in
T17M RHO retina and the progression of ADRP.
Conclusions: ATF4 deficiency dramatically slows down a loss of
physiological function and photoreceptors in T17M RHO retina
suggesting that the ATF4 gene could be a viable therapeutic target for
the treatment of ADRP.
Commercial Relationships: Yogesh Bhootada, None; Marina S.
Gorbatyuk, None
Support: NIH Grant R01EYO20905
Program Number: 378 Poster Board Number: C0149
Presentation Time: 8:30 AM–10:15 AM
Loss of mTORC1 & mTORC2 but nor mTORC1 or mTORC2
leads to reduction in cone function
Shan Ma1, 2, Aditya Venkatesh1, Claudio Punzo1. 1Ophthalmology,
University of Massachusetts Medical School, Worcester, MA;
2
Ophthalomology, Tianjin Medical University Eye Hospital, Nankai,
China.
Purpose: Photoreceptors are metabolically highly active requiring
phosphoinositide 3-kinase (PI3K) activity for long-term survival.
The mechanistic target of rapamycin (mTOR) is a key regulator of
cell metabolism downstream of PI3K integrating nutrient availability
and growth factor signals. mTOR is found in two distinct complexes
(mTORC1 & mTORC2) that are characterized by their unique
accessory proteins raptor and rictor respectively. This study analyses
the effect on cones of loss of mTORC1, which regulates mainly cell
metabolism in response to nutrient availability and growth factor
signals, and mTORC2, which regulates pro-survival mechanisms
in response to growth factors. Concomitant loss of both mTOR
complexes is analyzed as well.
Methods: Mice carrying conditional knockout alleles for raptor and
rictor were crossed to a cone-specific Cre recombinase line to ablate
mTORC1 and/or mTORC2 in cones. Cone function and survival was
followed by electroretinogram and histological analyses.
Results: Concomitant loss of mTORC1 and mTORC2 affected
cone function but not survival, while individual loss of mTORC1 or
mTORC2 had no effect on cone function or survival. Interestingly,
red/green opsin expression was reduced ventrally for a period of 3-4
months starting at 3 months of age for loss of mTORC1 and 4 months
of age for loss of mTORC2.
Conclusions: PI3K mediated pro-survival pathways are independent
of both mTOR complexes. Although mTOR is key in regulating cell
metabolism and PRs are metabolically highly active, mTOR does
not regulate cell metabolism and growth in cones, suggesting that the
metabolic transcriptome that is regulated by mTORC1 in other cells
is independent of mTOR in cones.
Commercial Relationships: Shan Ma, None; Aditya Venkatesh,
None; Claudio Punzo, None
Support: EY023570
Program Number: 379 Poster Board Number: C0150
Presentation Time: 8:30 AM–10:15 AM
Automated analysis of autofluorescent human RPE cell granules
using structured illumination microscopy
Nil Celik1, Gerrit Best2, Alena Bakulina3, Florian Schock2, Christoph
Cremer2, 4, Jürgen Hesser5, Stefan Dithmar1. 1Ophtalmology,
University Hospital, Heidelberg, Germany; 2Kirchhoff Institute
for Physics, University of Heidelberg, Heidelberg, Germany;
3
Application Scientific Computing, University of Heidelberg,
Heidelberg, Germany; 4Institute of Molecular Biology, University
of Mainz, Mainz, Germany; 5Department of Radiation Oncology,
University Medical Center, Mannheim, Germany.
Purpose: Characteristics of autofluorescent granules (AG) in retinal
pigment epithelium (RPE) cells like number of AG in single cells,
distribution across the fundus, AG size and position within RPE
cells are still not known. We have recently shown that Structured
illumination microscopy (SIM) enables three-dimensional high
resolution imaging of AG in RPE cells. Here we present the selfwritten software AGES (Analysis of Granules Established by SIM)
for automated analysis of 3D-SIM-image data to characterize AG
within RPE.
Methods: Highly resolved SIM images were processed with the
newly developed software AGES for differentiation and analysis
of intracellular AG. As AG are heterogeneous in brightness, an
algorithm applying a rising threshold is used. AGES enables
breaking down clusters of densely-packed AG into their composing
granules. 3D-SIM images from macular and temporal RPE cells were
examined (two donors, 32 years and 91 years) using three different
laser excitation wavelengths (488, 568 and 647 nm). We analyzed
number, size, position and fluorescence intensity of AG in 10-20 cells
per ocular region of each donor.
Results: AGES is able to detect AG separately, even in complex
structures and AG being next to each other. The mean number of AG
per macular cell was 77,9 ± 37,8 (32-year-old donor) and 112,0 ±
55,0 (91-year-old donor) and per temporal cell 44,4 ± 18,7 (32-yearold donor) and 67,5 ± 29,3 (91-year-old donor). The granula diameter
(1,00 ± 0,23 mm) and the granula volume (0,61 ± 0,44 mm 3) at all
sites were in average similar for both donors. AG were distributed
towards the cell edge.
Conclusions: 3D-high-resolution-SIM and the self-written software
AGES allows detailed examination and automated analysis of AG in
RPE cells. For the first time characteristics of AG like exact number
and localization within single RPE cells are determinable.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Nil Celik, None; Gerrit Best, None;
Alena Bakulina, None; Florian Schock, None; Christoph Cremer,
None; Jürgen Hesser, None; Stefan Dithmar, None
Support: Supported by Gertrud Kusen Foundation
Program Number: 380 Poster Board Number: C0151
Presentation Time: 8:30 AM–10:15 AM
Role of female sex hormones in collagen gel contraction mediated
by retinal pigment epithelial cells
Tomoko Orita, Kazuhiro Kimura, Koh-hei Sonoda. Ophthalmology,
Yamaguchi University, Ube, Japan.
Purpose: Contraction of collagen gels mediated by retinal pigment
epithelial (RPE) cells has been studied as a model of proliferative
vitreoretinopathy (PVR). The effects of sex hormones on the
contractility of RPE cells cultured in a three-dementional collagen gel
were investigated.
Methods: Mouse RPE cells were cultured in a type I collagen
gel with 17β-estradiol, progesterone or dehydroepiandrosterone.
Collagen contraction induced by transforming growth factor–β2
(TGF-β2) was evaluated by measurement of gel diameter. Expression
of α–smooth muscle actin (α-SMA) as well as phosphorylation of
Smad2 and myosin light chain (MLC) were examined by immunoblot
analysis. Matrix metalloproteinase (MMP) release was evaluated by
gelatin zymography. Fibronectin and interleukin-6 secretion were
measured with immunoassays.
Results: The female sex hormones 17β-estradiol and progesterone
inhibited TGF-β2–induced collagen contraction mediated by RPE
cells, whereas the male sex hormone dehydroepiandrosterone
had no such effect. The TGF-β2–induced release of MMP-2 and
MMP-9 from RPE cells was also inhibited by 17β-estradiol and
progesterone, and the MMP inhibitor GM6001 attenuated TGF-β2–
induced collagen contraction. Expression of the mesenchymal
markers α-SMA and fibronectin, interleukin-6 release, and Smad2
and MLC phosphorylation induced by TGF-β2 were all inhibited by
17β-estradiol and progesterone. Immunohistochemical analysis also
detected nuclear immunoreactivity for estrogen and progesterone
receptors in proliferative fibrocellular membranes of PVR patients.
Conclusions: Female sex hormones inhibited TGF-β2–induced
collagen contraction mediated by RPE cells. This action appeared
to be mediated through inhibition both of MMP, α-SMA,
and fibronectin expression as well as of Smad2 and MLC
phosphorylation. Female sex hormones might thus inhibit progress of
PVR.
Commercial Relationships: Tomoko Orita, None; Kazuhiro
Kimura, None; Koh-hei Sonoda, None
Program Number: 381 Poster Board Number: C0152
Presentation Time: 8:30 AM–10:15 AM
Atomic force microscopy (AFM) and fluorescence imaging of
ARPE-19 cells subjected to sub-lethal oxidative stress
Tadeusz J. Sarna1, Michal Sarna1, 2, Anna K. Pilat1, Magdalena M.
Olchawa1. 1Biophysics, Jagiellonian University, Krakow, Poland;
2
Medical Physics and Biophysics, AGH University of Science and
Technology, Krakow, Poland.
Purpose: Retinal pigment epithelium (RPE), being exposed to
intense visible light from focal irradiation and high oxygen tension,
is at risk of oxidative stress, exacerbated by accumulation of the age
pigment lipofuscin. It has been postulated that chronic oxidative
stress in this retinal tissue could contribute to the pathogenesis of agerelated macular degeneration (AMD). To monitor sub-lethal oxidative
stress in ARPE-19 cells, subjected to photodynamic action mediated
by phagocytized human RPE lipofuscin or selected photosensitizers,
atomic force microscopy (AFM) and fluorescence imaging were
employed.
Methods: Purified lipofuscin granules obtained from RPE of human
donors were introduced into ARPE-19 cells by phagocytosis. Control
cells, lipofuscin loaded cells or cells with added rose Bengal (rB)
or Merocyanine 540 (MC 540) were exposed, for selected time
intervals, to blue or green light, respectively. Agilent 5500 atomic
force microscope working in either ac mode or force spectroscopy
mode, was used for obtaining cell images and for analyzing
mechanical properties of the cells. Standard immunofluorescence
staining was employed to visualize actin fibers and microtubule
filaments of control cells and cells subjected to photodynamic
treatment.
Results: Immunofluorescence imaging revealed that early changes
associated with photodynamic stress comprised disorganization of
the architecture of the cell cytoskeleton. Simultaneous examination
of the cells by AFM showed that lipofuscin-mediated photodynamic
stress brought about significant decrease in the formation of actin
stress fibers. Importantly, such cells exhibited substantially modified
nanomechanical properties, as demonstrated by the measured changes
in the distribution of the cell Young’s modulus. Similar results were
obtained with cells subjected to photodynamic treatment mediated by
rB and MC 540.
Conclusions: Our results indicate that AFM can be used for sensitive
early detection of changes of cultured RPE cells subjected to sublethal oxidative stress.
Commercial Relationships: Tadeusz J. Sarna, None; Michal
Sarna, None; Anna K. Pilat, None; Magdalena M. Olchawa, None
Support: National Science Center (grant Maestro 2013/08/A/
NZ1/00194).
Program Number: 382 Poster Board Number: C0153
Presentation Time: 8:30 AM–10:15 AM
TALEN-induced knockout of Kif17 in zebrafish
Tylor Lewis, Jason Bader, Peter Volberding, Jonathan Bostrom,
Ross F. Collery, Brian A. Link, Joseph C. Besharse. Cell Biology,
Neurobiology & Anatomy, Medical College of Wisconsin,
Milwaukee, WI.
Purpose: Previous work using both dominant-negative Kif17 and
morpholino-mediated knockdown of Kif17 has shown a crucial
role for the kinesin-II family member in the initial development of
photoreceptor OS. However, these methods limited the analysis to
the initial 5 dpf. To further establish the function of Kif17 within
photoreceptors, permanent knockout is required. A line, kif17sa0119,
that was reportedly null for Kif17 was found to have delayed
peripheral OS morphogenesis at 3 dpf but recovered by 5 dpf.
However, the “null” mutant was found to be hypomorphic with only
50% reduction in Kif17 levels. We, therefore, sought to produce a
true knockout allele by gene targeting.
Methods: We used transcription activator-like effector nucleases
(TALENs), a recent technology in genetic editing, to target Kif17
exon 1 to generate a double-stranded break. Repair by the errorprone non-homologous end joining was expected to create frameshift mutations leading to a functional Kif17 knockout. Mutations
were analyzed through genomic PCR and sequencing and mRNA
expression was analyzed by qPCR. Retinas were then analyzed
morphologically using a combination of histology and optical
coherence tomography (OCT), an emerging technique that allows
for the high-resolution imaging of a retina through measuring
backscattered or reflected light both in real time and in vivo.
Results: A line of zebrafish, kif17mw405, was established containing
an 11 base pair deletion early in exon 1 of Kif17 that leads to a
premature stop-codon at the 21st codon, generating a functional Kif17
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
knockout. Contrary to previous work, Kif17 knockouts exhibited
normal OS formation within the first 6 days of development.
However, there is progressive disorganization of the outer retina,
particularly observed as the loss of the ordered tiering of cone OS at
10 weeks.
Conclusions: Kif17 knockout does not appear to significantly affect
OS morphogenesis as previously suggested. However, there appears
to be a long-term, progressive outer retinal disorganization phenotype
that could not be studied with the previous transient methods. With an
established stable line of Kif17 knockout fish, long-term evaluation
of the progression of disorganization, as well as investigation of the
underlying mechanisms, is possible.
Commercial Relationships: Tylor Lewis, None; Jason Bader,
None; Peter Volberding, None; Jonathan Bostrom, None; Ross F.
Collery, None; Brian A. Link, None; Joseph C. Besharse, None
Support: NEI R01 EY03222
Program Number: 383 Poster Board Number: C0154
Presentation Time: 8:30 AM–10:15 AM
Synergistic interaction between endothelial cells and retinal
pigment epithelium
Magali Saint-Geniez1, 2, Carrie Spencer1, Stephanie Abend1, Kevin
McHugh1, 3. 1Schepens Eye Research Institute, Mass Eye and Ear,
Boston, MA; 2Department of Ophthalmology, Harvard Medical
School, Boston, MA; 3Department of Biomedical Engineering,
Boston University, Boston, MA.
Purpose: Previous studies have demonstrated the critical
interdependence of choroidal endothelial cells (EC) and retinal
pigment epithelial (RPE) cells. For example, we recently showed
that production of soluble VEGF isoforms by RPE is necessary for
choriocapillaris survival. Here we analyze the reciprocal interaction
between RPE and EC using a long-term coculture system and
determine the effect of RPE-EC interaction on cell behavior and
matrix deposition.
Methods: ARPE-19 and HUVECs were seeded on opposite sides
of polyester transwells and cultured for up to 4 weeks in low
serum conditions.Cell viability was quantified using a trypan blue
assay. Cell morphology was evaluated by H&E staining, SEM and
immunohistochemistry. RPE barrier function was determined by
recording of the transepithelial resistance and localization of ZO-1.
RPE phagocytic function was examined by performing an 18-hour
fluorescent bead assay. Gene expression analysis was performed on
both RPE and ECs by qPCR. Quantification of extracellular matrix
deposition was performed on acellular transwells stained for collagen
IV, fibronectin, and fibrillin. EC angiogenic activity was quantified in
a 2D tube-formation assay.
Results: Coculture with RPE sustains primary human EC survival
and proliferation in low serum for up to 4 weeks. Presence of ECs
significantly improves RPE barrier and phagocytic functions with
no effect on RPE survival or morphology. Gene expression analysis
reveals that coculture with ECs strongly induces RPE expression
of matrix- and visual cycle-associated genes such as RPE65 and
CRALBP. Presence of ECs leads to the accumulation of BrM-like
material on the RPE basal side as measured by electron microscopy
and immunohistological analysis. Expression of PEDF and
Thrombospondin-1 by RPE was also significantly upregulated in coculture condition, suggesting that RPE acquired an anti-angiogenic
status. Respectively, gene expression and tube formation studies
confirmed that co-culture with RPE significantly decreased the
angiogenic phenotype of ECs.
Conclusions: This study demonstrates that long-term RPE-EC
coculture improves RPE differentiation and deposition of a BrMlike matrix while inhibiting the ECs angiogenic potential and further
emphasizes the importance of RPE-EC interaction in the maintenance
of retinal homeostasis.
Commercial Relationships: Magali Saint-Geniez, None; Carrie
Spencer, None; Stephanie Abend, None; Kevin McHugh, None
Support: NIH Director’s New Innovator Award 1DP2OD006649
124 Retinal Development
Sunday, May 04, 2014 1:30 PM–3:15 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 691–723/C0234–C0266
Organizing Section: Retinal Cell Biology
Program Number: 691 Poster Board Number: C0234
Presentation Time: 1:30 PM–3:15 PM
Characteristics of Normal Foveal Development in Infants and
Young Children as Imaged Using Hand-Held Optical Coherence
Tomography
Helena Lee, Ravi Purohit, Aarti Patel, Eleni Papageorgiou, Mashal
Bibi, Viral Sheth, Gail Maconachie, Rebecca McLean, Frank A.
Proudlock, Irene Gottlob. Ophthalmology, University of Leicester,
Leicester, United Kingdom.
Purpose: To characterise the time course of normal foveal
development in vivo in full term infants and children using hand-held
high resolution spectral domain optical coherence tomography (HHSDOCT).
Methods: 256 children with a mean age of 2.2 years (range 0-6.9
years) and 39 older children and adults with a mean age of 15 years
(range 7.1-27 years) were recruited. Each participant had a full
ophthalmological examination and HH-SDOCT scans. The OCT
scans were segmented using a customised macro in ImageJ. The
thickness and angle of each retinal layer at the fovea and parafovea
were quantified and correlated with log gestational age (logGA) and
visual acuity (VA).
Results: The central macular thickness (CMT) increases linearly with
logGA by 85% between birth and three years of age, after which it
plateaus. This relationship is described by: CMT (mm) = 61log(GA-1)
+ 111. In the parafovea, there is a more gradual 20% increase in
retinal thickness over the same time period. The foveal outer segment
(OS) and inner segment (IS) of the photoreceptors and the outer
nuclear layer (ONL) also follow a linear pattern, with a 330%,
24% and 55% increase in thickness respectively, between birth and
eighteen months of age, after which they plateau. In the parafovea,
this increase is more gradual with the OS (76%), IS (12%) and ONL
(15%). The foveal outer plexiform (OPL), inner nuclear (INL), inner
plexiform (IPL) and ganglion cell layers (GCL) decrease in thickness
with GA. Interestingly, the parafoveal thickness of the retinal nerve
fibre layer (RNFL), GC complex (GCL and IPL) initially decrease in
thickness until two years of age, followed by a gradual increase. The
age adjusted CMT, ONL and IS are significant predictors of VA, with
r 2 = 0.739 and p = 0.000 for CMT and r2 = 0.748 and p= 0.000 for
the ONL and IS. The age adjusted angle between the fovea and the
upper border of the ONL at 1000 mm is also a significant predictor
of VA, with r 2 = 0.777 (p = 0.001) and 0.765 (p = 0.045) for the
temporal and nasal angles respectively.
Conclusions: We have characterised the time course of normal foveal
development in infants and young children using the HH-SDOCT
and several predictors of visual acuity have been identified. This is
important as the HH-SDOCT will play an increasingly prominent
diagnostic and prognostic role in children with retinal pathology.
Commercial Relationships: Helena Lee, None; Ravi Purohit,
None; Aarti Patel, None; Eleni Papageorgiou, None; Mashal
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Bibi, None; Viral Sheth, None; Gail Maconachie, None; Rebecca
McLean, None; Frank A. Proudlock, None; Irene Gottlob, None
Support: Medical Research Council, London, UK (grant number:
MR/J004189/1), Ulverscroft Foundation, Leicester, UK and
Nystagmus Network UK
Program Number: 692 Poster Board Number: C0235
Presentation Time: 1:30 PM–3:15 PM
Identifying surface markers to distinguish cells types in the
human fetal retina
Jennifer Aparicio, Anthony Choi, Victor Liao, David Cobrinik.
Ophthalmology, Children’s Hospital Los Angeles, Los Angeles, CA.
Purpose: In light of recent advances in stem cell biology and
prospects of cell transplantation in the field of ophthalmology, it is
increasingly important to characterize retinal cells, especially in the
early stages of eye development. Our primary objective is to define
surface markers to enable isolation of pure cell populations in the
human fetal retina and human embryonic stem cell (ES)-derived
retinal tissue for further study.
Methods: To demarcate unique cell types in the developing
human retina, we screened a fetal retina at 20 weeks gestation,
with a commercially available cell surface marker antibody panel.
Dissociated retinal cells were co-labeled with CD133 and each of
251 monoclonal antibodies, and analyzed by flow cytometry. CD133,
a marker of progenitors and photoreceptors, was employed to aid
in defining populations marked by panel antibodies. Dissociated
ES-derived retinal cells were analyzed for expression of a subset
of markers present in fetal retina. In addition, immunofluorescence
microscopy was used to examine the pattern of expression of many of
the antibodies in fetal retina 15 to 21 fetal weeks in age.
Results: Of the surface markers analyzed by flow cytometry, 112
were expressed in some cells in the human fetal retina. The 55
markers most frequently expressed on retinal cells exhibited one of
eight different patterns of expression when analyzed along with cell
size and CD133 expression. Patterns detected by antibodies to CD73,
CD15, and CD44 are consistent with patterns previously described
in the murine retina, a photoreceptor precursor marker, and early and
late progenitor markers, respectively. Furthermore, 49-day-old ESderived retina exhibited expression of 45 of 50 antigens expressed in
fetal retina, suggesting that those not expressed are displayed on more
mature cell types. Immunofluorescence analysis demonstrated that
some surface markers differed in their expression levels from central
to peripheral retina indicative of cells of differing maturational states.
Conclusions: Commonly available cell surface antigens can be
used to separate disassociated single cell suspensions of retinal cells
into distinct subpopulations. These markers may enable purification
of retinal cell types from heterogeneous populations, including
photoreceptor precursors, ganglion cells, and interneurons from both
human fetal retina and ES-derived retina.
Commercial Relationships: Jennifer Aparicio, None; Anthony
Choi, None; Victor Liao, None; David Cobrinik, None
Program Number: 693 Poster Board Number: C0236
Presentation Time: 1:30 PM–3:15 PM
Cup-to-disc and arteriole-to-venule ratios in preterm birth
Da Ye Choi, Jaeryung Kim, Kyung-Ah Park, In-Jeong Lyu, Sei Yeul
Oh. Ophthalmology, Samsung medical center, Seoul, Republic of
Korea.
Purpose: To investigate the influence of preterm birth on the optic
disc and retinal vessels by measurements of cup-to-disc (C/D) ratio
and arteriole-to-venule (A/V) ratio.
Methods: Eighty-three eyes of 42 preterm birth were included in this
study. In the age and sex-matched control group, 83 eyes of 42 full-
term birth were used. Fundus color photographs were taken. ImageJ
software was used to calculate C/D and A/V ratios from the fundus
images.
Results: Fundus photographs were taken at an age of 8.01 ± 2.22
years for the preterm group and 8.01 ± 2.13 years for the control
group. The mean gestational age of the preterm group was 27.57
weeks (range, 24 to 34 weeks). The preterm group had significantly
larger C/D ratios and smaller A/V ratios (mean ± standard deviation;
0.46 ± 0.12 and 0.71 ± 0.09, respectively) than the control
group (mean ± standard deviation; 0.36 ± 0.07 and 0.82 ± 0.08,
respectively) (p < 0.001 and p < 0.001, respectively) after age, sex
and spherical equivalent refractive error were adjusted.
Conclusions: Preterm birth is significantly associated with larger
C/D ratio and smaller A/V ratio. These findings show the effect of
preterm birth on the development of optic disc and retinal vessel
development.
Commercial Relationships: Da Ye Choi, None; Jaeryung Kim,
None; Kyung-Ah Park, None; In-Jeong Lyu, None; Sei Yeul Oh,
None
Program Number: 694 Poster Board Number: C0237
Presentation Time: 1:30 PM–3:15 PM
Contrasting Foveal Specialization in Disorders Associated with
Foveal Hypoplasia
Melissa A. Wilk1, Brian Higgins2, Robert F. Cooper3, Drew H. Scoles4,
Kimberly E. Stepien2, C Gail Summers5, 6, Alfredo Dubra2, 7, Deborah
M. Costakos2, Joseph Carroll1, 2. 1Cell Biology, Neurobiology,
& Anatomy, Medical College of Wisconsin, Milwaukee, WI;
2
Ophthalmology, Medical College of Wisconsin, Milwaukee, WI;
3
Biomedical Engineering, Marquette University, Milwaukee, WI;
4
Biomedical Engineering, University of Rochester, Rochester, NY;
5
Ophthalmology & Visual Neurosciences, University of Minnesota,
Minneapolis, MN; 6Pediatrics, University of Minnesota, Minneapolis,
MN; 7Biophysics, Medical College of Wisconsin, Milwaukee, WI.
Purpose: While foveal specialization has been well characterized
in albinism, less is known regarding foveal morphology in other
disorders associated with foveal hypoplasia. Here we sought to
quantify foveal specialization in patients with aniridia or a history of
premature birth using spectral domain optical coherence tomography
(SD-OCT) and adaptive optics scanning light ophthalmoscopy
(AOSLO), and compare these findings to those of patients with
albinism.
Methods: Subjects with a diagnosis of albinism (n=5), aniridia
(n=3), or a history of premature birth (n=3, birth was between 25
and 30 weeks’ gestation) were recruited for this study. Volumetric
SD-OCT scans of the macula were acquired, and custom MATLAB
software was used to derive estimates of foveal pit depth, diameter,
and volume. Additionally, high-resolution linear SD-OCT scans were
acquired and manually segmented to obtain measurements of relative
foveal cone inner and outer segment (IS and OS, respectively) length.
Images of the photoreceptor mosaic and foveal avascular zone
(FAZ) were acquired using AOSLO. When possible, cone density
was measured using a semi-automated cone counting program,
and FAZ area and diameter were measured using semi-automated
segmentation.
Results: Despite having reduced FAZ areas, all 3 subjects with
a history of premature birth displayed normal foveal pit metrics,
normal foveal cone OS elongation, and normal cone packing.
Consistent with previously reported results from 32 subjects with
albinism and the additional 5 subjects reported here, the subjects with
aniridia had variable OS lengthening. Clear evidence of cone packing
was seen in one subject with aniridia, though due to the presence of
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
severe nystagmus, it was not possible to quantify cone density in the
remaining two subjects.
Conclusions: Contrary to previous observations in patients with
a history of premature birth, our subjects displayed normal foveal
specialization. Further work contrasting foveal specialization across
these disorders may be useful to better understanding normal foveal
development. While previous work from our group has shown that it
is possible to obtain high quality images in patients with nystagmus,
AOSLO hardware improvements and some form of eye tracking are
needed to enable imaging of patients with severe nystagmus, such as
that seen in aniridia.
Commercial Relationships: Melissa A. Wilk, None; Brian Higgins,
None; Robert F. Cooper, None; Drew H. Scoles, None; Kimberly
E. Stepien, None; C Gail Summers, None; Alfredo Dubra, Canon
USA, Inc. (C), US Patent 8,226,236 (P); Deborah M. Costakos,
None; Joseph Carroll, None
Support: Supported by Vision for Tomorrow, Research to
Prevent Blindness, Burroughs Wellcome Fund, and NIH Grants
P30EY001931, UL1RR031973, T32EY014537, & T32GM007356.
Supported, in part, by an unrestricted grant to the Department
of Ophthalmology & Visual Neurosciences at the University of
Minnesota from Research to Prevent Blindness, Inc., New York, NY.
Program Number: 695 Poster Board Number: C0238
Presentation Time: 1:30 PM–3:15 PM
Viability of human fetal retina/RPE sheets after 4 days cold
storage
Magdalene J. Seiler1, Gabriel Nistor2, 1, Norman D. Radtke3, Robert
B. Aramant1. 1Anatomy & Neurobiology, Univ of California, Irvine,
Irvine, CA; 2California Stem Cell Inc., Irvine, CA; 3Retina Vitreous
Resource Center, Louisville, KY.
Purpose: For development of clinical retinal transplantation trials,
it would be important to maintain viability of the donor tissue for
several days after dissection, and to ship the tissue in temperature
controlled containers.
Methods: Permission to use fetal tissue for research was obtained
from the Western Institution Review Board, Norton Healthcare
Research office, and the hSCRO committee of UC Irvine. Retina
together with its RPE was dissected from 6 fetal eyes (age 10.5 – 12.5
weeks post-conception, 4 donors) that were received at 1 day (d) after
abortion. All tissues had been placed into cold custom-made CO2independent hibernation medium with B27 supplements immediately
after harvest. Eyecups were treated with dispase for 15-20 minutes to
separate choroidal vessels from RPE. After dissection, retinas with
RPE were cut into 3-5 tissue pieces (size 2-6 mm2). Of each eye,
pieces were either fixed immediately, or placed inside plastic nozzles
in hibernation medium in shipping tubes (18 pieces). The tubes were
placed into shipping containers, and their temperature monitored
(between 8 and 2.5 sC). After shipping for 1, 2, or 3 d, tissues were
fixed (= 4 d after harvest). Cell viability in the retinas was tested by
TUNEL staining (counting TUNEL-stained(+) cells per mm2 and %
TUNEL-stained cells of total cells in 10 μm cryostat cross-sections).
This analysis was performed for all cells, and separately for the
inner retinal layers (containing differentiating neurons) and outer
neuroblastic layers (that would develop into photoreceptors and other
retinal cells).
Results: Of the 18 pieces placed into shipping containers, 14 pieces
retained retina-RPE contact. - A low percentage of cells (0.3%) were
TUNEL-positive after dissection. Although the number of TUNEL+
cells increased with time, the percentage of TUNEL+ cells remained
very low (up to 0.9% on d 2 of shipping). In the inner retinal layers,
there was a higher percentage of TUNEL+ cells (0.5-1.9%) than
the 0.3-0.6% TUNEL+ cells in the outer neuroblastic layers. The
number of TUNEL + cells increased significantly in the inner layers
after 1 d shipping, whereas the number of TUNEL+ cells in the outer
neuroblastic layers did not increase significantly (from 0.3 to 0.6%)
until 3 d of shipping (= 4 d after harvest).
Conclusions: These results demonstrate that intact fetal-derived
sheets of retina with RPE remain viable for several days with careful
dissection and handling.
Commercial Relationships: Magdalene J. Seiler, Ocular
Transplantation LLC (C), Ocular Transplantation LLC, patent #
5,941,250; # 8,057,483 (P); Gabriel Nistor, None; Norman D.
Radtke, None; Robert B. Aramant, Ocular Transplantation LLC
(E), Ocular Transplantation LLC patent # 5,941,250; # 8,057,483 (P)
Support: Lincy Foundation
Program Number: 696 Poster Board Number: C0239
Presentation Time: 1:30 PM–3:15 PM
In vivo retinal development using hand held ultra-high resolution
spectral domain optical coherence tomography in premature and
full term infants
Samira Anwar1, 2, Aarti Patel1, Helena Lee1, Frank A. Proudlock1,
Jonathan Cusack3, Irene Gottlob1. 1Ophthalmology Group,
University of Leicester, Leicester, United Kingdom; 2Department of
Ophthalmology, University Hospitals of Leicester, Leicester Royal
Infirmary, Leicester, United Kingdom; 3Department of Neonatology,
University Hospitals of Leicester, Leicester Royal Infirmary,
Leicester, United Kingdom.
Purpose: To describe in vivo retinal changes during development in
premature infants and normal aged matched controls using hand-held
spectral domain optical coherence tomography (HH-SDOCT).
Methods: Cross sectional and longitudinal images HH-SDOCT were
collected from 33 premature infants ranging in age from 31 – 42
weeks gestational age. Participants were recruited from the neonatal
unit while also undergoing ophthalmic screening for retinopathy of
prematurity (ROP). Control infants ranged in age from 37 weeks
to 41 weeks gestation and were recruited from the maternity unit.
Premature infants were examined at 1-2 weekly intervals until up to
42 weeks. Normal controls were examined within 1 week of birth.
No premature infants had received treatment for ROP at time of
scanning. HH-SDOCT (Bioptigen, 2.6μm axial resolution) scans
were performed without sedation and after dilatation of pupils.
Customised Image J manual segmentation of retinal images including
thickness and angle of each layer centrally and at 1000μm nasally
and temporally was performed and quantified. Statistical analysis was
completed using SPSS v20.
Results: The central retina in the premature group progressively
thinned by 1.74μm/week in contrast to 5μm/week thickening of the
nasal and temporal retina located at 1000μm from the foveal centre.
Changes in central retina were due to a combination of inner layer
migration (reduction of approximately 4.4μm/week) in conjunction
with increased thickening of the outer retinal layers. Changes in
temporal and nasal retina consisted of thickening of both inner layers
and photoreceptor layers. Increase in thickness was primarily seen in
nuclear layers (GCL and INL) whereas plexiform layers did not show
any significant changes. The premature group demonstrated thicker
central retina in comparison to age-matched controls because of
disrupted inner layer migration. Outer retinal photoreceptive elements
were easier to define in the normal group compared to the premature
group.
Conclusions: Premature infants maintain thicker inner retina
centrally with decreased inner retinal migration compared to agematched normal infants. Outer retinal layers were thinner in the
premature group suggesting slower development of these layers.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Samira Anwar, None; Aarti Patel,
None; Helena Lee, None; Frank A. Proudlock, None; Jonathan
Cusack, None; Irene Gottlob, None
Support: Medical Research Council, London, UK (grant number:
MR/J004189/1) Ulverscroft Foundation, Leicester, UK Nystagmus
Network UK
Program Number: 697 Poster Board Number: C0240
Presentation Time: 1:30 PM–3:15 PM
Microphthalmia-associated Transcription Factor (MITF) affects
optic vesicle cell proliferation and retinal pigment epithelium
maturation in human ES cell (hESC) cultures
Anna Petelinsek1, Elizabeth E. Capowski1, Sara Howden2, Lynda S.
Wright1, Isabel Pinilla Lozano5, 6, Kyle Wallace1, Eric Clark1, Joe
Phillips1, David M. Gamm3, 4. 1University of Wisconsin-Madison,
Madison, WI; 2Morgridge Institute for Research, Madison, WI;
3
Department of Ophthalmology and Visual Sciences, University
of Wisconsin, Madison, WI; 4McPherson Eye Research Institute,
Madison, WI; 5Ophthalmology, University Hospital Lozano Blesa,
Zaragoza, Spain; 6IIS Aragon, Aragon Health Sciences Institute,
Zaragoza, Spain.
Purpose: Loss of MITF expression results in microphthalmia and
RPE defects in rodent models, but its role in human retinogenesis
remains unknown. We created a MITF double knockout (dKO) hESC
line to examine the consequences of its absence during early retinal
development.
Methods: The second common exon of MITF was targeted by two
rounds of BAC-mediated homologous recombination to inactivate
both alleles in WA09 hESCs. Correct targeting and gene inactivation
were confirmed by genomic PCR, RT-PCR, and Western blots. Light
microscopy, immunocytochemistry (ICC), RT-PCR, and RT-qPCR
were then performed at different stages of retinal cell development
to examine the impact of MITF loss on the production, proliferation,
and maturation of early neuroretinal cells and RPE.
Results: RT-PCR and ICC analyses confirmed the absence of MITF
transcripts and protein in dKO-MITF hESCs. Upon differentiation,
ICC revealed that dKO-MITF hESCs sequentially adopted anterior
neuroectoderm and early eye field fates in a manner similar to
isogenic control WA09 hESCs. However, optic vesicle-like structures
(OVs) generated from dKO-MITF hESCs were significantly
smaller than control OVs (>60% reduction; p<0.0001). In addition,
expression levels of PAX6, RX, and SIX6 were reduced in dKO-MITF
vs. control OVs (54, 76, and 79% reductions, respectively), as was
the number of proliferating cells (Ki67+ cells: 15.4% vs. 23.4%; p≤
0.01). Following onset of VSX2 expression, cell proliferation within
dKO-MITF OVs matched that of control OVs. Furthermore, dKOMITF OVs produced CRX/Recoverin+ precursors in a temporal and
spatial pattern indistinguishable from control OVs. However, RPE
generated from dKO-MITF hESCs failed to fully mature; instead,
dKO-MITF RPE remained unpigmented and lacked typical RPE
organization even after several months in culture.
Conclusions: Using a genetically engineered hESC line, we showed
that MITF is specifically involved in human OV proliferation and
RPE maturation. Such information may lead to improvements in
retinal cell production in vitro and/or provide insight into retinal
development at stages previously inaccessible in humans.
Commercial Relationships: Anna Petelinsek, None; Elizabeth E.
Capowski, None; Sara Howden, None; Lynda S. Wright, None;
Isabel Pinilla Lozano, None; Kyle Wallace, None; Eric Clark,
None; Joe Phillips, None; David M. Gamm, None
Support: Foundation Fighting Blindness Wynn-Gund Research
Acceleration Award; NIH RO1EY21218 and P30HD03352; E.
Matilda Ziegler Foundation for the Blind;Retina Research Foundation
(Kathryn and Latimer Murfee and Emmett A. Humble Chairs);
McPherson Eye Research Institute (Sandra Lemke Trout Chair)
Program Number: 698 Poster Board Number: C0241
Presentation Time: 1:30 PM–3:15 PM
Prenatal and Postnatal Nutrient Effects in Neonatal Rat Growth
and Retinal Development
Yuta Saito, Emi Ozawa, Haruo Takahashi. Ophthalmology, Showa
University, Tokyo, Japan.
Purpose: In preterm human infants, risk of retinopathy of
prematurity (ROP) has been linked to small for gestational age, low
circulating levels of insulin-like growth factor-1 (IGF-1) and slow
postnatal weight gain. To prevent severe ROP, postnatal nutrition in
preterm infants is very important.
The aim of this study is to investigate prenatal and postnatal nutrient
effects on body weight gain, retinal vascularization and IGF-1 in
plasma in neonatal rat pups.
Methods: Sprague-Dawley rat dams were fed either an unrestricted,
isocaloric normal protein (20%) or low protein (10%) diet to cause
pup growth restriction from 7 days before gestation. The same diet
was continued after the birth of pups. Neonatal rat pups were divided
in two groups after birth, smaller litters (7 rats) and larger litters (14
rats) to cause postnatal growth retardation. On day 8, after measuring
body weight, the rat pups were sacrificed and blood samples
were collected. Retinas were dissected, stained with adenosine
diphosphatase and flat-mounted. The total retinal area (TRA) and
vascularized retinal areas (VA) were measured. Concentration of
IGF-1 in plasma was measured with ELISA. These results were
compared in 20%-7rats, 20%-14rats, 10%-7rats and 10%-14rats
groups. Statistical analyses were performed with Mann-Whitney’s
U test and Kruskal-Wallis test. P value <0.05 was considered
significantly.
Results: The pups from dams fed the low protein diet weighted
significantly lighter at birth (5.8±0.1g vs. 6.8±0.2g P<0.001). The
results on day 8 were shown in table.
Conclusions: Prenatal and postnatal undernutrition may delay
postnatal retinal vascularization and retinal development, and cause
low concentration of IGF-1 in Plasma.
Commercial Relationships: Yuta Saito, None; Emi Ozawa, None;
Haruo Takahashi, None
Support: KAKENHI Number 25870737
Program Number: 699 Poster Board Number: C0242
Presentation Time: 1:30 PM–3:15 PM
Cc2d2a is utilized in assembling subdistal appendages required
for ciliogenesis
Shobi Veleri. Neurobiol-Neurodegen & Repair Lab, NEI, Bethesda,
MD.
Purpose: Meckel syndrome is a lethal form of syndromic ciliopathy
with pleiotropic clinical features like retinal dystrophy, mental
retardation, polydactyly. The patients with mutations in CC2D2A
(Coiled-Coil & C2 Domain containing 2A) displayed Meckel
syndrome. CC2D2A protein is localized to the cilia basal body. The
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
patient derived fibroblasts have basal body, however, they failed to
develop cilia indicating a mechanistic disconnect in the basal body
due to CC2D2A mutation. To examine this disconnect we have
generated Cc2d2a loss of function diseases models in the mouse.
Methods: The Cc2d2a-knockout (Cc2d2a-/-) mouse was generated
by targeted deletion of exons 6 to 8 relying on homologous
recombination. Immunochemistry and electron microscopy were
employed to examine the basal body from mice fibroblasts.
Results: The Cc2d2a-/- mouse is embryonic lethal. It displays
developmental defects like situs inversus, heterotaxy, polydactyly,
anophthalmia, hydrocephalus and liver fibrosis. Occasionally,
the Cc2d2a-/- mouse survived for a month but was with severe
hydrocephalus and retinal dystrophy. Analysis of the embryonic
node, embryonic fibroblasts, and kidney tubules showed that cilia
biogenesis was disrupted in the Cc2d2a-/- mouse. Embryonic
fibroblasts (MEFs) isolated from Cc2d2a-/- mice lack cilia
even though contain mother centriole (MC or basal body) and
pericentriolar proteins. In addition, MEFs have reduced levels of
Odf2 (associated with subdistal appendages (SDA)) and ninein.
Transmission electron microscopy revealed lack of SDAs in
Cc2d2a-/- MEFs and consistent with this observation immuno-EM
showed Cc2d2a localization onto SDA.
Conclusions: Our studies show that Cc2d2a function is critical for
embryonic development. We conclude that Cc2d2a is essential for the
assembly of SDA, which is anchoring cytoplasmic microtubules and
prime MC for axoneme biogenesis.
Commercial Relationships: Shobi Veleri, None
Program Number: 700 Poster Board Number: C0243
Presentation Time: 1:30 PM–3:15 PM
Fishing for evolutionarily conserved Pax6 eye enhancers in mice
Jena Chojnowski1, Jorn Lakowski1, 2, Kenji Johnson1, James D.
Lauderdale1. 1Cellular Biology, University of Georgia, Athens,
GA; 2Neurosciences & Mental Health, University College London,
London, United Kingdom.
Purpose: To identify evolutionarily conserved cis-regulatory
elements that mediate different aspects of PAX6 expression in
the developing vertebrate eye. The PAX6 transcription factor
plays several roles in eye development, including control of cell
proliferation, maintenance of the retinogenic potential of progenitor
cells, and cell fate specification. These roles in turn are mediated by
modular cis-regulatory elements that are widely spaced within the
Pax6 locus. Although a number of elements have been identified,
the regulatory mechanisms governing several aspects of PAX6
expression remain poorly understood.
Methods: We have taken a comparative approach using mouse and
zebrafish to investigate Pax6 transcriptional regulation. Whereas
mouse has a single Pax6 transcript unit, zebrafish have two Pax6
genes (Pax6a and Pax6b) and Pax6 regulatory elements have
been partitioned between them. Mice harboring either Pax6a or
Pax6b bacterial artificial chromosome (BAC) reporter transgenes
were generated. Reporter gene expression was assessed in eyes of
developing mouse embryos and compared to endogenous PAX6
expression.
Results: The Pax6a and Pax6b BAC reporter transgenes exhibit
overlapping and distinct patterns of expression in the developing
mouse eye. Pax6a expression more closely replicates that of the
mouse gene, and is expressed in the surface ectoderm, lens, retina,
and retinal pigmented epithelium of the developing optic cup. As
development progresses, Pax6a transgene expression is detected in
retinal ganglion cells, amacrine cells, horizontal cells, and Müller
glia of the mature retina and also in anterior structures of the
eye. Interestingly, the Pax6b transgene exhibits a more restricted
spatiotemporal expression pattern in the developing eye suggesting
the action of distinct cis-regulatory modules.
Conclusions: Many if not most of the cis-acting regulatory sequences
governing Pax6 transcription in both mice and zebrafish have
been functionally conserved throughout evolution. This functional
conservation in combination with comparative genomic data provides
a robust framework for the identification of novel PAX6 regulatory
elements and a deeper understanding of Pax6 regulatory mechanisms.
Commercial Relationships: Jena Chojnowski, None; Jorn
Lakowski, None; Kenji Johnson, None; James D. Lauderdale,
None
Program Number: 701 Poster Board Number: C0244
Presentation Time: 1:30 PM–3:15 PM
The Effect of Titanium Dioxide Nanoparticles on the Developing
Retina
Yuhao Li1, Yajie Wang1, Zizi He1, Yangwu Fang1, Yang Xu2, Yanan
Chen1. 1Department of Pathology, Nankai University School of
Medicine, Tianjin, China; 2State Key Laboratory of Medicinal
Chemical Biology, College of Chemistry, Nankai University, Tianjin,
China.
Purpose: To investigate whether TiO2 NPs was toxic on zebrafish
embryos and developing retina via aqueous exposure.
Methods: Embryonic zebrafish were exposed to TiO2 NPs at a
concentration of 1mg/L from 1-4 cell stage to 72 hpf. The mortality
and hatching rate were calculated at 24 hpf and 48 hpf respcetively.
An mRNA probe for atonal homolog 7 (atoh7) was used as a
marker to explore whether the neurogenesis initiated on time.
Differentiation of ganglion cells, cones and rods was determined
using immunohistochemistry at 72hpf with Zn12, Zpr1 and Zpr3
antibodies respectively. Microglia in the brain and retina were labeled
using an mRNA probe for fms.
Results: TiO2 NPs exposure did not induce any embryonic
developmental malformations. The onset of neurogenesis and
neuronal differentiation were not delayed following TiO2 exposure.
The location and number of microglia didn’t have a significant
difference between the TiO2 NPs exposed and control group.
Conclusions: TiO2 NPs exposure at 1mg/L doesn’t disrupt the
development of zebrafish embryos and retinal neurogenesis.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Amanda G. Kautzman, None; Irene
E. Whitney, None; Benjamin E. Reese, None
Support: NIH Grant EY-19968
Commercial Relationships: Yuhao Li, None; Yajie Wang, None;
Zizi He, None; Yangwu Fang, None; Yang Xu, None; Yanan Chen,
None
Support: Chinese National Natural Science Foundation (81250017,
81301080), National Key Technology R&D Program of China
(2012BAI08B06)
Program Number: 703 Poster Board Number: C0246
Presentation Time: 1:30 PM–3:15 PM
Transcriptional Activation Differs Significantly Between the Two
Isoforms of Isl1
Amanda G. Kautzman, Irene E. Whitney, Benjamin E. Reese.
Neuroscience Research Institute, University of California Santa
Barbara, Santa Barbara, CA.
Purpose: Islet1 (Isl1) is a LIM homeodomain transcription factor
that plays an essential role in retinal development. It forms a well
characterized transcriptional complex with two other proteins, Lhx3
and Ldb1 (Isl1:Lhx3:Ldb1). It has, however, a splicing site that
generates two isoforms, Isl1α and Isl1 β, and the Isl1β isoform lacks
a critical portion of a protein-binding domain with which Isl1 binds
to Lhx3. We recently demonstrated that the two isoforms of Isl1 are
expressed in developing retinal tissue, and that some classes of retinal
ganglion cell express only the β isoform. The present study sought
direct evidence that these two isoforms function distinctly in their
ability to regulate gene expression, using a luciferase reporter with
Isl1:Lhx3:Ldb1 DNA recognition elements in the promoter region to
assay transcriptional activation.
Methods: HEK293T cells, which endogenously express Ldb1,
were transiently transfected with multiple combinations of plasmids
designed to express Lhx3, Isl1α, Isl1β, or GFP. Cells were also
co-transfected with a luciferase reporter that has been previously
shown to be activated specifically by the Isl1:Lhx3:Ldb1 complex.
Cell lysates were assayed for luciferase activity, in three biological
replicates, each experiment being performed in triplicate.
Results: The activation of luciferase by Isl1α:Lhx3:Ldb1 was 5
fold greater than Isl1β:Lhx3:Ldb1. A one-way ANOVA and posthoc comparisons showed the luciferase activation in Lhx3+Islα
overexpressing cells to be significantly different from Lhx3+Isl1β
and all other conditions, while activation by Lhx3+Isl1β was
indistinguishable from Lhx3 alone.
Conclusions: These results demonstrate a functional difference
between the two isoforms of Isl1; furthermore, they suggest that the
β isoform may not be capable of forming a complex with Lhx3 and
Ldb1. This may suggest that Isl1β containing-complexes have unique
gene targets from Isl1α. Given our previous finding that Isl1α and
Isl1β are differentially expressed in subsets of retinal ganglion cells,
these results further suggest that these isoforms likely play distinct
roles in neuronal differentiation during development.
Program Number: 704 Poster Board Number: C0247
Presentation Time: 1:30 PM–3:15 PM
Primary Cilium Regulates iPS Cell Derived RPE Maturation
Juliet Hartford, Helen May-Simera, Jason Silver, Janine Davis,
Kiyoharu J. Miyagishima, Vladimir Khristov, Omar Memon, Andrea
Li, Sheldon S. Miller, Kapil Bharti. National Eye Institute, National
Institutes of Health, Bethesda, MD.
Purpose: The retinal pigment epithelium (RPE) is a ciliated
monolayer of cells situated adjacent to retinal photoreceptors.
The RPE is critical for maintaining the health and integrity of
photoreceptors. Ciliopathies are a class of disorders that affect cilia
formation or functioning and lead to photoreceptor degeneration.
The role of cilia proteins in photoreceptor development is well
understood, however, its function in the RPE is not known. The goal
of this study is to understand the role of the primary cilium in RPE
development and function in mouse and human models.
Methods: RPE cells in WT and two different cilia mutant mice
(Bbs8-/- and Mkks-/-) were analyzed using immunohistochemistry,
scanning electron microscopy, and qPCR. Primary cilium in human
induced pluripotent stem (iPS) cell derived RPE was manipulated
using cilia inducers and inhibitors. Immunohistochemistry,
scanning and transmission electron microscopy, gene expression,
electrophysiology, intracellular calcium measurements, and fluid
transport were used to determine the maturity of iPS cell derived
RPE.
Results: Ciliopathy mutant mice displayed persistent upregulation
of developmental transcription factors MITF and PAX6 in the RPE.
This was consistent with incomplete maturation of RPE in mutant
mice. iPS cell derived RPE treated with primary cilia agonists and
antagonists reinforced this result. Induction of primary cilium in
iPS cell derived RPE improved melanogenesis, induction of apical
processes, barrier resistance across the monolayer, and ability to
transport fluid. All these properties are consistent with improved
maturation of RPE cells. In contrast, suppression of cilium function
dramatically reduced RPE maturation.
Conclusions: Disrupted cilia function results in delayed/improper
RPE maturation that could contribute to retinal degeneration in
ciliopathy patients. Cilium induction in iPS cell derived RPE leads
to physiologically and phenotypically stable RPE cells. We propose
that the primary cilium helps mature RPE cells through modulation
of RPE developmental pathways and that manipulation of these
pathways can help generate mature RPE cells that serve as more
effective disease and cell-therapy models.
Commercial Relationships: Juliet Hartford, None; Helen MaySimera, None; Jason Silver, None; Janine Davis, None; Kiyoharu
J. Miyagishima, None; Vladimir Khristov, None; Omar Memon,
None; Andrea Li, None; Sheldon S. Miller, None; Kapil Bharti,
None
Support: NIH Intramural Grant
Program Number: 705 Poster Board Number: C0248
Presentation Time: 1:30 PM–3:15 PM
Midkine-a protein localization in the embryonic and adult retina
of the zebrafish
Travis D’Cruz, Esther Gramage, Peter F. Hitchcock. Ophthalmology
and Visual Sciences, University of Michigan, Ann Arbor, MI.
Purpose: Midkine-a (Mdka) is a retinoic acid-induced, heparinbinding growth factor with multiple functions in neural development
and repair. In the zebrafish retina, actively dividing retinal progenitor
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
cells and Müller glia express mdka mRNA during development,
while in the adult, mdka expression is restricted to the horizontal cells
and is robustly regulated by the circadian rhythm. The purpose of
this study was to determine the protein localization of Mdka in the
developing and adult retinas of zebrafish.
Methods: Whole embryos and larvae and eye cups from adults were
fixed with 4% paraformaldehyde and embedded in OCT. Sections
were obtained from embryos at 30 and 48 hours post fertilization
(hpf), larvae at 72, 96 and 120 hpf, and adult eyes. Fluorescence
immunohistochemistry using polyclonal antibodies raised against
Mdka, cell type-specific antibody markers and confocal microscopy
were used to determine the localization of Mdka. Specificity
of the antibody was determined by the selective loss of Mdka
immunolabeling in sections and Western blots from embryos injected
with morpholino oligonucleotides targeted to mdka.
Results: At 30 hpf, Mdka is localized throughout the basal processes
of neuroepithelial cells in the undifferentiated retina. At 48 and
72 hpf, it becomes restricted to the presumptive inner and outer
plexiform layers. At 96 and 120 hpf, when the larval retina is fully
differentiated and mdka expression is restricted to Müller glia and
horizontal cells, Mdka protein is in the outer nuclear layer, but does
not co-localize with cone-specific markers or the Müller glia marker
glutamine synthetase. At the same time, Mdka forms a single small
plaque of protein at the apical surface of each horizontal cell nucleus.
In adults, the horizontal cell staining persists and is regulated by the
circadian rhythm.
Conclusions: Matching the complex pattern of gene regulation,
Mdka protein localization follows a complex temporal and spatial
pattern in the zebrafish retina. Our results suggest that Mdka protein
may be secreted by Müller glia into the extracellular space of the
outer nuclear layer. However, for horizontal cells, the protein is
accumulated in a specific location inside the cell and regulated by the
circadian rhythm.
Commercial Relationships: Travis D’Cruz, None; Esther
Gramage, None; Peter F. Hitchcock, None
Support: NIH Grant EY007060
Program Number: 706 Poster Board Number: C0249
Presentation Time: 1:30 PM–3:15 PM
Regulation of RPE phenotype by Annexin A8 and Wnt signalling
Katharina Lueck, John Greenwood, Stephen E. Moss. Cell Biology,
UCL Institute of Ophthalmology, London, United Kingdom.
Purpose: Fenretinide (FR), a retinoic acid derivative, is capable
of trans-differentiating retinal pigment epithelial (RPE) cells into
a neuronal-like phenotype in culture. Microarray analysis pre- and
post-FR treatment revealed down-regulation of Annexin (Anx)
A8 and various proteins involved in Wnt signalling in transdifferentiated cells. AnxA8, a member of a superfamily of calciumdependent phospholipid-binding proteins, is expressed in RPE cells
and involved in membrane and cytoskeletal organisation and cell
proliferation. The purpose of this study was to analyse the role of
AnxA8 and its relationship with Wnt signalling in epithelial transdifferentiation.
Methods: At 10% confluence, human RPE cells were treated with
3% charcoal dextran-treated foetal bovine serum (FBS) for 24 h. 3
mM FR or vehicle (0.1% dimethylsulfoxide) was added to the cells
every day for 7 days. As a second approach, AnxA8 was suppressed
in RPE cells using short interfering RNA (siRNA). Cells were then
analysed for expression of AnxA8, neuronal markers (Calbindin,
Calretinin) and Wnt signalling proteins (β-Catenin, Frizzled-1,
Frizzled-4, Wnt2b, Wnt3a) using immunofluorescence staining,
qPCR and western blot analysis.
Results: FR and AnxA8 siRNA treatment both induced a decrease
in AnxA8 expression and inhibited cell proliferation. FR also led
to trans-differentiation of ARPE-19 cells into neuron-like cells and
a concomitant up-regulation of neuronal markers. Additionally,
expression of proteins involved in Wnt signalling was decreased.
The effect of FR was partially reversible by activating Wnt signalling
using recombinant Wnt3a or SB216763, a glycogen synthase kinase3β inhibitor.
Conclusions: These data imply an important role for AnxA8 in
maintaining RPE phenotype. Down-regulation of AnxA8 appears
to be sufficient for neuronal trans-differentiation of RPE cells and
the expression of neuronal markers. Further, the interdependence of
AnxA8 and Wnt proteins suggests that AnxA8 might be an important
regulator in Wnt signalling.
Commercial Relationships: Katharina Lueck, None; John
Greenwood, None; Stephen E. Moss, None
Support: BBSRC
Program Number: 707 Poster Board Number: C0250
Presentation Time: 1:30 PM–3:15 PM
ICK ciliary kinase is essential for ciliogenesis in retinal/neuronal
progenitors and regulation of ciliary protein transport at the
ciliary tip
Takahisa Furukawa, Taro Chaya, Yoshihiro Omori. Molecular
and Developmental Biology, Inst for Protein Rsrch, Osaka & JST,
CREST, Osaka, Japan.
Purpose: We previously showed that Mak is required for ciliary
length regulation in retinal photoreceptor cells. Recent reports
showed that mutations in human MAK cause retinitis pigmentosa.
By contrast to cell type-specific expression of Mak, another murine
ortholog of Chlamydomonas LF4, Intestinal Cell Kinase (ICK),
shows ubiquitous expression including in the developing CNS.
However, the exact biological functions of ICK have not yet been
elucidated.
Methods: We generated and analyzed retina- or brain-specific ICKdeficient mutant mice as well as conventional ICK-deficient mutant
mice.
Results: In ICK-null mutant mice, we observed some phenotypes
of defective Hh signaling including polydactyly, shortened leg
bones, and developmental defects of the cerebellum, hippocampus
and retina. At the cellular level, we found that ICK is essential for
ciliogenesis at least in retinal and neural progenitor cells and MEFs.
Conclusions: Our results show that ICK controls protein transport at
the ciliary tip and plays an essential role in ciliogenesis in retinal and
neuronal progenitors but not in mature neurons, proposing a role for
ICK in the regulation of IFT.
Commercial Relationships: Takahisa Furukawa, None; Taro
Chaya, None; Yoshihiro Omori, None
Support: This work was supported by CREST from Japan Science
and Technology Agency, Grant-in-Aid for Scientific Research and
Specially Designated Research Promotion and Scientific Research
on Innovative Areas “Intracellular Logistics” from the Ministry of
Education, Culture, Sports and Technology of Japan .
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 708 Poster Board Number: C0251
Presentation Time: 1:30 PM–3:15 PM
The role of interleukin-33 expression in retinal tissue
Jobu Sugita1, Yosuke Asada1, Hiroyuki Kawano2, Nobuyuki Ebihara1,
Akira Murakami2, Susumu Nakae3, Akira Matsuda1. 1ophthalmology,
Laboratory of Ocular Atopic Diseases, Juntendo University Graduate
School of Medicine, Tokyo, Japan; 2ophthalmology, Department of
Ophthalmology, Juntendo University Graduate School of Medicine,
Tokyo, Japan; 3ophthalmology, Frontier Research Initiative, Institute
of Medical Science, University of Tokyo, Tokyo, Japan.
Purpose: Interleukin-33 (IL-33) is a IL-1 family cytokine, known to
have pro-fibrotic function as other Th2 cytokines. It is also proposed
that IL-33 work as an alarmin in response to cellular injury. In this
study, we investigated IL-33 expression in retinal tissue and its role
for retinal fibrotic responses.
Methods: Immunohistochemical analysis and Western blotting
analysis of mouse retinal tissue (BALB/C strain) were carried out
with anti-mouse IL-33 antibody (R&D systems). Organ culture of
mouse retinal tissue was carried out using OPTI-MEM medium
(Invitrogen) in 96 well culture dishes, and IL-33 concentration in the
supernatant was quantified by mouse IL-33 ELISA kit (e-bioscience).
IL-33 expression after needle injury of the retina was evaluated
with realtime PCR. The effect of recombinant IL-33 injection to the
vitreous cavity was analyzed using IL-33 knockout mouse.
Results: Immunohistological analysis showed that IL-33 expression
was observed in the nucleus of Muller cells of retinal tissue. Western
blotting analysis of retinal tissue confirmed IL-33 protein expression.
IL-33 protein produced from organ culture of retinal tissue increased
from 403pg/ml at 1 hour to 1761pg/ml at 24 hour. Statistically
significant IL-33 mRNA upregulation was observed 24 hour after
needle injury. Recombinant IL-33 injection to the vitreous induced
TGF-beta1, TIMP-1 and COL1A1 expression in the retinal tissue.
Conclusions: Profibrotic cytokine IL-33 is expressed in mouse
retinal tissue and may have some roles for fibrotic responses of the
eye.
Commercial Relationships: Jobu Sugita, None; Yosuke Asada,
None; Hiroyuki Kawano, None; Nobuyuki Ebihara, None; Akira
Murakami, None; Susumu Nakae, None; Akira Matsuda, None
Program Number: 709 Poster Board Number: C0252
Presentation Time: 1:30 PM–3:15 PM
Ectopic BMP4 alters Neural Retina and Retinal Pigmented
Epithelium Specification
Vijay K. Kalaskar1, James D. Lauderdale2. 1Biomed & Health
Sciences Inst, University of Georgia, Athens, GA; 2Cellular Biology,
University of Georgia, Athens, GA.
Purpose: To evaluate the role of BMP4 signaling in neural retina and
retinal pigmented epithelium (RPE) development by ectopic Bmp4
expression in a mouse whole embryo culture system.
Methods: Mouse embryos were obtained from our breeding colony
with noon on the day of plug discovery designated as embryonic
day 0.5 (E0.5). Wild-type (WT) embryos of CD1-C57BL/6J genetic
background were implanted with affi-gel agarose beads treated either
with recombinant BMP4 or Noggin proteins in the eye region of
embryos at E10.5 and cultured in a standardized serum free medium.
Contralateral eyes implanted with BSA protein treated beads were
used as control. Eye tissues from these embryos cultured for 10
– 18 hours were analyzed for development and differential gene
expression using immunofluorescence, western blot, qRT-PCR and in
situ hybridization.
Results: Ectopic expression of Bmp4 in the ocular region altered
neural retinal specification and affected the development of RPE
pigmentation. The neural retina showed significant down-regulation
of specific markers such as Vsx2 (Chx10) and Pax6. While in the
RPE, the pigmentation was affected in a stage-dependent manner.
When the ocular tissue was exposed to BMP4 before the stage of
visible pigmentation (~30-32 somite stage (ss)), the development of
pigmentation was inhibited and when exposed after the initiation of
pigmentation (~34-35 ss), the RPE showed decreased pigmentation.
Further evaluation of the ocular tissue revealed significant changes
in the expression of early genes such as Rx, Six3 in the retina and
Wnt13, Otx2, Mitf and other downstream pigmentation genes in the
RPE in the BMP4 treated eyes compared to the BSA treated eyes.
Conclusions: BMP4 alters the expression of early genes important
in neural retinal specification and RPE pigmentation indicating a
potential role for BMP4 in RPE and retinal development.
Commercial Relationships: Vijay K. Kalaskar, None; James D.
Lauderdale, None
Support: 1) Children’s Glaucoma Foundation, 2) Sharon Stewart
Aniridia Research Trust
Program Number: 710 Poster Board Number: C0253
Presentation Time: 1:30 PM–3:15 PM
Hedgehog signaling regulates cell movements underlying choroid
fissure formation
Kristen Kwan, Emily Wirick. Human Genetics, University of Utah,
Salt Lake City, UT.
Purpose: The choroid fissure is a transient, yet critical structure
through which retinal axons exit and vasculature enters the eye.
Disruption of choroid fissure formation or fusion results in uveal
coloboma. Loss-of-function mutations in the Hedgehog (Hh)
receptor patched2 result in colobomata in humans and zebrafish.
These mutations result in overactive Hh signaling, yet the specific
mechanisms by which overactive signaling leads to colobomata are
unknown. Previous work demonstrated that Hh signaling plays a
key role in patterning the dorsal-ventral axis of the eye, however,
it is unknown whether it plays a more direct role controlling cell
movements during eye morphogenesis.
Methods: We developed 4-dimensional live imaging techniques
for visualizing and tracking cell movements during optic cup
morphogenesis. For cell tracking, embryos are labeled for membranes
(EGFP-CAAX) and nuclei (H2A-mCherry); 4D datasets are acquired
via laser scanning confocal microscopy. Cell tracking is performed
using custom LongTracker manual tracking software, and results
visualized using FluoRender. The photoactivatable fluorophore
Kaede is used to quantify movements of specific cell populations
contributing to the ventroanterior retina, including the anterior margin
of the choroid fissure. Overactivation of Hh signaling is achieved via
expression of activated Smoothened or use of the zebrafish patched2
(blowout) mutant.
Results: We find that cells moving into the optic vesicle during
a specific time period that we term late evagination populate the
ventroanterior retina, including the anterior margin of the choroid
fissure. We hypothesized that activated Hh signaling would affect
late evagination, thereby disrupting choroid fissure formation. We
find that activated Hh signaling, either via expression of activated
Smoothened or using the zebrafish patched2 mutant, inhibits late
evagination cell movements: cells do not migrate out of the brain,
and as a result, choroid fissure formation is disrupted, resulting in
colobomata.
Conclusions: We have identified the cellular origin of the anterior
choroid fissure. Overactive Hh signaling affects a very early step of
optic cup and choroid fissure formation, suggesting a direct effect on
morphogenesis and cell behavior. We are currently determining the
effect of overactive Hh signaling on actin-based single-cell behaviors,
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
as well as whether Hh is acting via canonical or non-canonical
pathways.
Commercial Relationships: Kristen Kwan, None; Emily Wirick,
None
Support: University of Utah Research Foundation, Knights Templar
Eye Foundation
Program Number: 711 Poster Board Number: C0254
Presentation Time: 1:30 PM–3:15 PM
Laminin β2 and γ3 Chains Regulate Retinal Progenitor Cell
Division Polarity
Dmitri Serjanov1, 2, Galina Bachay1, 2, Dale D. Hunter1, 2, William
J. Brunken1, 2. 1Ophthalmology, SUNY Downstate Medical Center,
Brooklyn, NY; 2SUNY Eye Institute, Brooklyn, NY.
Purpose: Basement membranes are highly organized extracellular
matrices that are important sources of developmental cues. Laminin,
a heterotrimeric molecule, is an indispensable organizational
component of basement membranes. Mutations in laminin genes
lead to defective CNS and ocular development in mice and humans.
This study investigates the role of two constituents of CNS laminins,
laminin β2 and γ3 chains in the regulation of the retinal progenitor
cell polarity as characterized by angles of division, cell cycle
dynamics and the consequent neurogenesis.
Methods: Retinae from P3 WT, laminin β2-/- and laminin γ3-/- mice
were used in this study. Immunohistochemistry was performed using
centrosomal and mitotic markers. Retinal progenitor cell angles of
division were visualized through 3D-reconstruction of the dividing
nuclei. Division angles were calculated in 3D by measuring the angle
between a line joining opposing centrosomes and the plane of the
apical retinal surface.
Results: It has been previously shown that deletion of the laminin
β2 and γ3 chains results in alteration of the cell cycle of retinal
progenitor cells, retinal ganglion cell development, Müller glial
cell polarity and photoreceptor development. Here, we demonstrate
different cell division dynamics between developmentally older
and younger regions of the retina as well as a disruption in laminin
mutants. In WT retinae, cell divisions in younger regions are
preferentially symmetrical and gradually become more asymmetric
as the retina becomes older. In laminin β2-/-retinae, the divisions in
all regions are skewed, with noticeable division abnormalities. In
laminin γ3-/- retinae, younger regions are unaffected, whereas older
regions are more symmetrically oriented than in WT retinae. There
is also a high incidence of centrosomal abnormalities in laminin
β2-/-retinae, such as multipolar cells and centrosomes with multiple
centrioles.
Conclusions: Together with our previous findings, these data suggest
that the ILM provides orientation cues and that laminin deletions lead
to premature progenitor pool depletion, resulting in overproduction of
early-born cells at the expense of later-born ones.
Commercial Relationships: Dmitri Serjanov, None; Galina
Bachay, None; Dale D. Hunter, None; William J. Brunken, None
Support: NEI Grant EY12676, RPB Challenge Grant to Department
of Ophthalmology
Program Number: 712 Poster Board Number: C0255
Presentation Time: 1:30 PM–3:15 PM
Increased dendritic branching in direction selective retinal
ganglion cells in nob1 mice
Hung-Ya Tu1, 5, April Bang5, Adam R. McQuiston4, Chuan-Chin
Chiao2, 3, Ching-Kang J. Chen5. 1Institute of Molecular Medicine,
National Tsing Hua University, Hsinchu, Taiwan; 2Department of
Life Science, National Tsing Hua University, Hsinchu, Taiwan;
3
Institute of Systems Neuroscience, National Tsing Hua University,
Hsinchu, Taiwan; 4Department of Anatomy and Neurobiology,
Virginia Commonwealth University, Richmond, VA; 5Department
of Biochemistry and Molecular Biology, Virginia Commonwealth
University, Richmond, VA.
Purpose: Persistent retinal waves in nob1 mice result in abnormal
ganglion cell projections to the brain. However, it remains unclear
whether dendritic morphology and synaptic connectivity are
also altered in the retina. Therefore, we examined the dendritic
development of a genetically identified retinal ganglion cell in the
nob1 background.
Methods: Transgenic mice expressing GFP under the control of a
thyrotropin-releasing hormone receptor promoter (Gong et al., 2003;
Rivlin-Etzion et al., 2011) were obtained from Dr. William Guido
and crossed into the nob1 background. Intrinsic membrane properties
of GFP-positive ganglion cells were recorded by whole-cell current
clamp with potassium-based internal solution containing biocytin.
The dendritic morphologies of recorded cells were visualized by
dye-conjugated streptavidin. Image stacks were acquired in a Zeiss
LSM-710 at 0.5 μm z-interval and traced in the Neurolucida program
for quantitative morphometric analyses.
Results: The wild type GFP-positive ganglion cells displayed
characteristic intrinsic membrane properties and were bistratified with
dendrites co-fasiculated with cholinergic amacrine cells as previously
reported (Rivlin-Etzion et al. J Neurosci 31:8760-8769, 2011). In
the nob1 background, membrane potentials of these cells oscillated
with a characteristic peak frequency of 4-6 Hz, first observed around
postnatal day (P) 15 (4.1±0.1 Hz, n=7) and persisted into adulthood
(5.9±0.2 Hz at P28, n=11; p < 0.001). Cells in heterozygous female
(S+) mutants possessed hyper synaptic activity but not rhythmic
oscillations as observed in homozygous female and mutant male
animals. The numbers of dendritic branching points in adult ganglion
cells in the nob1 and S+ backgrounds were significantly higher than
those of the control animals. We also examined GFP-positive cells in
adult mice lacking Gβ5 and R7 RGS proteins where ERG b-waves
were similarly missing. We found that they oscillated similarly to
nob1 mice (4.9±0.4 Hz; n=11; p > 0.05) but surprisingly did not
display differences in their dendritic morphologies.
Conclusions: The results from the nob1 mice suggest that rhythmic
and arrhythmic hyperactivities in the retina facilitate dendritic
branching in this bistratified ganglion cell type. The lack of such
a phenotype in mice lacking Gβ5 and R7 RGS proteins implies
that these proteins may have a cell-autonomous role in dendritic
development and/or plasticity.
Commercial Relationships: Hung-Ya Tu, None; April Bang, None;
Adam R. McQuiston, None; Chuan-Chin Chiao, None; ChingKang J. Chen, None
Support: NIH Grant EY013811; NIH Grant EY022228
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 713 Poster Board Number: C0256
Presentation Time: 1:30 PM–3:15 PM
Characterization of Retinal Structure and Function in Mice
Carrying Ezh2 Deficiency Specifically in Retinal Ganglion Cells
Lin Cheng1, 2, Honghua Yu2, Naihong Yan2, 3, Honghao Zhou1,
Dongfeng Chen2. 1Institute of Clinical Pharmacology, Central South
University, Changsha, China; 2Schepens Eye Research Institute,
Department of Ophthalmology, Harvard Medical School, Boston,
MA; 3Department of Ophthalmology and Ophthalmic Laboratories,
West China Hospital, Sichuan University, Chengdu, China.
Purpose: Previously in our lab we detected photoreceptor
degeneration in Chx10-Cre-Ezh2flox/flox mice, suggesting that
histone methylase Ezh2 plays an essential role in the retinal
photoreceptor development and function. In this study, we sought
to investigate the role of Ezh2 in retinal ganglion cell (RGC)
development and function.
Methods: To analyze the role of Ezh2 in RGC development, we
generated RGC specific deficiency of Ezh2 by crossing Math5cre with Ezh2flox/floxt mice. Retinal functions were assessed
by electroretinography (ERG) in animals aged 1- 8 months. The
thickness of the ganglion cell complex (GCC) was measured with
spectrum domain-OCT. RGCs and optic nerve fibers were counted
in anti-βIII-tubulin immunolabeled retinal flat-mounts and optic
nerve cross sections, respectively. The expression levels of various
retinal cell markers, such as photoreceptor (recoverin) and ganglion
cell (β-III-tubulin) markers were quantitatively assessed by both
immunolabeling and real time RT-PCR. cDNA microarray was
performed to determine the gene expression profile in Ezh2-deficient
mice. In addition, purified RGC were cultured and their cell survival
and neurite outgrowth were determined. In the above studies,
Ezh2flox/flox mice were used as control animals.
Results: Math5Cre-Ezh2flox/flox mice showed no significant
difference in ERG a wave and b wave responses and GCC thickness.
The retinal lamination and staining pattern of recoverin+ and β-IIItubulin+ cells were visibly comparable between the knockout and
control mice. Moreover, we did not observe significant differences
in wildtype and Math5-Ezh2-/- mice in RGC survival and neurite
outgrowth ability in culture.Results of the cDNA microarray showed
that 0.3% of genes were upregulated and 0.05% of genes were
downregulated in RGCs.
Conclusions: No apparent developmental or functional defects were
observed in Math5Cre-Ezh2-/- mice, suggesting that Ezh2 may not
play a significant role in RGC development or function.
Commercial Relationships: Lin Cheng, None; Honghua Yu, None;
Naihong Yan, None; Honghao Zhou, None; Dongfeng Chen, None
Support: Department of Veterans Affairs (1I01RX000110),
Department of Defense (W81XWH-09-2-0091), Lion’s Foundation
Grants to D.F.C. and K.S.C., China Scholarship Council (CSC).
Program Number: 714 Poster Board Number: C0257
Presentation Time: 1:30 PM–3:15 PM
Expression and role of classical cadherins in the mammalian
retina
Irina De la Huerta1, 2, Xin Duan2, Masahito Yamagata2, Joshua R.
Sanes2. 1Ophthalmology, University of California San Francisco,
San Francisco, CA; 2Center for Brain Science and Department of
Molecular and Cellular Biology, Harvard University, Cambridge,
MA.
Purpose: The circuitry of the mammalian retina depends on the
formation of correct synapses during development. Classical
cadherins are homophilic adhesion molecules present at synapses in
the central nervous system and known to stabilize neuronal contacts.
In this study we describe the expression of classical cadherins in the
mouse retina and examine the roles of selected cadherins in synaptic
specificity.
Methods: In situ hybridization was used to determine the expression
of 18 classical cadherins in the mouse retina during development.
The cadherins that marked relatively small subsets of retinal ganglion
cells (RGCs), or of bipolar or amacrine interneurons known to
synapse with RGCs, were selected for further study. We used mouse
lines in which a marker was knocked in after the start codon of each
cadherin gene of interest to further characterize the connections made
by cadherin-expressing neurons and to determine the effect of loss of
cadherin expression.
Results: We began by examining the expression of all classical
cadherins in the mouse retina during development. Seven cadherins
were expressed in RGCs and/or in bipolar and amacrine interneurons
that are known to synapse with RGCs in the inner plexiform layer of
the retina (IPL). The IPL is composed of 5 sublayers (S1-5), of which
S1-3 are the OFF sublaminae while S4-5 are the ON sublaminae.
We traced the projections of neurons expressing the seven cadherins
of interest and determined that four cadherins (cdh 4, 8, 13, and 22)
marked predominantly OFF-projecting RGCs while two cadherins
(cdh 6 and 10) were expressed by ON-OFF RGCs. Cadherin 8
also marked a subset of OFF bipolar cells while cadherin 9 was
expressed by a subset of ON bipolar cells. Analysis with previously
validated markers showed that the cdh8- and cdh9-positive bipolar
cells are types 2 and 5 respectively. Patterns of cadherin expression
are established before eye opening and are therefore independent of
visual experience. In ongoing work we are using loss- and gainof-function studies to elucidate the roles of cdh8 and chd9 in the
synaptic specificity of ON- and OFF- bipolar cells.
Conclusions: Classical cadherin expression distinguishes subtypes of
RGCs and interneurons during development. Certain cadherins may
play a role in targeting retinal interneurons to the appropriate synaptic
sublaminae in the IPL.
Commercial Relationships: Irina De la Huerta, None; Xin Duan,
None; Masahito Yamagata, None; Joshua R. Sanes, None
Support: NIH Grants NS29169 and EY019355 and a National
Science and Engineering Research Council of Canada Grant to
I.D.l.H.
Program Number: 715 Poster Board Number: C0258
Presentation Time: 1:30 PM–3:15 PM
Differential Cell Adhesion in Organization of the Cone Synapse
Peter G. Fuerst. 1Biology, University of Idaho, Moscow, ID;
2
WWAMI Medical Education Program, University of Idaho, Moscow,
ID.
Purpose: Development of the retina’s functional circuitry involves
the coordinated interactions of multiple cell types. Despite the limited
number of cell types in the retina compared to other parts of the
brain the retina contains some of the nervous system’s most complex
synapses. For example, development of cone synapses involves
multiple bipolar and horizontal cells contacting and making synapses
with distinct cone photoreceptors, each of which in turn is contacted
by multiple bipolar cells of different types.
Methods: In this study we assay the localization of cadherin,
protocadherin and IGF superfamily adhesion molecules, as well
as PDZ containing scaffolding molecules and catenins at the cone
synapse. Functional analysis of these molecules was focused on
gain and loss of function of the IGF superfamily gene Dscam
(Down Syndrome Cell Adhesion Molecule). The influence of cell
density, cell death and disorganization of downstream circuitry was
also assayed by utilizing bax, bcl2 and cleaved caspase 3 null mice
and by conditional disorganization of inner retinal circuitry. The
integrity of the cone synapse was assayed by immunohistochemistry,
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
3-dimensional electron microscopy and electroretinography (ERG)
analysis.
Results: A complex localization pattern of cell adhesion molecules
was observed. For example some bipolar cells make contacts at
both cone and rod synapses and localization of some cadherins
was observed on the bipolar cell dendrites contacting cone but not
rod synapses. Functional analysis of Dscam function in the outer
plexiform layer indicated that this gene is required to provide
heteroneuronal recognition within cell types, while ectopic expression
resulted in the formation of ectopic synapses in the outer nuclear
and photoreceptor layers. A role for cell density and the integrity of
downstream circuitry was found with respect to the contacts between
some bipolar cells and the cone synapse.
Conclusions: Our data are consistent with the differential adhesion
hypothesis of development. As more players underlying adhesionmediated organization of the retina are discovered an elegant
picture of the mechanism underlying retinal synaptic organization is
emerging.
Commercial Relationships: Peter G. Fuerst, None
Support: NIH Grant EY020857
Program Number: 716 Poster Board Number: C0259
Presentation Time: 1:30 PM–3:15 PM
Abnormal synaptic transmission between photoreceptors and
bipolar cells in DHDDSK42E/K42E mice
Rong Wen1, Byron L. Lam1, Ziqiang Guan2, Zhengying Wang1,
Ning Wang1, Yihui Chen1, Yiwen Li1. 1Bascom Palmer Eye Institute,
University of Miami, Miami, FL; 2Biochemistry, Duke University
Medical Center, Durham, NC.
Purpose: We recently identified a single-nucleotide mutation
c.124A>G in the DHDDS gene encoding dehydrodolichol
diphosphate synthase (DHDDS), which changes a highly conserved
Lys42 to Glu and is responsible for 12% of autosomal recessive RP
(arRP) cases in patients of Ashkenazi Jewish (AJ) origin. The present
work characterizes electrophysiological changes in recently created
DHDDSK42E/K42E mice.
Methods: Transgenic mice with DHDDSK42E genotype were created
by the knock-in (KI) technology and bred into homozygosity. Lipids
were extracted from plasma and dolichols were measured by liquid
chromatography-mass spectrometry (LC-MS). Scotopic full-field
ERGs were recorded from 3 month old DHDDSK42E/K42E mice and
compared to age-matched wild-type (wt) animals.
Results: The DHDDSK42E/K42E genotype was confirmed by PCR. A
characteristic shortening of dolichol length distribution was found
in the plasma of DHDDSK42E/K42E mice, similar to what was found
in patients. Dolichol 17 (D17) became the dominant species in the
mutant mice instead of dolichol 18 (D18) in wt animals. As a result,
the DHDDSK42E/K42E mice have much higher plasma D17/D18 ratio.
The ERG a-wave in the DHDDSK42E/K42E mice was smaller than
a-wave of the wt controls, and the b-wave was disproportionally
smaller with the b- to a-wave amplitude ratio being close to 1. In
contrast, the ratio was more than 2 in the wt controls.
Conclusions: These results indicate that abnormal dolichol
biosynthesis by the K42E DHDDS mutation leads to impaired
synaptic transmission between photoreceptors and bipolar cells.
Previous studies using artificial membrane suggest a potential role of
dolichols in facilitating vesicle fusion. Our results provide the first in
vivo evidence supporting a biological function of free dolichols in the
activities of synaptic vesicles.
Commercial Relationships: Rong Wen, None; Byron L. Lam,
None; Ziqiang Guan, None; Zhengying Wang, None; Ning Wang,
None; Yihui Chen, None; Yiwen Li, None
Support: NIH grants R01EY018586, P30-EY014801, LIPID MAPS
Collaborative Grant GM-069338, Department of Defense grant
W81XWH-09-1-0674, Adrienne Arsht Hope for Vision fund, and
Research to Prevent Blindness, Inc.
Program Number: 717 Poster Board Number: C0260
Presentation Time: 1:30 PM–3:15 PM
Identification of early retinal bipolar cell-specific genes
Ko Park1, Joseph A. Brzezinski1, Tatiana Eliseeva1, Kenneth Jones2.
1
Ophthalmology, Universith of Denver, Aurora, CO; 2Biochemistry
and Molecular Genetics, University of Colorado School of Medicine,
Aurora, CO.
Purpose: The mechanisms that control bipolar cell formation
during retinogenesis are incompletely understood. Mice that lack the
transcription factor Blimp1 (Prdm1) form bipolar cells at the expense
of photoreceptors shortly after birth. To discover early bipolar cell
regulators, we identified genes that were precociously upregulated in
Blimp1 mutant retinas.
Methods: Postnatal day (P) 2 retinas from five Blimp1 conditional
null mice and five controls were processed for high throughput
RNA-sequencing. Samples were sequenced to an average depth of
30 million reads. The reads were mapped to annotated transcripts
and normalized as reads per kilobase exon per million mapped
reads. Genes that were statistically different between conditions and
that changed at least 40% were considered candidate regulators.
Candidates were further evaluated by in situ hybridization and
immunohistochemistry.
Results: RNA-seq comparison between P2 Blimp1 conditional
null and control retinas revealed approximately 84 significantly
upregulated genes. This included several known bipolarspecific genes, such as Vsx1 and Scgn; which we validated by
immunohistochemistry. We also characterized the expression of a
novel upregulated gene, Tmem215, which codes for a transmembrane
protein of unknown function. We first observed Tmem215 expression
by in situ hybridization in the central retinas of P2 mice. Expression
spread to the periphery by P5 and became more robust in adult
retinas. Tmem215 expression was confined to a narrow region within
the inner retina, consistent with bipolar cell localization.
Conclusions: We have identified several potential early bipolarspecific genes by searching for precociously upregulated genes in
Blimp1 mutant retinas. The expression of one of these candidates,
Tmem215, closely paralleled the spatial and temporal pattern of
bipolar cell genesis and maturation. It remains unclear what role
Tmem215 plays in bipolar cell development.
Commercial Relationships: Ko Park, None; Joseph A. Brzezinski,
None; Tatiana Eliseeva, None; Kenneth Jones, None
Program Number: 718 Poster Board Number: C0261
Presentation Time: 1:30 PM–3:15 PM
Genomic Control of Horizontal Cell Regularity
Patrick W. Keeley1, 2, Benjamin E. Reese1, 3. 1Neuroscience Research
Institute, Univ of California, Santa Barbara, Santa Barbara, CA;
2
Molecular, Cellular, and Developmental Biology, Univ of California,
Santa Barbara, Santa Barbara, CA; 3Psychological and Brain
Sciences, Univ of California, Santa Barbara, Santa Barbara, CA.
Purpose: Retinal neurons are often arranged in nonrandom mosaics,
as their somata are distributed to minimize proximity to neighboring
cells of the same type. The mosaic of horizontal cells (HC) is an
exemplar of such a distribution, but little is known of molecular
determinants controlling its patterning. We have previously shown
that different strains of mice vary in the regularity of their HC
mosaics, indicating a genetic component to this trait.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Methods: The present study adopted a forward genetic approach to
seek candidate genes controlling nerve cell patterning, quantifying
the regularity of the HC mosaic in 25 genetically diverse recombinant
inbred strains of mice, each of known genotype, being derived from
the C57BL/6J and A/J inbred laboratory strains. For each strain (n ~
3 mice), we sampled the population of horizontal cells at eight retinal
locations in every mouse and calculated four spatial statistics: nearest
neighbor regularity index (NNRI), Voronoi domain regularity index
(VDRI), effective radius (ER), and packing factor (PF).
Results: The regularity indexes varied across the 25 RI strains,
increasing by 24% from the lowest to the highest strain, while the
coefficient of variation (CoV) within each strain showed relatively
little variation (average CoV=0.04). The estimated heritability of
these indexes was ~0.5, indicating a sizeable proportion of the
variation in trait values across all 95 mice could be ascribed to an
effect of genotype. The two regularity indexes correlated highly
with one another across the recombinant inbred strains (r=0.80), and
each was significantly correlated with PF (r=0.78 and 0.81 for NNRI
and VDRI, respectively). These indexes were moderately correlated
with ER (r=0.59 and 0.46), while showing no significant correlation
with cell density (r=-0.27 and -0.15). Using quantitative trait loci
mapping, we identified two genomic loci, on Chromosomes 1 and 14,
that modulate the regularity of the HC mosaic. Together, these two
quantitative trait loci account for ~44% and ~31% of the interstrain
variation in NNRI and VDRI, respectively.
Conclusions: Using the population of horizontal cells, we show
that mosaic regularity and packing are heritable traits, and that these
traits can be mapped to discrete genomic loci. Further interrogation
of these loci should identify candidate gene variants responsible for
this variation in mosaic patterning, ultimately revealing the molecular
mechanisms of mosaic assembly.
Commercial Relationships: Patrick W. Keeley, None; Benjamin
E. Reese, None
Support: NIH Grant EY-19968
Program Number: 719 Poster Board Number: C0262
Presentation Time: 1:30 PM–3:15 PM
The bHLH transcription factors Ascl1a and NeuroD function in
a regulatory feedback loop with the Notch pathway to regulate
proliferation of photoreceptor progenitors
Scott M. Taylor1, Karen Alvarez-Delfin2, Carole Saade2, James M.
Fadool2, Peter F. Hitchcock1. 1Ophthal & Visual Sciences, University
of Michigan, Ann Arbor, MI; 2Biological Science, Florida State
University, Tallahassee, FL.
Purpose: Development of photoreceptors in the vertebrate retina
requires precise regulation of cell cycle entry and exit. In the
retina of the embryonic zebrafish, the bHLH transcription factor
NeuroD mediates exit of photoreceptor progenitors from the cell
cycle (Ochocinska and Hitchcock, 2009). The purpose of this study
was to determine the mechanisms through which NeuroD governs
photoreceptor genesis in the zebrafish retina.
Methods: First, genetic mosaic analysis was performed to determine
if NeuroD functions cell autonomously in the developing retina.
Second, morpholino-induced NeuroD loss-of-function (LOF) was
used in combination with in-situ hybridization and qRT-PCR to
determine which molecules/pathways are regulated by NeuroD.
Third, LOF approaches were used for putative target proteins to
experimentally test the hypothesized relationships with NeuroD and
with each other.
Results: Genetic mosaic analysis revealed that NeuroD functions
non-cell autonomously in the developing retina, and therefore
subsequent experiments were focused on identifying mechanisms
that could mediate this function. In-situ hybridization and qRT-PCR
revealed that expression of notch1a, her4, ascl1a and hes6 increase
following NeuroD LOF. Inhibition of the Notch pathway using
the gamma secretase inhibitor DAPT rescued the NeuroD LOF
phenotype and restored ascl1a expression to normal levels. Ascl1a
LOF resulted in the loss of neuroD expression, increased Notch
pathway activity, and increased ascl1a expression. Both Ascl1a
and NeuroD LOF resulted in increased cyclinD1 and cyclinB1
expression, indicating a shared mechanism of cell cycle regulation.
Conclusions: Taken together, these data indicate that within
photoreceptor progenitors the Notch pathway functions upstream of
Ascl1a, which, in turn, governs the expression of NeuroD, which
provides feedback inhibition onto the Notch pathway and ascl1a. The
data also show that loss of Ascl1a maintains retinal progenitors in a
proliferative, undifferentiated state and that Ascl1a strongly regulates
its own expression.
Commercial Relationships: Scott M. Taylor, None; Karen
Alvarez-Delfin, None; Carole Saade, None; James M. Fadool,
None; Peter F. Hitchcock, None
Support: NIH T32 EY013934, R01 EY07060-22, F32 EY023129-02
Program Number: 720 Poster Board Number: C0263
Presentation Time: 1:30 PM–3:15 PM
The ponli enhancer activates transcription in the zebrafish green,
red and blue cone photoreceptors
Wei Fang1, 2, Xiangyun Wei1, 2. 1Department of Ophthalmology,
University of Pittsburgh School of Medicine, Pittsburgh, PA;
2
Department of Developmental Biology, University of Pittsburgh
School of Medicine, Pittsburgh, PA.
Purpose: The ponli gene encodes a membrane-associated apical
polarity protein. The Ponli protein is restrictively expressed the
inner segment membrane regions of green, red, and blue cone
photoreceptors in zebrafish. Ponli is expected to play a critical role
in the cone mosaic patterning in the zebrafish. It is unknown how
the distinct expression pattern of the ponli gene is established and
maintained in the zebrafish retina. Thus, we set out to identify the cisregulatory elements of the ponli gene, which we hypothesize should
be conserved among different teleost species that express the ponli
gene.
Methods: The conserved cis-elements were predicted by aligning the
sequences of ponli genes of five teleost species using the ClustalW2
sequence alignment program. The functions of these predicted
cis-elements were investigated via mutation analyses in vivo. The
expression levels and patterns of reporter genes and the endogenous
ponli gene were analyzed by confocal microscopy, real-time PCR,
and Western blotting.
Results: We showed that the promoter of the ponli gene resides
in a 130-bp sequence upstream of its transcription start site and its
enhancer is located in the 5.7-kb first intron. These cis-regulatory
elements drive specific expression of mCherry in green, red, and blue
cones in the Tg(ponli130-bp promter+5.7-kb enhancer:HA-mcherry)pt118b transgenic
fish. The first intron of the ponli gene contains a 200-bp region
that is sequentially and functionally conserved among five teleosts.
This critical region is likely a central part of the ponli enhancer that
determines the cell-type specificity. In addition, the expression level
of the ponli cis-regulatory elements is moderate, thousands times
lower than that of the green opsin promoter.
Conclusions: The ponli enhancer provides us with an entry point
to study how vertebrate cone and rod photoreceptors differ in their
biochemical, structural, and functional properties by expressing
distinct gene profiles. The ponli enhancer may potentially serve as a
handle to reveal specific interactions between cis- and trans-acting
transcriptional regulatory elements in these cones. In addition, the
ponli enhancer provides us with a unique tool to specifically and
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
moderately express transgenes in the zebrafish green, red, and blue
cones.
Commercial Relationships: Wei Fang, None; Xiangyun Wei, None
Support: NIH Core Grant P30EY008098, NIH R01 Grant
EY016099, RPB Wasserman Merit Award (to X.W.)
Program Number: 721 Poster Board Number: C0264
Presentation Time: 1:30 PM–3:15 PM
Immunocytochemical analysis of misplaced rhodopsin-positive
cells in the developing rodent retina
Klaudia Szabo1, Arnold Szabo1, Anna Enzsoly2, Agoston Szel1, Ákos
Lukáts1. 1Human Morphology & Dev Biol, Semmelweis University,
Budapest, Hungary; 2Department of Ophthalmology, Semmelweis
University, Budapest, Hungary.
Purpose: During the first postnatal weeks of the developing rodent
retina, rhodopsin can be detected in a number of neuron-like cells
in the inner retina. In the present study we aimed to characterize the
morphology, number and staining characteristics of this peculiar
population.
Methods: Misplaced rhodopsin-positive cells (MRCs) were analyzed
on the retinas of four rodent species, labeled with various rhodopsinspecific antibodies. To investigate their possible relation with nonphotoreceptor cells, sections were double-stained against distinct
retinal cell types, while the possibility of photoreception was assessed
by counter staining with cone- and melanopsin specific antibodies
and proteins of the phototransduction cascade. The possibility of
synapse formation and apoptosis were also investigated.
Results: In all species studied, misplaced cells comprised a few
percent of all rhodopsin-positive elements. Their ratio was relatively
constant with a decline from the end of the second week, and
MRCs disappeared near completely from the retina by P24. MRCs
resembled resident neurons of the inner retina; outer segmentlike processes were seen only rarely. MRCs expressed no other
photopigment types and showed no colocalization with any of the
bipolar, horizontal, amacrine and ganglion cell markers used. While
all MRCs colabeled for arrestin and recoverin, other proteins of the
phototransduction cascade were only detectable in a minority of the
population. Only a few MRCs were shown to form synaptic-like
endings.
Conclusions: Our results showed that during development a
significant percentage of the hodopsin-expressing cells are displaced
to the inner retinal layers. Although most MRCs lack morphological
features of photoreceptors, they contain some, but not all elements of
the phototransduction cascade, indicating that they are most probably
misplaced rods that failed to complete differentiation and integrate
into the photoreceptor mosaic.
Commercial Relationships: Klaudia Szabo, None; Arnold Szabo,
None; Anna Enzsoly, None; Agoston Szel, None; Ákos Lukáts,
None
Support: Hungarian Scientific Research Fund (OTKA #73000),
TÁMOP- 4.2.1.B-09/1KMRB2010-0001.
Program Number: 722 Poster Board Number: C0265
Presentation Time: 1:30 PM–3:15 PM
F-Spondin, a Neuronal Guidance Molecule and a Regulator of
Amyloid Beta Generation is Expressed Discretely in a Subset of
Cone Photoreceptors within the Retina
Rupalatha Maddala1, Paulo Ferreira2, Vasanth Rao3.
1
Ophthalmology, Duke University Medical Center, Durham, NC;
2
Opthalmology & Cell Biology, Duke University Medical Center,
Durham, NC; 3Opthalmology & Pharmacology, Duke University
Medical Center, Durham, NC.
Purpose: F-Spondin, a floor plate enriched multidomain extracellular
matrix-associated protein is known to play a critical role in axonal
pathfinding and in neuronal migration, differentiation and survival
by regulating cell adhesive interactions. Additionally, F-spondin is
involved in the regulation of proteolysis of amyloid precursor protein
and ApoER2 and generation of amyloid beta. Here we report the
discrete distribution profile of F-spondin in the photoreceptor layer of
both the mouse and human retina.
Methods: F-Spondin expression and distribution profiles in
the retinas of mice (C57BL6) were determined by immunoblot,
immunofluorescence and RT-PCR analyses. F-spondin colocalization
with rhodopsin, cone arrestin, M-opsin, S-opsin and peanut
agglutinin was determined using mouse retinal sections and flat
mounts. The levels of F-spondin in cone dystrophy and cone-only
mouse models were determined in the Ranbp2 coneless (Tg-HRGPcre:Ranbp2Flox/Flox) and Nrl null mice respectively, using immunoblot
and immunofluorescence analyses.
Results: F-spondin exhibits a mosaic pattern and distributes
discretely to the outer and inner segments of a subset of peanut
agglutinin (PNA) labeled photoreceptor cells (cones) but not to
the rhodopsin presenting cells (rod photoreceptors), based on the
results of immunofluorescence analysis. Unlike PNA, F-spondin is
not localized to the synaptic terminals of photoreceptors. F-spondin
appears to colocalize with S-opsin at both the dorsal and ventral
regions of mouse retina. Developmentally, F-Spondin expression in
the mouse retina coincides with the morphogenesis of photoreceptor
outer segments. Compared to wild-type retinas, the retinas of Ranbp2
coneless and Nrl null mice, the levels of F-spondin were decreased
and increased, respectively.
Conclusions: Taken together, the observation of novel, discrete
and intense co-distribution of F-spondin with cone photoreceptors
suggests a critical role for F-spondin in photoreceptor morphogenesis,
survival or function.
Commercial Relationships: Rupalatha Maddala, None; Paulo
Ferreira, None; Vasanth Rao, None
Support: R01 EY12201, R01EY 18590, P30-EY005722 and RPB.
Program Number: 723 Poster Board Number: C0266
Presentation Time: 1:30 PM–3:15 PM
An analysis of photoreceptor basal body positioning in zebrafish
with mutations in cytoplasmic Dynein 1 and Dynactin
Joseph Fogerty, Brian D. Perkins. Cole Eye Institute, Cleveland
Clinic, Cleveland, OH.
Purpose: The photoreceptor outer segment is an elaborate primary
cilium specialized for photon detection. Anchoring of the basal
body at the apical membrane is a prerequisite for outer segment
extension, but little is known about the processes that regulate this
event. We hypothesized that the microtubule motor cytoplasmic
dynein 1 is required for basal body localization prior to photoreceptor
ciliogenesis. Cannonball mutant zebrafish carry a nonsense mutation
in the heavy chain subunit of cytoplasmic dynein 1 (dync1h1), and
the mikre oko mutant has a nonsense mutation in the p150Glued
subunit of Dynactin (dctn1a), a key Dynein1 regulatory complex.
Previous studies on these mutants showed inner segment organelle
positioning defects as well as impaired outer segment morphogenesis.
We utilized these mutants to evaluate the requirement of Dynein1
based motility for proper basal body positioning.
Methods: Cannonball and mikre oko mutant fish were crossed
to the Tg(-5actb2:cetn2-GFP) transgenic line, which expresses a
centrin-GFP fusion protein from the beta-actin promoter and labels
basal bodies. Basal body positioning relative to the outer limiting
membrane was assayed at multiple time points in frozen sections
counterstained with phalloidin.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: Both cannonball and mikre oko fish underwent a significant
degree of retinal degeneration by 4dpf, with rounded nuclei and
disorganized lamination frequently observed. The apical actin
network was disorganized and often absent in mutant fish, but GFP+
basal bodies were present in areas with an intact outer limiting
membrane. Where basal bodies were observed, they were positioned
at the expected distance from the OLM.
Conclusions: Basal bodies in cannonball and mikre oko fish localize
properly to the apical membrane in those areas of retina that establish
an apical actin network. The patches of retina lacking phalloidin
reactivity have no observable basal bodies and may represent areas
that lose cell polarity after exhaustion of the maternal protein.
Commercial Relationships: Joseph Fogerty, None; Brian D.
Perkins, None
Support: EY017037
146 Glia
Sunday, May 04, 2014 3:15 PM–5:00 PM
S 310E-H Paper Session
Program #/Board # Range: 825–831
Organizing Section: Retinal Cell Biology
Program Number: 825
Presentation Time: 3:15 PM–3:30 PM
Quiescent Retinal Glia are Protective, but their Activation
Increases Vulnerability to Acute RGC Injury In Vivo
Jeremy M. Sivak2, 1, Izzy Livne-Bar2. 1Ophthalmology and Vision
Sciences, University of Toronto, Toronto, ON, Canada; 2Vision
Sciences, Toronto Western Hospital, UHN, Toronto, ON, Canada.
Purpose: Astrocytes and related Müller glia play critical homeostatic
roles in the inner retina, and rapidly activate with disease or injury.
However, the positive and/or negative influence of activation on
retinal ganglion cells (RGCs) has remained controversial. We tested
the influence of quiescent and activated astrocytes on RGC survival
using an acute injury model, combined with pharmacologic methods
and direct transplantation of primary cells.
Methods: An acute mouse excitotoxic RGC damage model was
combined with pharmacologic inhibitors of glial activation, induced
gliosis, or direct injection of quiescent primary astrocytes. The
drugs withaferin A (WFA) and SB203580 (SB) target intermediate
filaments, and p38α and β MAPKs, respectively, to block glial
reactivity. In some experiments prominent glial reactivity was
induced prior to retinal injury by mechanical corneal debridement, as
recently reported (PMID: 20048155). Alternatively, quiescent retinal
astrocytes were injected intravitreally prior to retinal injury.
Results: Administration of WFA or SB strongly reduced GFAP
staining in retinal astrocytes and Müller cells, and significantly
blocked RGC death by approximately 80% (p<0.01). Conversely,
mechanically induced gliosis exacerbated subsequent RGC death
by over 7 fold (p<0.01), which was blocked by WFA or SB. Finally,
intravitreal transplantation of quiescent retinal astrocytes two weeks
prior to injury inhibited RGC death by nearly 80% (p<0.01).
Conclusions: We have used a variety of strategies to consistently
demonstrate that retinal astrocytes are protective to RGCs when
quiescent, but that activation increases vulnerability to acute injury in
vivo.
Commercial Relationships: Jeremy M. Sivak, None; Izzy LivneBar, None
Support: CIHR, GRSC, David & Sandra Smith Fellowship
Program Number: 826
Presentation Time: 3:30 PM–3:45 PM
Idiopathic glial blooms in aged human retinas
Malia M. Edwards1, Scott D. McLeod1, Imran A. Bhutto1, Amanda
Hardin2, Johanna Seddon2, Gerard A. Lutty1. 1Wilmer Eye Institute,
Johns Hopkins University, Baltimore, MD; 2Ophthalmology, Tufts
University School of Medicine, Boston, MA.
Purpose: Four decades ago, Foos discovered glial protrusions
into the vitreous and epiretinal glial membranes using
electron microscopy (1). The present study used wholemount
immunohistochemistry to characterize what appear to be similar
glial structures, termed glial blooms herein, in age-related macular
degeneration (AMD) and aged control retinas.
Methods: Retinal wholemounts from donors with and without AMD
were stained with markers for retinal astrocytes and activated Müller
cells (GFAP), Müller cells (vimentin, glutamine synthetase) and
microglia (IBA-1). Retinal vessels were labeled with UEA lectin.
Imaged retinas were cryopreserved and sections labeled with laminin
to determine the location of glial blooms in relation to the inner
limiting membrane (ILM). Images were collected using a Zeiss 710
Meta confocal microscope.
Results: Epiretinal glial blooms of varying size were observed in
all retinas regardless of disease state. In some retinas, only focal
tufts of glial processes were noted on the vitreal surface over large
vessels. Others contained large, multicellular glial blooms that
extended radially in the vitreous. The most extreme cases had
complete epiretinal glial membranes with compact networks of
cells and processes creating multiple layers. While blooms were
observed in all areas of the retina, they were most prominent over
large vessels and in the peripapillary region. All epirteinal glial
blooms and membranes contained vimentin and GFAP-positive
cells although these proteins were seldom co-localized and vimentin
staining extended beyond that for GFAP. While glutamine synthetase
was prominent in Müller cells within the retina, labeling in blooms
was confined to fine processes at the base. Microglia were found
in blooms but did not concentrate in the retina below them. The
astrocyte template was normal below some blooms but attenuated
below others. Cross sections identified small breaks in the ILM
above large vessels through which glial cells entered the vitreous.
Conclusions: Epiretinal glial blooms are a common occurrence in
aged retinas and, in many cases, subclinical. While all retinal glia
are found in blooms, the extension of vimentin labeling beyond that
of GFAP suggests that Müller cells form the leading edge. Although
these structures may be benign, their increased growth and spread
along the vitreal/retinal interface could exert tractional forces on the
retina.
1. Foos, 1972, Investigative Ophthalmology 11, 801-808.
Commercial Relationships: Malia M. Edwards, None; Scott D.
McLeod, None; Imran A. Bhutto, None; Amanda Hardin, None;
Johanna Seddon, None; Gerard A. Lutty, None
Support: NIEI/NIH EY009357, EY016151 (GL) and EY01765
(Wilmer), The Beckman Foundation (GL & JS), and RPB (Wilmer)
Program Number: 827
Presentation Time: 3:45 PM–4:00 PM
Energy deprivation alters Müller cells ability to protect retinal
ganglion cells
Miriam Kolko1, 2, Anne Katrine Toft-Kehler1, Rupali Vohra1,
Sridevi Gurubaran Iswariyaraja1, Dorte Skytt1. 1Neuroscience and
Pharmacology, University of Copenhagen, Copehnhagen, Denmark;
2
Ophthalmology, Roskilde University Hospital, Roskilde, Denmark.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: To study the effect of energy deprivation in Müller cells
ability to maintain their function as well as their ability to protect
retinal ganglion cells (RGC).
Methods: The human Müller cell line, MIO-M1 and primary
mouse Müller cells were used to study changes in glutamate uptake,
glutamate release, excitatory amino acid transporter (EAAT) protein
expression, ATP levels and glycogen content, when cells were
compromised from energy. Moreover, a co-culture system of primary
RGC cultures and primary Müller cells, separated by an insert, was
used to evaluate the role of Müller cells in RGC survival.
Results: EAAT1 and EAAT2 proteins were up-regulated in energydeprived Müller cells and glutamate uptake was significantly
increased in the absence of glucose, as opposed to conditions with
sodium depletion or in the presence of the EAAT-inhibitor tfb-TBOA,
in which glutamate uptake was decreased. The intracellular glycogen
content decreased in a time-dependent manner, whereas the ATP
levels were sustained following energy deprivation. Co-cultures of
RGC and Müller cells revealed better survival of glutamate treated
RGC in the presence of Müller cells compared to controls. Energy
deprivation of Müller cells enhanced their ability to protect RGC.
Conclusions: The present findings revealed an up-regulation of
EAAT1 and EAAT2 in energy compromised Müller cells as well
as an increased ability to remove glutamate from the extracellular
space. Co-cultures of RGC and Müller cells revealed a protective
role of Müller cells when RGC were exposed to glutamate. Hence,
energy failure may result in an increased ability to protect RGC from
glutamate-induced excitotoxicity, whereas malfunction of glutamate
uptake in Müller cells may contribute to RGC death.
Commercial Relationships: Miriam Kolko, None; Anne Katrine
Toft-Kehler, None; Rupali Vohra, None; Sridevi Gurubaran
Iswariyaraja, None; Dorte Skytt, None
Support: Velux Foundation
Program Number: 828
Presentation Time: 4:00 PM–4:15 PM
Wnt-signaling and the formation of Muller glia-derived
progenitor cells in the chick retina
Donika Gallina, Lillia N. Steffenson, Andrew J. Fischer.
Neuroscience, The Ohio State University, Columbus, OH.
Purpose: One of the signaling pathways that may regulate the
activity of retinal glia and Müller glia-derived progenitor cells
(MGPCs) is the Wnt pathway. During ocular development, Wntsignaling is known to have many important functions that involve
patterning the anterior optic structures and suppressing retinal
development. Studies in zebrafish have shown that activation of
Wnt-signaling is required for MGPC-mediated retinal regeneration
in vivo. Studies in mammalian retina have shown that Wnt-signaling
stimulates the Müller glia to re-enter the cell cycle in retinal explants
in vitro. We investigate the role of the Wnt pathway in the formation
of MGPCs in the avian retina in vivo.
Methods: : Immunocytochemistry was used to study the expression
pattern of nuclear β-catenin (a read-out of Wnt-signaling) in mature
and damaged chick retinas. Wnt- signaling inhibitors (XAV939) or
a combination of Wnt-signaling activators (GSK3β-inhibitors) were
injected into the vitreous chamber of normal and damaged eyes.
Retinas were processed for cell proliferation and glial reactivity.
qRT-PCR was used to measure retinal levels of different Wnt-related
and progenitor genes. Retinal damage was induced by an intraocular
injection of N-Methyl-D-aspartate (NMDA).
Results: We found that nuclear β-catenin is present in many neurons,
but not Müller glia in mature retina. In damaged retina nuclear
β-catenin is expressed by MGPCs (need confirmation). Furthermore,
NMDA-induced retinal damage leads to activation of Wnt-signaling,
indicated by the regulation of transcripts of Wnt-target genes.
Inhibition of Wnt-signaling suppressed the formation of proliferating
MGPCs, and activation of Wnt-signaling expanded the population of
the proliferating MGPCs.
Conclusions: We propose that Wnt-signaling is important for the
proliferation of MGPCs in damaged avian retina in vivo. Our data
suggest a role of Wnt-signaling in the regeneration of the chick retina.
Commercial Relationships: Donika Gallina, None; Lillia N.
Steffenson, None; Andrew J. Fischer, None
Support: EY022030-02
Program Number: 829
Presentation Time: 4:15 PM–4:30 PM
Metabolic changes associated with Müller cells in a transgenic
rabbit model of retinal degeneration
Rebecca L. Pfeiffer, Bryan W. Jones, Robert E. Marc. Ophthalmology,
Moran Eye Center at the University of Utah, Salt Lake City, UT.
Purpose: Müller cells play a central role in retinal metabolism via the
glutamate cycle. During retinal degeneration Müller cells are among
the first to demonstrate changes, reflected in alterations of metabolic
signatures and morphology. The timing, extent and regulation of
these changes is not fully characterized. To address this issue, we
evaluated Müller cell metabolic phenotypes at multiple stages of
retinal remodeling.
Methods: Samples were collected post-mortem from both WT and
P347L rabbits. The retinas were then divided into fragments, fixed
in buffered aldehydes, and embedded in epoxy resins. Tissues were
sectioned at 200nm followed by classification with computational
molecular phenotyping (CMP) using an array of small and
macromolecular signatures (aspartate (D), glutamate (E), glycine (G),
glutamine (Q), glutathione (J), GABA (yy), taurine (T), CRALBP,
Glutamine Synthetase (GS), and GFAP). Levels of amino acid or
protein were quantified by selecting a region of interest either within
the Müller cell population or surrounding neurons and evaluating the
intensity of the signal within that region.
Results: CMP reveals overall decreases in GS levels over the course
of degeneration. Of notable importance, we saw that in regions of
near complete photoreceptor loss neighboring Müller cells may
express independent variation in metabolic signatures of E, Q, and
GS. Also observed in these Müller cells, ratios of GS:E and GS:Q
are not consistent with the ratios seen in WT retina. These results are
inconsistent with the current models of both E to Q metabolism and
microenvironment regulation of Müller cell phenotypes.
Conclusions: These observations indicate two conclusions. First,
although the degenerate state of the retina is the likely trigger
inducing Müller cells to express altered metabolic signatures, the
rate at which the metabolic state changes is not purely a product of
the surrounding environment, but also a stochastic change within
individual Müller cells. Second, although it is commonly accepted
that GS is the primary enzyme which converts Q to E as part of the
glutamate cycle, in degenerate retina alternative pathways may be
utilized following decrease in GS.
Commercial Relationships: Rebecca L. Pfeiffer, None; Bryan W.
Jones, None; Robert E. Marc, Signature Immunologics (E)
Support: NIH Grant EY02576, NIH Grant EY15128, NIH Grant
EY14800, Research to Prevent Blindness, Thome Foundation,
Signature Immunologics, Inc.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 830
Presentation Time: 4:30 PM–4:45 PM
Hedgehog-signaling stimulates the formation of Müller gliaderived retinal progenitors
Levi Todd, Andrew J. Fischer. The Ohio State University, Columbus,
OH.
Purpose: Müller glia can be stimulated to de-diferentiate, proliferate
and form Muller glia-derived progenitor cells (MGPCs) that can
regenerate retinal neurons. The identitiy of the pathways that regulate
the de-differention of mature Müller glia and proliferation of MGPCs
remain largely unknown. Hh-signaling is known to influence the
proliferation of neural progenitors and astrocytes in the developing
and mature nervous system. Thus, the purpose of this study was to
investigate whether Hh-signaling influences the formation of MGPCs
in the chick retina in vivo.
Methods: Immunocytochemistry was used to study Shh expression
in mature and damaged chick retinas. The Hh-signaling inhibitor
KAAD-cyclopamine or recombinant human Shh (rhShh) were
injected into the vitreous chamber of normal and damaged eyes.
Retinas were processed for cell proliferation, and glial reactivity.
qRT-PCR was used to measure retinal levels of different Hh-related
genes, cytokines and progenitor genes. Retinal damage was induced
by an intraocular injection of N-Methyl-D-aspartate (NMDA).
Results: Data is provided that Hh-signaling is active in mature intact
retinas, influencing glial reactivity, and Hh-signaling is dramatically
increased in damaged retinas. rhShh stimulates the formation
of MGPCs in damaged retinas, but not in undamaged retinas.
Conversely, inhibition of Hh-signaling with KAAD-cyclopamine
reduces numbers of proliferating MGPCs in damaged retinas.
Conclusions: This work implicates Hh-signaling as a key regulator
of the formation and proliferation of MGPCs.
Commercial Relationships: Levi Todd, None; Andrew J. Fischer,
None
Support: NIH Grant 5R01EY022030-02
Program Number: 831
Presentation Time: 4:45 PM–5:00 PM
PTEN/mTor-signaling and the formation of Müller glia-derived
retinal progenitors
Chris Zelinka, Zachary A. Goodman, William A. Bishop, Andrew J.
Fischer. Neuroscience, Ohio State University, Columbus, OH.
Purpose: The Akt/mTor pathway is an important cell-signaling
pathway that can influence many cellular processes including growth
and proliferation. During the development of the eye, Akt/mTorsignaling is known to have important context-specific functions.
However, there is a paucity of information regarding the roles that
Akt/mTor-signaling plays in the mature retina. Accordingly, we
investigate the roles of the Akt/mTor pathway in the formation of
Müller glia-derived progenitor cells (MGPCs) in the retina in the
chick model system in vivo.
Methods: : Immunocytochemistry was used to characterize the
expression of pS6 (an effector and readout of the Akt/mTor pathway)
in developing, mature and damaged chick retinas. The PTENinhibitor (VO-OHpic trihydrate) or mTor-inhibitor (rapamycin) were
injected into the vitreous chamber of normal and damaged eyes.
Retinas were processed for cell proliferation and glial reactivity.
Retinal damage was induced by an intraocular injection of N-MethylD-aspartate (NMDA).
Results: pS6 is widely expressed by differentiating cells in
embryonic retina, but there are minimal levels in a mature, normal
retina. Upon excitotoxic damage, pS6 in Müller glia is highly upregulated within 4hrs after damage and remains elevated for 2 days.
This up-regulation of pS6 was blocked by rapamycin. Treatment with
PTEN-inhibitor increased, while treatment with rapamycin inhibited,
the number of proliferating MGPCs.
Conclusions: Akt/mTor-signaling in mature Müller glial regulates
the ability of these glia to become proliferating progenitor-like
cells. We conclude that the Akt pathway is not active in a normal
retina, the Akt/mTOR pathway is rapidly activated in Müller glia in
damaged retinas, and the activation of this pathway is required for the
formation of MGPCs.
Commercial Relationships: Chris Zelinka, None; Zachary A.
Goodman, None; William A. Bishop, None; Andrew J. Fischer,
None
Support: EY-022030-02
161 AMD and CNV: Preclinical and Clinical Studies
Sunday, May 04, 2014 3:15 PM–5:00 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 1173–1205/C0201–C0233
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 1173 Poster Board Number: C0201
Presentation Time: 3:15 PM–5:00 PM
The Apolipoprotein E4 Targeted Replacement (APOE4 TR)
Mouse Model of Age-Related Macular Degeneration (AMD)
Exhibits Anti-Retinal Autoreactivity
Albert H. Alhatem1, Nataliya Lenchik1, 2, Sarka Beranova-Giorgianni4,
Mikael Klingeborn5, David D. New1, Francesco Giorgianni4, Ivan
Gerling2, Marko Radic3, Catherine Bowes Rickman5, 6, Alessandro
Iannaccone1. 1Ophthalmology, Hamilton Eye Institute, Univ.
Tennessee HSC, Memphis, TN; 2Medicine/ Endocrinology, Univ.
Tennessee HSC, Memphis, TN; 3Microbiology, Immunology and
Biochemistry, Univ. Tennessee HSC, Memphis, TN; 4Pharmaceutical
Sciences, University of TN Health Science Center, Memphis, TN;
5
Dept. Ophthalmology/Duke Eye Center, Duke University Medical
Center, Durham, NC; 6Cell Biology, Duke University, Durham, NC.
Purpose: To test the hypothesis that auto-antibodies (AAbs)
recognizing ocular tissue antigens develop in the APOE4 TR mouse
model of AMD.
Methods: APOE4 TR mice aged at least 65 weeks (~15 mo) develop
an AMD-like phenotype after being fed high fat cholesterol-enriched
diet (HFCD) for 8-10 wks (Malek et al. PNAS 2005; 102: 11900-05).
Western blots (WBs) were performed by reacting sera collected at 24
mos from APOE4 TR-HFCD mice and control APOE4 TR mice on
normal diet (ND) against retina/RPE/BM/choroid tissue lysates of
adult C57BL/6 mice. Lysates (10 mg) were loaded on gels, incubated
with 5mL of serum, and developed. The intensity of the bands seen
on WB was quantified with the Odyssey system and compared by
student’s T-test. Immunohistochemistry (IHC) was performed against
anti-mouse IgG antibody by fluorescence microscopy following
incubation of adult C57BL/6 mouse retina sections with mouse
sera. To identify the autoantigens, APOE4 TR-HFCD sera were
immunoprecipitated, followed by 2-dimension electrophoresis (2DE)
and liquid chromatography-tandem mass spectrometry (LC-MS/
MS), performed on spots seen on 2DEs with APOE4 TR-HFCD sera
following previously reported methods (Lenchik et al. ARVO 2013,
Abs. 4103).
Results: Significantly more intensely reactive bands were observed
in sera of APOE4 TR-HFCD compared to APOE4 TR-ND mice.
The most robust reactivities were seen at 17kDa (p= 0.005 after
1-hr incubation, p= 0.000007 after 3 hrs) and 13kDa (p= 0.005,
1 hr; p=0.001, 3 hrs). Additional reactivities were seen after 1-hr
incubation at 79kDa (p= 0.013), 47kDa (p= 0.028), and 20kDa
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
(p=0.046). IHC studies showed moderate and diffuse staining of
C57BL/6 mouse retinal sections at the outer nuclear layer level. No
staining was seen with APOE4 TR-ND sera. Differentially reactive
spots were also observed on 2DE between APOE4 TR-HFCD and
APOE4 TR-ND sera, and IDs are being investigated by LC-MS/MS.
Conclusions: As we have previously shown in the serum of human
AMD (Iannaccone et al. Adv. Exp. Med. Biol. 2012; 723:11-6), AAbs
recognizing retinal targets develop in this animal model of AMD,
suggesting a stereotyped response to AMD-like retinal degenerative
events that incites a secondary autoimmune component and that has
the potential to further retinal damage.
Commercial Relationships: Albert H. Alhatem, None; Nataliya
Lenchik, None; Sarka Beranova-Giorgianni, None; Mikael
Klingeborn, None; David D. New, None; Francesco Giorgianni,
None; Ivan Gerling, None; Marko Radic, None; Catherine Bowes
Rickman, None; Alessandro Iannaccone, None
Support: Grants from NEI/NIH R01 EY022706 (AI), R01
EY019038 (CBR) and P30 EY005722 (Duke Eye Center); Research
to Prevent Blindness, Inc. New York, NY (Physician Scientist Award
to AI, unrestricted grants to UTHSC Ophthalmology/Hamilton
Eye Institute and Duke Eye Center); Edward N. & Della L. Thome
Memorial Foundation Award (CBR).
Program Number: 1174 Poster Board Number: C0202
Presentation Time: 3:15 PM–5:00 PM
Dual inhibition of angiopoietin-2 and vascular endothelial growth
factor-A with Crossmab RG7716 suppressed laser-induced
choroidal neovascularization in a non-human primate model
Gemmy C. Cheung1, Veluchamy A. Barathi1, Bo Bo Tun1, Say Wei
Yeo1, Pei Pei Gan1, Chan lwin Nyein1, Jorg Regula2, Guido Hartman2.
1
Singapore Eye Research Institute, Singapore, Singapore; 2Pharma
Research & Early Development, Hoffmann La Roche, Basel,
Switzerland.
Purpose: RG7716 is a bispecific antibody developed with CrossMab
technology to tightly bind VEGF-A on one arm and angiopoietin
(Ang)-2 on the other arm. We evaluated in vivo efficacy of
RG7716 in a nonhuman primate model of laser-induced choroidal
neovascularization (CNV).
Methods: CNV was induced by laser photocoagulation on Day 0
in both eyes of 30 cynomolgus monkeys. Laser-induced lesions
were confirmed; their severity was scored using fluorescein
angiography from grade 1 (no hyperfluorescence) to grade 4 (bright
hyperfluorescence and leakage) by a masked investigator on Day 14.
Intravitreal injection was administered in both eyes on Day 15. Three
active-treatment groups (high-dose RG7716, 90ug/0.05mL; low-dose
RG7716, 30ug/0.05mL; and anti-Ang-2, 90ug/0.05mL), one active
control group (anti-VEGF-A [ranibizumab, the best characterized
anti-VEGF inhibitor], 30ug/0.05mL, equimolar to high-dose
RG7716), and one inactive control group (isotype at 90ug/0.05mL)
were included. Change in CNV grade was determined by comparing
fluorescein angiography on Days 28 vs 14. Aqueous humor was
collected from all eyes at baseline and on Days 15 and 32. Cytokine
analysis of aqueous was performed by Multiplex ELISA system
measuring IL-6, IL-8, MCP1, VEGF-A, PDGF-B, Ang-2 and bFGF.
Results: A single intravitreal injection of high- and low-dose
RG7716 reduced mean CNV lesion grade by 0.99-fold and -0.68 fold
respectively. Treatment with anti-VEGF-A (Ranibizumab) resulted
in reduction of mean CNV lesion grade by 0.63-fold. anti ANG2
treatment results in a reduction of mean CNV lesion grade by -0.46
–fold. At an equimolar number of binding sites, RG7716 reduced
lesion severity significantly more than anti-VEGF alone (analysis of
variance followed by Tukey’s multiple comparison, P<0.05). Ang-2
and VEGF levels were significantly downregulated by RG7716, as
were the proinflammatory cytokines IL-6, IL-8, and MCP-1.
Conclusions: Dual inhibition of ANG-2 and VEGF-A resulted in
the suppression of pre-formed laser-induced CNV in the non-human
primate model. . Due to the suppression of CNV formation and
reduction of inflammation as measured by cytokines in aqueous
humor, RG7716 may be a feasible treatment for CNV associated with
age-related macular degeneration or other causes.
Commercial Relationships: Gemmy C. Cheung, Bayer (C), Bayer
(F), Bayer (R), Cisthera (F), GlaxoSmithKline (F), Novartis (R),
Roche (F); Veluchamy A. Barathi, Roche (F); Bo Bo Tun, None;
Say Wei Yeo, None; Pei Pei Gan, None; Chan lwin Nyein, None;
Jorg Regula, Roche (E); Guido Hartman, Roche (E)
Support: Roche-Singapore Translational Research Award
Program Number: 1175 Poster Board Number: C0203
Presentation Time: 3:15 PM–5:00 PM
Optimizing Biodegradable Scaffolds for Developing iPS Cell
Derived RPE Tissue
Vladimir Khristov2, Juliet Hartford1, Qin Wan2, Mostafa R. Lotfi2,
Kiyoharu J. Miyagishima2, Arvydas Maminishkis2, Juan Amaral3,
Sheldon S. Miller2, Janine Davis1, Kapil Bharti1. 1Unit on Ocular
Stem Cell and Translation Research, National Eye Institute, National
Institutes of Health, Bethesda, MD; 2Section on Epithelial and Retinal
Physiology and Disease, National Eye Institute, National Institutes
of Health, Bethesda, MD; 3Laboratory of Retinal Cell and Molecular
Biology, National Eye Institute, National Institutes of Health,
Bethesda, MD.
Purpose: Age-related macular degeneration (AMD) is a leading
cause of vision loss in the United States. The disease is thought to
originate by a malfunctioning retinal pigment epithelium (RPE),
which accompanies photoreceptor death. The RPE is a monolayer
of cells that performs multiple roles required for survival and
proper functionality of the photoreceptors, including fluid level
regulation, ion transport, nutrient replenishment, and phagocytosis of
photoreceptor outer segments. Potential therapies for AMD consist
of replacing the diseased RPE layer. Previous work has shown that
transplanting RPE cells on a scaffold rather than cell suspension
provides greater viability and integration into the host retina.
Methods: We used a triphasic WNT and TGF treatment protocol
to generate fully differentiated and functional RPE from iPS cells.
The resulting RPE was cultured as a polarized monolayer on two
different types of biodegradable scaffolds: a dual layer poly lactoco-glycolic acid (PLGA) and a cross-linked electro-spun collagen
scaffold. We compared cell viability on collagen and PLGA scaffolds
and monitored the degradation of the scaffolds and cell growth over
time. RPE tissue structure and functionality was confirmed through
electron microscopy, immunostaining, gene expression analysis, fluid
transport assays, and electrophysiological analysis.
Results: iPS cell derived RPE was successfully cultured as
electrically intact monolayers on biodegradable scaffolds.
As compared to collagen, PLGA provides a better substrate
for RPE scaffold and helps cells form a confluent monolayer.
Electrophysiological experiments show that RPE cells on PLGA
develop resistance >100 ohms/cm2, suggesting tight electrical
contacts between neighboring cells. RPE cells on PLGA show
improved responses to changes in intracellular calcium and for their
ability to transport fluid from apical to basal side on the monolayer.
Conclusions: Successful validation of artificial RPE tissue indicates
its suitability for implantation into the subretinal space. We are
currently testing these scaffolds in animal models. This work will
provide a GMP-ready protocol for generating RPE tissues on a
scaffold for transplantation as treatment for diseases like AMD.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Vladimir Khristov, None; Juliet
Hartford, None; Qin Wan, None; Mostafa R. Lotfi, None;
Kiyoharu J. Miyagishima, None; Arvydas Maminishkis, None;
Juan Amaral, None; Sheldon S. Miller, None; Janine Davis, None;
Kapil Bharti, None
Program Number: 1176 Poster Board Number: C0204
Presentation Time: 3:15 PM–5:00 PM
Anti-C5 mAb: In Vivo Effects Following Intravitreal
Administration to Cynomolgus Monkeys
Mark Milton, Laura Dill Morton, Birgit Jaitner, David A. Shaw,
Timothy MacLachlan. Novartis Institutes For BioMedical Research,
Cambridge, MA.
Purpose: To characterize the in vivo effects of LFG316, a
recombinant anti-C5 IgG1 monoclonal antibody after intravitreal
(IVT) administration to cynomolgus monkeys
Methods: Male and female cynomolgus monkeys were administered
repeated (0, 3, or 5 mg/eye every two weeks) 50 mL intravitreal
injections of LFG316 for either 3 or 6 months
Results: After intravitreal administration, LFG316 distributed
slowly out of the eye and into the systemic circulation with the
maximum serum concentrations of total LFG316 occurring at ~ 5
days post dose. The shape of the total LFG316 serum concentration
versus time curve was typical for the extravascular administration
of a monoclonal antibody. At all dose levels, LFG316 could be
detected throughout the dose interval and the duration of the study.
Appearance of LFG316 in serum is accompanied by a similar
molar increase in total C5 serum concentration; indicating that
LFG316 in serum is mostly complexed to C5. Consequently, there
was no evidence of pharmacodynamic activity (as measured by the
hemolysis of rabbit red blood cells by serum samples) from systemic
exposure to LFG316, since apparent free C5 concentration in serum
(approximated by total C5 minus total LFG316) is not affected. AntiLFG316 antibodies were detected in 1/24 and 3/20 monkeys in the
3-month and 6-month studies, respectively. There were no treatmentrelated mortalities or findings regarding clinical observations, body
weights, estimated food consumption, ophthalmoscopy, intra-ocular
pressure, electroretinography, electrocardiography, blood pressure,
immunophenotyping, hematology, clinical chemistry, urine analysis,
organ weights or histopathology that could be attributed to treatment
with LFG316
Conclusions: LFG316 was well tolerated in cynomolgus monkeys
after multiple intravitreal injections up to a dose of 5 mg/eye every
other week for up to 6 months. LFG316 distributed from the eye into
the serum, bound to C5 in the serum but the amount of target (C5)
capture was low, leading to no observable impact on the activity of
the alternative complement pathway in the serum
Commercial Relationships: Mark Milton, Novartis (E); Laura Dill
Morton, Novartis (E); Birgit Jaitner, Novartis (E); David A. Shaw,
Novartis (E); Timothy MacLachlan, Novartis (E)
Program Number: 1177 Poster Board Number: C0205
Presentation Time: 3:15 PM–5:00 PM
AAV2.Flt23k Intraceptor Inhibits VEGF and CNV in Mice
Xiaohui Zhang, Hironori Uehara, Subrata K. Das, Austin Bohner,
Balamurali K. Ambati. Moran Eye Center, University of Utah, Salt
Lake City, UT.
Purpose: To determine the efficacy and safety of long-term
expression of a virus mediated intraceptor vascular endothelial
growth factor (VEGF) inhibitor, AAV2.Flt23k, on murine choroidal
neovascularization (CNV) induced by laser photocoagulation.
Methods: To evaluate the long-term expression of AAV2 mediated
gene therapy, AAV2.AcGFP was subretinal injected 1 ml (5x108 vg)
and screened by Heidelberg Spectralis at 2 weeks, 1 month, 3 months
and 6 months. In an experimental model of laser induced CNV,
AAV2.Flt23k, and control AAV2.AcGFP and PBS were subretinal
injected one month prior to laser induction. After two weeks, CNV
volume was measured. VEGF level was measured with ELISA. To
evaluate safety, electroretinography (ERG) and OCT retinal thickness
were assessed 6 months after subretinal injection.
Results: The AAV2.AcGFP expression was at 2 weeks and sustained
expression for at least 6 months. The mean CNV volume was
significantly smaller in the AAV2.Flt23k (7.60 ± 1.15 × 104 mm3)
group compared with control mice treated with AAV2.AcGFP
(19.81 ± 4.10 × 104 mm3, p<0.01) or PBS (16.11 ± 4.34 × 104 mm3,
p<0.05). The mean VEGF protein levels decreased significantly in
the treated group (376.9 ± 69.5 pg/mg, p < 0.05) compared with the
control group AAV2.AcGFP (597.5 ± 52.1 pg/mg). Retinal electrical
function and structural architecture were not affected by treatment.
Conclusions: AAV2.Flt23k can effectively long term express and
inhibit laser-induced CNV in mice through downregulation of VEGF.
These findings provide a promising approach for treatment of agerelated macular degeneration.
Commercial Relationships: Xiaohui Zhang, None; Hironori
Uehara, None; Subrata K. Das, None; Austin Bohner, None;
Balamurali K. Ambati, None
Program Number: 1178 Poster Board Number: C0206
Presentation Time: 3:15 PM–5:00 PM
Effective targeting of the PI3K/Akt/mTOR pathway: A promising
therapeutic option for the treatment of ocular neovascularization/
inflammation/oedema
Temitope Sasore, Breandan N. Kennedy. Conway Institute of
Biomolecular and Biomedical Science, University College Dublin,
Dublin, Ireland.
Purpose: There is a clinical need to develop improved
pharmacological therapeutics for ocular neovascularization, retinal
inflammation and retinal oedema associated with diabetic retinopathy
(DR), age-related macular degeneration (AMD) and diabetic macular
oedema. In this study, we investigate the in vivo anti-angiogenic
efficacy of a panel of PI3K/Akt/mTOR inhibitors, alone and in
combination.
Methods: Standard and real-time reverse transcription polymerase
reaction was used to examine the developmental expression pattern
of PI3K/Akt/mTOR target genes in the zebrafish. Tg(fli1:EGFP)
transgenic zebrafish were treated with drugs from 2-5 days post
fertilisation and inhibitory effects on developmental angiogenesis of
intersegmental vessels (ISV) and hyaloid vessels (HV) quantified.
Optokinetic response assay assessed effects on visual behaviour.
Human retinal pigment epithelial cell number was examined using
MTT assay and the localization of the tight junction protein ZO-1
was determined by immunocytochemistry.
Results: RT-PCR and qRT-PCR results demonstrate that PI3K/Akt/
mTOR genes are expressed in zebrafish from 6 hours post fertilisation
(hpf) to 5 days post fertilisation (dpf). 10 uM of individual drugs had
moderate anti-angiogenic activity, but 5 uM combinations of specific
PI3K plus mTOR inhibitors ranked as the most efficacious kinase
inhibitors in zebrafish. In addition, some combinations exhibited
little to no effect on retinal morphology, whilst the effect on visual
function is currently being examined. The most active inhibitors do
not reduce RPE cell number. In addition, immunofluorescence study
is currently being used to examine the effect on cell permeability.
Conclusions: In summary, chemical screens in zebrafish identify
a combination of mTOR and PI3K inhibitors that are safe and
effective inhibitors of developmental angiogenesis. Therefore,
further investigations of the PI3K pathway and drug combinations
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
hold promise for the identification of better therapeutics for ocular
neovascularization.
Commercial Relationships: Temitope Sasore, None; Breandan N.
Kennedy, None
Program Number: 1179 Poster Board Number: C0207
Presentation Time: 3:15 PM–5:00 PM
Localized RPE removal with a loop instrument in rabbits
Boris V. Stanzel1, Fabian Thieltges1, Zengping Liu2, 1, Norbert
Braun3, Warapat Wongsawad4, 1, Sudawadee Somboonthanakij4, 1,
Ralf Brinken1, Frank G. Holz1. 1Ophthalmology, University of Bonn,
Bonn, Germany; 2Southwest Eye Hospital, he Third Military Medical
University, Chongqing, China; 3Geuder AG, Heidelberg, Germany;
4
Ophthalmology, Wat Raikhing Hospital, Wat Raikhing, Thailand.
Purpose: To surgically create a localized RPE wound with minimal
trauma to adjacent structures.
Methods: Following a 23G core vitrectomy, 2 bleb retinal
detachments (bRD) were raised with balanced salt solution (BSS) in
23 eyes of 21 pigmented and 2 albino rabbits. The RPE underneath
the bRD was scraped with 3 prototypes of a novel custom-made
instrument (Geuder) in a total of 17 eyes, 6 eyes with bRDs alone
served as controls. The instrument consisted of an extensible loop
folded within a 20G oval nozzle, which could widen and move
forward upon release to debride a targeted RPE surface underneath
a bRD. The respective loop designs differed in material and
thickness (Prolene 0.1 and 0.06mm vs. metal wire 0.1mm). bRD
were stabilized with various concentrations of hyaluronic acid (HA,
Gelbag®, C. Zeiss Meditec), or BSS alone. All surgical procedures
were analyzed by video. The eyes were enucleated following
perfusion fixation immediately after the procedure and processed for
standard histology. Serial sections were evaluated by lightmicroscopy
by two blinded observers seeking for lacerations in BM/ CC and
hemorrhages, as well as integrity of the outer retina.
Results: Prolene loops yielded smooth and continuous sliding over
the targeted RPE by 3 surgeons, while the metal wire got trapped
after some extension. Using 0.1 prolene with a single forward/
backward stroke, an area of ca. 2.5x1.5mm was 70% devoid of
RPE, yet showed few minuscule BM lacerations and random
choriocapillaris blood clots. A single scrape with 0.06 prolene
resulted in unsatisfactory RPE denudement, whilst repeated scraping
maneuvers caused more BM defects and hemorraghes compared
with the 0.1 prolene loop. The metal loop resulted in incomplete
RPE removal and intraoperative subretinal hemorraghes. Controls
showed normal histology. Injection of ≥ 0.25% HA into bRDs did not
result in (collapsing) movements of the overlying retina during RPE
scraping, reflected in pilot histologic data with normal outer retinal
architecture. Blebs filled with 0.1% HA or BSS alone collapsed upon
manipulation, suggesting photoreceptor trauma.
Conclusions: A novel instrument with an extensible 0.1mm prolene
loop enabled satisfactory RPE removal, albeit with occasional
minuscule BM breaks and CC hemorrhages. This technique may
serve as surgical model for geographic atrophy and/ or RPE
replacement strategies.
Commercial Relationships: Boris V. Stanzel, Geuder AG (F),
Geuder AG (P); Fabian Thieltges, None; Zengping Liu, None;
Norbert Braun, Geuder AG (E), Geuder AG (P); Warapat
Wongsawad, None; Sudawadee Somboonthanakij, None; Ralf
Brinken, Geuder AG (P); Frank G. Holz, None
Support: Dr. Ruediger Foundation, Frankfurt/ Germany and Gerok/
BONFOR funds, University of Bonn/ Germany (O-137.0015) to
BVS. Chinese Scholarship Council (No. 2008627116) to ZL.
Program Number: 1180 Poster Board Number: C0208
Presentation Time: 3:15 PM–5:00 PM
Characterization of a spontaneous neovascular mouse model
Eiichi Hasegawa1, Deeba Husain1, Bo Chang2, Demetrios G. Vavvas1,
Robert J. D’Amato3, Joan W. Miller1, Kip M. Connor1. 1Angiogenesis
Laboratory, Department of Ophthalmology, Massachusetts Eye &
Ear Infirmary, Harvard Medical School, Boston, MA; 2The Jackson
Laboratory, Bar Harbor, ME; 3Department of Surgery, Vascular
Biology Program, Boston Children’s Hospital, Harvard medical
School, Boston, MA.
Purpose: Vision loss due to vascular disease of the retina is a
leading cause of blindness in the world affecting all age groups.
Neovascularization is a hallmark of complex, polygenic diseases,
such as age-related macular degeneration (AMD), which affects 1
in 3 individuals over the age of 65. The growth of abnormal blood
vessels leads to debilitating vision loss and eventual blindness. The
novel mouse strain neoretinal vascularization (NRV)2 mice show
spontaneous early chorio-retinal neovascularization. The purpose of
this study is to characterize the induction of pathologic angiogenesis
in this mouse model.
Methods: The present studies adhered to ARVO’s Statement for
the Use of Animals in Ophthalmic and Vision Research. The NRV2
mice were examined from postnatal Day 17 (p17) to 3months.
The phenotypic changes were evaluated by fundus photography,
fluorescein angiography, optical coherence tomography ( MicronIII
Retinal Imaging Microscope; Phoenix). Immunohistochemical
and electron microscopic analysis of retina/choroid sections was
performed. The pathological neovascularization was imaged by
confocal microscopy and reconstructed using three-dimensional
image analysis (Amira).
Results: We found that the NRV2 mice developed multifocal retinal
depigmentation in the posterior fundus. Several of these depigmented
areas showed vascular leakage by fluorescein angiography. Disease
progression in NRV2 mice revealed that depigmentation and
vascular leakage beginning around p21, increased and peaked at
p25, and persisted for more than 3 months. Histological analysis
revealed disruption of the retinal pigment epithelium (RPE) and the
neovascular structures in the photoreceptor cell layer and at the RPE
Bruch’s interface.
Conclusions: We have further characterized a model of spontaneous
angiogenesis that occurs in the NRV2 mouse strain. This non-injury
model of retinal neovascularization will be useful in elucidating the
molecular causes of retinal neovascular disease and in developing
therapeutics.
Commercial Relationships: Eiichi Hasegawa, None; Deeba
Husain, None; Bo Chang, None; Demetrios G. Vavvas, None;
Robert J. D’Amato, None; Joan W. Miller, Alcon (C), Imagen
Biotech, Inc. (C), ISIS Pharmaceuticals, Inc. (C), KalVista
Pharmaceuticals (C), Maculogix, Inc. (C), Massachusetts Eye
and Ear Infirmary (P), ONL Therapeutics, LLC (C), Regeneron
Pharmaceuticals, Inc. (C); Kip M. Connor, None
Support: This study was generously supported by the Massachusetts
Lions Eye Research Fund, Inc., National Institutes of Health (NIH)
grants R01EY022084–01/S1 (KMC), Research to Prevent Blindness
Special Scholar Award (KMC)
Program Number: 1181 Poster Board Number: C0209
Presentation Time: 3:15 PM–5:00 PM
Polyethylene Glycol (PEG) Induced Mouse Model of Dry Age
Related Macular Degeneration (AMD)
Valeriy V. Lyzogubov, Nalini S. Bora, Puran S. Bora. Ophthalmology,
Jones Eye Institute - UAMS, Little Rock, AR.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Age-related macular degeneration (AMD) is a leading
cause of irreversible blindness worldwide. Understanding the
mechanisms responsible for dry AMD during its initial phases will
be highly beneficial in designing new therapeutic strategies for the
treatment of dry AMD. The aim of this study was to characterize dry
AMD-like changes in mouse RPE and retina after PEG treatment.
Methods: We injected male C57BL/6 mice subretinally with
PBS, 0.025, 0.25, 0.5 and 1.0 mg of PEG-400 and the animals
were sacrificed on day 5. Eyes were harvested and processed for
histological analysis. In all other experiments 0.5 mg PEG was
injected and animals were sacrificed on days 1, 3, 5 or 14. Paraffin
sections were used for TUNEL staining. Immunofluorescent staining
using paraffin sections were utilized to detect active Caspase 3
(Casp3act), Atg12 and proliferating cell nuclear antigen. Plastic
sections were analyzed using light and electron microscopy.
Measurement and quantification of morphometric parameters were
performed using ImageJ program. Gene expression was studied in
PEG injected eyes and PBS controls using RNA microarray.
Results: Subretinal injection of 0.5 mg PEG caused a 32%
reduction of outer nuclear layer (ONL) thickness, 61% decrease
of photoreceptor outer and inner segment length, 49% decrease of
nuclear density in the ONL and 31% increase of RPE cell density
by day 5 post-injection. Maximum TUNEL positive nuclei in the
ONL were detected at day 5 after PEG injection. Histological
signs of apoptosis were observed in the ONL by light and electron
microscopy. Degeneration, autophagy and proliferation of RPE cells
was noted in PEG injected eyes. RNA microarray demonstrated that
several genes including Mmp9, Htra1, Casp1, Lgals3, C3, Serping1,
Cfi, Lpl, Cp, fgf2 were up-regulated in PEG injected eyes compared
to PBS controls.
Conclusions: Intraocular administration of PEG leads to
morphological changes in RPE and retina consistent with dry AMD.
Furthermore, PEG treatment up-regulated several genes reported to
be involved in human AMD. Collectively, these results suggest that
this simple and fast model will be useful to investigate dry AMD
pathogenesis and treatment.
Commercial Relationships: Valeriy V. Lyzogubov, None; Nalini S.
Bora, None; Puran S. Bora, None
Support: Department of Ophthalmology Research foundation and
Philip Palade, Pat & Willard Walker Eye Research Center, Jones Eye
Institute, Little Rock, AR.
Program Number: 1182 Poster Board Number: C0210
Presentation Time: 3:15 PM–5:00 PM
Response to supplementation with lutein and zeaxanthin in
subjects with familial history of AMD: the LIMPIA Study
Marie-Noelle Delyfer1, 2, Marie B. Rougier1, Catherine P. Garcher4,
Helene Savel5, Genevieve Chene5, Stéphane Etheve6, Wolfgang
Schalch6, Cecile DelCourt2, 3, Jean-Francois Korobelnik1, 2.
1
Ophthalmology, Hopital Pellegrin, Bordeaux, France; 2Univ.
Bordeaux, ISPED, Centre INSERM U897-EpidemiologieBiostatistique, Bordeaux, France; 3ISPED, Centre INSERM U897Epidemiologie-Biostatistique, INSERM, Bordeaux, France; 4Service
d’Ophtalmologie, CHU de Dijon, Dijon, France; 5Pôle de Santé
Publique, Unité de soutien méthodologique à la recherche clinique
et épidémiologie (USMR), CHU de Bordeaux, Bordeaux, France;
6
Research and Development, DSM Nutritional Products, Kaiseraugst,
Switzerland.
Purpose: To assess the effect of nutritional supplementation on
plasma lutein and zeaxanthin in healthy subjects with familial history
of AMD.
Methods: The Limpia Study is a randomized clinical trial performed
in 120 healthy subjects aged 40-70 years with at least one parent
affected by neovascular AMD. Included subjects were randomly
assigned to receive Nutrof® Total (2 gels/day, representing lutein 10
mg/day, zeaxanthin 2 mg/day, long chain omega 3 fatty acids 540 mg/
day, vitamin C 180 mg/day, vitamin E 30 mg/day, zinc 15 mg/day,
copper 1 mg/day and resveratrol 1 mg/day) or placebo for 6 months,
and were subsequently followed for 6 additional months after the end
of supplementation. Plasma lutein and zeaxanthin were measured by
normal-phase HPLC, from fasting blood samples.
Results: Baseline plasma lutein concentrations were 0.38 mmol/l
± 0.15 in the supplemented group and 0.37 mmol/l ± 0.16 in the
placebo group. It increased in the supplemented subjects during the
supplementation period (change from baseline +0.58 mmol/l ± 0.41 in
the supplemented group and -0.02 mmol/l ± 0.09 in the placebo group
at 3 months, p<0.0001 and +0.59 mmol/l ± 0.39 versus -0.04 ±0.12 at
6 months, p<0.0001). Three months after the end of supplementation,
the supplemented group was still different from placebo (change from
baseline +0.02± 0.10 versus -0.02 ± 0.12, p=0.03), but this difference
between groups was no longer significant at 6 months (+0.01±
0.10 versus -0.02 ± 0.11, p=0.09). Baseline plasma zeaxanthin
concentrations were 0.08 mmol/l ± 0.04 in the supplemented
group and 0.09 ± 0.04 in the placebo group. It increased in the
supplemented subjects during the supplementation period (change
from baseline +0.14 ± 0.09 versus -0.01± 0.03, p<0.0001 at 3 months
and +0.14 ± 0.09 versus -0.01± 0.03, p<0.0001 at 6 months), and,
remained slightly higher after the end of supplementation (+0.01
± 0.04 versus -0.01± 0.03, p=0.002, 3 months after the end of
supplementation and +0.01 ± 0.03 versus -0.01± 0.03, p=0.003, 6
months after the end of supplementation).
Conclusions: This study documents an increase of more than 150%
in plasma L and Z with Nutrof supplementation, in subjects at high
genetic risk for AMD. A minimal difference in plasma L and Z
between supplemented and placebo groups persisted several months
after the end of supplementation.
Commercial Relationships: Marie-Noelle Delyfer, Alcon (C), Carl
Zeiss Meditec. (C), Laboratoires Théa. (C), Novartis (C); Marie
B. Rougier, Allergan (C), Bausch+Lomb (C), Kemin (C), Thea (C);
Catherine P. Garcher, Alcon (C), Allergan (C), Bausch and Lomb
(C), Bayer Pharma (C), Laboratoires Théa (C), Novartis (E);
Helene Savel, None; Genevieve Chene, None; Stéphane Etheve,
None; Wolfgang Schalch, None; Cecile DelCourt, Bausch&Lomb
(C), Laboratoires Théa (C), Novartis (C); Jean-Francois
Korobelnik, Alcon (C), Allergan (C), Bayer Pharma. (C), Carl Zeiss
Meditec (C), Laboratoires Théa (C), Novartis (C)
Support: Thea
Clinical Trial: NCT01269697
Program Number: 1183 Poster Board Number: C0211
Presentation Time: 3:15 PM–5:00 PM
Investigating a link between photodegradation of bisretinoid and
cross-linking of protein
Janet R. Sparrow1, Jilin Zhou2, Keiko Ueda2. 1Opthalmology and
pathology, Columbia University, New York, NY; 2Ophthalmology,
Columbia University, New York, NY.
Purpose: The pathogenesis of AMD is thought to begin with the
RPE and subjacent Bruch’s membrane, the latter undergoing agerelated changes especially in the submacular region. Non-enzymatic
collagen cross-linking is a feature of aging Bruch’s membrane
and disturbances in extracellular matrix turnover are considered to
contribute to Bruch’s membrane thickening with age. We investigated
a constituent of RPE lipofuscin as a source of cross-linking agents.
Methods: As models for cross-linking, RNase A (13.7 kDa); and
the peptides somatostatin (1638 Da) and N-acetyl renin substrate
tetradecapeptide (1801 Da) were incubated with photodegraded A2E
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
with and without aminoguanidine. Cross-linking was evaluated by
SDS-PAGE. After incubating collagen IV with photodegraded A2E,
matrix metalloproteinases (MMP) activity was assayed by modified
zymography with densitometric quantification and by measuring
hydroxyproline. Positive controls were glyoxal and methylglyoxal.
Results: Incubation of RNase A (5 days; 37°C) with 430 nmirradiated A2E, conditions known to release GO and MG, resulted in
cross-linking that was evidenced by the presence of higher molecular
weight oligomers when analyzed by SDS-PAGE. Inclusion of the
dicarbonyl scavenger aminoguanidine protected against cross-linking.
RNase A activity assay also showed that enzyme function was
diminished by exposure of RNase A to photoA2E. Higher molecular
weight bands on SDS-PAGE were also observed with lysine-free
(renin substrate) and arginine-free (somatostatin) peptides. Assaying
by zymography revealed that modification of collagen IV under
conditions of A2E photodegradation reduced MMP-2 and MMP-9
cleavage of collagen IV.
Conclusions: Photodegradation of the RPE lipofuscin fluorophores
A2E and all-trans-retinal dimer have been shown to release the
dicarbonyls glyoxal and methylglyoxal that are responsible for
modifications of protein by advanced glycation endproducts (AGE).
Release of these dicarbonyls from RPE cells could contribute to
cross-linking and age-related thickening of the underlying Bruch’s
membrane. It is also potentially significant that AGE-related proteins
are detected in drusen.
Commercial Relationships: Janet R. Sparrow, None; Jilin Zhou,
None; Keiko Ueda, None
Support: Supported in part by grants from the National Eye Institute/
NIH EY12591, EY019007 (Core Support for Vision Research); and
unrestricted funds from Research to Prevent Blindness (New York,
NY) to the Department of Ophthalmology, Columbia University
Program Number: 1184 Poster Board Number: C0212
Presentation Time: 3:15 PM–5:00 PM
Histopathological characterization of different lesion types of
polypoidal choroidal vasculopathy in HTRA1 transgenic mice
Alex Jones, Sandeep Kumar, Shixian Wang, Zachary Berriochoa,
Yingbin Fu. Ophthalmology, University of Utah, Salt Lake City, UT.
Purpose: Polypoidal choroidal vasculopathy (PCV) is characterized
by a branching vascular network (BVN) with terminal polypoidal
dilations (polyps) in the choroid. The etiology and pathogenesis
of PCV are largely unknown. We recently generated the first PCV
model by transgenically expressing human HTRA1 in mouse retinal
pigment epithelium (RPE). By in vivo imaging, HTRA1 mice
exhibit a number of angiographic features that are associated with
PCV (e.g. polyps,BVN, pigment epithelium detachment (PED), late
geographic hyperfluorescence (LGH)). This PCV model provides an
unprecedented opportunity to correlate phenotypes charaterized by in
vivo imaging with the correspoding histopathological features. Our
purpose is to understand the pathogenesis of different types of lesions
and their progression.
Methods: HTRA1 mice were injected with a mixture of FITCdextran and indocyanine green (ICG). Guided by ICG angiography
(ICGA), fluorescein angiography and flatmount-fluorescent
microscopy, we surgically excised different types of lesions.
The excised lesion-containing tissues were fixed overnight and
resin-embedded. Thin plastic sections (1um) were stained with
Richardson’s stain.
Results: The small-polyp lesion shows a cluster of dilated thinwall vessels with broken vessel walls, which “pushes” RPE and
retinal layers upward. A small PED is observed above the thin-wall
vessel. The large PCV lesion with polyps and BVN is associated
with extensive exudation of plasma proteins. The artery wall is thick
and hyalinized, which obstructs the vessel. The large PCV lesion
causes extensive RPE degeneration accompanied by photoreceptor
degeneration. In contrast, the small PCV lesion causes milder RPE
degeneration with no obvious signs of photoreceptor degeneration.
This may explain why PCV patients with larger lesions have higher
rates of lesion progression and severe complications. The LGH lesion
contains both dilated-thin broken vessels, which presses into the RPE
and photoreceptor, and hyalinized vessels. RPE vacuoles are detected
while photoreceptors are mostly intact. Macrophages and neutrophils
were present in both large PCV lesion and LGH.
Conclusions: PCV is the result of degenerative changes of choroidal
vessels triggered by the proteolytic activity of HTRA1. The presence
of inflammatory cells in large lesions suggests inflammatory
processes are involved in the progression of PCV.
Commercial Relationships: Alex Jones, None; Sandeep Kumar,
None; Shixian Wang, None; Zachary Berriochoa, None; Yingbin
Fu, None
Support: This work was supported by the Career Development
Award from Research to Prevent Blindness (RPB), C.M.Reeves &
M.A. Reeves Foundation, E. Matilda Ziegler Foundation for the
Blind, and an unrestricted grant to the Department of Ophthalmology
at the University of Utah from RPB. We thank Balamurali Ambati
for technical assistance on the Spectralis Multi-Modality Imaging
System.
Program Number: 1185 Poster Board Number: C0213
Presentation Time: 3:15 PM–5:00 PM
Relative Afferent Pupillary Defect Detected In Asymmetric
Macular Disease Using An Automated Binocular Pupillometer
Shiraaz Rahman, Dilraj S. Grewal, Pooja Bhat, Morgan Fallor,
Nicholas J. Volpe, Rukhsana Mirza. Northwestern University,
CHICAGO, IL.
Purpose: To evaluate the presence of relative afferent pupillary
defect (RAPD) in asymmetric macular disease and quantify it using
an automated binocular infrared computerized pupillometer (RAPDx,
Konan Medical USA Inc., Irvine, CA), We also correlated the intereye difference in magnitude of RAPD with the inter-eye difference
in macular lesion size measured on Fundus Auto-fluorescence (FAF)
and Color Fundus Photos (FP).
Methods: 22 patients with asymmetric macular disease (20 with
age-related macular degeneration, 1 with pattern dystrophy, 1 with
pigment mottling) were prospectively enrolled and their pupil
responses were tested using the RAPDx. Testing was conducted
under dark room conditions using the full field stimulus testing
strategy extending to ~18 degrees from fixation. They also underwent
FP (Topcon, Tokyo, Japan) and FAF (Heidelberg, Carlsbad, CA).
Patients with glaucomatous optic neuropathy, other optic nerve
disease, or retinal vascular diseases were excluded. The RAPD score,
an index of the direction and magnitude of pupil response asymmetry
of the two eyes was calculated. The surface area of macular lesions
visualized on both FP and FAF was measured using ImageJ software
(NIH, Bethesda, MD) after determining an appropriate scale
(11.36um/pixel for FAF and 13.56 μm/pixel for FP).
Results: Mean RAPD score was 0.55 ± 0.66 units, mean FAF lesion
size was 17.45 ±15.52mm2 and mean lesion size on FP was 10.67
±9.63mm2. Mean inter-eye difference in lesion size on FAF was
9.01 ±10.50mm2 and on FP was 5.48 ±4.97mm2. RAPD score had a
significant positive correlation with the inter-eye difference in lesion
size on both FAF (r=0.75, p<0.001) and FP (r=0.68, p<0.0002). 8/22
patients (36.3%, 4 male, 4 female) had an RAPD score ≥0.5 and were
considered positive for an APD. Among these 8 patients, there was
a trend towards correlation of RAPD score with inter-eye difference
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
in lesion size on FAF (r=0.61, p=0.05) and FP (r=0.38, p=0.17). 1/22
had an APD on the swinging flash light test.
Conclusions: Macular pathology has the potential to cause clinical
and sub-clinical RAPD when a sufficient portion of the retina is
involved and there is a high degree of asymmetry. The inter-eye
difference in the surface area of macular lesions correlates with the
RAPD score, detected using an automated pupillometer.
Commercial Relationships: Shiraaz Rahman, None; Dilraj S.
Grewal, None; Pooja Bhat, None; Morgan Fallor, None; Nicholas
J. Volpe, None; Rukhsana Mirza, None
Program Number: 1186 Poster Board Number: C0214
Presentation Time: 3:15 PM–5:00 PM
Choroidal neovascularization can help photoreceptors to survive
in late AMD
Antje K. Biesemeier, Sylvie Julien, Ulrich Schraermeyer. Section
of Experimental Vitreoretinal Surgery, Center for Ophthalmology
Tuebingen, Tuebingen, Germany.
Purpose: To study the ultrastructure of choroidal neovascular vessels
(CNV) of age-related macular degeneration (AMD) donor eyes and
their influence on the surrounding tissue.
Methods: Perimacular sections of eight late wet AMD donor eyes
(mean age 81+/- 10 years) were investigated with light and electron
microscopy. The ultrastructures of neovascular and non neovascular
vessels were analyzed with respect to pathological alterations.
Signs for pathology were e.g. extreme swelling of endothelial
cells, collapse of luminal space, invasion of endothelial projections
into the vessel lumen building a labyrinth-like structure, changes
in fenestration, necrotic cells or loss of cell junctions. We tried to
discriminate perfused from not perfused vessels by morphological
criteria and presence of erythrocytes, e.g. leaky vessels which did not
show an edema or blood leakage were judged to be non-perfused if
they did not show a thrombotic reaction.
Results: The donor eyes showed different stages of destruction,
ranging from areas with normal subretinal ultrastructure to areas with
retinal scars. In 33 % of the sections showing occult CNV, we found
small sites of functional neovascular vessels which were in close
distance to groups of locally surviving photoreceptors (yellow vessels
in Fig. 1). In contrast, areas with retinal scars were associated with
probably non-perfused damaged vessels (red vessels in Fig. 1).
Conclusions: It is supposed that intact CNV in late AMD are formed
in a kind of wound healing reaction to facilitate retinal metabolism
and survival. Only if CNV vessels became unfunctional and/or leaky,
edema is formed causing photoreceptor death. Only this stage can
be diagnozed by an ophthalmologist during patient care. Whether
also in early AMD subclinically neovascularization may support
photoreceptor survival remains to be investigated.
Figure 1: Toluidin-blue stained section of a wet AMD eye. The
retina is already extremely degenerated in most parts of the image.
Few islands of surviving photoreceptor inner segments (arrows) or
remnant subretinal space (arrow heads) appear in close association
with morphologically intact, probably perfused neovasular blood
vessels (yellow). Vessels which are stained red in the image were
found to contain leaky sites and were thus judged to be non-perfused.
Commercial Relationships: Antje K. Biesemeier, None; Sylvie
Julien, None; Ulrich Schraermeyer, None
Support: DFG grant BI 1551/2-1
Program Number: 1187 Poster Board Number: C0215
Presentation Time: 3:15 PM–5:00 PM
Effects of COMP-Ang1 on Neovascularization in a Mouse Model
of Age-related Macular Degeneration
Nathan Lambert, Xiaohui Zhang, Judd Cahoon, Hironori Uehara,
Balamurali Ambati. University of Utah, Salt Lake City, UT.
Purpose: To determine the effect of COMP-Ang1 on vessel
proliferation and VEGF levels in a mouse model of AMD
Methods: C57Bl6 mice received 1mL subretinal injections of PBS
or a viral vector (AAV2) expressing LacZ, GFP, or COMP-Ang1.
1 month later mice underwent laser-induced CNV (532-nm diode
laser spots, 140mW, 100msec, 50mm). Laser lesions were induced
at locations of 3, 6, 9, and 12 o’clock 2-3 disc diameters from the
optic nerve in each eye. One week after laser CNV, mice eyes
were enucleated, the choroid was stained for CD31 markers using
immunohistochemistry, and then prepared as choroidal flatmounts.
CNV volume was assessed using confocal microscopy and quantified
using Image J software.
Results: CNV volume was significantly decreased in mice with
AAV2.COMP-Ang1 treatment compared with AAV2.GFP, AAV2.
LacZ, and PBS groups. The CNV volume for the various groups was
as follows:
+AAV2.COMP-Ang1 6.36 ± 1.94 X 104 mm3 (n=31)*
+AAV2.LacZ 15.75 ± 4.91 X 104 mm3 (n=38)
+AAV2.GFP 21.32 ± 4.57 X 104 mm3 (n=30)
+PBS 16.12 ± 3.155 X 104 mm3 (n=40)
COMP-Ang1 decreased laser CNV volume nearly 2.5x when
compared to PBS (P=0.017) and AAV2.LacZ (P=0.1) and over 3.3x
compared to AAV2.GFP (P=0.003).
Conclusions: These findings provide evidence that AAV2 expression
of COMP-Ang1 decreases laser CNV volume in a mouse model.
Some limitations to this study include size and shape of laser-induced
CNV growth. Further studies will address the effects COMP-Ang1 on
VEGF levels.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Marco T. Birke, None; Erion Lipo,
None; Mehreen Adhi, None; Kerstin Birke, None; Rajendra
Kumar-Singh, None
Support: The Ellison Foundation, The Virginia B Smith Trust, The
National Institute of Health/NEI (EY021805 and EY013837), The
Department of Defense/ TATRC and grants to the Department of
Ophthalmology at Tufts University School of Medicine from the
Lions Eye Foundation and Research to Prevent Blindness, The Paul
and Phyllis Fireman Foundation
Commercial Relationships: Nathan Lambert, Fight For Sight (F);
Xiaohui Zhang, None; Judd Cahoon, None; Hironori Uehara,
None; Balamurali Ambati, None
Support: Fight for Sight Summer Student Fellowship
Program Number: 1188 Poster Board Number: C0216
Presentation Time: 3:15 PM–5:00 PM
AAV Mediated Expression of Human PRELP inhibits
Complement Activation, Choroidal Neovascularization and
Deposition of Membrane Attack Complex in Mice
Marco T. Birke, Erion Lipo, Mehreen Adhi, Kerstin Birke, Rajendra
Kumar-Singh. Ophthalmology, Tufts University, Boston, MA.
Purpose: Activation of complement has been previously associated
with age related macular degeneration (AMD). We wished to
investigate the potential use of human proline/arginine-rich end
leucine-rich repeat protein (PRELP) as an inhibitor of complement
deposition and laser induced choroidal neovascularization (CNV) in a
murine model of AMD.
Methods: We constructed an AAV2/8 vector expressing human
PRELP from a chicken beta actin promoter. PRELP-mediated
inhibition of complement was examined using FACS lysis
assays. PRELP-mediated inhibition of in vitro angiogenesis
was measured using tube formation assays with HUVEC cells.
AAV2/8 coding for PRELP was injected into the subretinal space
of normal adult mice and the human PRELP protein was localized
by immunocytochemistry. A separate group of injected mice were
utilized for laser induced CNV. At 7 days post laser, eyes were
harvested and stained for lectin or formation of the membrane attack
complex (MAC).
Results: In FACS lysis assays, PRELP reduced normal human
serum (NHS) mediated lysis of Hepa-1c1c7 cells by 18.7+/-3.95%.
Unexpectedly, in HUVEC tube formation assays, PRELP enhanced
the formation of master junctions by 210% and enhanced formation
of master segments and meshes by 230% and 290% respectively.
Immunofluourescence analyses of PRELP expression in retinal
sections identified the RPE and the photoreceptor outer segments
as initial sites of AAV2/8 infection and expression, but indicated
secretion of PRELP and it’s subsequent migration to the inner retinal
layers. PRELP reduced laser induced CNV by 60+/-13.1% and
deposition of MAC at the site of CNV lesion by 25.5+/-12.3%.
Conclusions: PRELP is a potent inhibitor of complement and laser
induced CNV in a model of AMD. Our results have implications for
the development of complement inhibitors as a therapy for AMD.
Program Number: 1189 Poster Board Number: C0217
Presentation Time: 3:15 PM–5:00 PM
Progressive dysfunction of the retinal pigment epithelium and
retina due to increased VEGF-A levels
Alexander G. Marneros1, Mohammad Dahrouj2, Zsolt Ablonczy2.
1
Massachusetts General Hospital/Harvard Medical School, Boston,
MA; 2Medical University of South Carolina, Charleston, SC.
Purpose: Increased VEGF-A has been implicated in the pathogenesis
of choroidal neovascularization (CNV) in neovascular age-related
macular degeneration (AMD), while its role in nonexudative
AMD is not well defined. Mice with increased VEGF-A develop
age-dependent morphological features of both forms of AMD,
with an early breakdown of the retinal pigment epithelium (RPE)
barrier function and a progressive generalized degeneration of
the RPE and subsequently of the photoreceptors that occurs also
at sites where no CNV is present. This suggests that an increase
in VEGF-A has a direct adverse effect on RPE and photoreceptor
function independently of the CNV-promoting proangiogenic effect
of VEGF-A. Here, we correlated morphological RPE and retinal
abnormalities in mice with increased VEGF-A levels with functional
defects in these tissues to provide evidence for a direct role of
VEGF-A in the development of age-dependent pathologies as seen in
nonexudative AMD.
Methods: Eyes of mice with increased VEGF-A were used for
morphological and ultrastructural analyses. The morphologic
abnormalitites were correlated with functional RPE and retinal
defects in these mice using electroretinograms (ERGs), OCT and
fundus imaging, fluorescent angiography, rhodopsin measurements,
and retinoid profiling.
Results: Here we show that morphological RPE abnormalities and
retinal thinning correlate with progressive age-dependent attenuation
of visual function with abnormal electroretinograms and reduced
retinal rhodopsin levels. Retinoid profiling revealed a progressive
reduction of 11-cis and all-trans retinal in the retinae of mice with
increased VEGF-A, with a relative accumulation of all-trans retinal,
consistent with an impaired retinoid transport between the RPE and
photoreceptors due to a VEGF-A-induced RPE barrier breakdown.
Conclusions: These findings provide evidence that increased
VEGF-A leads to a progressive RPE and retinal dysfunction that
occurs independently of CNV lesion formation. The data support a
central role of increased VEGF-A not only in the pathogenesis of
neovascular but also of nonexudative AMD.
Commercial Relationships: Alexander G. Marneros, None;
Mohammad Dahrouj, None; Zsolt Ablonczy, None
Support: NEI EY019297, NEI EY019065
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1190 Poster Board Number: C0218
Presentation Time: 3:15 PM–5:00 PM
Intravenous immunoglobulin treatment inhibits choroidal and
corneal neovascularization via FcyR1
Reo Yasuma, Sasha Bogdanovich, Younghee Kim, Tetsuhiro Yasuma,
Takeshi Mizutani, Ana Bastos-Carvalho, Benjamin Fowler, shengjian
li, Bradley D. Gelfand, Jayakrishna Ambati. Ophthalmology,
University of Kentucky, Lexington, KY.
Purpose: The antibody bevacizumab is widely used to treat ocular
neovascularization. Interestingly, bevacizumab has been shown to
block mouse models of neovascularization, despite the fact that
the Fab domain does not recognize mouse VEGF-A. Therefore,
we hypothesized that antibodies can inhibit angiogenesis via their
Fc domain. Intravenous immunoglobulin treatment (IVIG) is an
Fc-containing, widely-used therapeutic used to treat many diseases.
Here, we tested whether IVIG is anti-angiogenic via the Fc receptor
FcyR1 in choroidal and corneal neovascularization.
Methods: To induce choroidal angiogenesis, laser photocoagulation
was performed on both eyes using a 532-nm laser. Seven days after
treatment, eyes were enucleated and CNV lesions were labeled
with FITC-conjugated isolectin B4, and volumes determined using
a confocal microscope. For the corneal angiogenesis model, 11-0
nylon sutures were placed into the corneal stroma between the
corneal apex and the limbus. On day 10 after surgery, the mean
percentage CD31+Lyve–1– blood vessel areas on corneal flat mounts
was calculated. IVIG was administrated by tail vein or intravitreous
injection. Experiments were performed in wild-type and Fcgr1–/–
mice.
Results: Both systemic and intraocular IVIG inhibited angiogenesis
in wild-type, but not Fcgr1–/– mice, compared with PBS-injected
control group, in both of choroidal and corneal neovascularization
models.
Conclusions: Intravenous immunoglobulin treatment is a novel antiangiogenic agent for ocular neovacularization that functions via an
unexpected FcyR1-mediated mechanism.
Commercial Relationships: Reo Yasuma, None; Sasha
Bogdanovich, None; Younghee Kim, None; Tetsuhiro Yasuma,
None; Takeshi Mizutani, None; Ana Bastos-Carvalho, None;
Benjamin Fowler, None; shengjian li, None; Bradley D. Gelfand,
None; Jayakrishna Ambati, University of Kentucky (P)
Support: NIH/NEI, Doris Duke Charitable Foundation, Burroughs
Wellcome Fund, Ellison Medical Foundation, Carl Reeves
Foundation, Harrington Discovery Institute, Research to Prevent
Blindness, Foundation Fighting Blindness, Dr. E. Vernon & Eloise C.
Smith Endowed Chair
Program Number: 1191 Poster Board Number: C0219
Presentation Time: 3:15 PM–5:00 PM
The Fc fragments of Bevacizumab or IVIG suppress choroidal
neovascularization
shengjian li, Nagaraj Kerur, Sasha Bogdanovich, Reo Yasuma,
Younghee Kim, Takeshi Mizutani, Ana Bastos-Carvalho, Jayakrishna
Ambati. Department of Ophthalmology and Visual Sciences,
University of Kentucky, lexington, KY.
Purpose: Choroidal neovascularization (CNV) in humans is
often treated with full-length bevacizuman or the Fab fragment
ranibizumab. Although bevacizumab does not bind or block mouse
VEGF it has been reported to suppress mouse CNV. We tested the
hypothesis that this anti-angiogenic effect of bevacizumab in mice
was due not to mouse VEGF targeting but due to a novel effect of its
Fc fragment. We tested Fab and Fc fragments of bevacizumab as well
as of those of “intravenous human immunoglobulin” (IVIG), a widely
used clinical therapeutic in mouse CNV.
Methods: Commercially supplied bevacizumab or IVIG was digested
with Papain, and the further purified Fab and Fc fragments were
tested in the laser injury CNV model in WT and Fcgr1-deficient mice.
CNV size was quantified by volumetric assessment.
Results: Bevacizumab-Fc and IVIG-Fc suppressed CNV in WT
mice, whereas bevacizumab-Fab and IVIG-Fab did not do so.
Bevacizumab-Fc and IVIG-Fc did not suppress CNV in Fcgr1deficient mice.
Conclusions: These data identify a novel anti-angiogenic function
of the Fc fragment of human immunoglobulin-containing therapeutic
preparations and clarify earlier puzzling data of the bioactivity of
bevacizumab in mice.
Commercial Relationships: shengjian li, None; Nagaraj Kerur,
None; Sasha Bogdanovich, None; Reo Yasuma, None; Younghee
Kim, None; Takeshi Mizutani, None; Ana Bastos-Carvalho, None;
Jayakrishna Ambati, University of Kentucky (P)
Support: R01EY018350, R01EY018836, R01EY020672,
R01EY022238, R21EY019778, and RC1EY020442, Doris Duke
Distinguished Clinical Scientist Award, Burroughs Wellcome
Fund Clinical Scientist Award in Translational Research, Ellison
Medical Foundation Senior Scholar in Aging Award, Foundation
Fighting Blindness Individual Investigator Research Award, Carl
Marshall Reeves Foundation, Dr. E. Vernon Smith and Eloise C.
Smith Macular Degeneration Endowed Chair, and Research to
Prevent Blindness departmental unrestricted grant; S.D.F by AIRC
(Associazione Italiana Ricerca sul Cancro) grant IG11420 and Italian
Ministry for Scientific Research, project PON01_02342; J.Z.B. by
NIH K08EY021521 and University of Kentucky Physician Scientist
Award; B.J.F. and S.B. by NIH T32HL091812 and UL1RR033173;
Y.H. by Alcon Research Award; A.B.C. by the Programme for
Advanced Medical Education (sponsored by Fundação Calouste
Gulbenkian, Fundação Champalimaud, Ministério da Saúde and
Fundação para a Ciência e Tecnologia, Portugal) and Bayer Global
Ophthalmology Research Award; Y.H. by Alcon Japan Research
award; N.K. by Beckman Initiative for Macular Research; B.D.G.
by American Heart Association and International Retinal Research
Foundation; B.K.A. by NIH R01EY017182 and R01EY017950,
VA Merit Award, and Department of Defense; O.V. by Ministry
of Health, Czech Republic - conceptual development of research
organization (Institute for Clinical and Experimental Medicine –
IKEM, IN 00023001)
Program Number: 1192 Poster Board Number: C0220
Presentation Time: 3:15 PM–5:00 PM
Sorsby Fundus Dystrophy S156C-TIMP3 mutation promotes
angiogenesis and choroidal neovascularization via a FGF
receptor-1 signaling pathway
Jian H. Qi1, Heidi Stoehr2, Bela Anand-Apte1. 1Ophthalmic Research,
Cole Eye Institute, Cleveland Clinic Lerner College of Medicine at
CWRU, Cleveland, OH; 2Institute of Human Genetics, University of
Regensburg, Regensburg, Germany.
Purpose: Sorsby Fundus Dystrophy (SFD), an inherited, early onset
macular degenerative disorder with choroidal neovascularization
(CNV) which closely resembles age-related macular degeneration
(AMD) is caused by mutations in the TIMP3 gene. The objective of
this study was to investigate the mechanism by which SFD-related
S156C-TIMP3 mutation promotes angiogenesis or CNV.
Methods: In vitro angiogenesis was induced by FGF, VEGF or the
conditioned medium (CM) from human retinal pigment epithelial
(RPE) cells expressing wild-type (WT), S156C-TIMP3 or empty
vector, and analyzed for tube formation and/or cell proliferation. The
laser-induced CNV responses in Timp3 S156C/S156C knock-in mice
and their corresponding WT littermates were quantitated. VEGFR-
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
2,FGFR-1 tyrosine phosphorylation and MAP kinase phosphorylation
in ECs or the RPE-choroid tissue following ligand stimulation or
laser treatment were analyzed by Western blots.
Results: The expression of S156C-TIMP3 in RPE cells exhibited
increased glycosylation and decreased MMP inhibitory activity of
the mutant protein. Increased levels of bFGF and Heparan Sulfate
(HS) but not VEGF were observed in the CM of mutant cells. The
CM from mutant RPE cells induced increased tube formation in PAE/
FGFR-1 cells with a bFGF dependency. Proliferation, tube formation,
FGFR-2 tyrosine phosphorylation and MAP kinase activation were
increased in mouse aortic endothelial cells from either heterozygous
TIMP3+/S156 or homozygous TIMP3S156C/S156C following
bFGF stimulation relative to WT control cells. Consistent with
the results from in vitro experiments, Timp3 S156C/S156C mice
showed increased glycosylation and reduced MMP inhibitory activity
of mutant protein, increased tyrosine phosphorylation of FGFR-2
and increased MAP kinase activation in RPE-choroids after a laser
burn. Furthermore, at 2 weeks post-laser induction, the area of
CNV at Bruch’s membrane (BM) rupture sites was observed to be
significantly larger in mutant mice compared to their WT controls.
Conclusions: S156C-TIMP3 may up-regulate angiogenesis and CNV
by increasing bFGF-FGFR-2 signaling.
Commercial Relationships: Jian H. Qi, None; Heidi Stoehr, None;
Bela Anand-Apte, None
Support: 5 R21EY022768-02
Program Number: 1193 Poster Board Number: C0221
Presentation Time: 3:15 PM–5:00 PM
Anti-histamine receptor h4 treatment reduced choroidal
neovascularization independent of vascular endothelial growth
factor
Ryo Ijima, Hiroki Kaneko, Fuxiang Ye, Shu Kachi, Hiroko Terasaki.
Ophthalmology, Nagoya Univ Grad School of Med, Nagoya, Japan.
Purpose: To examine the relationship between vascular endothelial
growth factor (Vegf) and histamine receptor h4 (Hrh4) for the
treatment of choroidal neovascularization (CNV) in mice.
Methods: In wild-type and Hrh4-/- mice, laser photocoagulation was
performed in the retina to induce experimental CNV (laser-CNV).
Protein expressions of Vegf were examined and CNV volumes were
measured in mice after inducing laser-CNV. Anti-Vegf neutralizing
antibody (1μg) or isotype IgG control combined with JNJ7777120
(1μg), Hrh4 antagonist, was injected intravitreously after inducing
laser-CNV, and CNV volumes were compared.
Results: Laser-CNV volume in Hrh4-/- mice was significantly
smaller than that in wild-type mice. However, mouse Vegf level
in wild-type mice RPE 3 days after laser-photocoagulation was
not significantly different from that in Hrh4-/- mice. Anti-VEGF
neutralizing antibody mixed with JNJ7777120 significantly reduced
CNV volume in wild-type mice compared to isotype IgG antibody
mixed with JNJ7777120.
Conclusions: CNV is suppressed by Hrh4 reduction via Vegfindependent mechanism.
Commercial Relationships: Ryo Ijima, None; Hiroki Kaneko,
None; Fuxiang Ye, None; Shu Kachi, None; Hiroko Terasaki, None
Program Number: 1194 Poster Board Number: C0222
Presentation Time: 3:15 PM–5:00 PM
Intravitreal (IVT) injection exacerbates murine choroidal
neovascularization (CNV) area by a MYD88 dependent
mechanism
Elizabeth Fassbender, Yubin Qiu, Siyuan Shen, Amber Woolfenden,
Andrea Delpero, Yong Kim, Bruce D. Jaffee, Stephen H. Poor.
Ophthalmology, Novartis Institutes for Biomedical Research,
Cambridge, MA.
Purpose: To compare the effect of route of administration of vehicle
and the role of innate immunity in the mouse laser-induced CNV
model.
Methods: We performed an analysis of CNV area in mice dosed
vehicle by different routes of administration.
CNV area in age and sex matched C57/BL6 mice were investigated
after systemic or IVT administration of a toll receptor 3 activator,
Polyinosinic:polycytidylic acid (poly I:C) or vehicle. A single 50mg
dose or vehicle was administered i.v. at day -1 or day 2. A single IVT
injection of 1.5 or 3.8mg poly I:C or vehicle was injected immediately
after laser.
CNV area was assessed in littermate age and sex matched MYD88
KO and toll receptor (TLR) 2 KO mice.
Laser pulses were applied with an Iridex Oculight® GLx 532nm
laser, 3 lesions/eye, 6 lesions/mouse, 9-15 mice/group yielding 54-90
data points per treatment group. On day 7 after laser, blood vessels
were labeled with an i.v. injection of FITC-ConA and mice were
euthanized. Eyes were enucleated and fixed in paraformaldehyde.
Posterior ocular segments were dissected and mounted onto slides.
CNV lesions were captured by fluorescent microscopy and CNV area
was analyzed with Axiovision software. Analysis and exclusions were
completed on masked data. Statistics were analyzed by an unpaired
two tailed t test in Graph pad prism Vs6.
Results: CNV area was increased 42% in mice injected IVT with
vehicle compared to mice receiving vehicle systemically (n=21
IVT vehicle studies, mean CNV area=17.0 ± 5.5 mm2 x 10-3; n=20
systemic vehicle studies, mean CNV area=12.8 ± 3.4 mm2 x 10-3,
p=0.0066).
CNV area in mice administered i.v. (n=2 studies) or IVT (n=3
studies) poly I:C was similar in size to CNV area in mice injected
with saline (p>0.05).
CNV area was similar in male MYD 88 KO mice and littermate
controls (n=3 studies, p>0.05). Both male and female MYD88 KO
mice injected with IVT saline had smaller CNV than control mice
(male n=2 studies, mean % CNV inhibition in KO compared to
WT=47%, p≤0.0038; females n=2 studies, mean % CNV inhibition in
KO compared to WT=48%, p≤0.0042). CNV area was similar in size
between TLR2 KO mice and controls injected IVT with saline (n=2
studies, p>0.05).
Conclusions: IVT injection exacerbates CNV area compared to noninjected mice. IVT injection increases CNV area through a MYD88dependent mechanism, not through TLR2. Activation of TLR3 does
not alter CNV area.
Commercial Relationships: Elizabeth Fassbender, Novartis
(E); Yubin Qiu, Novartis (E); Siyuan Shen, Novartis (E); Amber
Woolfenden, Novartis (E); Andrea Delpero, Novartis (E); Yong
Kim, Novartis (E); Bruce D. Jaffee, Novartis (E); Stephen H. Poor,
Novartis (E)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1195 Poster Board Number: C0223
Presentation Time: 3:15 PM–5:00 PM
Pathophysiological roles of endogenous calcitonin generelated peptide in mouse model of laser induced choroidal
neovascularization
Yuichi Toriyama1, 2, Yasuhiro Iesato1, 2, Akira Imai1, 2, Yuka IchikawaShindo1, 2, Akiko Kamiyoshi2, Takayuki Sakurai2, Takayuki Shindo2,
Toshinori Murata1. 1Department of Ophthalmology, Shinshu
University School of Medicine, Matsumoto, Nagano, Japan;
2
Department of Cardiovascular Research, Shinshu University
Graduate School of Medicine, Matsumoto, Nagano, Japan.
Purpose: Calcitonin gene-related peptide (CGRP) is a neuropeptide
that has a variety of physiological functions. Recently, angiogenic
potency of CGRP is reported in tumor angiogenesis model. We also
reported the angiogenic potency of endogenous CGRP in eye using
oxygen-induced retinopathy model in CGRP knockout mice (ARVO
2013). On the other hand, CGRP is also involved in the regulation
of inflammation; CGRP directly acts on macrophages and regulate
their production of cytokines. Choroidal neovascularization (CNV)
is a common symptom of age-related macular degeneration (AMD).
In the pathophysiology of CNV, both angiogenic and inflammatory
molecules play important roles. In the current study, we analyzed
the pathophysiological roles of endogenous CGRP in CNV by
introducing laser-induced CNV in CGRP knockout mice.
Methods: CNV was induced by laser photocoagulation (200mW,
50μm, 50msec). Four laser burns were placed in eyes of wild-type
C57BL/6 mice (WT) and homozygous knockout mice of CGRP
(KO). Fourteen days after laser injury, fluorescein angiography (FA)
was performed. Areas of CNV were determined in retinal pigment
epithelium-choroid-sclera (choroidal) flat mounts using perfused
fluorescein-labeled dextran. Macrophage infiltration was evaluated
by immunostaining of choroidal flat mounts on Day 7. F4/80 positive
cells around CNV were counted.
Results: Significantly larger CNV lesions with greater leakage
on FA developed in KO(23458±1679mm2) compared to
WT(17232±1122mm2) on day14. Laser photocoagulation scar area
was not different between KO and WT. KO mice showed increased
infiltration with F4/80-positive cells around the laser irradiated area
in comparison with WT.
Conclusions: Endogenous CGRP suppresses the development
of CNV thorough regulation of macrophage infiltration and
inflammation. CGRP could be a promising therapeutic target of
AMD.
Commercial Relationships: Yuichi Toriyama, None; Yasuhiro
Iesato, None; Akira Imai, None; Yuka Ichikawa-Shindo, None;
Akiko Kamiyoshi, None; Takayuki Sakurai, None; Takayuki
Shindo, None; Toshinori Murata, None
Program Number: 1196 Poster Board Number: C0224
Presentation Time: 3:15 PM–5:00 PM
Lacking Smad3 attenuates development of Argon laser-induced
CNV in mice
Hiroki Iwanishi, Takayoshi Sumioka, Yuka Okada, Osamu Yamanaka,
Shizuya Saika. Opthalmology, Wakayama Medical University,
Wakayama-City, Japan.
Purpose: To evaluate the effects of lacking Smad3 on the
development of experimental choroidal neovascularization (CNV)
following argon-laser irradiation in mice. Roles of ALK1 and ALK 5
in vascular formation were examined in cell culture.
Methods: Male wild type (WT) and Smad3-null (KO) mice of 8-12
week-old were used. (1) Argon laser-photocoagulation (5 shots, 75
μm spot size, 0.1 sec. duration, 150 mW) was performed in an eye
of WT (n = 5) and KO (n = 5) mice. The size of CNV was measured
at 2 weeks by software-assisted fluorescence angiography after
sacrifice and enucleation. (2) Laser photocoagulation (15 shots) was
performed in an eye of WT (n = 5) and KO (n = 5) mice. Total RNA
was extracted from retino-choroidal tissue at day 3 post-treatment.
Real-time RT-PCR was ran for mRNAs of VEGF, IL-6, F4/80
macrophage antigen, TGFb1 and MCP-1. (3) Effects of adding an
ALK5 inhibitor (SB431542) on CD31-labeled vessel-like structure
formation by HUVECs in a co-culture system with fibroblast feeder
layer.
Results: Laser-induced CNV formation was dramatically suppressed
by lacking Smad3 in mice. Loss of Smad3 suppressed mRNA
expression of VEGF, IL-6 and F4/80 in retino-choroidal tissue.
TGFb1-accelerated neovascularization (vessel-like structure
formation) by HUVECs was further augmented by adding an ALK5
inhibitor SB431542, suggesting ALK5/Smad3 signal counteracts
neovascularization promotion of HUVECs in cell culture condition.
Conclusions: Blocking TGFb/Smad3 signaling is a potential strategy
for therapeutic approach to CNV-related disorders, including AMD
by interfering macrophage-related inflammation.
Commercial Relationships: Hiroki Iwanishi, None; Takayoshi
Sumioka, None; Yuka Okada, None; Osamu Yamanaka, None;
Shizuya Saika, None
Program Number: 1197 Poster Board Number: C0225
Presentation Time: 3:15 PM–5:00 PM
Ultrastructural Evaluation of Retinal Neovascular Proliferation
in Rats with Advanced Light-Induced Retinal Degeneration
Leandro B. Teixeira1, 3, Nader Sheibani2, 3, Daniel M. Albert2, 3,
Richard R. Dubielzig1, 3. 1Pathobiological Science, University of
Wisconsin-Madison, Madison, WI; 2Department of Ophthalmology
and Vision Science, University of Wisconsin-Madison, Madison,
WI; 3McPherson Eye Research Institute, University of WisconsinMadison, Madison, WI.
Purpose: To characterize the ultrastructural morphology of retinal
neovascularization secondary to light-induced retinal damage in
albino rats.
Methods: Eyes from 5 adult female Albino Wistar rats exposed to
12h of cyclic diffuse cool white 3,000 lux intensity light for 1 month
were enucleated after two months of exposure to ambient light and
fixed in 2.5% glutaraldehyde. The posterior segments of the globes
were routinely processed for transmission electron microscopy.
Results: All animals presented lesions consistent with chronic
light-induced retinopathy, e.g., complete loss of photoreceptors and
atrophy of the outer plexiform layer with the inner nuclear layer
directly contacting RPE. Multiple vascular profiles traversed the
inner plexiform and nuclear layers, contacting hyperplastic RPE
that proliferated along the length of the vessels. In other areas RPE
was attenuated and vessels came in close proximity to Bruch’s
membrane. Proliferating vessels were small (5-15μm in diameter),
with patent lumen and formed clusters that were denser near RPE.
Vessels presented 3 distinct phenotypes. The first type, consistent
with a retinal phenotype, presented complete luminal coverage
with robust endothelial cells, tight junctions, thick (100-300 nm)
basement membrane and pericytes. The second type, resembling
choriocapillaris, presented delicate endothelium with fenestrations,
thin (20-50 nm), irregular and discontinuous basement membrane and
no pericyte coverage. The third type presented mix features of retinal
and choriocapillaris phenotype with thick basement membranes,
endothelial cell tight junctions and fenestrations and variable pericyte
coverage. Even after multiple step sections through the tissues we
did not visualize vascular profiles crossing Bruch’s membrane and/or
extending into the choriocapillaris in any of the samples analyzed.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Conclusions: Chronic retinal light induced retinopathy in rats is
characterized by severe atrophy of the outer retina and neovascular
proliferation. As the retina atrophies the proliferating vessels get
in close contact with RPE. We hypothesize that the variations
in vascular morphology reported are driven by the changes in
the perivascular environment and close proximity to RPE cells.
Understanding this phenomenon of vascular morphology shift can
be beneficial not only to basic vascular research but also to clinical
ocular neovascular diseases.
Commercial Relationships: Leandro B. Teixeira, None; Nader
Sheibani, None; Daniel M. Albert, None; Richard R. Dubielzig,
None
Support: This work was supported by the National Institutes of
Health grant P30-EY016665 (Core Grant for Vision Research) and
an unrestricted department award from the Research to Prevent
Blindness. Additional Contributions: We are grateful for the support
from the McPherson Eye Research Institute, University of Wisconsin.
Program Number: 1198 Poster Board Number: C0226
Presentation Time: 3:15 PM–5:00 PM
PAMP Stimulation of Macrophages Promotes Neovascular
Remodeling in Experimental Choroidal Neovascularization
Priyatham S. Mettu1, Peter Saloupis1, Scott W. Cousins1, 2.
1
Ophthalmology/Duke Eye Center, Duke University School of
Medicine, Durham, NC; 2Immunology, Duke University School of
Medicine, Durham, NC.
Purpose: Neovascular remodeling (NVR), the transformation of
capillaries into branching arterioles with perivascular fibrosis, is
a major cause of persistent disease activity in spite of anti-VEGF
therapy in NV AMD. Pathogen-associated molecular patterns
(PAMPs) are microbe-associated molecules that can stimulate
activation of macrophages via binding to cell-surface patternrecognition receptors. We explored the hypothesis that PAMP
stimulation of macrophages promotes NVR in experimental laserinduced choroidal neovascularization (CNV).
Methods: We performed laser-induced CNV in wild-type C57BL/6J
mice (8 mo. old, n=18). At the time of laser, half of these mice (n=9)
underwent PAMP stimulation with low-dose lipopolysaccharide (10
μg, well below dose associated with systemic toxicity), while the
other half (n=9) received saline control. After two weeks, fluorescein
angiography (FA) was performed, and eyes were removed and
processed for choroidal flatmount and immunohistochemistry.
Macrophage activation following PAMP stimulation was assessed by
RT-PCR of select cytokines in isolated splenic monocytes.
Results: PAMP-stimulated mice demonstrated a mean lesion size
of 3.7 ± 0.8 disc areas (DA) by propidium iodide cellular flatmount,
as compared to 2.0 ± 0.7 DA for control exposed mice (p < 0.01).
PAMP-stimulated mice also demonstrated larger lesions by TRITClectin vascular morphology flatmounts, 2.3 ± 0.3 DA, as compared
to 1.4 ± 0.1 DA for control mice (p < 0.02). NVR was readily
observed in CNV of PAMP-stimulated mice and was characterized by
prominent leakage by FA as well as large-caliber branching arterioles
with vascular loops on vascular morphology flatmounts. In contrast,
saline-exposed mice had small-caliber capillaries with few arterioles,
as well as mild FA leakage. Preliminary studies suggest that PAMPstimulated mice demonstrate increased ratio of SMA+ smooth
muscle cells / CD31+ endothelial cells, as well as increased F4/80+
macrophage frequency. Additionally, splenic monocytes from PAMPstimulated mice had increased activation state, with upregulated
expression of cytokines potentially relevant to NVR, including NOS2, MMP-9, and PDGF.
Conclusions: PAMP-stimulation of macrophages appears to promote
NVR in experimental CNV. Importantly, this highlights a potential
role for therapies directed against activated macrophages in the
treatment of persistent disease activity in NV AMD.
Commercial Relationships: Priyatham S. Mettu, Salutaris Medical
Devices (R), Valeant Ophthalmics (R); Peter Saloupis, None; Scott
W. Cousins, AbbVie (C), Alcon (C), Heidelberg Engineering (C),
Kala (C), Pfizer (C), Salutaris Medical Devices (C), Sanofi-Fovea
(C), Valeant Ophthalmics (C)
Support: NIH Grant R01-EY018880 (SWC), NIH Grant K12EY016333 (PSM), Research to Prevent Blindness (PSM)
Program Number: 1199 Poster Board Number: C0227
Presentation Time: 3:15 PM–5:00 PM
Functional role of TLR in choroidal neovascularization
Yin Shan Eric Ng1, 4, Meihua Ju1, Daiju Iwata1, Richard Foxton1,
Shannon Bunker1, Monica Belich2, Gerald Gough2, Peter S.
Adamson3, David T. Shima1. 1Ocular Biology and Therapeutics,
UCL Institute of Ophthalmology, London, United Kingdom;
2
Biopharmaceuticals R&D, GlaxoSmithKline, Stevenage, United
Kingdom; 3Ophthiris Discovery Performance Unit, GlaxoSmithKline,
Stevenage, United Kingdom; 4Vascular Biology, The Schepens Eye
Research Institute, Boston, MA.
Purpose: Strong evidence suggests a critical role of inflammation,
including the innate immune system, and its dysregulation in the
development of sub-retinal choroidal neovascularization (CNV)
associated with aged-related macular degeneration. We identified
toll-like receptor 2 (TLR2) as one of the molecules involved in the
development of CNV, and compared the therapeutic potential of
TLR-2 blockage to that of VEGFR2 blockage in experimental models
of CNV.
Methods: A novel murine model of spontaneous CNV (sCNV),
which is VEGF-dependent and driven by inflammation was used
for assessing the effects of intravitreally delivered neutralizing
monoclonal antibodies against TNFα, IL1R, TLR2 and VEGFR2.
A laser-induced murine CNV (lCNV) model was also used for
anti-TLR2 study. TLR2, macrophage and CNV were analysed by
immunostaining. CNV number and area evaluated by fluorescein
angiography and immunostaining.
Results: Among the neutralizing antibodies tested in an initial pilot
experiment, a single intravitreal anti-TLR2 treatment reduced the
number of CNV per eye in a sCNV model at day 7 post treatment,
although the inhibition was not statistically significant due to the
small sample size. In a dose escalation experiment, 2 intravitreal
anti-TLR2 treatments 7 days apart significantly reduced both the
number and area of sCNV in a dose-dependent manner at day 14,
and combination of anti-VEGFR2 and anti-TLR2 treatments did not
result in significant additive effects in the different dosages tested.
Immunostaining of the lesions showed that the number of F4/80positive macrophage associated with the sCNV was dramatically
reduced by anti-TLR2, whereas anti-VEGFR2 had less obvious effect
on sCNV-associated macrophage number. However, intravitreal antiTLR2 treatment was not effective in suppressing lCNV, suggesting
a differential role of TLR2 in this laser damage- and wound healing
–driven CNV model.
Conclusions: We have identified TLR2 and its direct functional role
in the development of sCNV, and this pathway perhaps is feeding
into the VEGF-A pathway because of the lack of additive effect.
Furthermore, TLR2 is involved in the recruitment of macrophage
to the sCNV, suggesting that it may be an attractive therapeutic
target for neovascular AMD via both anti-inflammation and indirect
inhibition of the VEGF-A pathway.
Commercial Relationships: Yin Shan Eric Ng, GlaxoSmithKline
(F); Meihua Ju, None; Daiju Iwata, None; Richard Foxton,
None; Shannon Bunker, None; Monica Belich, GlaxoSmithKline
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
(E); Gerald Gough, GlaxoSmithKline (E); Peter S. Adamson,
GlaxoSmithKline (E); David T. Shima, GlaxoSmithKline (F)
Support: NIHR Biomedical Research Centre at Moorfields Eye
Hospital NHS Foundation Trust and UCL Institute of Ophthalmology
London; GSK
Program Number: 1200 Poster Board Number: C0228
Presentation Time: 3:15 PM–5:00 PM
Effect of Intravitreal Imatinib on Laser-induced Rat Model of
Choroidal Neovascularization
Homayoun Nikkhah2, 1, Hamid Ahmadieh2, 1, Ramin Nourinia2, 1,
Mozhgan Rezaei Kanavi2, 1, Bagher Hosseini2, 1, Mohammad Reza
Oveisi3, Naficeh Sadeghi3, Seyed Mohsen Khandaghy Meybodi2,
Mohammadreza Rahimi2, Mehdi Yaseri2. 1Ophthalmology, Shahid
Beheshti University of Medical Sciences, Tehran, Islamic Republic
of Iran; 2Ophthalmic Research Center, Shahid Beheshti University of
Medical Sciences, Tehran, Islamic Republic of Iran; 3Department of
Drug and Food Control, School of Pharmacy, Tehran University of
Medical Sciences, Tehran, Islamic Republic of Iran.
Purpose: To evaluate the safety and efficacy of intravitreal injection
of a tyrosine kinase inhibitor, directed against platelet-derived growth
factor and vascular endothelial growth factor receptors, in a rat model
of choroidal neovascularization (CNV).
Methods: Phase I: Fifty-four pigmented rats were divided into six
groups. Intravitreal injection of imatinib consisting of 330mg/5ml,
250mg/5ml, 165mg/5ml, 80mg/5ml and 40mg/5ml was performed in the
right eye of each rat in groups 1 to 5 respectively. In group 6, 5ml of
balanced salt solution (BSS) was injected intravitreally.
Electroretinography (ERG) was performed at baseline and at
weeks one and four. After euthanizing the animals, the right eyes
were enucleated for histologic analysis. Based on the ERG records
and histologic results, the maximum safe dose of Imatinib was
determined.
Phase II: Thirty-one rats were used. Experimental CNV was
induced by laser photocoagulation in the right eye of each rat. In
21 rats, the highest safe dose of imatinib determined in phase I was
injected intravitreally. BSS was injected in the remaining 10 rats.
Fluorescein angiography was performed after 4 weeks; then the rats
were euthanized and their right eyes were sent for histopathologic
evaluation. Eventually, CNV area in tissue sections was measured
using ImageJ software and compared between treatment and control
groups.
Results: Phase I: ERG showed no statistically significant difference
between Imatinib-injected eyes and the controls. However, histologic
analysis disclosed focal retinal atrophic changes in the eyes receiving
165mg, 250mg, and 330mg/5mL of the drug. In the eyes that received
40mg and 80mg/5mL Imatinib, retinal histology was unremarkable and
immune-reactivity for glial fibrillary acidic protein was not different
from the controls. Maximum safe dose of Imatinib was determined to
be 80mg/5ml.
Phase II: In fluorescein angiography, there was no significant
difference between the treatment and control groups in terms of
leakages from CNV. Moreover, CNV area in the treatment group was
not statistically different from the controls.
Conclusions: Intravitreal imatinib alone is not effective in inhibition
of experimental CNV in a rat model.
Commercial Relationships: Homayoun Nikkhah, None; Hamid
Ahmadieh, None; Ramin Nourinia, None; Mozhgan Rezaei
Kanavi, None; Bagher Hosseini, None; Mohammad Reza Oveisi,
None; Naficeh Sadeghi, None; Seyed Mohsen Khandaghy
Meybodi, None; Mohammadreza Rahimi, None; Mehdi Yaseri,
None
Program Number: 1201 Poster Board Number: C0229
Presentation Time: 3:15 PM–5:00 PM
Therapeutic Effect of IBI302, a bispecific Fc-fusion protein, on
Age-related Macular Degeneration
Jing Wang1, Qiuhui Liu1, Qilin Wang1, Xia Dong1, Jia Li2, Michael
Yu2, Yan Luo1. 1State Key Laboratory of Ophthalmology, Zhongshan
Ophthalmic Center, Sun Yat-sen University, Guangzhou, China;
2
Innovent Biologics, Inc., Suzhou, China.
Purpose: To evaluate the therapeutic activity and its mechanism
of action of IBI-302, a bispecific Fc-fusion protein that effectively
inhibits VEGF and complement, on the laser-induced choroidal
neovascularization (CNV) in mice and oxidative stress in vitro
cultured human RPE cells.
Methods: C57BL/6J mice were induced CNV by laser
photocoagulation and received an intravitreal injection of
phosphate-buffered saline (PBS) and IBI-302 solution respectively.
Seven days after intravitreal injection, areas of CNV and leakage
were measured by FFA, ICGA and fluorescein-labeled dextran
perfusion. Numbers of infiltrating macrophages and neutrophils
and expression of MAC in the RPE-choroid-sclera complex were
detected by immunohistochemistry. The integrity of human primary
RPE monolayer barrier was established by measurement of transepithelial resistance (TER). Oxidative stress was induced with t-butyl
hydroperoxide (t-BHP). Normal human serum(NHS) was added for
complement activation. Cells in each group were then treated with
1μg/ml IBI302 and PBS for 4hrs, respectively. Both in vivo and
vitro study, the protein levels of VEGF, TNF-α, CCL-2, C3a, C5a
and sC5b-9 were assayed by ELISA. VEGF-Trap and CID, a soluble
complement receptor, were used as controls.
Results: Both area of CNV and leakage were significantly suppressed
by IBI302 treatment compared with PBS, has much better efficacy
than VEGF-Trap and CID. IBI302 treatment significantly inhibited
the infiltration of macrophages and neutrophils in the CNV area
and the deposition of MAC in the CNV lesion. IBI302 treatment
downregulated the protein expressions of VEGF, CCL-2, TNF-α and
C3a, C5a, MAC both in mice with CNV and cultured RPE under
oxidative stress. IBI302 also inhibited the decrease of TER.
Conclusions: IBI302 could reduce the CNV area and protect RPE
cells from oxidative stress and complement activation by inhibiting
the expression of VEGF, inflammation-related molecules including
TNF-α, CCL-2 and complement activation-related molecules
including C3a, C5a and MAC, then further inhibit the infiltration of
macrophages and neutrophils and the deposition of MAC.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
ICGA, FFA and IR images of mice seven days after intravitreal
injection. IBI302 significantly suppress the leakage and CNV area
comparing with VID and CID, however no statistic difference exist
in the IR images showing the primary laser damage is comparable
among the groups.
Commercial Relationships: Jing Wang, None; Qiuhui Liu, None;
Qilin Wang, None; Xia Dong, None; Jia Li, Innovent Biologics, Inc
(E); Michael Yu, Innovent Biologics, Inc (E); Yan Luo, None
Support: grants from the National Natural Science Foundation of
China (81170864, 81371020)
Program Number: 1202 Poster Board Number: C0230
Presentation Time: 3:15 PM–5:00 PM
Role of netrin-4 in a Mouse Model of Choroidal
Neovascularization
Christina Nuernberg1, Sabrina V. Klein1, Norbert Kociok1, AnnaKarina B. Maier1, Nadine Reichhart1, William J. Brunken2, Olaf
Strauss1, Manuel Koch3, Antonia M. Joussen1. 1Department of
Ophthalmology, University Medicine Berlin, Berlin, Germany;
2
Department of Cell Biology and Ophthalmology, SUNY Downstate
Medical Center, New York City, NY; 3Department of Biochemistry II,
University of Cologne, Cologne, Germany.
Purpose: Netrins are a laminin-like family of proteins that first
were characterized as axon guidance molecules in neurogenesis.
Interestingly, current research now draws parallels between molecular
mechanisms of neurogenesis and angiogenesis claiming that both
processes share molecular mechanisms.
In this work, we aimed to demonstrate whether netrin-4, the newest
member of the netrin-family, joins in the complex mechanisms of
choroidal neovascularization (CNV) as part of the pathogenesis of
wet Age-related macular degeneration (AMD).
Methods: Exploiting an established laser-induced mouse model of
CNV in Ntn-4-/- mice and C57BL/6J mice as controls, we examined
eyes 14 days after laser exposure by fluorescein angiography in
order to evaluate the leakage of nascent pathological choroidal
blood vessels. Whole flatmounts of the choroid were dissected and
stained with an endothelial cell staining (Isolectin IB4) and CD11b
as a myeloid cell marker. The size of the laser induced CNV area
was measured, the CD11b positive cells counted and statistically
analyzed. To prove that the Ntn-4-/- mice lack netrin-4 expression,
immunohistochemical stainings on cross sections on both groups
were performed in combination with an immunohistochemical
staining of endothelial cells.
Results: Quantification of the fluorescein leakage from choroidal
laser scars as a mete of pathological blood vessel formation showed
no significant differences between control and Ntn-4-/- mice. Two
weeks after laser coagulation the mean size of the CNV areas of
35824.33 ± 38428.49 μm in control mice (n = 26) did not differ
from that in Ntn-4-/- mice (n = 31) of 24209.96 ± 15199.35 μm (p =
0.064). Also the number of CD11b positive cells in CNV lesions in
both groups revealed no significant statistical difference (p = 0.45).
Likewise, there was no significant difference in VEGF expression in
the sclera/choroid or in the retina of laser-treated Ntn-4-/- mice when
compared to the control mice.
Conclusions: Our data show that the presence of netrin-4 does not
affect the formation of pathological blood vessels in this laserinduced mouse model of the wet form of AMD. We hypothesize that
netrin-4 does not play a significant role in the pathogenesis of the wet
form of AMD.
Commercial Relationships: Christina Nuernberg, None; Sabrina
V. Klein, None; Norbert Kociok, None; Anna-Karina B. Maier,
None; Nadine Reichhart, None; William J. Brunken, None; Olaf
Strauss, None; Manuel Koch, None; Antonia M. Joussen, None
Support: DFG SFB 612-B14
Program Number: 1203 Poster Board Number: C0231
Presentation Time: 3:15 PM–5:00 PM
PRI-724 Significantly Reduces Retinal Fibrosis in Models of CNV
and PVR
Andy Whitlock1, Rafal Farjo2, Geoffrey P. Lewis3, Steven K. Fisher3,
Gabriel Luna3, Takenao Odagami4, Tetsushi Inada4, Hiroyuki Kouji4.
1
Pre-Clinical, Ora, Andover, MA; 2EyeCRO LLC, Oklahoma City,
OK; 3Neuroscience Institute, University of California, Santa Barbara,
CA; 4Prism Pharma, Ltd, King of Prussia, PA.
Purpose: Fibrosis is a component of a number of ophthalmic diseases
such as late-stage wet AMD, diabetic retinopathy, proliferative vitreoretinopathy (PVR), and bleb closure/failure following trabeculectomy
surgery. While fibrosis is a multi-factorial process, the Wnt signaling
pathway is certainly implicated during the initiation of this process.
PRI-724 is a potent anti-fibrotic agent that has been shown to inhibit
both liver and pulmonary fibrosis in pre-clinical disease models.
PRI-724 also possesses significant anti-proliferative properties, and
is currently in human clinical trials for the treatment of both solid
and liquid tumors. PRI-724 acts by binding specifically to CREB
Binding Protein (CBP) and antagonizing β-catenin/CBP-mediated
transcription. This inhibition results in the down-regulation of many
genes required for cell proliferation. The purpose of this study was to
determine the efficacy and potential utility of this novel anti-fibrotic
drug in several models of retinal disease.
Methods: The appropriate dosing level and route of administration
(systemic/subcutaneous, intravitreal (IVT), or topical) was
determined in preliminary rabbit and rat pharmokinetic (PK) studies.
The anti-angiogenic effect of PRI-724 and its active metabolite, C-82,
were explored in a laser-induced CNV model in rats in comparison
to an anti-VEGF control. The direct anti-fibrotic effect of PRI-724
was examined in a retinal detachment model of PVR. Fibrosis was
measured in this model by quantifying both the number of antiphosphohistone labeled proliferating Muller cells, and the size and
number of subretinal glial scars.
Results: The most efficient route for retinal exposure was IVT
administration, which demonstrated measureable levels of compound
out to 7 days following a single injection of PRI-724 or C-82. In
the laser-induced CNV model, treatment with PRI-724, C-82, and
the anti-VEGF clinical comparator significantly reduced lesion size
compared to vehicle control. Treatment with PRI-724 in the rat
PVR significantly reduced Muller cell proliferation and glial scar
formation when compared to the vehicle control.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Conclusions: Given the favorable PK profile of this small molecule
and the effects on both vascular lesion size and sub-retinal fibrosis,
PRI-724 may possess clinical utility for the treatment of several
retinal fibro-vascular diseases. This would provide an added
improvement to the current anti-VEGF treatment paradigm.
Commercial Relationships: Andy Whitlock, Ora, Inc (E); Rafal
Farjo, EyeCRO (E); Geoffrey P. Lewis, UCSB (F); Steven K.
Fisher, UCSB (F); Gabriel Luna, UCSB (F); Takenao Odagami,
Prism Pharma (E); Tetsushi Inada, Prism Pharma (C); Hiroyuki
Kouji, Prism Pharma (E), Prism Pharma (P)
Program Number: 1204 Poster Board Number: C0232
Presentation Time: 3:15 PM–5:00 PM
Validation of a Rabbit Model of Choroidal Neovascularization
Induced by a Subretinal Injection of FGF-LPS
Tim T. Lam1, Paul Miller2, Susan Howard1, T Michael Nork2.
1
Metabolism, Covance laboratories, Madison, WI; 2OSOD, Madison,
WI.
Purpose: To evaluate a previously published model of choroidal
neovascularization (CNV) in Dutch-Belted rabbits (Ni et al 2005)
using a modified procedure.
Methods: 15 (3 in the first cohort and 12 in the second cohort)
young adult Dutch-Belted rabbits were used and a total of 18 eyes
were given subretinal injections of heparin-sepharose beads with
fibroblast growth factor and LPS (100 ng bFGF, 100 ng LPS in 50
ml of ~3% collagen gel, pH 7-7.2). Instead of a single step subretinal
injection with a 30G needle as previously published (Ni et al 2005),
a 2-step injection technique was used. After insertion of a 23G
cannula through the pars plana, a 41G needle (Dutch Ophthalmic
Research Center) was used to create a subretinal bleb with balanced
salt solution followed by subretinal injection of the mixture with a
blunt 26G needle through the same retinotomy. Bruch’s membrane
was then perforated at the visual streak with a sharp 30 gauge
needle. Angiography with either sodium fluorescein or FITC-dextran
were taken periodically. In the first cohort of eyes, the lesions were
followed for 13 weeks. In the second cohort of 12 eyes, 6 were
control and 6 were treated with intravitreous ranibizumab once on
Week 3. Area of the lesions on weeks 3 and 5 was quantified with
ImageJ.
Results: Approximately 60% of all injected eyes showed noticeable
CNV-like lesions at 2 weeks and thereafter. In the first cohort of
4 eyes, the lesions showed growth in the first 6 weeks and then
stabilized. All those lesions remained similar at Week 13. In the
second cohort of eyes, similar CNV-like lesions were noted.
Treatment with ranibizumab inhibited the growth of CNV-like lesions
as evaluated with FITC-dextran angiography on Week 5 (P<0.001).
Conclusions: Persistent CNV-like lesions were produced in DutchBelted rabbits using a procedure modified from an earlier publication.
The model was validated using ranibizumab as a positive control.
Commercial Relationships: Tim T. Lam, Covance Laboratories
(E); Paul Miller, OSOD (C); Susan Howard, Covance (E); T
Michael Nork, OSOD (C)
Program Number: 1205 Poster Board Number: C0233
Presentation Time: 3:15 PM–5:00 PM
Evidence for Anti-Retinal Auto-Antibodies (AAbs) in the
Complement Factor H Chimeric Transgenic (Cfh-Tg) Mouse
Model of Age-Related Macular Degeneration (AMD)
Alessandro Iannaccone1, Albert H. Alhatem1, Nataliya Lenchik2,
1
, Francesco Giorgianni3, David D. New1, Sarka BeranovaGiorgianni3, Ivan Gerling2, Rafael Ufret-Vincenty4, 5, Marko Radic6.
1
Ophthalmology/Hamilton Eye Institute, Univ Tennessee Health Sci
Ctr, Memphis, TN; 2Medicine/Endocrinology, Univ Tennessee Health
Sci Ctr, Memphis, TN; 3Pharmaceutical Sciences, Univ Tennessee
Health Sci Ctr, Memphis, TN; 4Ophthalmology, UT Southwestern
Medical Center, Dallas, TX; 5Neuroscience, UT Southwestern
Medical Center, Dallas, TX; 6Microbiology, Immunology and
Biochemistry, Univ Tennessee Health Sci Ctr, Memphis, TN.
Purpose: To determine if Cfh-Tg mice harboring the H402 Cfh variant
(Ufret-Vincenty et al. IOVS 2010; 51: 5878-87) develop circulating
AAbs recognizing ocular tissue antigens.
Methods: Methods: We compared sera by Western blots (WBs)
from 4 groups: 1) Cfh-Tg expressing the AMD-predisposing H402
Cfh variant (H-Cfh-Tg); 2) H-Cfh-Tg co-expressing the wild-type
(WT) transgene of human C-reactive protein (CRP), which interacts
with Cfh in inflammation (H-Cfh-Crp-Tg); 3) mice co-expressing
the WT Cfh variant (Y402) and the WT Crp gene (Y-Cfh-Crp-Tg);
and 4) control adult C57BL/6 mice. Adult C57BL/6 mouse retina/
RPE/BM/choroid tissue lysate (10 mg) was loaded on gels, incubated
with serum (5mL) and developed. WB band intensity was quantified
with the Odyssey system and compared by Kruskall-Wallis test.
Immunohistochemistry was done against anti-mouse IgG Ab by
fluorescence microscopy following incubation of adult C57BL/6
retina sections with mouse sera. To identify the autoantigens,
mouse sera were immunoprecipitated, followed by 2-dimension
electrophoresis (2DE) and Mass-Spectrometry (MS) was performed
on spots seen on 2DE following previously reported methods
(Lenchik et al. ARVO 2013, Abs. 4103).
Results: H-Cfh-Tg mice showed consistently the highest reactivity
compared to controls, Y-Cfh-Crp-Tg and H-Cfh-Crp-Tg. Significantly
more intense WB bands were seen in H-Cfh-Tg mice at 91kDa
(p=0.001), 17kDa and 13kDa (p=0.004), 31kDa (p=0.012), 54kDa
(p=0.039), and 22kDa (p=0.045). The outer nuclear (ONL), inner
nuclear (INL), and ganglion cell (GCL) layer of adult C57BL/6
mouse retina sections showed strong staining, with an apparent
perinuclear pattern, by both H-Cfh and H-Crp-Cfh sera. Differentially
reactive spots between H-Cfh-Tg and control mouse sera were
observed also on 2DE and preliminary IDs for some of the spots have
been obtained via MS, suggesting that antigens involved in apoptotic
mechanisms are targeted.
Conclusions: Similar to what we have seen in human AMD sera
(Iannaccone et al. Adv. Exp. Med. Biol. 2012; 723:11-6), AAbs
recognizing retinal targets develop also in this allele-specific AMD
mouse model. This suggests the existence of an autoimmune response
to AMD-like retinal degenerative events triggered by the H-Cfh
variant, which could be contributing to the pathology observed in the
H-Cfh-Tg AMD model as well as in human AMD.
Commercial Relationships: Alessandro Iannaccone, None;
Albert H. Alhatem, None; Nataliya Lenchik, None; Francesco
Giorgianni, None; David D. New, None; Sarka BeranovaGiorgianni, None; Ivan Gerling, None; Rafael Ufret-Vincenty,
None; Marko Radic, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Support: NEI/NIH grants R01 EY022706 (AI), R01 EY022652
(RLUV) and P30 EY020799 (UT Southwestern Ophthalmology);
Research to Prevent Blindness, Inc. New York, NY (Physician
Scientist Award to AI, unrestricted grants to UTHSC Ophthalmology/
Hamilton Eye Institute and UT Southwestern Ophthalmology).
Joseph Caron, UTHSC, assisted with the Kruskall-Wallis test
207 Photoreceptor Degeneration
Monday, May 05, 2014 8:30 AM–10:15 AM
S 310E-H Paper Session
Program #/Board # Range: 1254–1260
Organizing Section: Retinal Cell Biology
Program Number: 1254
Presentation Time: 8:30 AM–8:45 AM
The paradoxical role of CCL3 in acute and chronic retinal
degeneration
Hideo Kohno1, 3, Tsutomu Sakai1, Tadao Maeda2, Eric Pearlman2,
Krzysztof Palczewski3, Akiko Maeda2, 3. 1The department of
Ophthalmology, The Jikei university school of medicine, Tokyo,
Japan; 2The department of Ophthalmology and Visual Sciences, Case
Western Reserve University, Cleveland, OH; 3The department of
Pharmacology, Case Western Reserve University, Cleveland, OH.
Purpose: Accumulating evidence suggests that retinal inflammation
plays a key or important role in visual impairment or loss seen
in retinal degenerative diseases such as age-related macular
degeneration and retinitis pigmentosa. However, the link between
inflammation and retinal degeneration remains poorly defined. This
work was conducted to better elucidate the role of chemokines in
these retinal degenerative conditions.
Methods: Abca4-/-Rdh8-/- mice, which show acute (light induced)
and chronic (age related) retinal degeneration due to impaired
metabolism of all-trans-retinal, were employed in this study. First,
RNA array analysis of chemokines in light exposed retina of Abca4/-Rdh8-/- mice was performed. Second, the time course changes in
chemokine expression were tested by qRT-PCR analysis. Finally,
Ccl3-/-Abca4-/-Rdh8-/- mice were established by cross-breeding
between Ccl3-/-and Abca4-/-Rdh8-/- mice, and retinal phenotype of
acute and chronic retinal degeneration was compared.
Results: The chemokine Ccl3 showed the highest increase among all
tested chemokines by PCR-based RNA array analysis. Time coursed
qRT-PCR results revealed rapid increase of macrophage inflammatory
protein-1 (MIP-1) chemokines, including Ccl3 and Ccl4 compared to
other chemokines. Ccl3-/-Abca4-/-Rdh8-/- mice showed exaggerated
phenotype in acute light induced retinal degeneration compared to
Abca4-/-Rdh8-/- mice. On the contrary, Ccl3-/-Abca4-/-Rdh8-/mice showed attenuated retinal phenotype in age-related chronic
degeneration compared to Abca4-/-Rdh8-/- mice.
Conclusions: Inflammatory chemokine Ccl3 displayed distinct
effects in acute and chronic retinal degeneration in a mouse model
of retinal degeneration. This result suggests the key role of CCL3 in
mediating severity of inflammation and degenerative phenotypes in
retinal disorders.
Commercial Relationships: Hideo Kohno, None; Tsutomu Sakai,
None; Tadao Maeda, None; Eric Pearlman, None; Krzysztof
Palczewski, None; Akiko Maeda, None
Support: NIH (EY022658, EY019031, EY019880, EY021126, and
EY11373), Research to Prevent Blindness Foundation, Foundation
Fighting Blindness, and Ohio Lions Eye Research Foundation.
Program Number: 1255
Presentation Time: 8:45 AM–9:00 AM
Conversion of Photoreceptors into Glast-Positive Progenitor Cells
during Degeneration
Yvan Arsenijevic, Sarah Decembrini. Unit of Gene Therapy & Stem
Cell Biology, Jules-Gonin Eye Hospital, Univ Lausanne, Lausanne,
Switzerland.
Purpose: Several studies investigated whether Muller glial (MG)
cells of the retina have a regenerative potential to replace lost
photoreceptors during retinal degeneration. In Zebrafish, MG cells
can replace degenerating photoreceptors, whereas in mouse some
MG cells start a dedifferentiation program but do not complete the
acquisition of a photoreceptor fate. To date, few and unconfirmed
studies addressed the photoreceptor behavior during retinal
degeneration.
Methods: To that aim, we crossed the Crx-GFP with the Glast-DsRed
mouse lines expressing the green and red reporter genes in postmitotic photoreceptors and in adult MG cells respectively. Time-lapse
video, cell sorting, RT-PCR and immunohistochemical studies were
performed.
Results: We notably observed that during retinal development the
Glast transgene is expressed in retinal progenitors starting from
embryonic day (E) 14, attested by RT-PCR analysis of a sorted
Glast-positive population. Cell sorting study revealed the presence of
double positive cells showing that some Glast-positive cells generate
photoreceptors.
Photoreceptor degeneration was induced by injecting the neurotoxic
MNU compound in 2 month old mice. Retinas of treated mice were
collected after 1 day of treatment and embedded in culture to follow
the expression of Crx and Glast transgenes by time lapse video.
Using a fully automated cell tracking software, we traced migrating
green and red fluorescent cells in time and space up to 2 days.
Surprisingly, we observed that after treatment several displaced cells
expressing GFP (post-mitotic photoreceptors) started translating
the Glast-DsRed transgene. FACS analysis revealed that a low
percentage of Crx-Glast-positive cells are in S-phase.
Conclusions: So far, research focused prominently on the transition
of glia towards a neuronal fate during retinal degeneration and little
attention was reported on photoreceptor behavior. The present data
depict a new situation in which photoreceptors lose their specific
identity and start expressing progenitor-like markers in the attempt to
re-enter the cell cycle rendering the interpretation of regeneration, in
certain situation, challenging. This cell conversion needs also to be
analyzed in light of our recent data demonstrating the important role
of cell cycle proteins during the photoreceptor death process.
Commercial Relationships: Yvan Arsenijevic, None; Sarah
Decembrini, None
Support: FNS 31003A_138482
Program Number: 1256
Presentation Time: 9:00 AM–9:15 AM
Cellular ceramide acts as 2nd messenger mediating photoreceptor
cell death in mammalian retinal degeneration
Nawajes A. Mandal1, 2, Megan Stiles1, Eleanor Sun1, Tuan-Phat
Huynh1, Jeremy Tan1, Douglas Yasumura3, Michael T. Matthes3,
Matthew M. LaVail3. 1Ophthalmology/ DMEI, Univ of Oklahoma
Hlth Sci Ctr, Oklahoma City, OK; 2Physiology/ Cell Biology/ OCNS,
Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 3Beckman
Vision Center, University of California San Francisco, San Francisco,
CA.
Purpose: Ceramide is the key metabolite of cellular sphingolipid
biosynthesis, and its level in a cell is tightly regulated by the balanced
activity of catabolic and anabolic enzymes. Several environmental
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
factors and cell death signals can activate ceramide synthesis by
modulating its metabolic enzymes, and the elevated free ceramide in
a cell signals for apoptosis. We hypothesized that ceramide plays a
role of cellular 2nd messenger in photoreceptor apoptosis in various
forms of mammalian retinal degenerations (RDs).
Methods: In this study, we used well-characterized rat models of RD
such as light-induced RD (LIRD) in albino SD rats, mutant rhodopsin
rats (P23H-1), and RCS rats. We measured cellular ceramide levels at
different time points before and during the process of degeneration by
mass spectrometric analysis. We assayed the expression of the genes
involved by RT-PCR, and measured the activity of major enzymes, in
order to determine the association of ceramide production with retinal
degeneration and to identify the mechanism by which ceramide
levels increase in the retina during degeneration. We used ceramide
synthesis inhibitors in these retinal degeneration models to validate
the role of ceramide in the process of photoreceptor degeneration.
Results: We observed significant increases in ceramide level in the
retina in the LIRD model of SD rats before apoptosis and in P23H-1
and RCS rats during active photoreceptor cell death. In these models,
we found differences in the mechanism of ceramide production:
the de novo pathway of ceramide production was involved in the
LIRD model, and sphingomyelinase activation occurred along with
the activation of the de novo pathway in P23H1 and RCS retinas.
We also found inhibition of de novo ceramide biosynthesis prevents
photoreceptor cell death in light-induced retinal degeneration and in
P23H-1 rats.
Conclusions: We conclude that ceramide signaling is associated
with some forms of RD, which can be targeted for therapeutic
development.
Commercial Relationships: Nawajes A. Mandal, None; Megan
Stiles, None; Eleanor Sun, None; Tuan-Phat Huynh, None; Jeremy
Tan, None; Douglas Yasumura, None; Michael T. Matthes, None;
Matthew M. LaVail, None
Support: NIH grants EY022071, RR17703, EY12190, and
EY01919; and grants from Knight’s Templar Eye Foundation,
Foundation Fighting Blindness, and Research to Prevent Blindness
Inc. USA.
Program Number: 1257
Presentation Time: 9:15 AM–9:30 AM
Identification of Photoreceptor Compartment-Specific Tulp1
Binding Partners
Lindsey A. Ebke1, Gayle J. Pauer1, Belinda Willard2, Stephanie
A. Hagstrom1, 3. 1Cole Eye Institute, Cleveland Clinic, Cleveland,
OH; 2Proteomic Core Services, Cleveland Clinic Lerner Research
Institute, Cleveland, OH; 33Department of Ophthalmology, Cleveland
Clinic Lerner College of Medicine of Case Western Reserve
University, Cleveland, OH.
Purpose: Photoreceptors (PR) are highly polarized and
compartmentalized cells with large amounts of proteins synthesized
in the inner segment (IS) and delivered to the outer segment (OS)
and synaptic terminal. The photoreceptor-specific protein, Tulp1, is
localized to the IS and synapse and is hypothesized to be involved
in protein movement. To better understand the molecular processes
that regulate protein trafficking in photoreceptors, we aim to identify
compartment-specific Tulp1 binding partners.
Methods: Serial tangential sectioning of Long Evans rat retinas was
utilized to isolate the IS and synaptic PR compartments. The ganglion
cell layer was collected as a Tulp1 negative control tissue. Lysates
isolated from these compartments were immunoprecipitated using a
polyclonal Tulp1 antibody. Retinal lysates from the tulp1-/- mouse
were also immunoprecipitated to determine non-specific binding.
The resultant Tulp1-bound proteins were separated by SDS-PAGE,
protein bands excised, digested with trypsin and identified by liquid
chromatography tandem mass spectromety (LC-MS/MS) on an
LTQ-Orbitrap Elite hybrid mass spectrometer. Relative quantitation
was performed by comparing the normalized spectral counts for the
identified proteins across these samples.
Results: Potential Tulp1 binding partners were identified from the
specific PR compartments. In the IS, the primary proteins included
members of the phototransduction cascade and constituents of the
cytoskeleton involved in membrane dynamics. In the synaptic region,
the primary proteins included members of the ribbon active zone. A
separate subset of proteins were identified in both the IS and synapse.
These included members of the GTPase activating family of proteins.
None of these proteins were identified in the negative control
samples.
Conclusions: Tulp1 has two distinct compartment-specific
interactomes. Our results support the hypothesis that Tulp1 is
involved in the trafficking of proteins from the IS to the OS and the
continuous membrane remodeling and vesicle cycling at the synaptic
terminal. These findings may offer insight into possible mechanisms
underlying photoreceptor degeneration caused by mutations in
TULP1.
Commercial Relationships: Lindsey A. Ebke, None; Gayle J.
Pauer, None; Belinda Willard, None; Stephanie A. Hagstrom,
None
Support: NEI EY016072, Research to Prevent Blindness
Program Number: 1258
Presentation Time: 9:30 AM–9:45 AM
Prevention of photoreceptor cell degeneration in P23H rats after
allele-specific knockdown of mutant Rhodopsin RNA expression
using antisense oligonucleotide (ASO) treatment
Sue F. Murray1, Ali Jazayeri1, Matthew M. LaVail2, Michael T.
Matthes2, Douglas Yasumura2, Haidong Yang2, Michael McCaleb1,
Raechel Peralta1, Andy Watt1, Brett Monia1. 1Exploratory Research,
ISIS Pharmaceuticals, Carlsbad, CA; 2School of Medicine, UCSF,
San Francisco, CA.
Purpose: Reducing mutant mRNAs using antisense technology
can be a potentially effective therapeutic approach for dominantly
inherited diseases. To preserve the photoreceptor cell function
and structure in a rodent model of retinitis pigmentosa, P23H-1
transgenic rat, using a potent and selective allele-specific antisense
oligonucleotide (ASO) inhibitor targeting the mutant rhodopsin RNA.
Methods: Transgenic rats expressing the murine P23H gene (Line 1)
were administered either a mouse specific P23H ASO or non-specific
Control ASO in one eye on Day 10 and Day 22 by intravitreal
injection (VIT). The contralateral eye was injected with PBS and
used as a comparator control. On Day 45, ERG measurements were
taken followed by eye enucleation. Half of the rats were designated
for retinal outer nuclear layer analysis and the other half for mRNA
analysis.
Results: Using an ASO targeting the mouse mutant rhodopsin
mRNA, we demonstrated a slower progression of photoreceptor
degeneration and improved ERG measurements 30 days after
treatment. Eyes injected with P23H ASO had a 128 ± 22% improved
amplitude response (scotopic a-wave) as compared to the PBS,
while the control ASO, had a 37% ± 9 difference. In addition,
morphometric analysis of the outer nuclear layer showed an 18
± 8% or 5.7 ± 7% greater thickness following treatment with
P23H ASO or Control ASO, respectively. Eyes injected with the
P23H ASO had greater rat rhodopsin expression, 176 ± 17% as
compared to contralateral eye, consistent with the preservation of
the photoreceptor cells. In the control ASO group, the rat rhodopsin
expression showed very little difference from PBS eye (-2.6± 6%).
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Conclusions: Allele-specific ASO mediated knockdown of mutant
rhodopsin expression slowed the rate of photoreceptor degeneration
and preserved the function of the photoreceptor cells of the P23H
transgenic rat. Our data indicate that ASO treatment is a potentially
effective therapy for autosomal dominant retinitis pigmentosa.
Commercial Relationships: Sue F. Murray, ISIS Pharmaceuticals
(E); Ali Jazayeri, ISIS Pharmaceuticals (E); Matthew M. LaVail,
None; Michael T. Matthes, None; Douglas Yasumura, None;
Haidong Yang, None; Michael McCaleb, ISIS Pharmaceuticals
(E); Raechel Peralta, ISIS Pharmaceuticals (E); Andy Watt, ISIS
Pharmaceuticals (E); Brett Monia, ISIS Pharmaceuticals (E)
Program Number: 1259
Presentation Time: 9:45 AM–10:00 AM
Inhibition of Protein Kinase G Activity Improves Cone Survival
in Cyclic Nucleotide-gated Channel Deficiency
Hongwei Ma1, Arjun Thapa1, Lynsie Morris1, Mirja Koch2, Martin
Biel2, Stylianos Michalakis2, Xi-Qin Ding1. 1The Department of
Cell Biology, Univ of Oklahoma Health Sci Ctr, Oklahoma City,
OK; 2The Center for Integrated Protein Science Munich (CIPSM)
and Department of Pharmacy - Center for Drug Research, LudwigMaximilians-Universität München, Munich, Germany.
Purpose: Cone phototransduction mediated by cyclic nucleotidegated (CNG) channels is essential for central and color vision and
for visual acuity. Naturally occurring mutations in the cone CNG
channel subunit CNGA3 are associated with achromatopsia and cone
dystrophy. We have shown that cyclic guanosine monophosphate
(cGMP) is remarkably elevated in CNG channel-deficient retina. We
also observed an increased activity and expression of the cGMPdependent protein kinase G (PKG) in CNG channel deficiency. This
work examined whether the elevated PKG function plays a role in
cone cell death.
Methods: Wild type (C57BL/6), and three CNG channel-deficient
mouse lines, Cnga3-/-, Cnga3-/-/Nrl-/- (Cnga3 deficiency on a conedominant background), and Cnga3-/-/Pkg1-/- (deficiency of Cnga3
and Pkg1, which encodes mouse PKG1), were used in this study.
Inhibition of PKG activity was achieved by treating mice with
the PKG inhibitors KT5823 and Rp-8-Br-cGMPS. Retinal cGMP
level and PKG activity were analyzed by ELISA. Cone death was
evaluated by TUNEL on retinal cross sections, and cone density
and expression levels of cone specific proteins were evaluated by
immunohistochemical labeling and western blotting.
Results: Treatment with PKG inhibitors effectively reduced PKG
activity to the control levels. The TUNEL positive cells were
significantly reduced in Cnga3-/-/Nrl-/- mice treated with PKG
inhibitors. Cone density and expression levels of M-opsin and
S-opsin were greatly increased in PKG inhibitor-treated mice,
compared with vehicle-treated controls. In addition, the expression
levels of the endoplasmic reticulum stress marker p-eIF2α and
cleavage of the apoptotic protein caspase-7 were significantly
reduced in Cnga3-/-/Nrl-/- mice treated with PKG inhibitors. Moreover,
cone density was significantly increased in Cnga3-/-/Pkg1-/- mice
compared with age-matched Cnga3-/- controls.
Conclusions: This work demonstrates that PKG plays a role in cone
death. Thus, suppressing PKG activity may represent a new approach
to reduce cone cell death associated with cGMP accumulation,
including cone death caused by CNG channel deficiency and by
photoreceptor phosphodiesterase deficiency.
Commercial Relationships: Hongwei Ma, None; Arjun Thapa,
None; Lynsie Morris, None; Mirja Koch, None; Martin Biel,
None; Stylianos Michalakis, None; Xi-Qin Ding, None
Support: This work was supported by the NIH grants P20RR017703,
P30EY12190, R01EY019490, a research grant from the Oklahoma
Center for the Advancement of Science & Technology (OCAST), and
the Deutsche Forschungsgemeinschaft (DFG).
Program Number: 1260
Presentation Time: 10:00 AM–10:15 AM
Calcium dynamics in dying cone photoreceptors
Manoj M. Kulkarni1, 2, Robin Kemmler1, 3, Bernd Wissinger2, Thomas
Euler1, 2, Francois Paquet-Durand2. 1University of Tuebingen, Centre
for Integrative Neuroscience, Tuebingen, Germany; 2University of
Tuebingen, Institute for Ophthalmic Research, Tuebingen, Germany;
3
University of Tuebingen, Graduate School of Cellular & Molecular
Neuroscience, Tuebingen, Germany.
Purpose: Cone photoreceptors are the main source of human sight,
enabling color and high-resolution daylight vision. In inherited
retinal diseases, loss of cones may occur primarily or secondarily,
depending on whether the initial genetic defect affects cone or
rod photoreceptors. Here, we used cpfl1 mice, carrying a mutation
in the cone specific Pde6c gene, and rd1 mice with a mutation in
the rod specific Pde6b gene, as models for primary and secondary
cone degeneration, respectively. Since, it has been speculated that
both primary and secondary cone degeneration may be caused by a
cytotoxic overload of Ca2+, we set out to study cone Ca2+ dynamics
using 2-photon imaging and transgenic mice which express the
HR2.1:TN-XL Ca2+ biosensor in cones (Wei et al., J Neurosci.
32:6981-94; 2012).
Methods: Ca2+ imaging was performed on wild-type/TN-XL, as
well as on cpfl1/TN-XL and rd1/TN-XL crossbred animals. Different
staining techniques (TUNEL assay, cGMP immunofluorescence,
calpain activity assay) were used to further characterize cone
degeneration.
Results: Abnormal cGMP increases in cones, a presumed telltale
for Ca2+ accumulation, occurred only during primary, but not during
secondary cone degeneration. At post-natal day 30, genetically intact
cones in rd1/TN-XL retina showed no Ca2+ response when stimulated
by light, while a substantial number of mutant cpfl1/TN-XL cones
still displayed light responses. Accordingly, significant difference
in relative Ca2+ levels distribution was detected between cpfl1/TNXL and rd1/TN-XL cones. Currently we perform calibrated Ca2+
measurements to evaluate absolute cone Ca2+ levels in the three
mouse lines.
Conclusions: The HR2.1: TN-XL Ca2+ biosensor mouse line allows
studying cone Ca2+ regulation, not only with pharmacological
manipulations but also in genetic mutants. The observed lightevoked responses in cpfl1/TN-XL suggest residual cone PDE6
activity. Our preliminary data suggest dysfunctional Ca2+ regulation
during primary (cpfl1) but not in secondary (rd1) cone degeneration.
Therefore, modulation of Ca2+ signaling may be a suitable target for
neuroprotection therapy in primary cone degeneration.
Commercial Relationships: Manoj M. Kulkarni, None; Robin
Kemmler, None; Bernd Wissinger, None; Thomas Euler, None;
Francois Paquet-Durand, None
Support: EU (DRUGSFORD-HEALTH F2 2012-304963), NeuroOphthalmologische Gesellschaft: Mehr Forschen – Besser Sehen e.V.
(Tübingen), fortüne program (2015-0-0), DFG (CIN EXC 307)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
218 Stem Cell I
Monday, May 05, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 1358–1388/A0114–A0144
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 1358 Poster Board Number: A0114
Presentation Time: 8:30 AM–10:15 AM
The formation of primitive ocular structures and stratified neural
retina from human pluripotent stem cells
Carla B. Mellough1, Joseph F. Collin1, Mahmoud Khazim1, 2, Evelyne
Sernagor2, Nicholas Wride3, David Steel3, Majlinda Lako1. 1Institute
of Genetic Medicine and North East Stem Cell Institute (NESCI),
Newcastle University, Newcastle upon Tyne, United Kingdom;
2
Institute of Neuroscience, Newcastle University, Newcastle upon
Tyne, United Kingdom; 3Sunderland Eye Infirmary, Sunderland,
United Kingdom.
Purpose: Diseases of the outer retina are a leading cause of
irreversible blindness worldwide, yet no treatments currently exist
for many forms. The ability to generate retinal tissue for disease
modelling, drug screening or transplantation would be extremely
useful in order to resolve this important challenge. Human embryonic
stem cells (hESC) and induced pluripotent stem cells (hiPSC) can
be differentiated in vitro to develop towards retinal cells including
photoreceptors and retinal pigmented epithelium (RPE). Their ability
to self-organise into complex retinal tissue, similar to embryonic
human retina, has recently been demonstrated and represents a leap
towards in vitro modelling of disease phenotypes and the study of
human retinal ontogenesis. The reproducibility and efficacy of this
capability is however not well established across multiple hESC/
hiPSC lines. We have built upon our previous work to devise a simple
and reliable method that produces laminated retinal tissue in vitro
using one factor only.
Methods: hESC/hiPSC lines were expanded on mouse embryonic
fibroblasts then differentiated under 3-dimensional conditions
for 90 days in a ventral neural induction medium either alone or
supplemented with human insulin-like growth factor (IGF).
Results: The addition of IGF during differentiation orchestrates
the formation of ocular-like structures from hESC/hiPSC with high
frequency compared to controls. IGF-generated structures contain
not only RPE and neural retina, but other eye elements including
primitive lens and corneal epithelium. Retinal organisation is
reminiscent of developing human retina and comprises multiple
phenotypes including photoreceptors, amacrine and ganglion cells,
which establish synaptic connections and form visible plexiform
layers. Photoreceptors exhibit primitive rod- and cone-like inner
and outer segments and functional cyclic nucleotide gated channel
properties. Inhibition of IGF pathway signalling abolished the ability
of hESC/hiPSC to give rise to viable laminated retina, establishing an
important role for this pathway in retinal histogenesis.
Conclusions: The efficient derivation of ocular derivatives including
laminated retinal tissue can be achieved from hESC and hiPSC using
IGF, offering exciting new opportunities to study retinal development
and disease and to provide an expandable source of transplantable
derivatives for cell replacement studies.
Commercial Relationships: Carla B. Mellough, None; Joseph F.
Collin, None; Mahmoud Khazim, None; Evelyne Sernagor, None;
Nicholas Wride, None; David Steel, None; Majlinda Lako, None
Support: Fight for Sight (FFS) and Retinitis Pigmentosa Fighting
Blindness (RPFB).
Program Number: 1359 Poster Board Number: A0115
Presentation Time: 8:30 AM–10:15 AM
Identification and characterization of neural crest stem cells in
rat olfactory mucosa as a use for cell-mediated neuroprotection
as treatment of glaucoma
Maayke Kuijten1, 2, Claire Ginn2, Peng Khaw1, G Astrid Limb1,
Steve Brocchini1, 2. 1National Institute for Health Research (NIHR)
Biomedical Research Centre at Moorfields Eye Hospital NHS
Foundation Trust and UCL Institute of Ophthalmology, London,
United Kingdom; 2UCL School of Pharmacy, London, United
Kingdom.
Purpose: The olfactory mucosa (OM) is an area of constant neural
regeneration populated by olfactory ensheathing cells (OEC),
which may constitute a source of neurotrophic support for spinal
cord damage and glaucoma treatment. However, the heterogeneous
nature of these cells makes their isolation difficult for therapeutic
use. We examined the OM to identify neural crest stem cell (NCSC)
populations, from which OECs originate and which may constitute an
alternative source for neuroprotection in glaucoma.
Methods: We performed immunohistochemistry and confocal
microscopy to characterise the rat OM. Different cell sorting
strategies such as flow cytometry and differential adhesion were used
to separate different cell populations. Analyses were performed using
Western blotting and RT-PCR techniques.
Results: Confocal analysis of postnatal day 16 OM tissue showed
the presence of three different cell populations in the lamina propria
(LP). Positive staining for mesenchymal stem cell (MSC) markers
in this tissue indicates the presence of mesenchymal-like stem cells
and their location seems to indicate a MSC niche in the LP of OM
tissue. Expression of neural stem cell markers including Sox2, Pax6
and nestin was found in the LP, which could indicate the presence
of different stem cell populations. The NCSC markers Sox10, snail
and HNK-1 were also found, indicating the presence of a NCSC
population in the LP. Differential adhesion has been used as a means
of selecting different cell populations. In these cell populations,
expression of the NCSC genes p75, Sox10, HNK-1 and snail was
also seen. Expression of the above markers was further confirmed by
examination of mRNA and protein lysates of whole OM. In addition,
mRNA samples of the obtained cell population and OM tissue have
been investigated for neurotrophin expression, such as BDNF, CNTF,
GDNF, NGF, NT3 and NT4/5, which are all expressed.
Conclusions: The OM is a complex tissue that contains at least one
or possibly more stem cell populations residing in the LP. Based
on the gene expression profile, Western blotting of tissue and cell
lysates and staining of tissue and cell populations, neural crest stem
cells were shown to be present in the OM. In addition, the obtained
cell populations and whole OM express several neurotrophin genes,
suggesting their potential neuroprotective ability.
Commercial Relationships: Maayke Kuijten, None; Claire Ginn,
None; Peng Khaw, University College Moorfields (P); G Astrid
Limb, None; Steve Brocchini, None
Support: UCL School of Pharmacy, NIHR Moorfields Biomedical
Research Centre, Helen Hamlyn Trust, John Nolan, Michael and Ilse
Katz Foundation, Fight for sight
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1360 Poster Board Number: A0116
Presentation Time: 8:30 AM–10:15 AM
Biological and synthetic membranes for human induced
pluripotent stem cell-based transplantation therapy
Joe Phillips1, 2, Eric Clark1, Enio T. Perez1, Samantha T. Reshel1,
Patrick M. Barney1, Luke Beardslee3, Dyson Hickingbotham4,
Norman D. Radtke4, Magnus Bergkvist3, David M. Gamm2,
5 1
. Waisman Center, University of Wisconsin, Madison, WI;
2
McPherson Eye Research Institute, University of WisconsinMadison, Madison, WI; 3College of Nanoscale Science and
Engineering, University at Albany-SUNY, Albany, NY; 4University
of Louisville, Louisville, KY; 5Department of Ophthalmology and
Visual Sciences, University of Wisconsin-Madison, Madison, WI.
Purpose: Scaffolds may improve transplantation therapy by
providing an organized platform for cell delivery. In this study,
we evaluated two membranes for human induced pluripotent stem
cell (hiPSC)-based transplantation therapy, an acellular biological
membrane derived from porcine small intestine submucosa (SIS), as
well as a synthetic micro-patterned membrane (SU-8).
Methods: SIS membrane (Cook Biotech), a porous extracellular
matrix sheet roughly 15mm thick, was cultured using Snapwell
transwell inserts (Corning). SU-8 membranes were generated with
standard photolithography techniques, resulting in free-standing
porous microstructures that were 8mm thick with 5mm pores. Both
membranes were pretreated with laminin, and then coated with
hiPSC-derived RPE or hiPSC-derived neural retina (NR) cells.
Cellular gene and protein expression was monitored with RT-PCR
and immunocytochemistry. Cell growth, viability, and polarity were
analyzed by standard light and confocal microscopy. Subretinal
insertion of cell-impregnated membranes (1mm in diameter) into the
Long Evans rat retina was performed using a patented transplantation
device (Eyevation). Human cells within the rat retina were identified
with a human cytoplasm specific antibody (Stem Cell Inc.).
Results: Both membranes promoted cell adhesion and growth in
vitro. hiPSC-RPE formed polarized, pigmented monolayers and
expressed mature markers of RPE. hiPSC-NR cells, including
numerous RCVRN+ photoreceptors, grew in layers on both
membranes and expressed characteristic NR markers. Following
transplantation, the SIS membrane was generally well tolerated
by the host rat retina, although Muller glia became activated. Both
hiPSC-NR cells and hiPSC-RPE transplanted on the SIS membrane
survived for up to one month in vivo, the latest time point examined.
The SU-8 membrane has also been successfully transplanted and
analysis is currently underway.
Conclusions: We demonstrate that both synthetic and biological
scaffolds permit cell adherence, growth, and differentiation of
hiPSC-RPE and NR in vitro. Furthermore, both can be transplanted
into the rat retina. These scaffolds provide an organized structure for
hPSC-based cellular transplantation and may also improve in vitro
modeling.
Commercial Relationships: Joe Phillips, None; Eric Clark, None;
Enio T. Perez, None; Samantha T. Reshel, None; Patrick M.
Barney, None; Luke Beardslee, None; Dyson Hickingbotham,
Eyevation (P); Norman D. Radtke, Eyevation (P); Magnus
Bergkvist, None; David M. Gamm, None
Support: Foundation Fighting Blindness Wynn-Gund Translational
Research Award, Retina Research Foundation (Kathryn and Latimer
Murfee and Emmett A. Humble Chairs), McPherson Eye Research
Institute (Sandra Lemke Trout Chair), Carl and Mildred Reeves
Foundation, NIH P30HD03352, Muskingum County Community
Foundation, Choroideremia Research Foundation
Program Number: 1361 Poster Board Number: A0117
Presentation Time: 8:30 AM–10:15 AM
Generation and characterization of retinal ganglion cells from
glaucoma patient iPSCs
Yvonne Ou1, Karen L. Chu1, Erik M. Ullian1, 2. 1Ophthalmology,
University of California, San Francisco, San Francisco, CA;
2
Physiology, University of California, San Francisco, San Francisco,
CA.
Purpose: Glaucoma, the leading cause of irreversible blindness
worldwide, is a degenerative optic neuropathy characterized by
gradual loss of peripheral vision and eventual blindness. The
underlying cause of visual dysfunction is the progressive loss of
retinal ganglion cells (RGCs) and their axons. Recently, retinal
ganglion cell-like cells have been derived from human embryonic
stem cells and human induced pluripotent stem cells (hiPSCs).
However, the proportion of cells in these cultures that are iPSCderived RGCs (iPSC-RGCs) is typically low. Here we describe
a modified protocol based on existing methods to increase the
proportion of iPSC-RGCs derived from glaucoma patients in order to
establish a human glaucoma cell culture model.
Methods: Skin biopsies were obtained from glaucoma patients seen
at the UCSF Department of Ophthalmology’s Glaucoma Service.
Multiple hiPSC lines were established using nonintegrating episomal
reprogramming vectors consisting of OCT 3/4, c-Myc, KLF4, and
SOX2. The glaucoma patient-derived hiPSCs were differentiated into
RGCs in the presence of various BMP and Wnt inhibitors, as well
as IGF-1 and Sonic hedgehog (Shh). The retinal progenitor cells and
differentiated neurons produced were then assayed using RT-PCR and
immunofluorescence microscopy.
Results: Glaucoma patient-derived hiPSC lines were established and
then differentiated into retinal progenitors as assayed by expression
of eye-field transcription factors. The addition of DAPT, a Notch
signaling pathway inhibitor, increased the proportion of cells
expressing neuronal and RGC-specific markers. The inclusion of Shh
increased the proportion of Brn3a-expressing cells in a concentration
dependent manner (25.4% at 100 ng/ml vs. 11.4% at 10 ng/ml;
p<0.05). These glaucoma patient-derived iPSC-RGCs were also
capable of forming anatomic synapses by synaptic marker expression.
Conclusions: We have modified existing protocols to enhance the
differentiation of RGCs from glaucoma patient-derived hiPSCs,
providing an abundant source of cells for the establishment of an in
vitro cell culture model of human glaucoma.
Commercial Relationships: Yvonne Ou, None; Karen L. Chu,
None; Erik M. Ullian, None
Support: Alcon Research Institute Young Investigator grant,
Research to Prevent Blindness Career Development Award, NIH-NEI
Core Grant EY002162
Program Number: 1362 Poster Board Number: A0118
Presentation Time: 8:30 AM–10:15 AM
Spontaneously Differentiated Human Embryonic Stem Cell
derived Retinal Pigment Epithelium Cells express RPE-65 and
ZO-1 proteins
K V Chalam, Sankarathi Balaiya, William Scott, Lee R. Ferguson.
Ophthalmology, Univ of Florida-Jacksonville, Jacksonville, FL.
Purpose: To determine the protein expression profile of the retinal
pigment epithelium-65 (RPE65) and zonular occludins-1 (ZO-1)
markers during spontaneous maturation of precursor cells into human
embryonic stem cell derived RPE (hESC-RPE)
Methods: WA09-DL-11 feeder dependent human embryonic stem
cells (hESC) were seeded onto a confluent layer of Mitomycin
C inactivated mouse embryonic fibroblasts. hESC growth and
differentiation was monitored for the presence of pigmented
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
embryoid body (EB) development. Pigmented EB clumps were
isolated following dissection from hESC colonies. EBs were seeded
onto 6-well gelatin coated plates and allowed to form an expansive
monolayer of hESC-RPE cells. Cells were then passaged 2 – 3 weeks
after plating EB. Following passage cells were re-plated onto four
gelatin coated wells. Each well represented different time points of
extraction: 5, 13, 21, and 28 days post passage. Cells were lysed
and then centrifuged to acquire supernatant proteinaceous material.
Cellular lysate supernatant was then quantified and subjected to
western blot analysis. Antibodies to RPE65 and ZO-1 were used for
the analysis. Following western blot procedure densitometry was
performed to quantify protein expression at various time points.
Results: hESC-RPE post passage day 5 densitometry value was
1782.841 optical density units (odu) for RPE65 and 295.021 odu for
Zo-1. On day 13 there was a 1.4 fold increase for RPE65 (2556.024
odu) and 16.5 fold change in expression for ZO-1 (4856.589 odu).
Protein expression continued to rise for RPE65 and ZO-1, as day 21
levels showed a 1.6-fold (2832.953 odu) and 20.5-fold (6052.439
odu) increase, respectively, when contrasted to day 5 values. Finally
on day 28, expression profiles plateaued as RPE65 levels only
showed a 1.2-fold increase from baseline (2303.79 odu), while ZO-1
demonstrated a 20.4-fold increase from baseline (6019.196 odu).
Conclusions: Precursor marker characterization of hESC-RPE
demonstrates RPE-65 and ZO-1 expression as early as 5 days with
a plateau apparent at day 28. Further investigation into days prior to
day 5 and after day 28 are needed for precise delineation of onset of
expression for these two markers. This study demonstrates the pattern
of RPE65 and ZO-1 expression, which provides further insight into
the proper characterization of hESC-RPE cells as true RPE cells.
Commercial Relationships: K V Chalam, None; Sankarathi
Balaiya, None; William Scott, None; Lee R. Ferguson, None
Program Number: 1363 Poster Board Number: A0119
Presentation Time: 8:30 AM–10:15 AM
Small Molecules and Adult Ciliary Epithelium Cells
Reprogramming
Carolina B. Del Debbio, Dânia E. Hamassaki. Cell and
Developmental Biology, University of Sao Paulo, Sao Paulo, Brazil.
Purpose: The Ciliary Epithelial cells (CE) of adult mammalian eyes
are a quiescent population of cells able to proliferate and generate
neurospheres with retinal progenitor profile. Despite the potential
of CE to generate retinal progenitors, the efficiency is still low,
probably due to the inefficient reprogramming and the presence of
reminiscent epithelial parental properties. It is known that small
molecules have been effective in stem and iPS cells reprogramming
and, for that reason, we evaluate the effect of two small molecules
on CE reprogramming efficiency,the Compound C (CC), inhibitor of
AMP-activated protein kinase and bone morphogenetic protein, and
NSC23766, a Rac1 GTPase inhibitor
Methods: After 4 consecutive intraocular injections of the small
molecules (CC =10ng/eye, NSC = 200ng/eye) in adult Wistar rats,
we dissected the CE, sectioned in cryostat or extracted the RNA
and synthesized the cDNA. Later, the expression of the pluripotent
genes c-Myc, Oct4 and Sox2 was analyzed. For in vitro experiments,
CE cells were dissected and cultured in the presence of growth
factors (FGF and EGF), to form neurospheres, and small molecules.
Proliferation rates and progenitor profile were analyzed
Results: Our data indicated that CC injections increased the
expression of c-Myc and Oct4 (6 and 14 folds change (fc),
respectively), in comparison to controls (PBS), but no effect
was observed in Sox2 expression. Rac1 inhibition increased the
expression of c-Myc and Oct4 (5 and 9 fc), however, Sox2 expression
was decreased (4 fc). Furthermore, Rac1 inhibition increased CE
cells proliferation rates observed through Ki67 immunostaining
(32.8±0.20, control = 5.10±0.87), and quantitative PCR (5 fc).
Methyltransferase transcripts analysis indicated that both molecules
decreased the expression of DNMT1 and DNMT3b (CC = 1.5
and 1.2 folds; NSC = 1.6 and 2 folds), indicating the regulation of
epigenetic factors. Neurospheres treated in the presence of small
molecules showed an apparently increased expression of proliferation
transcripts (ki67 and cyclins), and decreased epithelial properties
genes (palmdelphin and Rab27), in comparison to controls
Conclusions: Our results suggest that the use of small molecules
can be efficient for CE reprogramming, and the information gleaned
from this study may provide valuable insight into the cellular and
molecular events that underlie the reprogramming response of CE
cells and the mechanism of retinal recovery
Commercial Relationships: Carolina B. Del Debbio, None; Dânia
E. Hamassaki, None
Support: FAPESP, CNPq, and Pró-Reitoria de Pesquisa/ USP
(NAPmir and NAPNA)
Program Number: 1364 Poster Board Number: A0120
Presentation Time: 8:30 AM–10:15 AM
Evaluation of Degree of Pigmentation as an Indicator of
Maturation status in Human iPSC-RPE
Hiroyuki Kamao1, 2, Michiko Mandai2, Katsutoshi Goto1, Junichi
Kiryu1, Masayo Takahashi2. 1Ophthalmology, Kawasaki Medical
School, Kurashiki, Japan; 2Laboratory for Retinal Regeneration,
RIKEN Center for Developmental Biology, Kobe, Japan.
Purpose: In transplantation of human induced pluripotent stem cell
derived retinal pigment epithelium (hiPSC-RPE), the determination
of maturation status of these cells is an essential factor and the degree
of pigmentation (dPG) can serve as a good indicator. The aim of
this study was to establish a method of evaluating dPG in a group of
hiPSC-RPE objectively and quantitatively.
Methods: Bright field images of hiPSC-RPE were recorded
sequentially (0w, 2w, 4w, 6w after hiPSC-RPE reached confluence)
under the same condition. Two observers, expert and inexpert,
determined dPG subjectively by observation the recoded images as
follows: a single cell dPG was classified into three different stages
(low, mediate, high) and the overall dPG was compared between
two cell groups to choose the one with higher dPG. The κ coefficient
was applied to assess inter-observer reproducibility. Subsequently,
dPG was objectively determined in single cells and cell groups by
converting the recorded images into those with 256 gray-levels and
the correlation with the subjective evaluation and time dependent
change of dPG was investigated.
Results: The κ coefficient was 0.96 and 0.87 in a single cell and
cell group observation respectively, meaning that inter-observer
reproducibility of dPG was excellent. The objective dPG as
gray-level of single cells highly correlated with the subjectively
determined dPG (low: 16.03, mediate: 71.11, high: 131.55, P <
0.001). However, in comparison between 2 cell groups, the subjective
determination of high dPG cell groups was not a complete match
with objective determination and sometimes the observers could not
distinguish dPG between 2 cell groups (dPG subjective/objective:
high/high 86%, high/low 3% undetermined 11%). The objective dPG
in cell groups increased in a time-dependent manner (0w: 27.21, 2w:
51.85, 4w: 80.25, 6w: 90.03, P < 0.001).
Conclusions: The determination of dPG in cell groups of hiPSCRPE using gray-scale image was reliable and excelled subjective
difficulties in monitoring hiPSC-RPE maturation indicated by dPG in
culture.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Hiroyuki Kamao, Fizer (F); Michiko
Mandai, None; Katsutoshi Goto, None; Junichi Kiryu, Fizer (F);
Masayo Takahashi, None
Program Number: 1365 Poster Board Number: A0121
Presentation Time: 8:30 AM–10:15 AM
Derivation, Characterization and Retinal Neural Induction of
Human Tenon’s -Derived iPS Cells
Jian Ge, Deng Fei, Liu Ying, Xiong Yunfan. Glaucoma, Zhongshan
Ophthalmic Center, Guangzhou, China.
Purpose: To determine if induced pluripotent stem cells (iPSCs)
derived from human Tenon’s capsule fibroblasts (HTFs) could
express retinal progenitor cell (RPC)-related genes with the capacity
to directly differentiate into retinal neurons in vitro.
Methods: HTFs harvested from fresh samples were reprogrammed
by retroviral transduction to iPSCs. The HTF-derived iPSCs (TiPSCs)
were characterized for pluripotency by morphology, gene expression,
surface antigens, alkaline phosphatase activity analysis and a
teratoma formation assay. Human ESC colonies were used as the
positive control. The resulting TiPSCs were induced to differentiate
into retinal cells by stepwise treatment with the defined factor
combination of Dkk1,Noggin,Lefty-A, DAPTand overexpression of
ATOH7, which follows the human retinal development timeline. The
induced retinal cells were analyzed with phase contrast microscopy,
real-time PCR, immunofluorescence, FACS analysis, and calcium
imaging analysis.
Results: The resulting TiPS colonies were indistinguishable from
human ESC colonies according to standard criteria. Upon retinal
differentiation, embryoid bodies (EBs) were formed from TiPSCs
by suspension culture in serum-free medium with the increase of
RPC-related gene expressions such as Pax6, Sox2 and Nestin. In
matrigel-coated culture, acquisition of neuroepithelial colonies at day
10 with an early eye field fate was enhanced through the addition
of DKK1, NOGGIN and Lefty-A, as determined by qPCR and
immunofluorescence. Further overexpression of ATOH7 combined
with DAPT-treated cultures, TiPSCs can generate retinal neural cells
that express Chx10,Brn3b, Atoh7, Syn,Crx,Mitf,GFAP,etc.
Conclusions: These findings demonstrate that iPSCs can be
generated from HTFs and through a stepwise neural differentiation
strategy, the TiPSCs can generate retina-specific cells in vitro, which
should aid in the investigation of ophthalmological regenerative
medicine.
Commercial Relationships: Jian Ge, None; Deng Fei, None; Liu
Ying, None; Xiong Yunfan, None
Support: NSFC Grant 81170846; NSFC Grant 30973266
Program Number: 1366 Poster Board Number: A0122
Presentation Time: 8:30 AM–10:15 AM
Epigenetic and Transcriptional Regulation of RAX in Retinal
Fate Determination using Human Induced Pluripotent Stem Cells
Akshayalakshmi Sridhar1, Sarah Ohlemacher1, Jason S. Meyer1, 2.
1
Biology, Indiana Univ Purdue Univ Indianapolis, Indianapolis, IN;
2
Stark Neurosciences, Indianapolis, IN.
Purpose: Human pluripotent stem cells (hPSCs) allow for the
unprecedented ability to study the earliest events in human retinal fate
determination in vitro. Previous studies have been successful in the
derivation of retinal cell types from hPSCs, however the mechanism
of retinal specification from an unspecified pluripotent population
remains to be clearly defined. Thus, efforts were undertaken to better
elucidate the transcriptional and epigenetic mechanisms underlying
retinal fate specification from hPSCs.
Methods: Following the differentiation of hPSCs to neural and
retinal lineages, expression patterns of candidate transcription factors
were characterized using immunocytochemistry and qRT-PCR. Based
on these studies, the transcription factor RAX (retinal and anterior
neural fold homeobox) was identified as influential in retinal fate due
to its early widespread expression followed by restricted expression
in retinal progenitor cells. To further investigate the role of RAX,
gene overexpression and shRNA mediated knockdown approaches
were undertaken. Furthermore, methylation analyses of CpG islands
in the RAX promoter region were investigated at various stages
of retinal development to better elucidate epigenetic modifications
associated with early retinal fate determination.
Results: Analyses by immunocytochemistry and qRT-PCR
demonstrated that the expression of RAX peaked early and then
became restricted to a subpopulation of cells, specifically retinal
progenitor populations identified by subsequent transcriptional
analysis. Additionally, the expression of RAX was subsequently lost
from non-retinal neural populations. Methylation analysis of the
RAX promoter region suggested that epigenetic mechanisms could
be regulating the expression of RAX in the establishment of a retinal
fate. The ability of RAX to regulate the adoption of a retinal fate was
subsequently tested through lentiviral-mediated overexpression and
shRNA experiments.
Conclusions: Overall, the results of this study help to elucidate the
role of RAX in the establishment of a retinal fate. These studies will
assist in the establishment of more efficient methods to generate
retinal cells from hPSCs for translational purposes, and serve to
further establish hPSCs as an important in vitro model system for
studies of the earliest stages of human retinal development.
Commercial Relationships: Akshayalakshmi Sridhar, None;
Sarah Ohlemacher, None; Jason S. Meyer, None
Support: BrightFocus Foundation Grant No. G2012027, Fight for
Sight
Program Number: 1367 Poster Board Number: A0123
Presentation Time: 8:30 AM–10:15 AM
c-Kit positive cells isolated from human fetus eyes are a new
population of stem cells
Guang-hua Peng1, 2, Pengyi Zhou2, Haiwei Xu1, Zhengqin Yin1.
1
Ophthalmology, Xinan Hospital, Chongqing, China; 2Ophthalmolgy,
Zhengzhou Unervisity, Zhengzhou, China.
Purpose: Despite intensive research on the potential use of retinal
progenitor cells in the treatment of degenerative eye diseases,
the definitive markers for these prognitors are still lacking. Our
current study focused on CD117 (c-kit), a cell surface marker for
haematopoietic stem cells and progenitor cells. Retinal progenitor
cells were isolated from human fetus and the c-kit + and SSEA4retinal cells were sorted out for further biological characterization
Methods: hRPCS were isolated from human retina of 10 to 16 weeks
gestational age (GA). Immunohistochemical staining was performed
using antibody against c-kit + to determine the distribution of c-kit+/
SSEA-4- cell in the eyes of fetus. The c-kit + / SSEA4- retinal
progenitor cells sorted out by flow cytometry were subjected to
cell-mediated immunity fluorescent and cell cycle analysis for their
capablity to proliferation and differentiation.
Results: A few c-kit + / SSEA4- cells were detected in the inner part
of fetal retina. Sorted c-kit + / SSEA4- cells expressed some retinal
stem cell markers including Pax6, Sox2 and Nestin. Over 80% of
the cells expressed ki67, and cell cycle analysis demonstrated that
more than 40% of the cells were in their proliferation phase. These
results clearly demonstrate proliferation property of these cells. When
cultured in differentiation medium, these cells expressed markers
found in photoreceptor cells and glial cells such as CRX, recoverin
and GFAP.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Conclusions: c-kit can be used as a surface marker for retinal
progenitor cells. The c-kit + / SSEA4- retinal progenitor cell isolated
fetal eyes exhibit the ability to self-renew and differentiate into
retinal cells.
Commercial Relationships: Guang-hua Peng, None; Pengyi Zhou,
None; Haiwei Xu, None; Zhengqin Yin, None
Support: National key basic research program of china(Project
No.2013CB967001 to Peng)
Program Number: 1368 Poster Board Number: A0124
Presentation Time: 8:30 AM–10:15 AM
Allogenic iPSC-derived RPE cell transplants induce immune
response in pigs
Elliott H. Sohn1, 2, Chunhua Jiao2, Robert Mullins2, Edwin M. Stone1,
2
, Budd A. Tucker2. 1Retina Service, Univ of Iowa Hosp & Clinics,
Iowa City, IA; 2Stephen A. Wynn Institute for Vision Research,
University of Iowa Dept of Ophthalmology, Iowa City, IA.
Purpose: Stem cell strategies focused on replacement of RPE
cells for the treatment of geographic atrophy are under intense
investigation. Although the eye has long been considered immune
privileged, a limited number of large animal studies focused on the
post-transplant immune response have been performed. The purpose
of this study was to determine if allogenic iPSC-derived RPE cells
delivered to the subretinal space of the pig would survive and fail to
induce an immune response in non-diseased eyes.
Methods: 250,000 iPSC-derived RPE cells, generated from GFPpositive outbred domestic swine, were injected subretinally into
12-week-old vitrectomized Yucatan mini swine (a subset of eyes
received BSS vehicle control only). Eyes were enucleated at 3
weeks post-op, fixed in 4% paraformaldehyde, cryosectioned
and immunostained with antibodies targeted against GFP, ZO1,
macrophages (BA4D5), CD45, GFAP, nestin, Ki67, and PCNA.
Vitreous samples extracted at the time of vitrectomy and again at
post-op week 3 were assayed for cytokine levels using a swine
cytokine Quantibody array kit (RayBiotech, Inc). Data were analyzed
using Student’s t-test and one-way ANOVA followed by Fisher’s
LSD test.
Results: GFP-positive cells expressing the RPE marker ZO-1
were identified in the subretinal space at 3 weeks post-injection.
Accompanying GFP-negative cells positive for CD45 and
macrophage markers were also identified. All cells were negative for
GFAP as well as the cell cycle markers nestin, Ki67, and PCNA. At
post-op week 3, vitreous TGF-beta1 levels were elevated in the iPSCRPE group compared to BSS controls and native vitreous. IL-12
levels were greater in post-op week 3 compared to native vitreous but
not post-op week 3 BSS controls.
Conclusions: Subretinal injection of allogenic iPSC-RPE cells into
wild-type mini-pigs can induce a positive immune response. These
findings suggest that immunologically matched or autogenic donor
cells may be required for clinical RPE cell replacement.
Commercial Relationships: Elliott H. Sohn, None; Chunhua Jiao,
None; Robert Mullins, None; Edwin M. Stone, None; Budd A.
Tucker, None
Program Number: 1369 Poster Board Number: A0125
Presentation Time: 8:30 AM–10:15 AM
Xeno-free 3D retinal differentiation of human inducedpluripotent stem cells
Ramesh Kaini, Anthony J. Johnson, Teresa A. Burke, Dallas Golden,
Heuy-Ching H. Wang. Ocular Trauma, USAISR, Fort Sam Houston,
TX.
Purpose: Advances made in recent years to generate different retinal
precursor cells from pluripotent stem cells have instilled hopes
for cell replacement therapy in retinal degeneration diseases and
retinal trauma. However, stem cells must be derived, maintained
and differentiated in xeno-free condition for potential clinical use
in human. In this study, we sought to differentiate human inducedpluripotent stem (iPS) cells towards neural retinal lineage and
produce clinical-grade retinal progenitor cells.
Methods: Commercially available iPS cells, IMR90-4, were
maintained in VitronectinXF coated culture plates with xenofree TeSR-E8 medium. iPS cells were dissociated and quickly
reaggregated using Sumilon PrimeSurface96V culture plates in
GMEM medium containing 20% KnockOut Serum Replacement
XenoFree . To start the xeno-free 3D differentiation, VitronectinXF
(10 ug/ml) was added in the retinal differentiation medium from
day 2 onwards to day 18. 3D cell aggregates were treated with Wnt
inhibitor, IWR-1-endo, for the first 12 days. KnockOut SR Growth
Factor cocktail was added on day 12 and Smoothened Agonist (SAG)
on day 15. Aggregates were then switched to DMEM/F12-Glutamax
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
medium with N2 supplement on day 18. Expression of different
markers of eye field and retinal progenitor cells were studied at
different time points.
Results: iPS cells maintained in vitronectinXF coated plates with
TeSR-E8 medium retained the expression of pluripotent markers,
including OCT4, Nanog, SSEA-3, and TRA-1-60 after five passages.
Aggregates of xeno-free 3D differentiation started expressing
different neural and eye field markers, including Otx2, Sox2, Rx,
LHX2, Six6, PAX6, MITF, and CHX10 at different time points of
differentiation. Retinal progenitor cell marker, CHX10 was observed
on day 16 onwards.
Conclusions: In this study, we observed that neural retinal lineage
cells can be derived from human iPS cells in a 3D culture system
using defined, xeno-free components. We are in the process of
developing stratified neural retina from iPS cells under defined, xenofree condition and generating different retinal precursor cells for cell
replacement therapy.
Commercial Relationships: Ramesh Kaini, None; Anthony J.
Johnson, None; Teresa A. Burke, None; Dallas Golden, None;
Heuy-Ching H. Wang, None
Support: National Research Council Research Associateship
Program, US Army Clinical and Rehabilitative Medical Research
Program (CRMRP)
Program Number: 1370 Poster Board Number: A0126
Presentation Time: 8:30 AM–10:15 AM
Ex vivo evaluation of intravitreal mesenchymal stem cell viability
using bioluminescence imaging
Marcelo J. Silva, Priscila C. Ferreira, Rubens C. Siqueira, Rodrigo
Jorge, Andre Messias, Maria L. Rodrigues, Rodrigo J. Calado,
Dimas T. Covas, Jayter S. Paula. Opthalmology, FMRP University of
São Paulo, Ribeirão Preto, Brazil.
Purpose: Bone marrow-derived mesenchymal stem cells (MSC)
therapy is a promising treatment for several degenerative diseases,
including retinopathies and glaucoma, however no reproducible
method of monitoring these cells into the eye has been established.
The aim of this study was to describe a successful bioluminescence
imaging (BLI) to detect viable luciferase-expressing MSC into the
eye.
Methods: Human donors’ MSC in culture were infected with 50ul
of luciferase lentiviral vector (3,000 viral particles/cell) prior to
intraocular injections. Three eyes of two rabbits were evaluated
through BLI after receiving 1x106 luciferase-expressing MSC
intravitreally with (E1) or without (E2) D-luciferin (40mg/ml - 300
mL of PBS), via pars plana. D-luciferin (40mg/ml – 300 mL of PBS)
without cells was injected in a third eye at beginning (E3) and after
one hour in E2.
Results: E1 showed high BLI radiance report and decay in eight
hours. After D-luciferin infusion, E2 also displayed high average
radiance, with similar decay rate of E1. No signal was observed
in E3. Figure 1 show the bioluminescence imaging acquired from
the three experimental eyes and the figure 2 show the distribution
of total amount of captured photons from the three eyes, using
bioluminescence, during the eight hours period.
Conclusions: Identification of cell location and viability is still an
important problem regarding the use of MSC for eye diseases. This is
maybe the first ex vivo study demonstrating BLI is useful and reliable
method to address these issues.
Commercial Relationships: Marcelo J. Silva, None; Priscila C.
Ferreira, None; Rubens C. Siqueira, None; Rodrigo Jorge, None;
Andre Messias, None; Maria L. Rodrigues, None; Rodrigo J.
Calado, None; Dimas T. Covas, None; Jayter S. Paula, None
Program Number: 1371 Poster Board Number: A0127
Presentation Time: 8:30 AM–10:15 AM
Upregulation of the Notch and Wnt signalling pathways by HB-EGF
in adult human Müller stem cells in vitro
Angshumonik Angbohang, Karen Eastlake, Silke Becker, Na Wu,
G Astrid Limb. National Institute for Health Research (NIHR)
Biomedical Research Centre at Moorfields Eye Hospital NHS
Foundation Trust and UCL Institute of Ophthalmology, UCL,
London, United Kingdom.
Purpose: Müller glial stem cells (MCSs) spontaneously regenerate
the retina of adult zebrafish after injury. MSCs can be isolated from
adult human eye and induced to proliferate in vitro by HB-EGF
stimulation. HB-EGF has been shown to initiate MSC derived retinal
regeneration by activating the Notch and Wnt signalling pathways
in the zebrafish. Since Notch inhibition in human MSC promotes
their differentiation into retinal ganglion cells (RGCs) in vitro, and
activation of the Wnt signalling pathway can promote MSC growth,
it would be important to investigate whether HB-EGF activation of
these cells may trigger interactive signals between the Notch and
Wnt signalling pathways that can control adult stem cell growth and
differentiation in vitro
Methods: Müller stem cells were cultured on matrigel coated flasks
in DMEM media containing 2% foetal calf serum in the presence
or absence of HB-EGF and γ-secretase inhibitor RO4929097.
mRNA and protein isolated from these cells were examined for
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
expression of molecules of the Notch and Wnt signalling pathways
and RGC marker Brn3b using RT-PCR and Western-blot analysis.
Morphological changes were examined by phase contrast microscopy.
Results: Culture of Müller stem cells with exogenous HB-EGF,
caused an increase in gene expression of the Notch downstream
target Hes1 and the canonical Wnt signalling components Wnt2b
and β-catenin. We found that the γ-secretase inhibitor RO4929097
induced down-regulation in HB-EGF gene expression, as well as
in gene and protein expression of the Notch downstream target
Hes1. MSCs cultured with RO4929097 acquired a neuronal-like
morphology, displaying a very thin elongated cytoplasm with bright
phase cell body and the formation of multiple neurites. It also
induced up-regulation of Brn3b gene expression, a marker of RGC.
The Wnt signalling ligand Wnt2b gene and protein expression were
also down-regulated in these cells by this inhibitor.
Conclusions: The results showed that HB-EGF causes activation
of both the Notch and Wnt signalling pathways in adult human
Müller stem cells. Since this appears to be an evolutionary conserved
interaction observed during the spontaneous retinal regeneration
observed in zebrafish, it would be important to investigate whether
activation of these pathways is suppressed in the adult human eye
and whether they can be modulated to induce endogenous retina
regeneration in the adult human eye.
Commercial Relationships: Angshumonik Angbohang, None;
Karen Eastlake, None; Silke Becker, None; Na Wu, None; G
Astrid Limb, None
Support: UCL Biomedicine Grand Challenge Studentship
Program Number: 1372 Poster Board Number: A0128
Presentation Time: 8:30 AM–10:15 AM
microRNAs regulate periodontal ligament-derived stem cell
retinal differentiation
Tsz Kin Ng1, 2, Kwong Wai Choy3, Hoi Kin Wong3, Chi Pui Pang1,
Herman S. Cheung2, 4. 1Department of Ophthalmology & Visual
Sciences, The Chinese University of Hong Kong, Kowloon, Hong
Kong; 2Geriatric Research, Education and Clinical Center, Miami
Veterans Affairs Medical Center, Miami, FL; 3Department of
Obstetrics & Gynaecology, The Chinese University of Hong Kong,
Shatin, Hong Kong; 4Department of Biomedical Engineering,
University of Miami, Miami, FL.
Purpose: To determine the microRNA (miRNA) signature of human
adult periodontal ligament stem cells (PDLSC) retinal differentiation.
Methods: Human adult PDLSC were induced to the retinal lineage
using the noggin-Dkk1-IGF-1 approach. The miRNA expression
was analyzed by the miRNA microarray technique and validated by
TaqMan assay. The predicted miRNA target genes were analyzed by
gene ontology.
Results: A total of 71 human miRNAs were differentially expressed
before and after retinal induction, which 44 of them were upregulated
and 27 were downregulated. Of the 5 selected miRNAs, 4 miRNAs
(hsa-miR-132, hsa-miR-29b, hsa-miR-630 and hsa-miR-7) were
validated. Gene ontology analysis of the predicted miRNA target
genes confirmed the induction treatment closely related to neuronal
differentiation processes.
Conclusions: The expression changes of miRNAs and their
target genes during retinal induction process revealed the genetic
and epigenetic regulatory mechanism for human adult stem cell
differentiation.
Commercial Relationships: Tsz Kin Ng, None; Kwong Wai Choy,
None; Hoi Kin Wong, None; Chi Pui Pang, None; Herman S.
Cheung, None
Support: The VA Merit Review Grant and the Senior VA Research
Career Scientist Award, Miami, and a block grant of the University
Grants Committee Hong Kong, Hong Kong
Program Number: 1373 Poster Board Number: A0129
Presentation Time: 8:30 AM–10:15 AM
Modulation of the Notch and Wnt signalling by TGF- β in adult
human Müller stem cells.
NA WU1, 2, Joseph Wiseman1, Yuan Lei2, Karen Eastlake1, Xinghuai
Sun2, G Astrid Limb1. 1National Institute for Health Research
(NIHR) Biomedical Research Centre at Moorfields Eye Hospital
NHS Foundation Trust and UCL Institute of Ophthalmology, UCL,
London, United Kingdom; 2Department of Ophthalmology and Visual
Science, Eye & ENT Hospital, Shanghai Medical College, Fudan
University, Fudan University, Shanghai, China.
Purpose: The transforming growth factor β (TGF- β) and the Wnt,
Notch signalling pathways all play crucial roles in many biological
processes, including embryonic development, cell fate differentiation
and cell proliferation. There is evidence for a crosstalk between the
Notch and Wnt pathways during retinal development, but it is not
known whether co-activation of these pathways may occur in adult
human Müller stem cells (hMSCs) or whether these can be modulated
by TGF- β.It was therefore the aim of this study to investigate the
effect of this cytokine on the expression of molecules involved in
these two pathways
Methods: Human Müller stem cells were cultured on matrigel using
DMEM containing foetal calf serum (FCS). hMSCs were cultured in
the absence or presence of the γ-secretase inhibitor DAPT plus bFGF
to induce their differentiation into RGCs. Exogenous TGF- β1 or
TGF- β3 were added to the differentiating or control cells which were
cultured for up to 7 days. mRNA and protein extracted from these
cells were examined by RT-PCR, western-blot and immuno-staining
techniques for the expression of molecules of the Notch and Wnt
signalling pathways. The hexosaminidase assay and Ki67 immunostaining were used to test the effect of TGF- β on cell proliferation.
Results: Notch inhibition by DAPT caused down-regulation in gene
expression of Hes1 and Wnt 2b. Similarly, TGF- β1 alone caused a
decrease in gene and protein expression of the Notch downstream
target Hes1 and the Wnt signalling ligand Wnt2b, while inducing
an increase of the canonical Wnt signalling intracellular component
β-catenin. Down-regulation of these genes by DAPT was not
modified by addition of TGF- β1 or TGF- β3 to the cultured cells.
It was also observed that both TGF- β1 and TGF- β3 had a similar
effect on the inhibition of cell growth.
Conclusions: These results provide evidence that TGF- β directly
induces down-regulation of the Notch target Hes1 and the Wnt
signalling ligand Wnt2b, as well as upregulation of β-catenin.
Furthermore, down-regulation of Notch by DAPT results in downregulation of Wnt2b, suggesting a crosstalk between these two
signalling pathways. However, TGF- β does not have additive
effects on the down-regulation of Hes1 and Wnt2b by DAPT. The
observations suggest that these two molecules may activate similar
intracellular pathways mediating neural differentiation of Müller stem
cells.
Commercial Relationships: NA WU, None; Joseph Wiseman,
None; Yuan Lei, None; Karen Eastlake, None; Xinghuai Sun,
None; G Astrid Limb, None
Support: MRC & National Natural Science Foundation of China
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1374 Poster Board Number: A0130
Presentation Time: 8:30 AM–10:15 AM
Comparison of viral and mRNA-reprogrammed human induced
pluripotent stem cells for retinal differentiation
Jason S. Meyer1, 2, Akshayalakshmi Sridhar1, Clara Iglesias1, Sarah
Ohlemacher1. 1Biology, Indiana Univ- Purdue Univ Indianapolis,
Indianapolis, IN; 2Stark Neuroscience Research Institute, Indiana
University, Indianapolis, IN.
Purpose: The derivation of human induced pluripotent stem
cells (hiPSCs) from patient-specific sources has allowed for the
development of novel approaches to studies of human development
and disease. However, traditional methods of generating hiPSCs
involve the risk of genomic integration or constitutive transgene
expression due to viral delivery. The delivery of reprogramming
factors by mRNA eliminates these risks and may provide a safer
alternative, but the efficient retinal differentiation of such cells has
yet to be demonstrated. Thus, efforts were undertaken to test the
ability and efficiency of mRNA-reprogrammed hiPSCs for retinal
differentiation.
Methods: Human fibroblasts were transfected daily with mRNAs
encoding for reprogramming genes, whereas similar cultures of
human fibroblasts were infected with retroviruses to deliver these
genes in parallel. Pluripotency was confirmed by live cell staining
for cell surface antigens and positive colonies were identified and
manually isolated, yielding stable lines of hiPSCs. New lines of
hiPSCs were differentiated to a retinal fate following established
protocols, and the efficiency of retinal specification from hiPSCs
was compared between the two systems at various stages of
differentiation.
Results: Both mRNA and retroviral methods of reprogramming
yielded stable lines of hiPSCs expressing numerous pluripotencyrelated characteristics as assessed by immunocytochemistry and
RT-PCR. Upon differentiation, no overt differences were found in
the ability of these cells to adopt a retinal fate as assessed by the
expression of retinal progenitor-associated genes. Within two months
of differentiation, many types of retinal cells could be derived from
both sources, including retinal pigment epithelium, photoreceptors,
and retinal ganglion cells.
Conclusions: The data presented demonstrates the feasibility of
utilizing mRNA-based reprogramming strategies to derive lines
of patient-specific hiPSCs for purposes of retinal differentiation.
Differences in the differentiation capacity of these cells from both
sources were not readily observed. Given that hiPSCs derived
through mRNA-based reprogramming strategies offer numerous
advantages because they lack the risks of genomic integration or
constitutive expression, such methods likely represent a promising
new approach for retinal stem cell research, particularly those for
translational purposes.
Commercial Relationships: Jason S. Meyer, None;
Akshayalakshmi Sridhar, None; Clara Iglesias, None; Sarah
Ohlemacher, None
Support: BrightFocus Foundation Grant# G2012027, Fight for Sight
Program Number: 1375 Poster Board Number: A0131
Presentation Time: 8:30 AM–10:15 AM
Differentiation and Characterization of Retinal Ganglion Cells
Derived from Human Pluripotent Stem Cells
Sarah Ohlemacher1, Jason S. Meyer1, 2. 1Biology, IUPUI,
Indianapolis, IN; 2Stark Neuroscience Research Institute, Indiana
University, Indianapolis, IN.
Purpose: Human pluripotent stem cells (hPSCs) possess the unique
ability to readily differentiate into any cell type of the body. As such,
they can serve as comprehensive and novel tools for drug screening,
disease modeling, and cell replacement therapies. Although previous
studies have demonstrated the ability to differentiate hPSCs to a
retinal lineage, the ability to derive retinal ganglion cells (RGCs)
from hPSCs has been largely overlooked to date. Establishing a
method to acquire RGCs from hPSCs would serve as a novel system
to study human retinogenesis as well as establishing a foundation for
the development of patient specific therapies for diseases of retinal
ganglion cells, such as glaucoma.
Methods: Following previously established protocols, hPSCs
were induced to differentiate towards a retinal fate and RGCs were
subsequently characterized by the RGC-associated transcription
factors BRN3 and Islet-1. The developmental process underlying this
RGC differentiation was further analyzed by immunocytochemistry
and RT-PCR analysis for the expression of expected RGC associated
characteristics.
Results: Within the first 40 days of differentiation, RGCs were
readily identifiable within differentiating cultures of hiPSCs based
on their progression through a CHX10-positive retinal progenitor
intermediary as well as their later adoption of the RGC-specific
transcription factor BRN3. Analysis of these differentiating cultures
at various timepoints by immunocytochemistry and RT-PCR analysis
revealed that these cells expressed numerous characteristics of
RGCs, including the expression of MATH5 and PAX6, as well as
morphological characteristics associated with RGCs. In addition,
treatment with intrinsic and/or extrinsic factors were tested for their
ability to modulate RGC specification.
Conclusions: The data presented within this study demonstrates
the ability of hiPSCs to serve as a reliable source of patientderived RGCs, as seen by their ability to proceed through predicted
developmental stages that yield a mature RGC population
possessing many RGC-associated characteristics. This protocol will
be advantageous for future studies into the normal and abnormal
development of RGCs and as such, will be instrumental as a tool to
study optic neuropathies that affect this cell population.
Commercial Relationships: Sarah Ohlemacher, None; Jason S.
Meyer, WARF (P)
Support: BrightFocus Foundation Grant #G2012027, Fight for Sight
Program Number: 1376 Poster Board Number: A0132
Presentation Time: 8:30 AM–10:15 AM
Age-related Gene Expression Changes in the Murine Optic Nerve
Lamina
Yan Guo, Zara Meharabyan, Steven L. Bernstein. Ophthalmology,
Univ of Maryland Sch of Medicine, Baltimore, MD.
Purpose: The optic nerve lamina (ONL) is a unique optic nerve
structure that borders the retina and optic nerve. The ONL is believed
to play an important role in many optic nerve diseases, including
nonarteritic anterior ischemic optic neuropathy (NAION) and open
angle glaucoma (OAG). We recently demonstrated that some cells
in the ONL possess the ability to develop into a number of different
cell types, suggesting pluripotency. We utilized a panel of genespecific primers to examine both the pluripotency and types of cell
differentiation in this region, and evaluated the relative changes in
gene expression in different age group.
Methods: 10 wild type mice (C57BL/6J) each at 15 day, 6 months
and >1year old were utilized for the analysis. The first two mm of
the optic nerve head was utilized for the ONL, as well as tissue from
the retina and posterior ON (posterior 3mm of optic nerve). mRNA
was extracted using Qiagen RNaeasy Micro kit, and unbiased linear
RNA amplification using a single chimeric primer and isothermal
amplification (Nugen) was performed, to enable sufficient material
for multiple assays. Following cDNA generation, we performed
quantitative PCR (qPCR). A variety of gene markers were used
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
including Nestin, Sox2, and Sox1 for progenitor cells, as well as a
number of genes for each glial cell type and glial progenitor line.
Reactions were performed using SYBR green Super mix on an
iCycler.
Results: qPCR revealed differential gene expression between the
lamina and optic nerve, as well as between the different age groups.
Gli-1, Nestin and SOX-2 gene expression were considerably elevated
in the lamina and ON, compared with the retina. In contrast, the
highest levels of Olig-2 and MBP gene products, corresponding to
mature oligodendrocytes, were found in the posterior ON region.
High levels of GFAP and s100β expression were detected in posterior
ON, less in the lamina, and minimal levels expressed in the retina.
Conclusions: The ONL is a progenitor cell niche, whose capacity for
self-renewal declines during aging. ONL gene expression is distinct
from either the retina or ON. Our results suggest that the ONL lamina
region possesses a multi-progenitor cell population that may give rise
to the different glial cell lines, as well as possess some capacity for
neuronal generation.
Commercial Relationships: Yan Guo, None; Zara Meharabyan,
None; Steven L. Bernstein, None
Support: EY-015304
Program Number: 1377 Poster Board Number: A0133
Presentation Time: 8:30 AM–10:15 AM
Is Mesenchymal Stem Cell Homing To The Injured Retina
Required For Visual Preservation In The RCS Rat?
Benjamin Bakondi, YuChun Tsai, Bin Lu, Sergey Girman, Lin Shen,
Melissa K. Jones, Shaomei Wang. Regenerative Medicine Institute,
Cedars-Sinai, Los Angeles, CA.
Purpose: Retinal degeneration is slowed by intravenous (IV)
injection of allogeneic bone marrow-derived mesenchymal stem
cells (MSCs) prior to the onset of degeneration in the RCS rat.
However, the mechanism(s) for MSC-mediated retinal protection
has yet been determined. It is unclear whether MSC homing to the
retina is required for vision rescue and is the subject of the current
investigation.
Methods: The predominant mechanism for MSC migration to tissue
injury is mediated through the CXCR4 chemokine receptor, which
was stimulated (SDF-1; 50ng/ml) or antagonized (AMD3100; 10uM)
for 30 minutes prior to infusion of allogeneic MSCs (1.6 x 106 cells)
into RCS rats on postnatal day 25. Optokinetic responses (OKR)
and electroretinograms (ERG) were performed on postnatal day 60.
For cell tracking experiments, MSCs were labeled with a lipophilic
fluorescent dye (PKH26 or PKH67; Sigma) prior to injection and cell
distribution was determined 24 and 72 hours post-injection via flow
cytometry and histological examination.
Results: SDF-1 pre-treatment enhanced the migration capacity of
MSCs in vitro, while AMD3100 pre-treatment reduced it, compared
with control (saline) treatment. OKR and ERG recordings showed
that injection of SDF-1 pre-treated MSCs preserved visual function
in RCS rats compared with injection of saline or AMD3100-treated
MSCs. Fluorescent donor cells were not detected at or near the injury
site at 24 or 72 hours, the typical timeframe for MSC chemotaxis.
MSCs were detected in the blood circulation, lungs, and bone
marrow.
Conclusions: SDF-1 pre-treatment of MSCs enhanced the
preservation of vision in the RCS rat. Further study is under way to
determine whether MSC homing to the retina is required for visual
preservation.
Commercial Relationships: Benjamin Bakondi, None; YuChun
Tsai, None; Bin Lu, None; Sergey Girman, None; Lin Shen, None;
Melissa K. Jones, None; Shaomei Wang, None
Support: NIH R01 EY020488-02, W81XWH-DOD, FFB, Fund
from the Regenerative Medicine Institute at Cedars-Sinai Medical
Center
Program Number: 1378 Poster Board Number: A0134
Presentation Time: 8:30 AM–10:15 AM
In vitro differentiation of human neuronal progenitor cells
towards retinal pigment epithelium-like cells
Volker Enzmann1, 2, Luca Tamó2, 3, Carolyn Trepp1, 2, Sebastian Wolf1.
1
Ophthalmology, University of Bern, Bern, Switzerland; 2Clinical
Research, University of Bern, Bern, Switzerland; 3Pulmonary
Medicine, University of Bern, Bern, Switzerland.
Purpose: Degeneration of the retinal pigment epithelium (RPE) is the
main etiology of several retinal diseases. The use of stem/progenitor
cells to replace the damaged tissue has been proposed recently. The
aim of the study was to investigate whether immortalized human
neuronal progenitor cells were able to differentiate towards RPE-like
cells.
Methods: ReNcells, human cortical neuronal progenitor cells
(Millipore, Temecula, USA), were used for differentiation. The
cells were incubated with RPE-conditioned medium (CM), pigment
epithelium-derived factor (PEDF) or retinoic acid (RA) for up to 14
days. Gene and protein expression of stem (nestin), neuronal (βIIItubulin), glial (GFAP) and RPE (RPE65, bestrophin) markers were
analyzed by qRT-PCR and immunohistochemistry (IHC). Additional
retinal genes (MITF, CRALBP, PAX-6, CHX-10) were investigated
by qRT-PCR. RPE-related function after differentiation was tested by
an in vitro phagocytosis assay.
Results: In comparison to undifferentiated ReNcells, nestin was still
slightly upregulated in all differentiated cells on the gene level but
downregulated on the protein level. On the other hand, βIII-tubulin
was upregulated on both levels under all treatment conditions. GFAP
gene and protein levels were increased significantly in RA-treated
cells only. RPE65 gene expression was upregulated in cells incubated
with CM or RA. Gene expression of bestrophin was strongly
upregulated under all conditions. IHC showed increased expression
of these RPE markers in all differentiated cells. Gene expression of
CHX-10, MITF and CRALBP was upregulated in all differentiated
cells, whereas PAX-6 was upregulated only in CM & RA-treated
samples. Importantly, gene expression of RPE-related genes was
frequently higher in differentiated cells than in the RPE controls. In
the phagocytosis assay ReNcells cultured with CM or PEDF showed
significantly higher phagocytosis rate than undifferentiated ReNcells.
Conclusions: The most efficient substance for differentiation
towards RPE-like cells appeared to be RPE-CM. The results show
that under the influence of growth factors neuronal progenitor cells
can differentiate into RPE-like cells. Therefore, these cells might
be a new source for regenerative treatment of degenerative retinal
diseases.
Commercial Relationships: Volker Enzmann, None; Luca Tamó,
None; Carolyn Trepp, None; Sebastian Wolf, None
Program Number: 1379 Poster Board Number: A0135
Presentation Time: 8:30 AM–10:15 AM
Efficient generation of retinal pigment epithelium cells from
human embryonic stem cells
Furong Gao1, Zongyi Li1, Weiye Li2, Lixia Lu1, Guotong
Xu1. 1Shanghai Tenth’s People Hospital, Department of
Ophthamology,Tongji Eye Institute and Department of Regenerative
Medicine, Tongji University School of Medicine, Shanghai, China;
2
Department of Ophthalmology, Drexel University College of
Medicine, Philadelphia, PA.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Retinal pigment epithelium (RPE) disorders usually
cause problems with vision. RPE derived from human embryonic
stem cells (hESCs) may become a promising therapeutic option
for transplantation in these retinal diseases. However, induction of
hESCs to RPE cells often takes several months with a low frequency.
The purpose of this study was to establish an improved method that
takes a short time and with a high efficiency.
Methods: ShhES2 cells were induced to differentiate into RPE by
the embryoid body (EB) formation method. We treated embryoid
bodies with a combination of CKI-7, SB431542, and two other
factors in N2B27 medium with or without PJ34. The EBs were
cultured for 3 days and then transferred to matrigel-coated dishes for
attached growth for two weeks. At last, the cells were maintained
in hESCs medium without bFGF for an additional 2 weeks. Then,
the cells were passaged for further differentiation. Assessment of
differentiation was performed using pigmentation formation, mRNA
and immunocytochemistry. The abilities of hESCs-derived RPE
cells in rescuing retinal structure and visual function of RCS rats
after subretinal transplantation were evaluated by electroretinogram
(ERG), histology (HE staining) and immunohistology (TUNEL
assay).
Results: Compared to the four factors group, PJ34, in combination
with the four factors increased the expression of transcripts of the
RPE cell markers MITF and RPE65 after 4 weeks of differentiation,
but decreased photoreceptor markers Rx and CHX10. In addition,
in the PJ34 group, pigmented areas with a cobblestone appearance
began to appear within the differentiating clusters after 4 weeks, and
by 6 weeks, almost all the ShhES2 differentiating cells contained
pigment granules and had a cobblestone appearance. In the
absence of PJ34 group, much less pigmented areas were observed.
Furthermore, the transplanted hESCs-derived RPE cell can improve
the retinal structure and function of RCS rats.
Conclusions: This study demonstrated that PJ34 can promote the
differentiation of hESCs toward an RPE fate at a high efficiency in
a short time. The subretinal transplantation of hESC-derived RPE
appears to improve the structure and function of degenerative retina
of RCS rats. These findings indicate a new short-term and efficient
protocol for differentiation of hESCs to RPE cells and thus may be
useful in the treatment of retinal degenerations.
Commercial Relationships: Furong Gao, None; Zongyi Li, None;
Weiye Li, None; Lixia Lu, None; Guotong Xu, None
Program Number: 1380 Poster Board Number: A0136
Presentation Time: 8:30 AM–10:15 AM
Molecular and Functional comparison of limbal iPS cell- and
retina-derived RGCs
Sharada Paudel Kattel, Sowmya Parameswaran, Iqbal Ahmad.
University of Nebraska Medical Center, Omaha, NE.
Purpose: The induced pluripotent stem (iPS) cells represent a viable
source of retinal progenitors for regenerative medicine for the retina
(Parameswaran et al., 2010, stem cells, 28:695-703). To address the
barrier of insertional mutagenesis associated with nuclieic-acidderived iPS cells, we reprogrammed limbal cells to pluripotency by
non-nucleic acid means (Balasubramanian et al., 2009, Stem Cells,
27:3053-3062), which is a robust source of retinal progenitors for
generating RGCs. The therapeutic utility of limbal iPS cell-derived
RGCs will depend upon its acquisition of properties of RGCs present
in the retina. Here we have compared the properties of limbal iPS
cell-derived RGCs with that of RGCs present in the adult rat retina.
Methods: Rat limbal iPS cells generation and their subsequent
neural differentiation were achieved as previously described
(Balasubramanian et al., 2009). Limbal iPS cell-derived retinal
progenitors were cultured in the presence of rat E14 retinal cell
conditioned medium (E14CM) to generate RGCs. RGCs from the
adult retina were obtained by the standard magnetic bead enrichment
method using Thy1 antibody. The two RGC populations were
subjected to immunocytochemical, Q-PCR and microarray analysis.
In addition, to evaluate their target specificity they were co-cultured
with superior/inferior colliculus (SC/IC) explants.
Results: The limbal iPS cell-derived RGCs, like their retinal
counterparts expressed regulators and markers of RGCs. In addition,
they expressed molecules necessary for axonal pathfinding. However,
their morphology was not as elaborate as RGCs enriched from
the adult retina. Their processes like that of RGCs from the adult
retina, could discriminate between SC and IC cells in establishing
contacts. Preliminary comparative microarray analyses revealed
reprogramming of the genome that approximated that of RGC
obtained from the adult retina.
Conclusions: Our preliminary comparative analysis suggests that
limbal iPS cell-derived RGCs embodies properties similar to that of
RGCs derived from the adult retina and therefore may also function
similarly.
Commercial Relationships: Sharada Paudel Kattel, None;
Sowmya Parameswaran, None; Iqbal Ahmad, None
Support: NEI, Research to Prevent Blindness
Program Number: 1381 Poster Board Number: A0137
Presentation Time: 8:30 AM–10:15 AM
Elucidating the role of FGF signaling using an iPS cell model of
retinal development
Eric Clark1, Ruchira Singh1, 3, Molly Wilson1, Jackie Meyer1, David
Kuai1, Joe Phillips1, David M. Gamm2, 3. 1Waisman Center, University
of Wisconsin, Madison, WI; 2Dept. of Ophthalmology and Visual
Sciences, University of Wisconsin, Madison, WI; 3McPherson Eye
Research Institute, University of Wisconsin, Madison, WI.
Purpose: Studies in animal models have implicated a regulatory loop
between FGF signaling and the transcription factor Visual System
Homeobox 2 (VSX2) in the maintenance of neural retinal progenitor
cell (NRPC) identity and regulation of retinogenesis. However, the
specific roles of FGF family members in these processes remain to be
elucidated. We used a human induced pluripotent stem cell (hiPSC)
model of retinogenesis to examine the expression and function of
specific FGFs during neural retina (NR) development.
Methods: hiPSCs derived from a patient with a functional null
mutation in VSX2 (R200Q) and an unaffected sibling underwent
targeted differentiation to obtain optic vesicle structures (hiPSCOVs). qRT-PCR was used to evaluate transcript levels of specific
FGFs and FGF receptors over time. qRT-PCR, Western blots, and
ICC were employed to evaluate the effect of exogenous FGF and
neutralizing antibody treatment on the expression of selected genes
and proteins involved in retinal development. Lastly, phosphorylation
assays were performed to determine the effect of FGFs on ERK1/2
activation.
Results: Time course studies revealed biphasic expression profiles
of FGF3, 8, and 9 in differentiating hiPSC-OVs, with peaks
corresponding to the formation of the eye field and OV. Furthermore,
expression levels of FGF3, 9, 19 were higher in WT vs. (R200Q)
VSX2 hiPSC-OVs. Continuous application of exogenous FGF9
to (R200Q)VSX2 hiPSC-OVs from d20-d90 led to increased
expression of NR genes and decreased expression of RPE markers.
Treatment of (R200Q)VSX2 hiPSC-OVs with exogenous FGF
during specific developmental time windows revealed a role for
FGF9 in proliferation and differentiation of NRPCs. In contrast,
treatment of WT hiPSC-OVs with neutralizing antibody did not have
a significant effect on the expression of NRPC markers, suggesting
that FGF9 deficiency is tolerated in the presence of functional
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
VSX2. Phosphorylation assays indicated that FGF9-mediated effects
on (R200Q)VSX2 hiPSC-OVs are likely mediated by ERK 1/2
activation.
Conclusions: Our results demonstrate that both VSX2 and FGF9
play roles in the proliferation and differentiation of NRPCs, and that
functional loss of VSX2 can be overcome by FGF9. These insights
increase our understanding of human retinogenesis and may prove
useful in the production of retinal cell types for therapeutic purposes.
Commercial Relationships: Eric Clark, None; Ruchira Singh,
None; Molly Wilson, None; Jackie Meyer, None; David Kuai,
None; Joe Phillips, None; David M. Gamm, None
Support: NIH R01 EY021218, NIH P30 HD03352, Foundation
Fighting Blindness Wynn-Gund Translational Research Award,
Retina Research Foundation
Program Number: 1382 Poster Board Number: A0138
Presentation Time: 8:30 AM–10:15 AM
Subpopulations of human umbilical cord mesenchymal stem cells
exhibit differential rescusing functions on retinal degeneration in
RCS rats
Li Wang1, 2, Haibin Tian1, 2, Peng Li1, 2, Zongyi Li1, 2, Chunpin Lian1, 2,
Qingjian Ou1, 2, Lixia Lu1, 2, Weiye Li1, 3, Guotong Xu1, 2. 1Department
of Ophthalmology of Shanghai Tenth Hospital, and Tongji Eye
institute, Tongji University School of Medicine, Shanghai, China;
2
Department of Regenerative Medicine and Stem Cell Research
Center, Tongji University School of Medicine, Shanghai, China;
3
Department of Ophthalmology, Drexel University College of
Medicine, Philadelphia, PA.
Purpose: To provides more information about the heterogeneity of
human umbilical cord mesenchymal stem cells (UCMSCs) and a
new strategy for UCMSCs treatment of retinal degeneration (RD) by
choosing appropriate subpopulations.
Methods: Two subsets of human umbilical cord mesenchymal stem
cells (UCMSCs), termed as UCMSC1 and UCMSC2, were isolated
by single cell cloning, specific surface markers were analyzed by
flow cytometry. Proliferating rate and gene expression were carried
out by MTT and RNA sequencing analysis. The gene levels of
growth factors and cytokines were measured by Real-time PCR.
Their differentiation abilities were confirmed by culturing UCMSC
in adipogenic, osteogenic and chondrogenic differentiation media.
UCMSC1 and UCMSC2 were transplanted into subretinal space of
RCS rats, their therapeutic functions were confirmed by analyzing
retinal nuclear layer thickness, apoptotic photoreceptors and
electroretinogram.
Results: Both the two subpopulations share similar membrane
marker phenotypes. However, UCMSC1 showed stronger ability
than UCMSC2 regarding proliferating rate, colony forming ability,
adipogenic and osteogenic potential, whereas the latter exhibited
increased chondrogenic ability. RNA sequencing analysis further
showed the differential gene levels relative to proliferation,
differentiation and immunoregulation in UCMSC1 and UCMSC2.
After transplanted into subretinal space of RCS rats, UCMSC1 had
stronger vision rescue function compared to UCMSC2, including
increased b¬-wave amplitude, retinal nuclear layer thickness, and
decreased apoptotic photoreceptors. Furthermore, When subjected to
interleukin-6 treatment in vitro, mimicking the transplanted MSCs
under inflammation condition in RD, UCMSC1 expressed much
higher levels of growth factors than UCMSC2, such as bFGF, CNTF,
EGF, HGF, PEDF, which indicates that the differential therapeutic
capacities of UCMSC subpopulations involve distinct paracrine
functions.
Conclusions: Two subpopulations with distinct morphology,
proliferation, differentiation potentials, marker expression and gene
expression were derived from human UCMSCs. In addition, the
different subpopulations of human UCMSCs had distinct therapeutic
functions on RD.
Commercial Relationships: Li Wang, None; Haibin Tian, None;
Peng Li, None; Zongyi Li, None; Chunpin Lian, None; Qingjian
Ou, None; Lixia Lu, None; Weiye Li, None; Guotong Xu, None
Program Number: 1383 Poster Board Number: A0139
Presentation Time: 8:30 AM–10:15 AM
MicroRNA Signatures Associated with the Differentiation of
Human Müller Glia with Stem Cell Characteristics towards a
Retinal Ganglion Cell Fate
Hari Jayaram1, 2, Megan F. Jones2, 1, Dan Frampton4, Karen
Eastlake2, 1, Silke Becker2, 1, Karl Matter3, G Astrid Limb2, 1. 1National
Institute for Health Research (NIHR) Biomedical Research Centre for
Ophthalmology at Moorfields Eye Hospital NHS Foundation Trust
and UCL Institute of Ophthalmology, London, United Kingdom;
2
Ocular Biology & Therapeutics, UCL Institute of Ophthalmology,
London, United Kingdom; 3Cell Biology, UCL Institute of
Ophthalmology, London, United Kingdom; 4Department of Infection
& Immunity, University College London, London, United Kingdom.
Purpose: MicroRNAs (miRNAs) are small, endogenous noncoding RNAs involved in the post-transcriptional silencing of gene
expression during development and disease. The purpose of this
study was to determine the miRNA signature associated with Notch
pathway downregulation and in vitro differentiation of Human Müller
Glia with Stem Cell Characteristics (hMSCs) towards a Retinal
Ganglion Cell (RGC) fate.
Methods: hMSCs were differentiated in vitro towards a retinal
ganglion cell fate using established protocols. Total cellular RNA
was isolated from both differentiated and undifferentiated cells
(Four replicates per condition) and the expression profiles were
interrogated using the Agilent miRNA microarray platform. Partek
Genomics Suite software was used to normalise the expression
data. Subsequent analysis was performed using ANOVA in order to
identify differentially expressed miRNAs using a 5% False Discovery
Rate (adjusted P-values < 0.05). Validation of the microarray results
was achieved by performing quantitative PCR.
Results: Analysis of the microarray data identified nineteen
differentially expressed miRNAs showing upregulation in
differentiated cells when compared to undifferentiated controls
(Fold Change >= 0.5, p<0.05). These included miR-199b, miR-151,
miR-204 and let-7i whose target genes include components of the
NOTCH pathway, and miR-222 that is known to be associated with
the development of neurite outgrowth.
Conclusions: The miRNA signature associated with differentiation
of hMSCs towards retinal ganglion cells is characterised by the
upregulation of miRNAs known to be involved with the NOTCH
pathway and neurite outgrowth. The identification of these targets
and their future experimental modulation may provide opportunity
for future research into the induction of in vivo retinal ganglion cell
differentiation by hMSCs.
Commercial Relationships: Hari Jayaram, None; Megan F. Jones,
None; Dan Frampton, None; Karen Eastlake, None; Silke Becker,
None; Karl Matter, None; G Astrid Limb, None
Support: Fight For Sight New Lecturers Award, United Kingdom
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1384 Poster Board Number: A0140
Presentation Time: 8:30 AM–10:15 AM
Adipose derived stem cell therapeutic potential for treatment of
diabetic retinopathy is modulated by both oxygen tension and
diabetic status of donor cells
Thomas A. Mendel1, 2, Stephen Cronk3, Jaymes Beech3, Alexander M.
Guendel2, Shayn Peirce3, Paul A. Yates2. 1Pathology, University of
Virginia, Charlottesville, VA; 2Ophthalmology, University of Virginia,
Charlottesville, VA; 3Biomedical Engineering, University of Virginia,
Charlottesville, VA.
Purpose: Adipose derived stem cells (ASCs) stabilize the retinal
microvasculature in the Akimba model of diabetic retinopathy (DR)
through both direct contact and paracrine effects. Factors influencing
the therapeutic efficacy are unknown, but critical to elucidate towards
translational application. In this study, we examine the impact of
oxygen tension and diabetic status of ASC donor on the secretome
ASC-derived-pericytes in vitro and how they alter therapeutic
efficacy in vivo.
Methods: ASCs were isolated from both diabetic Akimba (dASC)
and normo-glycemic wildtype mice (nASC) at 9 weeks of age. DiIlabeled passage 4 dASC and nASCs-pericytes were then injected
intravitreally in 6 week old Akimba mice, with eyes harvested 4
weeks later for analysis of ASC incorporation and impact on the
microvasculature. Supernatant from in vitro cultured dASCs and
nASCs-pericytes was collected and evaluated by multiplex ELISA
with respect to angiogenic secretome. In parallel, normoxic passage
5 human ASCs were subjected to hypoxic conditions (1% oxygen) or
left in normoxic conditions, with supernatant collected 24 hours later
and analyzed by multiplex ELISA.
Results: Pericytes derived from diabetic ASCs, in contrast to
normo-glycemic ASCs, were unable to prevent damage to the retinal
microvasculature that occurs in the Akimba DR mouse (Fig 1A). A
number of pro-angiogenic factors were found to be diminished in the
supernatant of dASCs as compared to nASCs, including IGFBP3 (Fig
1B). In vitro assays of proliferation and apoptosis were also altered
in dASCs as compared to nASCs. Similarly, hypoxia diminished
IBFBP3 expression in cultured hASCs (Fig 2).
Conclusions: Exogenous diabetic ASC-pericytes show diminished
ability to protect retinal microvasculature in a murine model of DR.
Diabetes impairs ASC-pericyte viability and alters the pro-angiogenic
secretome. Hypoxia similarly impacts ASC-pericyte paracrine
activity, reducing IGFBP3, a factor known to ameliorate oxygeninduced retinopathy. These findings may explain the inability of
native retinal pericytes to rescue the microvasculature in a diabetic
environment, and suggest autologous stem cell approaches to treating
DR may be problematic. It may be possible to enrich environmental
conditions as well as establish viability assays that can both predict
and enhance ASC therapeutic efficacy.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Thomas A. Mendel, U.S. Provisional
Patent Application Serial No. 61/684,375 (P); Stephen Cronk, None;
Jaymes Beech, None; Alexander M. Guendel, None; Shayn Peirce,
U.S. Provisional Patent Application Serial No. 61/684,375 (P); Paul
A. Yates, Genentech/Roche (C), Owner RetiVue LLC (I), RetiVue
LLC (E), U.S. Provisional Patent Application Serial No. 61/684,375
(P)
Support: NIH T32GM08715 and NIH EY022063-01
Program Number: 1385 Poster Board Number: A0141
Presentation Time: 8:30 AM–10:15 AM
The optic nerve lamina is a neural progenitor cell niche
Steven L. Bernstein1, Zara Mehrabyan1, Candace Kerr2, Sally
Temple3, Jeffrey Stern3, Yan Guo1. 1Ophthalmology and Visual
Sciences, Univ of Maryland Sch of Medicine, Baltimore, MD;
2
Department of Biochemistry and Molecular Biology, University of
Maryland-Baltimore, Baltimore, MD; 3Neural Stem Cell Institute,
Rensselaer, NY.
Purpose: The optic nerve (ON) is a myelinated CNS tract connecting
the eye and brain, comprised of retinal ganglion cell (RGC) axons.
RGC axons are unmyelinated in the retina but are myelinated
after traversing the optic nerve lamina (ONL). The reason for
this discontinuity is unknown but suggests that the ONL may
facilitate this process. One possibility is that the ONL is a source of
myelinating progenitor cells. This would be of great significance to
a number of ON diseases, including glaucoma, which may originate
from problems in the ONL. The ONL has an unusual vascularization
distinct from the rest of the ON, in that the ONL receives circulation
from the retina, underlying choroid, and intrinsic ON vasculature.
Thus, the ONL is uniquely poised in this area to provide a progenitor
cell niche. We evaluated the ONL as a potential source for progenitor
cells.
Methods: Rodent ONL vasculature was mapped using two
photon microscopy, and analyzed by gene expression and
immunohistochemically for nestin and other neural progenitor genes,
myelin components, oligodendrocyte and astrocyte precursors (Olig1, PDGFRα, NG2), and neuronal proteins. We generated ONL-cell
cultures and also utilized two mouse strains (ER-Cre-Nestin and
-PDGFRα), crossing them with ROSA26/LoxP mice with enhanced
yellow fluorescent protein (EYFP) to localize after progenitor
development. Human donor tissues of a variety of ages were analyzed
for nestin expression.
Results: Nestin expression strongly localizes to the ONL in the
young adult animal, with more differentiated, but still immature
NG2+ glial precursors directly behind the ONL. EDU-mitotic
labeling reveals mitotic nestin (+) cells with slow turnover. ER-CrePDGFR X ROSA double heterozygous mice demonstrated EYFP
oligodendrocytes banding directly under the ONL, in the anterior
ON. The ONL expresses genes for early neuronal, as well as glial
function. ONL-nestin expression declines during both human and
rodent aging, with a near absence in humans over 60y of age.
Conclusions: The ONL is a progenitor cell niche. This niche may
aid directionality of myelination/myelin barrier function, function
for necessary replacement of glial cells in the highly stressful
environment of the eye-nerve junction, and may generate postembryonic retinal neurons. The loss of the ONL progenitor niche may
play a key role in the progression of intrinsic ON diseases such as
glaucoma, where current treatment options are of limited usefulness.
Commercial Relationships: Steven L. Bernstein, None; Zara
Mehrabyan, None; Candace Kerr, None; Sally Temple, None;
Jeffrey Stern, None; Yan Guo, None
Support: NIH grants RO1EY015304 and EY019529
Program Number: 1386 Poster Board Number: A0142
Presentation Time: 8:30 AM–10:15 AM
Generation of ciliary epithelium from mouse ES and iPS cells
Hirofumi Kinoshita1, Kiyoshi Suzuma1, Jun Kaneko2, Michiko
Mandai2, Takashi Kitaoka1, Masayo Takahashi2. 1Department of
Ophthalmology, Nagasaki Univ School of Medicine, Nagasaki,
Japan; 2Laboratory for Retinal Regeneration, RIKEN Center for
Developmental Biology, Kobe, Japan.
Purpose: Recently, Eiraku et al reported a protocol for selforganizing optic-cup morphogenesis in three-dimensional culture.
With this procedure, we could differentiate mouse embryonic stem
(ES) cells or mouse induced pluripotent stem (iPS) cells into neural
retina, retinal pigment epithelium (RPE), as well as tissue derived
from neural ectoderm in the three-dimensional structure. Here, we
report a possibility that ciliary epithelium, which is essential for
production of aqueous humor, could be differentiated from mouse
ES/iPS cells.
Methods: Mouse ES/iPS cells (Rx knock-in GFP ES line mESRx+;
Nrlp-eGFP transgenic iPS line) were maintained in maintenance
culture as it might lead to undesirable differentiation of cells. Cells
were differentiated by using modified Eiraku’s differentiation
protocol, induced optic-cup-like structures were investigated
morphologically, or immunohistochemically. E16.5 and P5 of
C57BL/6J mice were evaluated as control.
Results: We could show autonomous formation of the optic-cuplike structure from a three-dimensional culture of mouse ES/iPS
cell aggregates by using modified Eiraku’s differentiation protocol.
To evaluate whether there is a development of ciliary epithelium in
this optic-cup-like structure, we identified immunohistochemistry
for ciliary marker. Prospective ciliary epitheliums, bilayer of
pigmented and non-pigmented epithelium (PE and NPE), were
seen in peripheral region of inner and outer epithelium in E16.5
mouse eye. Aquaporin1-positive staining was seen in the NPE.
Connexin43-positive staining was seen in the ciliary epitheliums (PE
and NPE), RPE and gap junction, which is structure between PE and
NPE. In P5 mouse eye, neural retina and ciliary epithelium became
morphologically distinguishable more clearly. Aquaporin1-positive
staining was seen in the NPE, especially in the anterior pars plicata.
Aquaporin4-positive staining was seen in the NPE, connexin43positive staining was seen in the ciliary epitheliums (PE and NPE),
RPE and gap junction. Differentiated structures derived from
mouse ES cells were Rx-GFP positive. In immunohistochemistry of
aggregates derived from mouse iPS cells, expression of aquaporin1,
connexin43 were positive in ciliary epithelium like structures.
Conclusions: We could differentiate the ciliary epithelium like
structures from mouse ES/iPS cells. These complexes showed
similarity of morphology and immunostaining of ciliary epitheliums
in vivo.
Commercial Relationships: Hirofumi Kinoshita, None; Kiyoshi
Suzuma, None; Jun Kaneko, None; Michiko Mandai, None;
Takashi Kitaoka, None; Masayo Takahashi, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1387 Poster Board Number: A0143
Presentation Time: 8:30 AM–10:15 AM
Subpopulations of monkey bone marrow mesenchymal stem cells
exhibit similar therapeutic functions on retinal degeneration in
RCS rats
Haibin Tian1, 2, Peng Li1, 2, Li Wang1, 2, Zongyi Li1, 2, Chunpin Lian1,
2
, Qingjian Ou1, 2, Lixia Lu1, 2, Weiye Li1, 3, Guo-Tong Xu1, 2. 1Tongji
University Eye institute, Tongji University Medical school, Shanghai,
China; 2epartment of Regenerative Medicine and Stem Cell Research
Center, Tongji University School of Medicine, Shanghai, China;
3
Department of Ophthalmology, Drexel University College of
Medicine, Philadelphia, PA.
Purpose: To provide more information about the heterogeneity of
monkey bone marrow mesenchymal stem cells (BMSCs) and to
confirm whether different subpopulations could be acquired by cell
membrane marker CD90, and their therapeutic functions on retinal
degeneration (RD) will be investigated.
Methods: Two subsets of monkey BMSCs, CD90+ and CD90-, were
isolated by flow cytometry, specific surface markers were further
confirmed by flow cytometry. Proliferating rate and the gene levels
of growth factors and cytokines were measured by MTT method
and real-time PCR. Their differentiation abilities were confirmed
by culturing BMSCs in adipogenic, osteogenic and chondrogenic
differentiation media. CD90+ BMSCs and CD90- BMSCs were
transplanted into subretinal spaces of RCS rats, their therapeutic
functions were confirmed by analyzing retinal nuclear layer
thickness, apoptotic photoreceptors and electroretinogram.
Results: Both the two CD90+ and CD90- subpopulations share
similar membrane marker phenotypes, both are positive for CD73,
CD44, CD29, and negative for CD45, CD34, MHCII. However,
differential expression of CD90 did not influence the characteristics
of the two subpopulations, regarding proliferating rate, colony
forming ability, growth factor generation, adipogenic osteogenic and
chondrogenic potentials. After transplanted into subretinal space of
RCS rats, both CD90+ and CD90- subpopulations showed similar
therapeutic functions, including increased b¬-wave amplitude, retinal
nuclear layer thickness, and decreased apoptotic photoreceptors.
Furthermore, when passaged in vitro, CD90- cells gradually acquired
membrane molecule CD90 after two passages, which suggested that
CD90- subpopulation could switch into CD90+ subpopulation when
cultured in vitro.
Conclusions: Subpopulations could be distinguished by cell
membrane markers. However, subpopulations may not always
show distinct characteristics, and membrane markers are not stable
when cells are cultured in vitro, which may account for their similar
therapeutic functions.
Commercial Relationships: Haibin Tian, None; Peng Li, None; Li
Wang, None; Zongyi Li, None; Chunpin Lian, None; Qingjian Ou,
None; Lixia Lu, None; Weiye Li, None; Guo-Tong Xu, None
Program Number: 1388 Poster Board Number: A0144
Presentation Time: 8:30 AM–10:15 AM
Enhanced Neurogenic Potential of Müller Cells in the Absence of
Ephrin-A2/A3
Ruilin Zhu1, 2, Kin-Sang Cho2, Yuan Fang3, Liu Yang1, Dongfeng F.
Chen2, 4. 1Ophthalmology, Peking University First Hospital, Key
Laboratory of Vision Loss and Restoration, Ministry of Education,
Beijing, China; 2Schepens Eye Research Institute, Massachusetts
Eye and Ear, Harvard Medical School, Boston, MA; 3Eye and ENT
hospital, Fudan University, Shanghai, China; 4Boston VA Healthcare
System, Boson, MA.
Purpose: We showed previously that ephrin-A2 and -A3 are negative
regulators for the growth of neural progenitor cells in the brain and
ciliary epithelium derived retinal stem cells.We hypothesize that
absence of ephrin-A2 and -A3 also increases Müller cell proliferation
and neurogenic potential in the adult.
Methods: Expression of ephrin-A2 and -A3 and their receptor
EphA4 in the retina and Müller cells was assessed by immunostaining
and real-time PCR.To label proliferating cells,purified Müller cells
of both wild-type and A2-/-A3-/- mice were treated with 0.5μM
BrdU for 24 hours in culture.Percentage of BrdU+ cells was then
recorded,and expression of retinal progenitor markers was evaluated
with real-time PCR.In another series of experiments,purified Müller
cells were induced to differentiate in the defined medium for 14 days
and stained with primary antibodies against a photoreceptor marker
recoverin or retinal ganglion cell marker β-III-tublin for evaluation of
their potential of trans-differentiation into retinal neurons.
Results: Expression of ephrin-A2/A3 and their receptor EphA4
was detected in both the retinas and purified Müller cell cultures.
Using double-immunolabeling of EphA4 and CRALBP,a marker
of Müller cells,in retinal sections we further demonstrate Müller
cell expression of EphA4. Moreover,results of real-time PCR
confirmed that ephrin-A3 and EphA4 expression is particularly
enriched in Müller cells.Expression of neural progenitor cell markers
Pax6,Chx10,Ngn2,Sox2 were significantly increased in Müller cells
derived from A2-/-A3-/- mice as compared to those of wild-type
mice.The percentage of proliferating Müller cells was significantly
higher in cultures derived from A2-/-A3-/- mice than that from
wild-type mice.Induction of neuron trans-differentiation also induced
significantly higher percentage of recoverin+ and β-III-tublin+ cells
in Müller cell cultures derived from A2-/-A3-/- mice.These data
suggest enhanced neurogenic potential of Müller cells in the absence
of ephrin-A2 and-A3.
Conclusions: Our results indicate that the adult mouse retina
expresses negative regulators for retinal stem cell growth,ephrin-A2/
A3,and their receptor EphA4 on Müller cells.The Müller cells derived
from A2-/-A3-/- mice show a higher proliferation and neurogenic
potentials in culture than those from wild-type mice.Thus,ephrin-A2
and -A3 contribute negatively to the regenerative behavior of Müller
cells in adult mice.
Commercial Relationships: Ruilin Zhu, None; Kin-Sang Cho,
None; Yuan Fang, None; Liu Yang, None; Dongfeng F. Chen, None
Support: Department of Veterans Affairs (1I01RX000110),
Department of Defense (W81XWH-09-2-0091), Lion’s Foundation
Grants to D.F.C. and K.S.C. National Natural Science Foundation of
China (No.81170837; NSFC81100667 )
237 Life and Death in the Retina/RPE
Monday, May 05, 2014 11:00 AM–12:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 1707–1744/A0076–A0113
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 1707 Poster Board Number: A0076
Presentation Time: 11:00 AM–12:45 PM
Elevated ocular A2E and bis-retinoid levels in a rat model of
Smith-Lemli-Opitz syndrome
Steven J. Fliesler1, 2, Christopher C. Goulah1, 2, Bruce A. Pfeffer1, 2,
Keiko Ueda3, Janet R. Sparrow3. 1Research Service, VAWNYHS,
Buffalo, NY; 2Ophthalmology and Biochemistry, University at
Buffalo and SUNY Eye Institute, Buffalo, NY; 3Ophthalmology and
Pathology & Cell Biology, Columbia University CP&S, New York,
NY.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: We previously showed that the retinal pigment epithelium
(RPE) in the AY9944-induced rat model of Smith-Lemli-Opitz
syndrome (SLOS) exhibits marked accumulation of phagosomes
and other lipid/membrane inclusions, compared to age-matched
control rats. We hypothesized that the RPE in this model would
contain substantially elevated amounts of A2E and other bisretinoids, relative to controls, which might contribute to the observed
pathology. We tested this hypothesis in the present study.
Methods: Sprague-Dawley rats were treated with AY9944 to
generate the SLOS rat model, as previously described (Fliesler et
al., Arch Ophthalmol. 2004); age-matched rats without AY9944
treatment served as controls. At ca. 10-11 wks postnatal, rat were
euthanized and eyes (N=4 per group/treatment) were harvested: for
biochemical analysis, eyes were flash frozen in liquid nitrogen and
stored at -80oC, while for histological analysis eyes were formalinfixed and stored at 4oC. Analysis of A2E and related bis-retinoids
as well as all-trans retinal was performed by HPLC as previously
described (Sparrow et al., Methods Molec Biol., 2010). Formalinfixed, OCT-embedded eyes were cryosectioned, and frozen sections
were mounted/coverslipped without staining; RPE autofluorescence
was assessed by confocal scanning laser fluorescence microscopy,
using 488 nm excitation and 500-600 nm emission.
Results: Compared to controls, SLOS rat eyes exhibited the
following fold-change increases (p<0.05): A2E, 1.52; isoA2E, 2.20;
total A2Es, 1.71; all-trans retinal, 1.57. RPE cells in SLOS rat eyes
also contained increased numbers of punctate, hyperfluorescent
inclusions, compared to age-matched controls, consistent in size
and distribution with phagosomes derived from ingested rod outer
segment tips.
Conclusions: RPE cells in the SLOS rat model contain elevated
levels of A2E and related bis-retinoids, consistent with the observed
increase in their phagosome content, compared to untreated controls.
These changes may contribute to the retinal dysfunction and
degeneration observed in the AY9944-induced SLOS rat model.
Commercial Relationships: Steven J. Fliesler, None; Christopher
C. Goulah, None; Bruce A. Pfeffer, None; Keiko Ueda, None;
Janet R. Sparrow, None
Support: NIH EY007361 (SJF), EY012951 (JRS); RPB Unrestricted
Grant (SJF); VAWNYHS facilities and resources (SJF, CCG & BAP).
Program Number: 1708 Poster Board Number: A0077
Presentation Time: 11:00 AM–12:45 PM
Transcriptomic analysis of oxysterol effects on a photoreceptorderived cell line, with relevance to Smith-Lemli-Opitz syndrome
Bruce A. Pfeffer1, 2, Paul Wong3, Libin Xu4, Zihua Hu5, Alison C.
Ziesel3, Sriganesh Rao1, 2, Ned A. Porter4, Steven J. Fliesler1, 2.
1
Research Service, VAWNYHS, Buffalo, NY; 2Ophthalmology,
Biochemistry, and SUNY Eye Institute, University at Buffalo,
Buffalo, NY; 3Ophthalmology, Emory University, Atlanta, GA;
4
Chemistry & Vanderbilt Institute of Chemical Biology, Vanderbilt
University, Nashville, TN; 5Center for Computational Research and
SUNY Eye Institute, University at Buffalo, Buffalo, NY.
Purpose: Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease
involving defective biosynthesis of cholesterol (CHOL) and aberrant
accumulation of its immediate precursor, 7-dehydrocholesterol
(7DHC), in body tissues and fluids. 7DHC gives rise to several
oxysterol by-products, some of which are cytotoxic, including
7-ketocholesterol (7kCHOL), as well as 7DHC-specific oxysterols,
notably 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD). Selective
degeneration and loss of photoreceptors (PRs) is a hallmark of the
AY9944-induced rat SLOS model, which we hypothesize is due to
the action of such oxysterols on PR cells. We previously showed
that such oxysterols accumulate in the retina in this model, but not
in untreated control rat retinas. We also have shown that EPCD is far
more toxic than 7kCHOL to 661W cells (a mouse PR-derived line),
and that RPE and rMC-1 cells are comparatively more resistant to
such cytotoxicity. To elucidate molecular mechanisms of oxysterolinduced PR cell death, we carried out microarray analysis on 661W
cells following incubations with oxysterols.
Methods: Cultured 661W cells were treated (N=3 replicates per
condition) with 7kCHOL, EPCD, or CHOL at defined concentrations,
vs. vehicle control (VC; hydroxypropyl-β-cyclodextrin). At selected
time points (5 and 24 h), prior to overt morphological signs of cell
death, RNA was harvested and subjected to gene microarray analysis.
Gene annotation analysis was performed using gene sets with ≥
2.0-fold change (FC) and false discovery rate (FDR) ≤ 0.05, with
particular emphasis on canonical pathways associated with stress
responses and cell death/ survival.
Results: Both 7kCHOL and EPCD up-regulated transcription
of nuclear receptors LXR and AHR, modulating their canonical
downstream effector genes (P≤0.05), including virtually complete
down-regulation of cholesterol pathway genes, as also seen in
microarray analysis of AY9944 rat retinas. Notably, key genes
associated with ER stress, mitochondrial dysfunction, autophagy, and
cell death/ survival were significantly dysregulated. Some oxysterolspecific (7kCHOL vs. EPCD) effects also were observed. While
CHOL (negative control) did not elicit such effects, it did exert some
differential gene expression changes compared to VC treatment.
Conclusions: Our results suggest that EPCD and 7kCHOL may
mediate some of their cytotoxic effects via divergent mechanisms.
Commercial Relationships: Bruce A. Pfeffer, None; Paul Wong,
None; Libin Xu, None; Zihua Hu, None; Alison C. Ziesel, None;
Sriganesh Rao, None; Ned A. Porter, None; Steven J. Fliesler,
None
Support: NIH EY007361 (SJF), RPB Unrestricted Grant (SJF), The
Parker Foundation (PW), Research to Prevent Blindness (PW), NIH
P30EY0006360 (PW), K99 HC073270-01 (LX), NIH ES013125
and HD064727 (NAP & LX), NSF CHE 0717067 (NAP & LX),
VAWNYHS facilities and resources (SJF, SR, & BAP)
Program Number: 1709 Poster Board Number: A0078
Presentation Time: 11:00 AM–12:45 PM
Vitamin E - TPGS inhibits P-glycoprotein in retinal cells through
modulation of the membrane dipole potential - more harm than
good? An in-vitro evaluation
Gibran F. Butt, Abubakar J. Habib, Benjamin M. Davis, Shereen
Nizari, Joana M. Galvao, Mark Tilley, Eduardo M. Normando, Li
Guo, M Francesca Cordeiro. Institue of Ophthalmology, UCL,
London, United Kingdom.
Purpose: Vitamin E –TPGS (TPGS) is an excipient used to solubilize
lipophilic molecules and improve bioavailability, the latter achieved
through inhibition of the multidrug efflux channel P-glycoprotein
(P-gp). In RGC-5 cells, we investigated whether TPGS (TPGS)
inhibits P-glycoprotein activity through modulation of the cell
membrane dipole potential. The effect of TPGS on cell viability
was also investigated, when it is co-administered with Dimethyl
sulfoxide (DMSO) a cellular insult. Finally, we investigated whether
the administration of CoQ10 affected cell viability where TPGS and
DMSO were co-administered.
Methods: All experiments were performed in the RGC-5 cell line.
P-gp activity was assessed using the lipophilic probe and P-gp
substrate Calcein AM. Changes in membrane dipole potential were
detected through wavelength ratiometric fluorescence measurements
using the electrochromic probe Di-8-anepps. The MTT assay was
used to measure cell viability.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: TPGS was found to significantly inhibit RGC-5 P-gp
activity in a dose dependent manner, EC50 3.748 μg/ml TPGS vs
0.47 μg/ml verapamil (control inhibitor).
The presence of TPGS altered the membrane dipole potential, with
results suggesting saturation of this effect at concentrations as low as
10 μg/ml.
DMSO reduced cell viability in a dose dependent manner. Coadministration of DMSO and TPGS to RGC-5s lead to significantly
greater reduction in cell viability compared to DMSO alone. In cell
cultures treated with CoQ10/TPGS and DMSO the cell viability was
greater in a fixed concentration range compared to TPGS and DMSO
alone.
Conclusions: DMSO is known to inhibit the antioxidant glutathione,
which is located in cell mitochondria. This leads to an increase in
cytotoxic compounds such as reactive oxygen species (ROS) and
consequently oxidized lipids. P-gp is responsible for the removal
of oxidized lipids and cytotoxic drug molecules from the cell and is
located in the plasma and mitochondrial membranes. The inhibition
of P-gp is shown here to decrease cell viability. The anti oxidant
CoQ10 demonstrates a neuroprotective effect in this context,
proposed to take effect in two possible ways: by reducing ROS; and
by minimizing mitochondrial dysfunction.
Commercial Relationships: Gibran F. Butt, None; Abubakar
J. Habib, None; Benjamin M. Davis, None; Shereen Nizari,
None; Joana M. Galvao, None; Mark Tilley, None; Eduardo M.
Normando, None; Li Guo, None; M Francesca Cordeiro, None
Program Number: 1710 Poster Board Number: A0079
Presentation Time: 11:00 AM–12:45 PM
A Study of Retinal Mitochondrial Changes in Aged Diabetic
(Goto-Kakizaki) Rats to Explore Possible Links with Alzheimer’s
Disease
Marianne Phillips1, Timothy Wong1, Shereen Nizari1, Yik L. Chan1,
Hannah Whittington2, Damian Cummings3, M Francesca Cordeiro1.
1
Visual Neuroscience Department, Institute of Ophthalmology, UCL,
London, United Kingdom; 2The Hatter Cardiovascular Institute,
UCL, London, United Kingdom; 3Department of Neuroscience,
Physiology & Pharmacology, UCL, London, United Kingdom.
Purpose: Evidence suggests an association between Alzheimer’s
Disease (AD) and Type 2 Diabetes Mellitus (T2DM). Metabolic
disturbances, including hyperglycaemia, decreased insulin receptor
sensitivity and mitochondrial dysfunction are common to both and
can cause neuronal damage and degeneration. Studies also indicate
that the retina may provide a non-invasive window to view the
neuropathology in these conditions – notably via retinal ganglion cell
layer (RGCL) thickness changes.
Aberrant mitochondrial activity and AD-like pathology had
previously been reported in the spontaneous T2DM Goto-Kakizaki
rat model (D), but retinal neurones had not yet been studied so were
of interest here.
Methods: Retinal sections were cut from 3, 12 and 18 month (m)
D eyes and age-matched controls (C). Markers of mitochondrial
permeability transition pore (mPTP) formation (VDAC and CypD),
pro-apoptotic activity and apoptosis (cytochrome c and caspase-3)
and electron transport chain activity (COX-1) were stained for, by
immunohistochemistry. Confocal microscopy images were obtained,
followed by grading to assess retinal changes in the 6 groups.
Results: When compared to age-matched C, D showed significantly
greater levels of staining for VDAC and caspase-3 – particularly at
12m (p<0.01 and p<0.001 respectively, with a minimum of n=3 per
group). Unexpectedly, COX-1 showed significantly increased staining
in D (p<0.001). CypD and cytochrome c showed a trend towards an
increase in staining in D. Interestingly, the greatest staining occurred
at 12m for all proteins, rather than the expected 18m.
Conclusions: As far as we are aware, this is the first study of retinal
AD changes in the GK model and results indicate aberrant retinal
mitochondrial activity, with increased mPTP formation and cell
death. These findings resemble previous mitochondrial studies in the
brain of other AD models, such as transgenic animals. They reinforce
the increasing realization that both the retina and T2DM animals may
model the neuropathology of AD. Thus, these results support T2DM
as a current and future model of AD, especially as the pathology
generated is sporadic – reflecting the majority of AD cases found
clinically.
Commercial Relationships: Marianne Phillips, None; Timothy
Wong, None; Shereen Nizari, None; Yik L. Chan, None; Hannah
Whittington, None; Damian Cummings, None; M Francesca
Cordeiro, None
Program Number: 1711 Poster Board Number: A0080
Presentation Time: 11:00 AM–12:45 PM
Neurotoxicity of Pannexin1 channel correlates with Panx1 gene
expression level in retinal ganglion cells
Valery Shestopalov1, 2, Alexey Pronin3, 1, Galina Dvoriantchikova1,
Breanne Prindeville1, Junior Tayou3, Vladlen Z. Slepak3.
1
Ophthalmology, Bascom Palmer Eye Institute University of Miami
Miller School of Medicine, Miami, FL; 2Vavilov Institute of General
Genetics, Moscow, Russian Federation; 3Molecular and Cellular
Pharmacology, University of Miami Miller School of Medicine,
Miami, FL.
Purpose: Activation of pannexin1 (Panx1) membrane channels
by purinergic P2X receptor agonists and ischemia were shown to
facilitate the loss of retinal ganglion cells (RGCs). Previously we
showed that RGCs expressed the highest level of Panx1 in the retina.
To correlate the activity of Panx1 with cellular resistance to ischemia,
we examined the effect of high levels of Panx1 on cell survival under
conditions of oxygen-glucose deprivation.
Methods: We engineered stable Neuro2A cell line with >100-fold
increased levels of Panx1. Cell permeation of Neuro2A lines by
ischemia was tested using fluorescent small molecule dyes; cell
death was measured using Annexin V/propidium iodide labeling.
Permeability to Ca2+ cations was measured using ratiometric timelapse imaging of fura2. Immunohistochemical and transcriptomic
analyses were used to examine expression of Panx1, P2X7 and P2X4
receptors in retinal ganglion cells and in cell lines. In situ detection
of Panx1 and P2X7 transcripts was performed using qPCR and
RNAscope technology.
Results: RGCs and Neuro2A cells over-expressing Panx1 were
significantly more susceptible to ischemia as compared to controls.
This high vulnerability correlated well with increased permeability
to small molecules and cations. Interestingly, adult RGCs showed
differential expression of Panx1, P2X7 and P2X4 receptors during
post-embryonic development. In situ detection of Panx1, P2X4 and
P2X7 transcripts confirmed elevated expression of Panx1 and P2X4
receptor in RGCs. This data suggest that in the retina Panx1 pairs
with P2X4 rather than with P2X7 receptor.
Conclusions: High expression level of Panx1 renders RGCs and
other neurons susceptible to ischemic injury. Our results suggest that
Panx1-associated mechanisms underlie high vulnerability of these
neurons to degenerative diseases induced by ischemia and increased
extracellular ATP.
Commercial Relationships: Valery Shestopalov, None; Alexey
Pronin, None; Galina Dvoriantchikova, None; Breanne
Prindeville, None; Junior Tayou, None; Vladlen Z. Slepak, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Support: NIH grants EY021517 (VIS), EY EY018666 (VZS),
EY020535 (BTS), P30 EY014801, RPB unrestricted grant and
Department of Defense (DOD- Grant#W81XWH-09-1-0675) to U.
Miami Ophthalmology
Program Number: 1712 Poster Board Number: A0081
Presentation Time: 11:00 AM–12:45 PM
A Nonsense Mutation in CEP250, a Mammalian-Specific
Homolog of Rootletin, Causes Usher Syndrome
Samer Khateb1, Lina Zelinger1, Liliana Mizrahi-Meissonnier1,
Carmen Ayuso2, Robert K. Koenekoop3, Uri Laxer4, Menachem
Gross5, Eyal Banin1, Dror Sharon1. 1Department of Ophthalmology,
Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
2
Department of Genetics, Instituto de Investigacion SanitariaFundacion Jimenez Diaz (IIS-FJD), ), CIBERER, ISCIII, Madrid,
Spain; 3Department of Human Genetics, McGill University Health
Centre, Montreal, QC, Canada; 4Department of Pulmonology,
Hadassah-Hebrew University Medical Center, Jerusalem, Israel;
5
Department of Otolaryngology - Head and Neck Surgery, HadassahHebrew University Medical Center, Jerusalem, Israel.
Purpose: To use the combination of homozygosity mapping and
whole exome sequencing to identify the disease-causing gene in
members of the same family presented with different phenotypes.
Methods: A consanguineous family (MOL0028) of Iranian Jewish
origin including six siblings affected with SNHL and retinal
degeneration without vestibular dysfunction. A combination of
homozygosity mapping and whole exome sequencing (WES) in
MOL0028 were applied on DNA samples of memebers with different
clinical phenotypes combined with electrophysiological tests and
laboratory molecular assays.
Results: We identified in family MOL0028 nonsense mutations
in two genes encoding ciliary proteins: c.3289C>T (p.Q1097*) in
C2orf71 (reported to cause nonsyndromic retinal degeneration)
and c.3463C>T (p.R1155*) in the centrosome-associated protein
CEP250 (encoding the C-Nap1 protein). The CEP250 gene has not
been associated thus far with any inherited human disease and the
c.3463C>T mutation was absent in 160 ethnicity-matched control
chromosomes and in about 13,000 chromosomes reported at the
exome variant server. Patients who were double homozygotes for
both mutations had SNHL accompanied by early-onset and severe
retinitis pigmentosa (RP), while patients who were homozygous for
the CEP250 mutation (and heterozygous for the C2orf71 mutation)
had SNHL with a relatively mild form of retinal degeneration.
No ciliary structural abnormalities in the respiratory system were
evident by electron microscopy analysis in patients with either
genotype combination. RT-PCR analysis in blood samples of patients
homozygous for the CEP250 mutation revealed expression of a
mutant transcript at levels similar to the expression of the wildtype
transcript in normal control individuals. The mutant transcript results
in the production of an abnormal truncated protein lacking the
NEK2-phosphorylation region. By evolutionary analysis we show
that C-Nap1 and rootletin are paralogues that arose by genomic
duplication about 500 million years ago.
Conclusions: Our data indicate that a homozygous nonsense
mutation in CEP250 causes a novel type of Usher syndrome,
characterized by early-onset hearing loss and a relatively mild retinal
degeneration. The more severe retinal involvement in the double
CEP250 and C2orf71 homozygotes may indicate an additive effect
caused by nonsense mutations in genes encoding two ciliary proteins.
Commercial Relationships: Samer Khateb, None; Lina Zelinger,
None; Liliana Mizrahi-Meissonnier, None; Carmen Ayuso, None;
Robert K. Koenekoop, None; Uri Laxer, None; Menachem Gross,
None; Eyal Banin, None; Dror Sharon, None
Support: FFB grant BR-GE-0510-0490-HUJ, Yedidut 1 grant, Israeli
Ministry of Health grant 3-00000-9177
Program Number: 1713 Poster Board Number: A0082
Presentation Time: 11:00 AM–12:45 PM
Suppressing Thyroid Hormone Signaling Preserves Cone
Photoreceptors in Mouse Models of Retinal Degeneration
Xi-Qin Ding1, Hongwei Ma1, Arjun Thapa1, Lynsie Morris1, T M.
Redmond2, Wolfgang Baehr3. 1Cell Biology, Univ Oklahoma Hlth
Sciences Ctr, Oklahoma City, OK; 2The Laboratory of Retinal Cell
and Molecular Biology, National Eye Institute, Bethesda, MD; 3John
A. Moran Eye Center, University of Utah, Salt Lake City, UT.
Purpose: Cone phototransduction and survival of cones in the human
macula is essential for color vision and for visual acuity. Progressive
cone degeneration in age-related macular degeneration, Stargardt
disease, and recessive cone dystrophies is a major cause of blindness.
Thyroid hormone (TH) signaling which regulates cell proliferation,
differentiation, and apoptosis plays a central role in cone opsin
expression and patterning in the retina. Here, we investigated whether
TH signaling affects cone viability in inherited retinal degeneration
mouse models.
Methods: Rpe65-/- mice (a model of severe Leber congenital
amaurosis or LCA) and Cpfl1 mice (severe recessive achromatopsia)
were used to determine whether suppressing TH signaling (with
anti-thyroid treatment) reduces cone death. Further, Cngb3-/- mice
(moderate achromatopsia) and Gucy2e-/- mice (moderate LCA)
were used to determine whether stimulating TH signaling (with
triiodothyronine (T3) treatment) deteriorates cones. The serum
T3 levels were analyzed by ELISA. Cone and rod survival were
evaluated by examining cone density and expression levels of cone
specific proteins using immunohistochemical and biochemical
approaches, and by examining morphological integrity of the retinas.
Results: Cone density increased about 6-fold in Rpe65-/- and cpfl1
mice following anti-thyroid treatment and decreased about 40% in
Cngb3-/- and Gucy2e-/- mice following T3 treatment. Anti-thyroid
treatment did not affect rod survival, manifested as unchanged outer
nuclear layer (ONL) thickness and the number of nuclei in ONL.
However, T3 treatment significantly reduced ONL thickness and the
number of nuclei in ONL in Cngb3-/- and Gucy2e-/- mice.
Conclusions: With multiple retinal degeneration mouse models,
we demonstrate that TH signaling regulates photoreceptor viability
in degenerating retinas. Suppressing TH signaling protects cones
whereas stimulating TH signaling has a negative effect on both cones
and rods. The findings of this study provide new insights into cone
preservation and therapeutic interventions.
Commercial Relationships: Xi-Qin Ding, None; Hongwei Ma,
None; Arjun Thapa, None; Lynsie Morris, None; T M. Redmond,
None; Wolfgang Baehr, None
Support: This work was supported by grants from the National
Center for Research Resources (P20RR017703), the National Eye
Institute (P30EY12190, R01EY019490, and R01EY08123), and the
Oklahoma Center for the Advancement of Science & Technology.
Program Number: 1714 Poster Board Number: A0083
Presentation Time: 11:00 AM–12:45 PM
The potential for restoration of retinal structure and function
following neural remodeling
Tian Wang1, Greg Field1, Francis Concepcion1, 2, Jeannie Chen1.
1
Zilkha Neurogenetic Institute, University of Southern California,
Los Angeles, CA; 2Department of Ophthalmology, University of
Washington, Seattle, WA.
Purpose: Most blinding disorders in humans are initiated by death
of photoreceptor cells. This is followed by retinal remodeling that
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
includes neuronal death, cell migration and rewiring of retinal
circuits. How these processes impact therapeutic efforts to restore
vision is unclear. To address this issue, we have generated a cyclicnucleotide gated channel knockout (CNGB1-/-) mouse line that is
capable of gene reactivation upon Cre-mediated recombination.
CNGB1-/- mice display a slow rate of retinal degeneration and the
stereotypic activation of neural remodeling markers during the retinal
degeneration process. By inducing CNGB1 protein expression at
different degeneration stages, we studied the potential of morphologic
and functional recovery after neural remodeling.
Methods: The insertion of the LoxP-flanked neomycin cassette
by homologous recombination precludes endogenous CNGB1
gene expression and leads to a slow rate of retinal degeneration.
The CNGB1-/- mice were bred with CAG-Cre/Esr1* (Jackson
lab #004682). The Cre/Esr1* fusion protein can be activated at
different ages by tamoxifen (TM) administration to remove the
neomycin cassette and restore the expression of CNGB1ΔCaM protein.
Phototransduction protein levels were examined by western blots
after TM administration. Retinal structures were investigated by light
microscopy as well as immunocytochemistry. Functional recovery
will be monitored by the electroretinogram and multielectrode array
recordings from retinal ganglion cells.
Results: After TM administration on one-month-old CAG-Cre/
Esr1*+/CNGB1-/- mice, CNGB1ΔCaM protein expression was
restored to similar level as wildtype CNGB1 protein, as were other
phototransduction proteins, and photoreceptor cell death was halted.
However, Müller cell activation and decrease of the ribbon synaptic
markers persists long after CNGB1 gene activation.
Conclusions: Cre-mediated CNGB1ΔCaM expression from the
endogenous locus restored expression of other phototransduction
proteins and halted photoreceptor cell death. These rescue effects
can also be achieved in later stages of retinal degeneration. However,
retinal remodeling persists after rescue. The functional consequences
of the persistence of retinal remodeling will be investigated.
Commercial Relationships: Tian Wang, None; Greg Field, None;
Francis Concepcion, None; Jeannie Chen, None
Support: Beckman Initiative for Macular Research, Whitehall
Foundation
Program Number: 1715 Poster Board Number: A0084
Presentation Time: 11:00 AM–12:45 PM
Retinitis Pigmentosa: Effects of homozygous vs. compound
heterozygous Pde6b mutations on the progression of retinal
degeneration
Francois Paquet-Durand1, Ayse Sahaboglu1, Dragana Trifunovic1,
Marius Ueffing1, Thomas Euler1, 2. 1Experimental Ophthalmology,
Institute for Ophthalmic Research, Tuebingen, Germany; 2Center
for Integrative Neuroscience, University of Tuebingen, Tuebingen,
Germany.
Purpose: Retinitis Pigmentosa (RP) is a group of hereditary retinal
diseases, characterized by mutation-induced rod photoreceptor
degeneration. Until today, no adequate treatment is available and the
photoreceptor degeneration mechanisms remain poorly understood.
Animal models used for the study of RP are usually homozygous for
the causative genetic defect. Yet, in the human situation compound
heterozygosity is the norm.
Methods: To study the effects of compound heterozygosity, we crossbred the Pde6b mouse mutants rd1 and rd10. We used the TUNEL
assay and a time-series to establish a time-line for the progression
of photoreceptor cell death in homozygous rd1/rd1 and rd10/rd10,
and in compound heterozygous rd1/rd10 retina. We then assessed
the numbers of surviving photoreceptors at different post-natal (PN)
time-points and correlated it with the length of the outer segments.
Immunofluorescence was employed to assess the accumulation of
cGMP, to indirectly estimate the activity of PDE6.
Results: The analysis of cell death markers in the different mutants
showed degeneration onsets ranging from PN11 (rd1), to PN14
(rd1/rd10), to PN18 (rd10). While the rd1 mouse degeneration was
characterized by a single peak of cell death at PN13, in the rd1/rd10
there were two peaks of cell death at PN15 and PN24, a phenomenon
that was even more marked in rd10 retina. Interestingly, the
degeneration speed in rd1/rd10 was higher than in both rd1 and rd10
homozygous mutants.
Conclusions: Our results suggest that, while the degeneration onset
is defined linearly by addition of genetic defects, the degeneration
kinetics follow a non-linear pattern that may result from a complex
interaction of PDE6 activity and outer segment length. Since many
human RP patients suffer from compound heterozygous mutations,
our study may have important implications for the translation
of experimental animal work into clinical trials, in particular for
determining the time-lines and optimal time-points for clinical
interventions.
Commercial Relationships: Francois Paquet-Durand, None; Ayse
Sahaboglu, None; Dragana Trifunovic, None; Marius Ueffing,
None; Thomas Euler, None
Support: DFG (PA1751/4-1); EU (DRUGSFORD:
HEALTH-F2-2012-304963); Alcon Research Institute; Kerstan
Foundation (RD-CURE, WP2-2)
Program Number: 1716 Poster Board Number: A0085
Presentation Time: 11:00 AM–12:45 PM
Inhibition of polyamine oxidase reduces hyperoxia-mediated
retinal neuro-vascular injury in a model of retinopathy of
prematurity
S. Priya Narayanan1, 2, Zhimin Xu1, 2, Tahira Lemtalsi1, 2, Robert
William Caldwell3, 2, Ruth B. Caldwell1, 4. 1Vascular Biology Center,
Georgia Regents University, Augusta, GA; 2Vision Discovery
Institute, Georgia Regents University, Augusta, GA; 3Deapartment of
Pharmacology and Toxicology, Georgia Regents University, Augusta,
GA; 4VA medical center, Augusta, GA.
Purpose: Retinopathy of prematurity (ROP) is a potentially blinding
eye disorder that affects infants born prematurely. It is one of the
most common causes of vision loss in childhood and can lead
to lifelong vision impairment and blindness. Using an oxygeninduced retinopathy (OIR) mouse model for ROP, we have shown
that deletion of the arginase2 significantly reduces neuronal injury
through the regulation of polyamine metabolism (Narayanan et
al ARVO 2012).The present study was undertaken to determine
whether inhibiting polyamine oxidase using MDL 72527 (N, Nʹ-Bis
(2, 3-butadienyl)-1, 4-butanediamine dihydrochloride) can reduce
neuronal, glial and vascular injury in the OIR retina.
Methods: Newborn wild-type (WT) mice were used. Pups were
exposed 70% oxygen from postnatal day 7 to 12, returned to room
air, and were sacrificed at different stages of OIR. Pups were
treated daily with MDL 72527 (39 mg/Kg of body weight, in saline,
intraperitoneal). Vehicle groups received intraperitoneal injections of
saline. Retinal cryostat sections were prepared for immunostaining
analysis and TUNEL labeling of apoptotic cells. Analysis of vasoobliteration was performed on retinal flatmounts. Fresh frozen retinal
samples were used for Western blotting analysis.
Results: During the hyperoxic phase of OIR, a significant increase
in the number of apoptotic cells was observed in the WT retina
compared to normoxic controls. Treatment using MDL 72527
significantly reduced the number of TUNEL positive cells in the
OIR retina compared to vehicle treated groups (p< 0.01, N=5).
Immunolabeling studies using synaptophysin antibody showed the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
presence of improved synaptic contacts in MDL 72527-treated OIR
retina compared to vehicle treated groups. Vaso-obliteration analyses
using retinal flatmounts showed significantly reduced avascular area
in MDL 72527-treated OIR retina compared to vehicle-treated (p<
0.05, N=6) retinas after 2 or 5 days of hyperoxia. Immunolabelling
studies using Iba1 antibody demonstrated that activation of microglia
was markedly reduced in MDL 72527-treated OIR retina. Studies on
signaling pathways showed the involvement of JNK/SAPK pathway
and cytochrome C in the polyamine oxidase mediated neuro-vascular
injury during OIR treatment.
Conclusions: Our data suggest that polyamine oxidase is crucially
involved in hyperoxia-induced neuro-vascular injury in the ROP
retina.
Commercial Relationships: S. Priya Narayanan, None; Zhimin
Xu, None; Tahira Lemtalsi, None; Robert William Caldwell,
None; Ruth B. Caldwell, None
Support: American Heart Association:11SDG7440088 (SPN); NEIEY11766, VA Merit Review(RBC)
Program Number: 1717 Poster Board Number: A0086
Presentation Time: 11:00 AM–12:45 PM
Serum Nampt/PBEF/visfatin levels correlate with incidence of
retinal vein occlusions
Simon Kaja, Anna A. Shah, Shamim A. Haji, Krishna Patel, Yuliya
Naumchuk, Nancy Kunjukunju, Nelson R. Sabates, Michael A.
Cassell, Abraham Poulose, Peter Koulen. Vision Research Center,
Department of Ophthalmology, University of Missouri - Kansas City,
Kansas City, MO.
Purpose: Nampt (nicotinamide phosphoribosyltransferase) / PBEF
(Pre B-Cell colony enhancing factor) / visfatin is a critical ratelimiting enzyme in the nicotinamide adenine dinucleotide pathway
critical for cellular energy metabolism. Furthermore, Nampt/PBEF/
visfatin has been shown to possess pro-inflammatory activity
as a cytokine and participate in the regulation of blood glucose
levels. Retinal vascular occlusions (RVOs) are disease conditions
characterized by pronounced ischemia and metabolic energy deficits.
Currently, there is currently no intervention that has emerged as the
standard of care for RVOs. Given the critical role of Nampt/PBEF/
visfatin in energy metabolism, and the increased energy demand
during traumatic cellular events such as ischemia, we hypothesized
Nampt/PBEF/visfatin serum levels may potentially serve as a
biomarker or predictor of treatment outcome for RVOs.
Methods: We quantified Nampt/PBEF/visfatin in serum obtained
from healthy controls and patients, who presented with RVO, using a
commercially available enzyme-linked immunosorbent assay.
Results: Nampt/PBEF/visfatin serum levels were 79% lower in
patients with a history of RVO compared with healthy volunteers
(P<0.05). There was no statistically significant difference among
types of RVOs, specifically branch retinal vein occlusions (n=7),
central retinal vein occlusions (n=5), hemi retinal vein occlusions
(n=3) and central retinal artery occlusions (n=3; P=0.69).
Conclusions: Low Nampt/PBEF/visfatin serum levels might serve as
a potential biomarker for increased risk for RVO and Nampt/PBEF/
visfatin deficiency may contribute to the etiology of RVO. Nampt/
PBEF/visfatin represents a potentially novel, druggable target that
can be exploited in future therapy development efforts for RVOs and
reduce the long-term complications associated with the condition,
such as macular edema, macular ischemia, neovascularization and
irreparable loss of vision.
Commercial Relationships: Simon Kaja, None; Anna A. Shah,
None; Shamim A. Haji, None; Krishna Patel, None; Yuliya
Naumchuk, None; Nancy Kunjukunju, None; Nelson R. Sabates,
None; Michael A. Cassell, None; Abraham Poulose, None; Peter
Koulen, None
Support: NIH grants EY014227, EY022774, AG010485, AG022550,
AG027956, RR022570, RR027093, Felix and Carmen Sabates
Missouri Endowed Chair in Vision Research, Challenge Grant from
Research to Prevent Blindness, Vision Research Foundation of
Kansas City
Program Number: 1718 Poster Board Number: A0087
Presentation Time: 11:00 AM–12:45 PM
Progression of Pro23His Retinopathy in a Miniature Swine
Model of Retinitis Pigmentosa
Patrick A. Scott, Juan P. Fernandez de Castro, Maureen A. McCall,
Henry J. Kaplan. Ophthalmology & Visual Sciences, University of
Louisville, Louisville, KY.
Purpose: We have recently generated transgenic P23H (TgP23H)
miniature swine founders that exhibit similar characteristics to
autosomal dominant retinitis pigmentsosa (RP) in humans. However,
morphological changes that occur in retinas in TgP23H swine
were not examined until 12 months of age. Here we tracked retinal
function and morphology from 1 month to 18 months in F1 progeny
of TgP23H miniswine founder 53-1.
Methods: Scotopic (rod-driven) and photopic (cone-driven) retinal
function were evaluated in TgP23H and Wt (Wild-type) littermates
using full field electroretinograms (ffERGs) at 1, 2, 3, 6, 9, 12, and
18 months of age. Miniswine were euthanized and their retinas
processed for morphological evaluation at the light and electron
microscopic level.
Results: Wt littermates reached mature scotopic and photopic
retinal function by 3 months, while TgP23H miniswine showed
abnormal scotopic ffERGs at the earliest time point, 1 month,
and depressed photopic ffERGs after 2 months. Rod and cone
photoreceptors exhibited morphological abnormalities and dropout
from the outer nuclear layer at 1 month, with only a monolayer of
cone photoreceptor somata remaining in the outer nuclear layer after
2 months. The retina showed progressive neural remodeling of the
outer retina that included dendritic retraction of rod bipolar cells
and glial seal formation by Müller cells that was evident by 3 and 9
months, respectively.
Conclusions: TgP23H offspring from miniswine founder 53-1
exhibit functional and morphological features similar to humans with
aggressive RP. TgP23H miniature swine are a useful large-eye model
to study pathogenesis and preservation of cone photoreceptors in
human RP.
Commercial Relationships: Patrick A. Scott, None; Juan P.
Fernandez de Castro, None; Maureen A. McCall, None; Henry J.
Kaplan, None
Support: Supported by University of Louisville, School of Medicine
Basic Research Grant (PAS); American Optometric Foundation and
Beta Sigma Kappa Optometric Honor Society (PAS); Fight For Sight
(PAS); NEI R21 EY020647 (HJK); KY Science and Engineering
Foundation (HJK); University of Louisville Clinical and Translational
Science Grant (HJK); The Kentucky Challenge Research Trust Fund
(HJK); Research to Prevent Blindness, New York, NY (HJK); KY
Science and Engineering Foundation (HJK); University of Louisville
Clinical and Translational Science Grant (HJK;, NIH EY018608
(MAMc), NIH HL076138-08 (JPF).
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1719 Poster Board Number: A0088
Presentation Time: 11:00 AM–12:45 PM
Regulation of Photoreceptor Survival and Tissue Remodeling by
the Wnt Pathway
Sapir Karli, Amit K. Patel, Krishna R. Surapaneni, BaoXang Li,
Hyun Yi, Abigail S. Hackam. Bascom Palmer Eye Institute, University
of Miami Miller School of Medicine, Miami, FL.
Purpose: A major obstacle for treating retinal degenerations is
pathologic tissue remodeling, which leads to aberrant neurite growth
and gliosis. We tested the hypothesis that activating Wnt signaling
specifically in Muller glia will increase photoreceptor survival and
decrease neuronal remodeling in the rd10 mouse model of retinal
degeneration. We also evaluated whether Wnt regulates remodeling
independently of photoreceptor protection.
Methods: Mice were subretinally injected with adenovirus
containing non-secreted Wnt activator β-cateninS33A or Wnt
inhibitor β-eng genes, both under control of the 2.2kb GFAP
promoter. GFP and PBS were injection controls. Photoreceptor
survival was measured by nuclei row counts, ERG’s and Western
blotting. Tissue remodeling was assessed by co-localization of the
GFP reporter in the adenovirus with GFAP or Ki67, and by Western
blotting for PKCα.
Results: GFP labeling indicated that the genes were delivered to at
least a third of the retina, and its expression co-localized with Muller
glia markers. β-cateninS33A increased Wnt signaling by 2.5-fold,
compared with control injections (n=3). Muller glia-specific Wnt
activation increased the number of photoreceptor rows by 44%
compared with Wnt inhibition (n=5, p<0.01), increased a-wave
amplitudes by 4.5-fold, and increased rhodopsin and cone transducin
levels by 2-5-fold (n=3). Furthermore, Wnt activation reduced
PKCα levels by 2-fold in both wildtype mice and rescued rd10 mice,
indicating reduced neuronal remodeling. In contrast, Wnt increased
PKCα by 1.8-fold in post-degeneration retinas showing increased
remodeling in absence of protection. There was also a significant
reduction in GFAP-positive activated Muller glia in retinas injected
with the Wnt activator compared with the Wnt inhibitor (n=3,
p<0.01), and no change in the number of proliferating Ki67-positive
Muller glia.
Conclusions: We demonstrated that Wnt plays a neuroprotective
role by increasing photoreceptor survival while concomitantly
decreasing neuronal remodeling during degeneration. In contrast,
Wnt signaling increases remodeling post degeneration, indicating that
its role in remodeling is independent of its protective functions. These
results will allow us to determine an appropriate Wnt activation
treatment window for retinal degeneration, during which there is
neuroprotection without tissue reorganization to keep the retina
functionally viable.
Commercial Relationships: Sapir Karli, None; Amit K. Patel,
None; Krishna R. Surapaneni, None; BaoXang Li, None; Hyun Yi,
None; Abigail S. Hackam, None
Support: the Karl Kirchgessner Foundation, NIH grant RO1
EY017837. Institutional support to BPEI was from a Research
to Prevent Blindness Unrestricted Grant and an NEI Center Core
Grant P30EY014801; Research to Prevent Blindness Ernest &
Elizabeth Althouse Special Scholar Award, the Karl Kirchgessner
Foundation, NIH grant RO1 EY017837, and a Fight for Sight Student
Fellowship. Institutional support to BPEI was from a Research to
Prevent Blindness Unrestricted Grant and an NEI Center Core Grant
P30EY014801; the Karl Kirchgessner Foundation, NIH grant RO1
EY017837. Institutional support to BPEI was from a Research to
Prevent Blindness Unrestricted Grant and an NEI Center Core Grant
P30EY014801; the Karl Kirchgessner Foundation, California Table
Grape Commission, NIH grant RO1 EY017837; Institutional support
to BPEI was from a Research to Prevent Blindness Unrestricted
Grant and an NEI Center Core Grant P30EY014801
Program Number: 1720 Poster Board Number: A0089
Presentation Time: 11:00 AM–12:45 PM
In vivo and post mortem analysis of the outer retina and RPE in
a mouse model for X-linked retinitis pigmentosa
Knut Stieger, Jutta U. Schlegel, Dorothee Röll, Brigitte Müller,
Baerbel Fuehler, Birgit Lorenz. Department of Ophthalmology,
Justus-Liebig-University Giessen, Giessen, Germany.
Purpose: Mutations in the Retinitis pigmentosa GTPase regulator
(RPGR) are associated with X-linked Retinitis pigmentosa (XLRP)
and account for up to 20% of all RP cases. No treatment option exists
to date. The aim of this study is to analyse morphological alterations
in the outer retina and RPE of a new mouse model for XLRP in vivo
and post mortem in order to further decipher the pathomechanism of
the disease.
Methods: A mouse model for XLRP was generated by introducing
a pathological mutation (2793delA), two silent mutations
(3071subT-A; 2650subT-C), as well as an ISceI recognition site into
C57BL6/129sv hybrid embryonic stem cells through homologous
recombination and breeding of the chimeric animals into a C57BL/6J
background. Time points for the analysis were 1, 3, 6, 9, 12, 15, and
18 months of age (M). Animals were examined in vivo by optical
coherence tomography (OCT) and funduscopy (MICRON III System,
Phoenix Research Inc.) in the peripapillary area and in the periphery.
Post mortem analysis included Hematoxylin/Eosin (H/E) staining of
paraffin sections, immunofluorescence staining (L/M-opsin, S-opsin,
centrin, RPGR) and electronmicroscopy (EM) analysis.
Results: OCT examinations revealed reduced visibility of the outer
retinal layers including the inner segment ellipsoid (Ise), and outer
segments as early as at 3 M in affected as well as carrier animals,
while the ONL thickness only decreased starting at 12 M. Focal white
spots were visible on the fundus image at 3 M. Interestingly, these
alterations were more pronounced in carriers. Post mortem analysis
showed progressive disorganisation of inner and outer segments of
the photoreceptors starting at 1 M and a reduction in the number of
photoreceptor nuclei starting at 12 M. Altered connecting cilium
structure and reduced opsin transport was visible early in the disease
process.
Conclusions: The newly generated mouse model shows early onset
of morphological alterations in the outer retina and RPE, which can
be visualized and quantified in vivo as well as post mortem. Since the
model has been designed specifically for studying new therapeutic
approaches in XLRP treatment, these morphological biomarkers can
now be used to analyse a possible treatment effect.
Commercial Relationships: Knut Stieger, None; Jutta U. Schlegel,
None; Dorothee Röll, None; Brigitte Müller, None; Baerbel
Fuehler, None; Birgit Lorenz, None
Support: ERC starting grant
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1721 Poster Board Number: A0090
Presentation Time: 11:00 AM–12:45 PM
A study of aged GK rats: is tau the missing link between diabetes
and Alzheimer’s Disease?
Timothy Wong1, Marianne Phillips1, Doris Chan1, Shereen Nizari1,
Damian Cummings3, M Francesca Cordeiro1, 2. 1Visual Neuroscience,
UCL Institute of Ophthalmology, London, United Kingdom; 2Western
Eye Hospital, Imperial College Healthcare Trust, London, United
Kingdom; 3Neuroscience, Physiology & Pharmacology, University
College London, London, United Kingdom.
Purpose: Recent research has shown that despite their different
features, diabetes and Alzheimer’s disease (AD) share similar
pathophysiology including abnormal glucose metabolism, insulin
resistance and impaired insulin signalling. AD is the most common
type of dementia and its pathological hallmarks include extracellular
β-amyloid plaques and intracellular neurofibrillary tangles (NFTs).
Diabetes is a metabolic condition characterized by insulin deficiency
(Type 1), insulin resistance and defective insulin signalling (Type 2).
Patients with Type 2 diabetes (T2DM) have an approximately 60%
increased risk of developing AD.
Goto Kakizaki (GK) rats are spontaneously diabetic and are
considered a model for T2DM in humans. As the brain and retina
share a common embryological origin, we have hypothesised that
AD pathology in the brains of AD transgenic mice also occurs
in the retina. Here, we investigate whether tau pathology and
neurofilamentopathy are present in the retinas of aged GK rats.
Methods: The eyes from 3-, 12- and 18-month old spontaneously
diabetic GK rats (n=4 for each age group) and age-matched controls
(n=3 for 3-month old; n=4 for 12- and 18-month old) were obtained
and embedded in paraffin blocks. Sections from these blocks were
stained with antibodies for tau, hyperphosphorylated tau (p-tau) and
NF-heavy (NFH). Immunohistochemistry images were visualized and
obtained. These images were graded for distribution and fluorescence
of the antibodies by three independent observers without knowledge
of diabetic or control status.
Results: Analysis showed an age-dependent increase of p-tau
(p<0.001) and NFH (p<0.001) in the diabetic retina, but not the
control retina. Increased levels of p-tau (p<0.01) and NFH (p<0.001)
were found in the diabetic retina as compared to the age-matched
control retina. No change of colocalization of p-tau and NFH was
found in the control or diabetic retina over time.
Conclusions: Recent evidence has suggested that neurodegeneration
is an early phenomenon in DM. This study demonstrated an increase
in tau-associated neurodegeneration in the diabetic retina over time
and as compared to the control retina. As far as we are aware, this is
the first study demonstrating AD tau pathology in the retinas of aged
diabetic GK rats.
Commercial Relationships: Timothy Wong, None; Marianne
Phillips, None; Doris Chan, None; Shereen Nizari, None; Damian
Cummings, None; M Francesca Cordeiro, None
Program Number: 1722 Poster Board Number: A0091
Presentation Time: 11:00 AM–12:45 PM
Human umbilical tissue-derived cells rescue phagocytosis in
cultured retinal pigment epithelial cells from Royal College
of Surgeons rat through secretion of receptor tyrosine kinase
ligands
George Inana1, Christopher Murat1, Xiang Yao2, Ian R. Harris3,
Jing Cao3. 1Ophthalmology, Bascom Palmer Eye Institute, Miami,
FL; 2Janssen Research and Development, San Diego, CA; 3Janssen
Research and Development, Spring House, PA.
Purpose: It is well established that retinal pigment epithelial (RPE)
cells of Royal College of Surgeons (RCS) rat exhibit impaired rod
outer segment (ROS) phagocytosis due to mutated Mertk gene. Lund
et al. have demonstrated that subretinal injection of human umbilical
tissue derived cells (hUTC) into the RCS rat eye improved visual
acuity and ameliorated retinal degeneration. However, the mechanism
of how hUTC improves vision in the RCS rat is unclear. Mertk is
a member of the receptor tyrosine kinase (RTK) family and plays
a crucial role in RPE phagocytosis. Basic fibroblast growth factor
(bFGF), a ligand of the FGF receptor, a member of RTK, was shown
to restore phagocytic function in cultured RPE cells from RCS rats.
This leads to our hypothesis that other RTK ligands may compensate
for the role of Mertk in RCS RPE cell, and hUTC may restore the
phagocytic function of RCS RPE cell through release of RTK ligands.
To test this hypothesis, we investigated the secretion of RTK ligands
by hUTC and determined their effects on phagocytosis.
Methods: The effect of hUTC on RCS RPE phagocytosis was
examined in cells pre-incubated with hUTC conditioned medium
(CM) for 24 h and subjected to phagocytosis. RNA-Seq based
transcriptome profiling was performed to identify gene expression
in hUTC and RCS RPE cells. Levels of expressed RTK ligands,
including brain-derived neurotrophic factor (BDNF), hepatocyte
growth factor (HGF), and platelet-derived growth factor type D
(PDGF-DD) were measured in hUTC CM by ELISA, and their
effects on RCS RPE phagocytosis were examined by pre-incubating
cells with the corresponding recombinant human proteins followed
by phagocytosis assay in the presence of the respective ligand
protein.
Results: hUTC CM significantly rescued phagocytosis in RCS
RPE cells, comparable to that of normal RPE cells. Transcriptome
profile analysis demonstrated that RCS RPE cells express multiple
RTK genes, while hUTC expresses genes for multiple RTK ligands.
Selected RTK ligands, BDNF, HGF and PDGF-DD, could be
detected in hUTC CM. Recombinant human BDNF, HGF, and
PDGF-DD had a statistically significant rescue effect on phagocytosis
in the RCS RPE cells.
Conclusions: These results suggest that hUTC could rescue RCS
RPE cell phagocytosis in a Mertk-independent manner, possibly
through secretion of RTK ligands.
Commercial Relationships: George Inana, Janssen Research and
Development (F); Christopher Murat, None; Xiang Yao, Janssen
Research and Development (E); Ian R. Harris, Janssen Research and
Development (E); Jing Cao, Janssen Research and Development (E)
Support: Janssen R&D, NIH Center Core Grant P30EY014801, RPB
Unrestricted Award to the Department
Program Number: 1723 Poster Board Number: A0092
Presentation Time: 11:00 AM–12:45 PM
The role of WNT singling activation and its blockage in the focal
retinal lesion of Ccl2-/-/Cx3cr1-/- mice with and without rd8
background
Jingsheng Tuo1, Yujuan Wang1, Rui Cheng2, Yichao Li3, Mei Chen4,
Haohua Qian3, Mones S. Abu-Asab1, Heping Xu4, Jian-Xing Ma2,
Chi-Chao Chan1. 1Laboratory of Immunology, National Eye Institute/
NIH, Rockville, MD; 2Department of Physiology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK; 3Core of
visual function, National Eye Institute, Bethesda, MD; 4Centre for
Vision & Vascular Science, Queen’s University Belfast, Belfast,
United Kingdom.
Purpose: The canonical Wnt signaling is activated by retinal
injury. Under disease conditions, the Wnt mediates inflammatory
responses. Inflammation has been detected in age-related macular
degeneration (AMD) retinas and Ccl2-/-/Cx3cr1-/- (DKO) mice with
or without rd8 background, a model with progressive AMD-like
lesions including focal photoreceptor/RPE degeneration and A2E
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
accumulation. We evaluated the effects of Wnt-β-catenin activation
and an antibody against LRP6, the co-receptor of Wnt on these two
models.
Methods: anti-LRP6 antibody (2F1, 1 μl of 5 μg/μL) was
intravitreally injected into the right eyes in 3 separate experiments
(DKOrd8, N=35; DKO, N=10). The left eyes were injected with
mouse IgG as controls. Fundoscopy was taken before injection and
sequentially monthly after injection. Two months after injection,
light-adapted ERG responses were recorded; then the eyes were
harvested for histopathology, the determination of retinal A2E, and
molecular analysis. The microarray of ocular mRNA of 92 Wnt
genes was compared between the treated and the control eyes. The
phosphorylated types of LRP6 and β-catenin and endogenous forms
of the proteins were assayed by Western blotting.
Results: For DKOrd8 mice, the fundus showed a slower progression
or alleviation of retinal lesions in the right eyes as compared to the
left eyes. Among 35 pairs of eyes, 26 (74.3%) were improved, 7
(20%) stayed the same and 2 (5.7%) remained progressing. Histology
confirmed the clinical observation. Light-adapted ERG of the treated
eyes exhibited larger amplitudes compared to control eyes (n=6),
with greater improvements under UV light stimulus. There was a
significantly lower A2E in the treated eyes compared to controls.
Microarray of 92 Wnt genes expression pattern was similar in
both eyes. Western blotting indicated local administration of 2F1
antibody to suppress the activation of Wnt pathway in the retina.
For DKO mice, the treatment improved ERG but less effect on RPE
degeneration.
Conclusions: The canonical Wnt signaling plays a role in the focal
retina lesion of both DKOrd8 and DKO mice; and intravitreal
anti-LRP6 antibody might be neuroprotective via deactivation of
canonical Wnt pathway.
Commercial Relationships: Jingsheng Tuo, None; Yujuan Wang,
None; Rui Cheng, None; Yichao Li, None; Mei Chen, None;
Haohua Qian, None; Mones S. Abu-Asab, None; Heping Xu,
None; Jian-Xing Ma, None; Chi-Chao Chan, None
Support: NEI intrmural fund
Program Number: 1724 Poster Board Number: A0093
Presentation Time: 11:00 AM–12:45 PM
FK962 enhances neurite elongation in cultured rat retinal
ganglion cells
Masaki Kakehi1, Chiho Yabuta1, Ayumi Yoshimatsu1, Yayoi
Kishimoto1, Thomas R. Shearer2, Mitsuyoshi Azuma1, 2. 1Senju
Laboratory of Ocular Sciences, Senju Pharmaceutical Co.,Ltd,
Kobe,Hyogo, Japan; 2Department of Integrative Biosciences, Oregon
Health & Science University, Portland, OR.
Purpose: Glaucoma is a progressive optic neuropathy, and a major
risk factor is elevated intraocular pressure (IOP). Various drugs to
reduce IOP are widely used to treat glaucoma, but control of IOP is
not fully successful. Neuroprotection is a new proposed treatment
for glaucoma. Damage to axons in the optic nerve disturbs axonal
transport and leads to retinal ganglion cell death. Endogenous
neurotrophic factors contribute to the regeneration of axons. Our
previous study showed that FK962 induces neurite elongation in
cultured trigeminal ganglion cells from rabbits and rats via induction
of glial cell-line derived neurotrophic factor. The purpose of the
present experiments was to test if FK962 promotes neurite elongation
in retinal ganglion cells. An in silico pharmacokinetics model was
also used to predict efficacy of topical application of FK962.
Methods: Neuronal cells (~75% retinal ganglion cells) and isolated
rat retinal ganglion cells were cultured with and without FK962, and
with (mixed cell culture) and without (pure culture) glial cells. Cells
with neurite elongation were counted after the cells were fixed and
immunolabeled with antibodies for neurofilaments or calcein AM.
For the in silico pharmacokinetics study, a modified cylindrical eye
model was used with parameters obtained from permeation studies
with rabbit sclera and sclera-choroid-retina preparations mounted
side-by-side in Ussing diffusion chambers.
Results: FK962 significantly increased the number of neuronal cells
with elongated neurites in mixed cell culture. FK962 also increased
the number of ganglion cells with elongated neurites in pure culture.
The in silico pharmacokinetics study predicted that 10-10 to 10-9
M FK962, which facilitated neurite elongation in vitro, would be
delivered to retina-choroid when a single dose of 0.0001% or 0.001%
FK962 was topically applied.
Conclusions: FK962 may be a good candidate for the therapy of
axonal degeneration caused by glaucoma and optic nerve injury.
Dr. Shearer receives a research contract and consulting fees from, and
Dr. Azuma is an employee of Senju Pharmaceutical Co., Ltd.
Commercial Relationships: Masaki Kakehi, Senju Pharmaceutical
Co.,Ltd (E); Chiho Yabuta, Senju Pharmaceutical Co.,Ltd (E);
Ayumi Yoshimatsu, Senju Pharmaceutical Co.,Ltd (E); Yayoi
Kishimoto, Senju Pharmaceutical Co.,Ltd (E); Thomas R. Shearer,
Senju Pharmaceutical Co.,Ltd (C); Mitsuyoshi Azuma, Senju
Pharmaceutical Co.,Ltd (E)
Program Number: 1725 Poster Board Number: A0094
Presentation Time: 11:00 AM–12:45 PM
Inner retinal layer thickness in eyes with retinitis pigmentosa
Yosuke Nagasaka, Yasuki Ito, Shinji Ueno, Hiroko Terasaki.
Opthalmology, Nagoya Univercity Guraduate School Of Medicine,
Nagoya, Japan.
Purpose: Retinitis pigmentosa (RP) is a disease that causes
photoreceptor degeneration and loss of photoreceptors. Reduction
of retinal thickness due to outer retinal layer loss was known, but
the thickness of inner retina of RP has not been elucidated. Several
studies reported the thickness of inner retina was thicker than that
of normal control but the thickness of each layer in inner retina has
not been investigated. In this study, we have evaluated inner retinal
thickness in eyes of RP using spectral domain optical coherence
tomography (SD-OCT).
Methods: SD-OCT images (Spectralis, Heidelberg Engineering,
Heidelberg, Germany) of 57 eyes of 29 patients of retinitis
pigmentosa (50.6±15.0 years) were analyzed. Thickness of total
neural retina, nerve fiver layer (NFL), ganglion cell layer (GCL),
inner plexiform layer (IPL), inner nuclear layer (INL) and other outer
layers were measured at the 1 and 2 mm superior, inferior, nasal,
or temporal to the fovea in 9 mm vertical and horizontal scan. As
a control, same measurements were performed in 13 normal eyes
of 13 age-matched subjects (51.6±9.7 years). Average thickness of
total neural retina and each layers of these 8 points were compared
between RP patients and normal controls.
Results: The average thickness of the total neural retina of RP
patients was 283.9±49.9mm and was significantly thinner than that
of normal controls of 322.8±23.2mm (p<0.01). The average NFL
thickness of RP patients and normal controls were 40.3±7.9 mm and
30.2±4.0 mm respectively. The GCL thickness of RP patients and
normal controls were 56.8±11.5 mm and 47.8±8.1 mm respectively.
The NFL and GCL thickness were significantly thicker in RP eyes
than normal eyes (p<0.001 and p<0.01 respectively). The IPL
thickness of RP patients and normal controls were 33.7±4.7 mm
and 34.6±3.0 mm respectively and were not significantly different
(p>0.05). The INL thickness of RP and normal controls were
48.0±7.2 mm and 40.5±5.0 mm respectively. The INL thickness
were significantly thicker in RP eyes than normal eyes (p<0.001).
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Thickness of other outer layers of RP patients was 105.0±36.4 mm
and was significantly thinner than normal eyes of 169.7.0±9.8 mm
(p<0.001).
Conclusions: Not only NFL and GCL thickness but also INL
thickness become thicker than normal controls in RP eyes. In eyes of
RP patients, inner retinal layers become thicker except for IPL.
Commercial Relationships: Yosuke Nagasaka, None; Yasuki Ito,
None; Shinji Ueno, None; Hiroko Terasaki, None
Support: Grant-in Aid for Scientific Research from the Ministry of
Education, Culture,Sports, Science, and Technology of Japan(Dr Ito,
C2159225)
Program Number: 1726 Poster Board Number: A0095
Presentation Time: 11:00 AM–12:45 PM
Pupil responses to selective wavelength (colored) light stimulation
in healthy and retinal degenerate mice
Corinne Kostic1, Sylvain Crippa1, Catherine Martin1, Randy H.
Kardon2, Yvan Arsenijevic1, Aki Kawasaki1. 1Dept Ophthalmology,
Univ Lausanne, Jules-Gonin Eye Hosp, Lausanne, Switzerland; 2Dept
Ophthalmology and Visual Science, University of Iowa Hospitals and
Clinics, Iowa, IA.
Purpose: Different light stimulus conditions were used to
characterize the pupillary light reflex (PLR) in different mouse
models.
Methods: Sv129 mice, Rho-/- and CNGA3-/- were subjected to short
stimuli of increasing intensity of red and blue light alternatively. The
PLR was recorded using the A2000 Neuroptics system. Maximal
contraction amplitudes as well as recovery phase were compared
between the different models.
Results: In wild type mice, increasing intensity of blue light
produced prolongation of the pupil contraction and a distinctive
change in the pupil response waveforms, particularly post-stimulus
recovery. Additionally the duration of dark adaptation influenced
both amplitude and recovery of the PLR. In comparison, Rho/- mice showed decreased pupil responses to all blue and red light
stimuli except the highest intensity blue stimulus. Pupil responses
of CNGA3-/- to blue or red light were similar to those of wild type
mice.
Conclusions: Loss of photoreceptors differentially affects the PLR to
light stimuli of different wavelengths and intensity.
Commercial Relationships: Corinne Kostic, None; Sylvain
Crippa, None; Catherine Martin, None; Randy H. Kardon, None;
Yvan Arsenijevic, None; Aki Kawasaki, None
Support: Provisu
Program Number: 1727 Poster Board Number: A0096
Presentation Time: 11:00 AM–12:45 PM
Dendritic degeneration as an early marker of neuronal
degeneration in retinal explants
Kate Binley1, Wai Siene Ng1, Bing Song2, James E. Morgan3, 1.
1
School of Optometry and Vision Sciences, Cardiff University,
Cardiff, United Kingdom; 2School of Dentistry, Cardiff University,
Cardiff, United Kingdom; 3Opthalmology, University Hospital of
Wales, Cardiff, United Kingdom.
Purpose: To determine the relationship between retinal ganglion cell
degeneration and retinal ganglion cell loss in a retinal explant model.
Methods: Adult C57Bl/6 mice retinas were prepared and cultured
as wholemounts for up to 14 days in Neurobasal-A culture medium
and maintained at 37°C in 95/5% O2/CO2. Retinas were fixed and
cryosectioned for nuclear staining, immunohistochemistry, and
TUNEL labelling. Neuronal viability was quantified by calcein-AM
staining. Single retinal ganglion cells were labelled diolistically (DiI
and DiO) using a hand-held gene gun (Bio-Rad) fired at 100 psi.
All cells were imaged confocally and their dendritic architecture
analysed by Sholl analysis. The pan-caspase inhibitor OPH001-01M
was applied to explants over 2 days to determine the extent to which
neuronal degeneration could be inhibited.
Results: Nuclear staining demonstrated a loss of 30% of retinal
ganglion cells over 14 days (p<0.1). Immunohistochemistry
confirmed persistent Thy-1 expression in the retinal ganglion cell
layer during this time. The ganglion cell layer showed a small
increase in apoptotic markers over 14 days: 2.5% active caspase-3
(2±1 cells/mm) and 5% TUNEL (5±3% ganglion cell layer).
Ganglion cell viability decreased by 35% over 14 days as measured
by calcein-AM staining (100±30 viable cells/mm2 after 14 days).
Sholl analysis revealed substantial dendrite loss in retinal ganglion
cells within 24 hours with a reduction in the area under the Sholl plot
of 30% (p<0.05). Following the addition of the pan-caspase inhibitor
immediately after retinal dissection neuronal degeneration was
prevented on the basis of Sholl plots (20% increased area under the
curve relative to control).
Conclusions: In the retinal explant model dendritic degeneration
may be an early marker of retinal ganglion cell loss. Significantly
these changes are seen over a week before substantial cell loss can
be detected. The explant model has potential as a sensitive assay for
neuroprotective interventions.
Commercial Relationships: Kate Binley, None; Wai Siene Ng,
None; Bing Song, None; James E. Morgan, None
Support: BB/F016352/1
Program Number: 1728 Poster Board Number: A0097
Presentation Time: 11:00 AM–12:45 PM
Human umbilical tissue-derived cells secrete bridge molecules
and rescue phagocytosis in cultured retinal pigment epithelial
cells from Royal College of Surgeons rat
Jing Cao1, Chris Murat3, Carol Anne Ogden1, Sandra SantulliMarotto1, Xiang Yao2, Ian R. Harris1, George Inana3. 1Janssen
Pharmaceutical R&D, Spring House, PA; 2Janssen Pharmaceutical
R&D, San Diego, CA; 3Ophthalmology, Bascom Palmer Eye
Institute, Miami, FL.
Purpose: Royal College of Surgeons (RCS) rat, a model of
retinal degeneration, carries a mutation in Mertk resulting in
diminished phagocytosis of rod outer segment (ROS) and leading
to photoreceptor cell death. Retinal pigment epithelium (RPE) cells
from RCS rats showed reduced phagocytosis in vitro. Interestingly,
these cells, when fed with ROS pretreated with human umbilical
tissue-derived cell (hUTC) conditioned medium (CM), showed
significantly restored phagocytosis of ROS, suggesting that hUTC
CM may prime ROS in a way that enhances its phagocytosis. It was
reported that isolated ROS possess externalized phosphatidylserine
(PS), whose blockade or removal reduces their binding and
engulfment by RPE in culture. PS exposure is a hallmark of apoptotic
cells. This leads to our hypothesis that hUTC could regulate RCS
RPE phagocytosis in a similar manner to apoptotic cell clearance
used by other phagocytes through secretion of bridge molecules.
Methods: Transcriptome profiling (RNA-Seq) was performed
to identify gene expression in hUTC and RCS RPE cells. Levels
of bridge molecules, including milk-fat-globule-EGF-factor 8
(MFG-E8), growth arrest-specific 6 (Gas6), thrombospondin (TSP)1, TSP-2 in hUTC CM were measured by ELISA, and their effects
on phagocytosis were examined by pre-incubating ROS with the
corresponding recombinant human proteins followed by addition of
ROS to RCS RPE cells for phagocytosis assay in the absence of the
respective bridge molecule protein.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: Transcriptome profile analysis of RCS RPE cells identified
multiple receptor genes involved in apoptotic clearance, such as
scavenger receptors and integrins. hUTC expresses a number of
bridge molecule genes including MFG-E8, Gas6, TSP-1 and TSP2. We detected high levels of MFG-E8, TSP-1, TSP-2 and low
amount of Gas6 in hUTC CM. Moreover, RCS RPE cells, fed with
ROS pretreated with the corresponding recombinant human bridge
molecule protein, showed significantly restored phagocytosis.
Conclusions: These results suggest that hUTC could rescue RCS
RPE cell phagocytosis in a Mertk-independent manner, through
secretion of bridge molecules.
Commercial Relationships: Jing Cao, Janssen Pharmaceutical
R&D (E); Chris Murat, None; Carol Anne Ogden, Janssen
Pharmaceutical R&D (E); Sandra Santulli-Marotto, Janssen
Pharmaceutical R&D (E); Xiang Yao, Janssen Pharmaceutical R&D
(E); Ian R. Harris, Janssen Pharmaceutical R&D (E); George
Inana, Janssen Pharmaceutical R&D (F)
Support: Janssen Pharmaceutical R&D
Program Number: 1729 Poster Board Number: A0098
Presentation Time: 11:00 AM–12:45 PM
Role of C/EBP homologous protein (CHOP) in the survival of
retinal ganglion cells after retinal ischemia/reperfusion injury
Sonali R. Nashine1, 2, Byung-Jin Kim1, 3, Abbot F. Clark1, 2, Iok-Hou
Pang1, 3. 1North Texas Eye Research Institute, University of North
Texas Health Science Center, Fort Worth, TX; 2Cell Biology and
Immunology, University of North Texas Health Science Center, Fort
Worth, TX; 3Department of Pharmaceutical Sciences, University of
North Texas Health Science Center, Fort Worth, TX.
Purpose: Retinal ischemia/reperfusion (I/R) causes apoptotic death
of retinal ganglion cells (RGC). CHOP is a pro-apoptotic protein and
a unfolded protein response (UPR) marker that plays a role in ERstress mediated apoptotic cell death. The purpose of this study was
to investigate the role of CHOP in mouse RGC survival following
retinal I/R injury.
Methods: Retinal I/R was induced in adult C57BL/6J (WT) and
CHOP-/- mice by cannulation of the anterior chamber of the left
eye with a needle connected to a reservoir of saline. Intraocular
pressure was increased to 120 mmHg for 60 min, after which the
needle was withdrawn to restore retinal circulation. Uninjured right
eyes served as controls. Expression of CHOP protein and other UPR
markers (p-eIF2α and BiP) in WT mice post-I/R was studied using
Western blot and immunohistochemistry. To compare RGC survival
between WT and CHOP-/- mice, retinal flat mount staining with
RGC marker, Brn3a was performed. Scotopic threshold response
electroretinography (STR-ERG) was performed at 0.03 mcd.s/m2
light intensity to evaluate retinal function.
Results: CHOP protein was up-regulated by 30 % in I/R injured
eyes (1.30 ± 0.11 arbitrary units (a.u.)) compared to control eyes (1 ±
0.07 a.u.) in WT mice three days after I/R injury (p < 0.05). Protein
levels of p-eIF2α and BiP also increased by 19% (I/R: 1.19 ± 0.15
a.u., Control: 1 ± 0.06 a.u.) and 11% (I/R: 1.11 ± 0.02 a.u., Control:
1 ± 0.03 a.u.) respectively (both p < 0.05). Co-localization of CHOP
and Brn3a confirmed the up-regulation of CHOP specifically in the
RGCs. In the uninjured control eyes, CHOP knockout did not affect
baseline RGC density or STR-ERG amplitude. I/R injury decreased
RGC densities and STR-ERG amplitudes in both WT and CHOP/- mice. However, survival of RGCs in I/R-injured CHOP-/- mouse
eyes (3337.1 ± 316.4 RGC/mm2) was 48% higher (p < 0.05) than that
of I/R-injured WT mouse eyes (2248.7 ± 225.9 RGC/mm2) three days
after I/R injury. STR-ERG amplitudes were 83 % higher in CHOP-/I/R eyes (18.6 ± 1.1 μV) compared to WT I/R eyes (10.1 ± 0.9 μV)
(p < 0.05).
Conclusions: Absence of CHOP partially protects against the loss
of RGCs and reduction in retinal function (STR-ERG) after I/R
injury. These results indicate that CHOP and thus ER-stress play an
important role in RGC apoptosis in retinal I/R injury.
Commercial Relationships: Sonali R. Nashine, None; Byung-Jin
Kim, None; Abbot F. Clark, None; Iok-Hou Pang, None
Support: U.S. Department of defense grant W81XWH-10-20-0003
Program Number: 1730 Poster Board Number: A0099
Presentation Time: 11:00 AM–12:45 PM
Atypical Degeneration Mechanisms of the Retinal Pigment
Epithelium upon Ablation of Ran-binding protein 2 (Ranbp2)
Dosuk Yoon1, Hemangi Patil1, Arjun Saha1, MdEmdadul Haque1,
Minzhong Yu2, Eugene Senda1, Sunny Qiu1, Neal S. Peachey2, Paulo
A. Ferreira1. 1Ophthalmology, Duke University Medical Center,
Durham, NC; 2Cole Eye Institute, Cleveland Clinic Foundation,
Cleveland, OH.
Purpose: The manifestation of several neurodegenerative and agingrelated disorders, such as age-related macular degeneration (AMD),
culminates with the degeneration of the retinal pigment epithelium
(RPE). However, the pathophysiological and cell-death mechanisms
underlying RPE degeneration upon a variety of intrinsic and extrinsic
stressors remain ill-defined. This study aims at testing the hypothesis
that compared to other cell types, Ranbp2 controls unique and shared
mechanisms of cell survival in the RPE.
Methods: We generated mice with selective ablation of Ranbp2 in
the RPE (cre-RPE::Ranbp2flox/flox). Longitudinal examination of RPE
degeneration was carried-out by molecular, proteomic, morphometric
and electrophysiological analyses between wild-type or creRPE::Ranbp2+/+ and cre-RPE::Ranbp2flox/flox. Markers of cell death
were used to discern cell death mechanisms in the RPE.
Results: Ranbp2 undergoes ablation at E18, but physiological EdU
labeling shows that Ranbp2 is dispensable to RPE cell proliferation
and RPE degeneration ensues upon RPE cells exit mitosis at ~P4.
RPE degeneration does not undergo the development of TUNEL+cells or activation of caspases. Instead, ablation of Ranbp2 triggers
the activation of a set of metalloproteinases in the mature RPE. This
mechanism causes the escape of dying RPE cells to the subretinal
space and profuse fluorescein leakage from the choriocapillaris.
Compared to cre-RPE::Ranbp2+/+, cre-RPE::Ranbp2flox/floxmice
present first a reduction of the amplitude of the light-adapted ERG;
by 8- and 24-weeks of age, light and dark-adapted ERGs become
reduced. The amplitude of the waveform of the direct current
(dc) ERGs of the RPE was markedly reduced with all its major
components (c-wave, fast oscillation, light-peak and off-response)
being greatly affected at 4-weeks of age. However, there were no
differences in the kinetics of dark adaptation between genotypes even
though there is a decrease of the transcriptional and translational
levels of Rpe65 in cre-RPE::Ranbp2flox/flox mice. Protein profiling
uncovered strong deregulation of proteostasis of ~12 proteins as early
as P14 in mutant RPE.
Conclusions: These studies unveil novel mechanisms underlying
the degeneration of RPE and with secondary pathological effects
on neighboring tissues. These studies will contribute to our
understanding of AMD pathogenesis and other aging-related
disorders.
Commercial Relationships: Dosuk Yoon, None; Hemangi Patil,
None; Arjun Saha, None; MdEmdadul Haque, None; Minzhong
Yu, None; Eugene Senda, None; Sunny Qiu, None; Neal S.
Peachey, None; Paulo A. Ferreira, None
Support: NIH support EY019492 & GM083165, Jules & Doris Stein
Research award (RPB)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1731 Poster Board Number: A0100
Presentation Time: 11:00 AM–12:45 PM
Crosstalk between autophagy and the ubiquitin-proteasome
pathway in human RPE cells
Jiao Zhan1, Jie-er He2, Fu Shang1, Mingxing Wu1, Xinyu Zhang1.
1
Zhongshan Ophthalmic Center, Guangzhou, China; 2The Second
Xiangya Hospital of Central South University, Changsha, China.
Purpose: The accumulation of damaged or misfolded proteins is
hypothesized to contribute to RPE dysfunction in age-related macular
degeneration (AMD). The ubiquitin-proteasome pathway (UPP) and
the autophagy-lysosome pathway (ALP) are the two major systems
for clearance of misfolded or damaged proteins. The aim of the
present study was to investigate how these two systems communicate
and coordinate with each other in RPE cells to eliminate intracellular
toxic misfolded or damaged proteins or their aggregates.
Methods: Cultured ARPE-19 cells were treated with proteasome
inhibitor MG132 and lysosomotropic agent Chloroquine (CQ)
respectively. The levels of ubiquitinated proteins and γ-tubulin were
analyzed by western blotting and immunofluorescence to monitor
protein aggregation. The activity of the ALP was determined by
LC3-II and LAMP1, the hallmarks of autophagy and the lysosome.
The fluorescent granules of LC3 and LAMP1 were confirmed
under fluorescence microscopy. The levels of HDAC6, p62 were
determined with immunofluorescence and Western Blotting.
Results: The levels of ubiquitinated protein aggregations significantly
increased and formed juxtanuclear aggresomes after the treatment
of MG132 in RPE cells, indicating that inhibition of the UPP caused
protein aggregation. The levels of LC3-II increased and aggregated
around the ubiquitinated proteins in MG132 treated cells. The levels
of HDAC6, p62 also increased in MG132 treated cells, indicating
inhibition of the UPP up-regulated autophagy activity. Treatment of
cells with CQ induced vacuole formation and secondary increases
in protein levels of LC3-II and P62. In addition, high mass ubiquitin
conjugates increased but levels of low mass ubiquitin conjugates
decreased oppositely upon CQ treatment. In contrast to proteasome
inhibition, the levels of HDAC6 decreased after CQ treatment.
Conclusions: Our results indicate that there is a crosstalk between
the UPP and the ALP in human RPE cells. When the UPP was
impaired, the ALP was induced to compensate for the limited activity
of the UPP. When the ALP was compromised, the UPP was also upregulated. The crosstalk and cooperation between the two proteolytic
pathways may play an important role in eliminating misfolded or
other forms of damaged proteins.
Commercial Relationships: Jiao Zhan, None; Jie-er He, None; Fu
Shang, None; Mingxing Wu, None; Xinyu Zhang, None
Support: National Natural Science Foundation of China81200670
Program Number: 1732 Poster Board Number: A0101
Presentation Time: 11:00 AM–12:45 PM
(R)- α-Lipoic acid treatments for preserving indocyanine green
and light-induced oxidative damage in ARPE-19 cells under
clinical vitrectomy conditions
Javier Araiz1, 3, Iñigo Corcostegui1, Ana Beloqui1, Ainhoa Bilbao2,
Vanessa Freire1, 2, Maria Miranda4, Itxaso Herrera1, Gonzalo
Castiella1, Gonzalo Corcostegui1, 3. 1R & D Dept, Instituto ClínicoQuirúrgico de Oftalmologia, Bilbao, Spain; 2Cell Biology and
Histology Dept, School of Medicine and Dentistry, University of
the Basque Country, Leioa, Spain; 3Ophthalmology Dept, School of
Medicine and Dentistry, University of the Basque Country, Leioa,
Spain; 4Biomedical Sciences Dept, CEU Cardenal Herrera University,
Moncada, Valencia, Spain.
Purpose: To evaluate the co-administration and pre-treatment with
(R)-α-Lipoic acid as protective agent against indocyanine green
(ICG) and light-induced oxidative damage in ARPE-19 cells using
a Constellation apparatus as light source in order to mimick clinical
vitrectomy conditions.
Methods: ARPE-19 cells were co-incubated along with ICG (0.5%
in culture medium) and subjected to light for 30 min following
either a (R)-α-Lipoic acid co-administration or pre-treatment
(0.05mM in culture medium) and compared with (R)-α-Lipoic aciduntreated cells under the same conditions (control). Cell viability
was measured colorimetrically (WST-8 reagent) at 0 and 24 h. The
determination of intracellular levels of reactive oxygen species
(ROS) and mitochondrial membrane potential (Ψm) was carried out
using, respectively, the fluorescent probes tetramethylrhodamine
methyl ester (TMRM) and 5-(and 6)-chloromethyldichlorodihydrofluorescein diacetate (CM-H2DCFDA) at 0 and 24 h.
Results: The results demonstrated that both the (R)-α-Lipoic acid coadministration and pre-treatment significantly decreased ROS levels
at 24 h (*p<0.05). However, the Ψm was significantly decreased
only with the (R)-α-Lipoic acid pre-treatment at times 0 and 24 h
(***p<0.001), but did not vary with the co-administration of the (R)α-Lipoic acid (p>0.05).
Conclusions: These results demonstrate the (R)-α-Lipoic acid
effectively preserves against ICG and light-induced oxidative damage
under clinical vitrectomy conditions. Whilst reducing ROS levels,
(R)-α-Lipoic acid pre-treatment induced a significant Ψm reduction
in ARPE-19 cells, suggesting a co-administration of (R)-α-Lipoic
acid during vitrectomy might be desirable in order to preserve longterm cell damage.
Commercial Relationships: Javier Araiz, None; Iñigo
Corcostegui, None; Ana Beloqui, None; Ainhoa Bilbao, None;
Vanessa Freire, None; Maria Miranda, None; Itxaso Herrera,
None; Gonzalo Castiella, None; Gonzalo Corcostegui, None
Program Number: 1733 Poster Board Number: A0102
Presentation Time: 11:00 AM–12:45 PM
Effect of glutathione ethyl ester, lipoic acid and progesterone on
photoreceptor survival in the retina of rd10 mice
Maria Miranda1, Soledad Benlloch-Navarro1, Violeta SánchezVallejo1, Laura Trachsel-Moncho1, Jose M. Soria1, Inmaculada
Almansa1, Javier Araiz2, 3. 1Ciencias Biomédicas, Univ CEU-Cardenal
Herrera, Moncada, Valencia, Spain; 2R & D Dept, Instituto ClínicoQuirúrgico de Oftalmologia, Bilbao, Spain; 3Ophthalmology Dept,
School of Medicine and Dentistry, University of the Basque Country,
Leioa, Spain.
Purpose: Retinitis Pigmentosa (RP) is a group of inherited retinal
degenerative diseases in which one of several different mutations
results in death of photoreceptor cells. Rd10 mice, an animal model
of RP, have a missense mutation in exon 13 of the beta subunit of
the rod phosphodiesterase (PDE6) gene, its rods express 40% of the
endogenous level of the PDE6 protein. In rd10 mice, rod loss starts
at post-natal day 18 (P18), peaks at P24 and is almost complete by
P35. In previous studies we have shown that GSH is decreased in
rd10 retinas just before the degenerative process becomes detectable.
Therefore, the purpose of this study was to investigate if three
substances (glutathione ethyl ester (GEE), lipoic acid (LA) and
progesterone) that have been shown to increase GSH concentrations
in other tissues are able to prevent photoreceptor cells from apoptosis
in rd10 mice.
Methods: Animals were treated in accordance to the ARVO
statement for the use of animals in ophthalmic and vision research.
Different doses of GEE, LA and progesterone were orally
administered to control and rd10 mice at different post-natal days
(P15, P17, P19 and P21). Mice were sacrificed at P21. Histological
evaluation was performed usin hematoxylin/eosin. Terminal
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling
(TUNEL) and glial fibrillary acidic protein (GFAP) staining were
also performed in retinal sections. Retinal GSH concentration was
determined by high-performance liquid chromatography (HPLC).
Results: Though GEE, LA and progesterone are able to increase
retinal GSH concentration, only progesterone was able to rescue
photoreceptors from death, mainly at the periphery of the retina.
Interestingly this effect was dose-related, being 150mg/kg the most
effective dose in improving cell survival in the retina of rd10 mice.
Progesterone was also able to decrease the c gliosis observed in the
retina.
Conclusions: We confirm, as we have previously shown in rd1
mice, that progesterone may have a beneficial effect in the delay of
photoreceptors death in rd10 mice, though this effect many not only
related to its capacity to increase retinal GSH concentrations.
Commercial Relationships: Maria Miranda, None; Soledad
Benlloch-Navarro, None; Violeta Sánchez-Vallejo, None; Laura
Trachsel-Moncho, None; Jose M. Soria, None; Inmaculada
Almansa, None; Javier Araiz, None
Support: Fundación Mutua Madrileña, Ayudas para el fomento de
la investigación científica en la Universidad CEU Cardenal Herrera
(PRCEU-UCH16/12)
Program Number: 1734 Poster Board Number: A0103
Presentation Time: 11:00 AM–12:45 PM
Inhibition of inflammasome rescues photoreceptor cell death
after retinal detachment
Keiko Kataoka, Hidetaka Matsumoto, Kimio Takeuchi, Joan W.
Miller, Demetrios G. Vavvas. Angiogenesis Lab, Massachusetts Eye
and Ear Infirmary, Boston, MA.
Purpose: Damage-associated molecular patterns (DAMPs) are
released from dead cells and activate molecular platforms containing
caspase-1, termed ‘inflammasome’, which lead to the release of
mature IL-1β in monocytes/macrophages. Meanwhile, IL-1β is
known to activate inflammatory responses and lead to cell death.
In this study, we investigated whether inflammasome was activated
in the mice eyes after retinal detachment (RD). Furthermore, we
examined whether inhibition of inflammasome could reduced
photoreceptor cell death after RD.
Methods: RD was induced in mice eyes, by injection of 4 ul of
sodium hyaluronate into the subretinal space of C57/BL6 mice.
Caspase 1 inhibitor, Ac-Tyr-Val-Ala-Asp-Chloromethylketone
(YVAD), was diluted with sodium hyaluronate to a final
concentration of 300 μM. The levels of total IL-1β and cleaved IL1β after RD in mice eyes were measured with ELISA and western
blotting respectively. To evaluate the quantity of cell death, collected
eyes were frozen in O.C.T Compound and cut into 8 μm thicksection. TUNEL staining was performed with ApopTag Fluorescein
In Situ Apoptosis Detection Kit.
Results: Total IL-1β including pro- IL-1β and cleaved IL-1β
increased as early as 6 hours after the induction of RD, peaked at 12
hours (6.3 fold compared to control) and was down-regulated after
24 hours. Cleaved IL-1β peaked at 24 hours after RD simultaneously
with the peak of TUNEL positive cells in outer nuclear layer.
Treatment with the Caspase1 inhibitor YVAD decreased cell death by
55.3%.
Conclusions: Retinal detachment induced inflammasome activation
in mice eyes. Inhibition of inflammasome significantly reduced
photoreceptor cell death after RD suggesting potential novel
neuroprotective strategies.
Commercial Relationships: Keiko Kataoka, None; Hidetaka
Matsumoto, None; Kimio Takeuchi, None; Joan W. Miller, None;
Demetrios G. Vavvas, None
Program Number: 1735 Poster Board Number: A0104
Presentation Time: 11:00 AM–12:45 PM
Dideoxy-nucleosides are anti-inflammatory and inhibit RPE
degeneration independent of reverse transcriptase inhibition
Benjamin Fowler, Younghee Kim, Yoshio Hirano, Nagaraj Kerur,
Valeria Tarallo, Bradley D. Gelfand, Jayakrishna Ambati. University
of Kentucky, Lexington, KY.
Purpose: NLRP3 inflammasome activation induces RPE cell death
in dry AMD, a devastating and currently untreatable blindness. In
the dry AMD model based on Alu RNA accumulation in the RPE,
cytotoxic non-coding Alu RNAs cause macular degeneration.
Although the precise details of how Alu RNAs induce inflammasome
activation are not fully understood, it is known that Alu RNA requires
endogenous reverse transcriptase for its life cycle. Therefore, we
hypothesized that nucleoside reverse transcriptase inhibitors (NRTIs)
block inflammasome activation and RPE cell death in cell culture and
animal models of dry AMD.
Methods: NLRP3 inflammasome activation was monitored by
western blot for Caspase-1 and phospho-IRAK4 (primary human
RPE cells), or western blot/ELISA for Interleukin (IL)-1 beta, IL-18,
and Caspase-1 (mouse BMDM/THP-1 monocytes/Raji TK+/- cells).
AZT phosphorylation was measured by LC-MS/MS. Inflammasome
priming was assessed by real time-quantitative PCR. ATP release
was assessed using a luciferase-based detection kit. P2X7 receptor
function was assessed by uptake of YO-PRO-1 dye in HEK293 cells
with stable expression of P2X7 after incubation with bzATP. For
the mouse model of dry AMD, RPE degeneration was induced by
subretinal injection of a plasmid expressing Alu RNA. Oral gavage of
NRTIs or controls in wild-type male C57BL6/J mice was performed
daily for one week after Alu administration. RPE degeneration was
assessed by fundus photography and ZO-1 staining of RPE flat
mounts.
Results: Multiple NRTIs blocked RPE cell death and inflammasome
activation induced by Alu RNA. Oral gavage delivery of the NRTI
stavudine, at a similar equivalent dose typically administered in
humans, prevented P2X7-dependent RPE degeneration in the Alu
RNA-induced mouse model of dry AMD. NRTIs blocked ATPmediated P2X7-dependent Caspase-1 and Interleukin-1 beta/-18
secretion, and YO-PRO-1 dye uptake. AZT inhibited inflammasome
and was not phosphorylated in thymidine kinase-deficient cells.
Conclusions: We have identified a novel anti-inflammatory action of
NRTIs characterized by P2X7 and NLRP3 inflammasome inhibition,
and demonstrated their in vivo ability to prevent RPE cell death
in a mouse model of dry AMD. The tested NRTI compounds have
been FDA-approved and widely used clinically for decades, and
are therefore ideal drug repurposing candidates in an unanticipated
treatment strategy for dry AMD.
Commercial Relationships: Benjamin Fowler, University of
Kentucky (P); Younghee Kim, None; Yoshio Hirano, None;
Nagaraj Kerur, None; Valeria Tarallo, None; Bradley D. Gelfand,
None; Jayakrishna Ambati, iVeena (F), University of Kentucky (P)
Support: NEI/NIH grant 5R01EY022238-02 NIH T32HL091812
UL1RR033173 Doris Duke Charitable Foundation, Burroughs
Wellcome Fund, Ellison Medical Foundation, Carl Reeves
Foundation, Foundation Fighting Blindness, Harrington Discovery
Institute, Research to Prevent Blindness, Dr. E. Vernon & Eloise C.
Smith Endowed Chair
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 1736 Poster Board Number: A0105
Presentation Time: 11:00 AM–12:45 PM
Compared with N-acetylcysteine (NAC), N-Acetylcysteine Amide
(NACA) Provides Increased Protection of Cone Function in a
Model of Retinitis Pigmentosa
Aling Dong, Rebecca Stevens, Sean Hackett, Peter A. Campochiaro.
Johns Hopkins University Wilmer Eye Institute, Baltimore, MD.
Purpose: Retinitis pigmentosa (RP) is a major cause of blindness
due to a large variety of mutations that lead to the death of rod
photoreceptors. After rods die, cones gradually die from progressive
oxidative damage. We previously found that N-acetylcysteine (NAC),
a well-known thiol antioxidant, reduces cone cell death and preserves
cone function in models of RP. N-acetylcysteine amide (NACA) is
a prodrug for NAC with increased bioavailability. In this study, we
compared NAC and NACA in a model of RP.
Methods: Starting at postnatal day (P) 14, rd10+/+ mice were given
normal drinking water (n=6) or water containing 7mg/ml NACA
or 20mg/ml NAC (n=8 for each group). Scotopic and photopic
electroretinograms (ERGs) were recorded at P35 and P50. Cone
density was measured at P50 in four 230 mmx 230 mm (512x512
pixels) areas located 0.5mm superior, temporal, inferior, and nasal
to the center of the optic nerve in retinal flat mounts stained with
fluorescein-labeled peanut agglutinin (PNA).
Results: At P35, mean peak scotopic ERG b-wave amplitude was
similar in rd10+/+ mice treated with 7mg/ml NACA or 20mg/ml
NAC, and were significantly greater (about 2-fold) than those in
controls. Mean peak photopic b-wave amplitude was 41% higher
(p=0.024) in NACA-treated mice than NAC-treated mice and both
were more than 3-fold higher than that in controls. At P50, mean
peak scotopic ERG b-wave amplitude was more than 5-fold higher
in NAC- or NACA-treated mice than in controls with mean b-wave
amplitudes significantly greater in NACA-treated mice compared
with NAC-treated mice at 10 of 11 stimulus intensities. Mean
photopic ERG b-wave amplitude was 50% higher (p=0.001) at all
3 stimulus intensities in NACA-treated versus NAC-treated mice
and more than 4-fold greater than controls. At P50, cone cell density
was significantly greater in 3 of 4 quadrants in NACA-treated mice
compared to NAC-treated mice.
Conclusions: Even with a substantially lower oral dose, NACA
showed significantly greater preservation of cone cell function and
cone survival compared with NAC in rd10+/+ mice.
Commercial Relationships: Aling Dong, None; Rebecca Stevens,
None; Sean Hackett, None; Peter A. Campochiaro, None
Support: NIH grant EY005951
Program Number: 1737 Poster Board Number: A0106
Presentation Time: 11:00 AM–12:45 PM
Investigating the influence of blast on cellularity in the retinal
ganglion cell layer in a mouse model of blast-induced traumatic
brain injury using a novel semi-automated technique
Adam Hedberg-Buenz1, 2, Matthew M. Harper1, 3, Mark Christopher4,
5
, Laura Dutca1, Todd Scheetz4, 5, Randy H. Kardon1, 3, Michael G.
Anderson1, 2. 1Center for the Prevention and Treatment of Visual
Loss, Veterans Affairs (VA) Health Care System, Iowa City,
IA; 2Department of Molecular Physiology and Biophysics, The
University of Iowa, Iowa City, IA; 3Department of Ophthalmology
and Visual Sciences, The University of Iowa, Iowa City, IA;
4
Biomedical Engineering, The University of Iowa, Iowa City, IA;
5
Wynn Institute for Vision Research, The University of Iowa, Iowa
City, IA.
Purpose: Blast-mediated injuries are the leading cause of combatrelated injury in modern warfare. Visual dysfunction has been
reported in Veterans with blast-mediated traumatic brain injury
(TBI). We have previously shown retinal ganglion cells (RGC) are
exquisitely sensitive to blast exposure. However, the magnitude of
RGC loss in blast-mediated injury is not yet understood. The purpose
of these experiments is to develop a method to quantify cellularity
and investigate the influence of blast on the retinal ganglion cell layer
(GCL) after blast-induced TBI.
Methods: C57BL/6J mice were exposed to an overpressure wave
(20 PSI) directed to the head using a custom-built blast chamber
(blast-injured). Mice placed in the chamber without blast were used
as controls (sham control). At 4 months post-blast, retinas from both
blast-injured (n=16) and sham control (n =12) eyes were mounted
whole, stained, and imaged by light microscopy. Images were
uniformly collected across the retina with equal sampling from the
central and peripheral retina. Images were quantitatively assessed for
cellularity in the GCL using custom-written macros in Image J.
Results: Retinas from both blast-injured and sham control mice had
greater cell densities in the central compared to peripheral retina.
In the peripheral retina, blast-injured mice exhibited a significant
decrease (p = 0.03) in cell density compared to controls using a
Students t-test. In the central retina, blast-injured mice exhibited a
trend of reduced cell density compared to controls (not significant).
Together, these results indicate exposure to blast causes cellular loss
in the GCL in this model. Additionally, this novel semi-automated
technique is able to detect subtle changes in cell density.
Conclusions: These results demonstrate that this mouse model of
blast-induced TBI recapitulates the neuronal loss in the GCL that
contributes to visual dysfunction in humans with TBI. This semiautomated technique provides a useful method to quantitatively
assess cellularity in the GCL. Extending our knowledge of RGC
susceptibility and mechanistic responses that influence their fate
following blast-injury will help in the development of improved
clinical testing and treatment of visual deficits to those suffering from
TBI.
Commercial Relationships: Adam Hedberg-Buenz, None;
Matthew M. Harper, None; Mark Christopher, None; Laura
Dutca, None; Todd Scheetz, None; Randy H. Kardon, None;
Michael G. Anderson, None
Support: Supported by the Department of Veterans Affairs; Veterans
Health Administration; Office of Research and Development;
Rehabilitation Research and Development Center for Prevention
and Treatment of Visual Loss; a Rehabilitation Research and
Development Career Development Award (MMH); an RRD Merit
Study Award (1I01RX000427-01).
Program Number: 1738 Poster Board Number: A0107
Presentation Time: 11:00 AM–12:45 PM
AAV-mediated activation of the Nrf2 pathway in the Nrf2deficient mouse
Katharine J. Liang, Kenton T. Woodard, R. Jude Samulski. Gene
Therapy Center, Univ of North Carolina Chapel Hill, Chapel HIll,
NC.
Purpose: Retinal degeneration is due at least in part to redox
deregulation. Under conditions of oxidative stress, the Nrf2
transcription factor activates the antioxidant response element,
driving expression of endogenous antioxidant genes. The Nrf2deficient mouse exhibits an increased susceptibility to exogenous
oxidative stress, but limited overt pathology. Because the Nrf2/- mouse exhibits an age-related retinal degeneration of incomplete
penetrance, we applied novel AAV vectors to dampen oxidative stress
in the light-stressed Nrf2-deficient mouse.
Methods: In order to increase uniformity of disease progression and
mimic environmental factors contributing to retinal degeneration, we
exposed Nrf2-deficient mice to a light-induced retinal degeneration
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
protocol. An AAV2.5-CBh-Nrf2 vector was engineered using a
chimeric capsid vector and promoter selected for retinal transduction
via intravitreal injection and delivered prior to light damage exposure.
Functional efficacy of AAV-Nrf2 was verified using in vitro luciferase
reporter and fluorescent ROS detection assays. Downstream gene
activation was assessed by western blot. Therapeutic efficacy was
assessed using ERG, fundus imaging, and OCT.
Results: AAV2.5 combines the improved transduction properties
of AAV1 with reduced antigenic cross-reactivity against antibodies
directed at both parental serotypes while keeping the receptor
binding properties of AAV2. AAV-Nrf2 successfully mediates Nrf2
expression, activates downstream genes, and suppresses reactive
oxygen species in vitro. In vivo, intravitreally delivered AAV-Nrf2
was sufficient to activate downstream gene expression up for up to
six months. Light-induced degeneration in Nrf2-/- mice and AAVmediated rescue is being characterized by ERG, fundus imaging, and
OCT.
Conclusions: AAV vector development combined with gene
replacement offers a unique opportunity to optimize and test both
efficient transgene delivery as well as potent therapeutic potential.
Our research provides a targeted molecular approach to drive
antioxidant gene expression, and offers a promising alternative to
currently limited therapeutics for multifactorial diseases involving
ROS deregulation.
Large subretinal deposits as seen on fundus (Micon IV) in a 15
month-old Nrf2-/- mouse.
OCT (Micron IV) of large subretinal deposit and degeneration in
Nrf2-/- mouse retina.
Commercial Relationships: Katharine J. Liang, None; Kenton T.
Woodard, None; R. Jude Samulski, None
Support: Ruth L. Kirschstein National Research Service Award
5-F30-AG044100-03
Program Number: 1739 Poster Board Number: A0108
Presentation Time: 11:00 AM–12:45 PM
Arginase 2 deletion limits hyperoxia-induced retinal vascular
injury through normalization of NOS function and upregulation
of arginase 1
Jutamas Suwanpradid1, 4, Modesto A. Rojas1, 4, Robert William
Caldwell2, Ruth B. Caldwell1, 3. 1Vascular Biology Center, Georgia
Regents University, Augusta, GA; 2Department of Pharmacology and
Toxicology, Georgia Regents University, Augusta, GA; 3VA Medical
Center, Augusta, GA; 4Vision Discovery Institute, Augusta, GA.
Purpose: We have shown that deletion of arginase 2 (A2) limits
vascular injury in the mouse model of oxygen-induced retinopathy
(OIR) by reducing hyperoxia-induced vaso-obliteration. This
protective effect is associated with decreased peroxynitrite formation
and reduced activation of microglia/macrophage cells (ARVO, 2012).
We have now examined the involvement of NOS uncoupling and A1
expression in this process.
Methods: Neonatal mice [lacking one copy of A1 (A1+/-), lacking
both copies of A2 (A2-/-), and wild type (WT)] were maintained in
hyperoxia (70% oxygen) from postnatal day 7 (p7) to p12 and then
returned to normoxia. Controls were maintained in normoxia. Mice
were sacrificed at different times. Retinas were processed for analysis
of NOS uncoupling by using diaminofluorescein (DAF) imaging of
nitric oxide (NO) and dihydroethidium (DHE) imaging of superoxide
(O2.-). Retinal samples were also processed for immunolocalization
and western blotting analysis of expression of arginase 1 and
activation of survival pathways in relation to retinal vascular injury
and repair.
Results: Formation of NO was reduced by 60% in the hyperoxiatreated WT retina as compared with the normoxia control (P<0.05).
NO formation was preserved in the A2-/- OIR retina (P<0.05).
Formation of O2.- was increased by 100% in WT OIR retinas as
compared with WT controls after 24 hours of hyperoxia (P<0.05).
This effect was blocked by treatment with the NOS inhibitor
(L-NAME) or by A2 deletion (P<0.05). The vaso-protective effects
of A2 deletion in the OIR retina were associated with marked upregulation of A1immunoreactivity. Furthermore, hyperoxia-induced
vaso-obliteration was markedly enhanced in the A1+/- OIR retina
as compared with WT OIR retina (P<0.05). These protective effects
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
of A2 deletion and A1 upregulation were accompanied by increased
formation of the autophagic marker microtubule associated protein
1 light chain 3 and activation of ERK and Akt survival pathways
(P<0.05).
Conclusions: Arginase 2 deletion prevents hyperoxia-induced retinal
vascular injury by attenuating NOS uncoupling mediated superoxide
formation which is associated with upregulation of A1 expression and
activation of autophagy and survival pathways.
Commercial Relationships: Jutamas Suwanpradid, None;
Modesto A. Rojas, None; Robert William Caldwell, None; Ruth B.
Caldwell, None
Support: NEI-R01-EY11766, VA Merit Review, Vision Discovery
Institute at Georgia Regents University
Program Number: 1740 Poster Board Number: A0109
Presentation Time: 11:00 AM–12:45 PM
PGC-1 alpha regulates human RPE oxidative metabolism and
anti-oxidant capacity
Jared Iacovelli1, 2, Glenn C. Rowe4, 3, Zoltan Arany4, 3, Magali SaintGeniez1, 2. 1The Schepens Eye Research Institute, Massachusetts Eye
and Ear Infirmary, Boston, MA; 2Opthalmology, Harvard Medical
School, Boston, MA; 3Medicine, Harvard Medical School, Boston,
MA; 4Cardiovascular Institute, Beth Israel Deaconess Medical
Center, Boston, MA.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of blindness in the elderly population of industrialized
countries. AMD is thought to occur, at least in part, from oxidative
damage to the retinal pigment epithelium (RPE). Thus, enhancement
of the anti-oxidant response of RPE is an attractive treatment for
AMD. The transcriptional co-activator, peroxisome proliferatoractivated receptor-gamma coactivator 1α (PGC1α), is powerful
mediator of mitochondrial biogenesis, oxidative metabolism, and
the cellular anti-oxidant response. This study examines the ability of
PGC1α to modulate oxidative metabolism of human RPE (ARPE-19)
and protect them from oxidative damage.
Methods: ARPE-19 were maintained in standard culture conditions.
To induce oxidative stress, confluent ARPE-19 were cultured in
serum-free media overnight and treated with hydrogen peroxide.
Oxidative stress and reactive oxygen species (ROS) were measured
by CM-H2DCFDA fluorescence. Cell death was analyzed by
LDH release. Gene expression of PGC-1α and selected oxidative
metabolism and anti-oxidant enzymes was analyzed by qPCR.
PGC-1α expression was increased using adenoviral delivery.
Mitochondrial respiration and fatty acid oxidation was monitored
using the Seahorse extracellular flux analyzer.
Results: Over-expression of PGC1α in ARPE-19 significantly
increased (p<0.01) all phases of mitochondrial respiration and
fatty acid oxidation in the presence of palmitate (p<0.05). This
increase corresponded with at least a 3-fold increase (p<0.05) in
gene expression of oxidative phosphorylation subunits including
COX5B, NDUFB5, and ATP5O. Over-expression of PGC1α also
induced significant increases (p<0.05) in anti-oxidant gene expression
including CAT, GPX1, PRDX3, SOD1, SOD2, and TXN2. Treatment
of confluent ARPE-19 with H2O2 increased ROS in a dose- and timedependent manner. 18-hr treatment of ARPE-19 with 1 mM H2O2
caused 40% cytotoxicity which was dramatically reduced to 10% by
PGC1α overexpression.
Conclusions: PGC1α is a powerful regulator of oxidative
metabolism and the antioxidant response in human RPE. Modulation
of its expression can reduce oxidant mediated cell death and may be a
useful tool to reduce oxidative damage to RPE in vivo.
Commercial Relationships: Jared Iacovelli, None; Glenn C.
Rowe, None; Zoltan Arany, None; Magali Saint-Geniez, None
Support: Brightfocus Foundation, NIH EY023682
Program Number: 1741 Poster Board Number: A0110
Presentation Time: 11:00 AM–12:45 PM
Tamoxifen Toxicity of the Retinal Pigment Epithelium is
Mediated by RIP Kinase and the NLRP3 Inflammasome
Leo A. Kim, Dhanesh Amarnani, Wen A. Tseng, Demetrios G. Vavvas,
Patricia A. D’Amore. Department of Ophthalmology, Massachusetts
Eye and Ear Infirmary, Boston, MA.
Purpose: To evaluate the mechanism of tamoxifen-induced cell
death in human cultured retinal pigment epithelial cells (RPE), and
to investigate the relative contribution of cell death mechanisms
including apoptosis, necroptosis, and pyroptosis to tamoxifen toxicity
of the RPE.
Methods: Human RPE cells (ARPE-19 cells) were cultured until
confluence and treated with 20 μM tamoxifen; subsequent cell death
was measured by detecting lactate dehydrogenase (LDH) release.
Lysosomal membrane permeabilization was assessed using acridine
orange staining. The roles of lysosomal enzymes cathepsin B and L
were examined by blocking their activity with Z-FF-FMK, CA074-Me, and Z-FY(t-Bu)-DMK. Caspase activity was evaluated by
caspase-1, 3-, 8-, and 9-specific inhibition using Z-YVAD-FMK,
Z-DEVD-FMK, Z-LETD-FMK, and Z-LEHD-FMK respectively.
Cells were primed with IL-1α and treated with tamoxifen and
IL-1β production was quantified via ELISA. Caspase activity was
verified with the fluorochrome-labeled inhibitor of caspases (FLICA)
probe specific for each caspase. Necroptosis was evaluated using
necrostatin-1 (Nec-1) to inhibit RIP1 kinase.
Results: Cell death occurred within two hours of tamoxifen treatment
of confluent ARPE-19 cells, and was accompanied by lysosomal
membrane permeabilization. Tamoxifen toxicity was shown to
occur through both caspase-dependent and non-caspase-dependent
cell death pathways. Blockage of cathepsin activity resulted in
a significant decrease in cell death, indicating that lysosomal
destabilization and cathepsin release are upstream of these cell death
pathways. Simultaneous treatment of ARPE-19 cells with caspase
inhibitors and Nec-1 resulted in a near complete rescue from cell
death.
Conclusions: Tamoxifen-induced cell death occurs through multiple
cell death mechanisms. Simultaneous inhibition of caspase-dependent
and caspase-independent cell death pathways is required to protect
cells from tamoxifen. Inhibition of upstream activators such as
cathepsin B/L may be a feasible approach to block multiple cell death
pathways.
Commercial Relationships: Leo A. Kim, None; Dhanesh
Amarnani, None; Wen A. Tseng, None; Demetrios G. Vavvas, US
Patent 201, 0137642 (P); Patricia A. D’Amore, None
Support: NEI/NIH Grant K-12-EY16335, Lions Eye Foundation to
LAK, and NIH Grant EY05435 to PAD
Program Number: 1742 Poster Board Number: A0111
Presentation Time: 11:00 AM–12:45 PM
Endoplasmic Reticulum-Associated Degradation (ERAD) of
Mutant Rod Opsin Disrupts Photoreceptor Protein Homeostasis
During Retinal Degeneration
Wei-Chieh Chiang1, Heike Kroeger1, Carissa Messah1, Douglas
Yasumura2, Michael T. Matthes2, Sanae Sakami3, Krzysztof
Palczewski3, Matthew M. LaVail2, Jonathan H. Lin1. 1Department
of Pathology, University of California, San Diego, La Jolla, CA;
2
Department of Ophthalmology, University of California, San
Francisco, San Francisco, CA; 3Department of Pharmacology, Case
Western Reserve University, Cleveland, OH.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Rod opsin is a G-protein coupled receptor exclusively
expressed by rod photoreceptors. Over 150 mutations in rod opsin
have been identified in hereditary forms of retinal degeneration. The
P23H rod opsin mutation leads to opsin protein misfolding, increased
ER stress, and the activation of ER stress-induced Unfolded Protein
Response (UPR) signal transduction network. The role of ER stress
and UPR signaling in rod cells is poorly understood. Here, we
investigated the function of UPR activation in P23H knock-in (P23HKIn) mice that recapitulate the gene dosage and retinal degeneration
disease phenotype seen in patients carrying P23H opsin alleles.
Methods: The rate of retinal degeneration in P23H-KIn homozygous
mice was determined by measuring the thickness of the retinal outer
nuclear layer at postnatal days 4, 6, 10, 15, 20, 30, 45, 60, and 90.
P23H-KIn mice were crossed with Xbp1-Venus (ERAI) reporter mice
for monitoring IRE1 pathway activation in retinas expressing P23H
opsin.
Results: Through analysis of P23H-KIn;Xbp1-Venus mice, we find
that rod cells expressing P23H opsin strongly activate the IRE1 signal
transduction pathway of the UPR to generate XBP1 transcription
factor. IRE1-XBP1 signaling upregulates many genes required
for ER-associated degradation (ERAD) of misfolded proteins in
the ER. We find that ERAD components physically associate with
P23H opsin in photoreceptors leading to pronounced P23H opsin
ubiquitination, and rapid degradation. By contrast to heterologous
cell culture studies of misfolded opsin, virtually no misfolded opsin
accumulates within photoreceptors as ER-retained aggregates.
Consistent with efficient elimination of P23H opsin from the ER of
photoreceptors, we find no increase in proapoptotic Chop, induced
by persistent ER protein aggregation. Surprisingly, despite ERAD
induction and rapid P23H opsin protein degradation, total cellular
levels of ubiquitinated proteins markedly increase during retinal
degeneration.
Conclusions: Our findings show that IRE1 branch of the UPR
promotes robust ERAD of P23H opsin in photoreceptors. We propose
that increased buildup of damaged, ubiquitinated proteins arises as
a consequence of extensive ERAD of P23H opsin, and disruption
of cellular protein homeostasis could lead to rod photoreceptor cell
death and retinal degeneration.
Commercial Relationships: Wei-Chieh Chiang, None; Heike
Kroeger, None; Carissa Messah, None; Douglas Yasumura,
None; Michael T. Matthes, None; Sanae Sakami, None; Krzysztof
Palczewski, None; Matthew M. LaVail, None; Jonathan H. Lin,
None
Support: WWC: Fight-for-Sight postdoctoral fellowship. MML:
R01EY001919, P30EY002162, and Foundation Fighting Blindness.
JHL: RO1EY020846 and BrightFocus Foundation for AMD.
Program Number: 1743 Poster Board Number: A0112
Presentation Time: 11:00 AM–12:45 PM
Effects of Ethanol on ARPE-19 cells: Autophagy and Oxidative
Stress
Luis Bonet1, Miguel Flores-Bellver1, Sara Saez-Atienzar1, 2, Natalia
Martínez1, Javier Sancho-Pelluz1, Jorge M. Barcia1, Joaquin Jordan3,
Maria F. Galindo2, Francisco J. Romero1. 1Neurobiology and
Neurophysiology, Catholic University of Valencia, Valencia, Spain;
2
Neuropsicopharmacology, Complejo Universitario Universidad
de Albacete, Albacete, Spain; 3Ciencias Medicas, Universidad de
Castilla la Mancha, Albacete, Spain.
Purpose: Despite the well-known pathological effects of ethanol
(EtOH) consumption on the nervous system, how alcohol exposure
affects retinal cells has yet to be clarified. Retinal pigment
epithelium (RPE) plays a crucial role in the physiology of the
retina due to its location and metabolism. Oxidative damage has
been demonstrated as a pathogenic mechanism in several retinal
diseases, and reactive oxygen species (ROS) are important byproducts of ethanol metabolism. ROS production seems to promote
autophagy, a mechanism designed to degrade damaged organelles and
proteins. Recently, autophagy has been proposed as a cytoprotective
mechanism against EtOH-induced toxicity in liver and brain cells.
Thus, our goal is to study the effect of EtOH in ARPE-19 cells, and
the role of autophagy
Methods: ARPE-19 cells were seeded and then exposed to different
EtOH concentrations (80 mM, 200 mM, 400 mM, and 600 mM).
Autophagy was measured by analyzing LC3 punctae with a GFPLC3 plasmid and different protein expression (p62 and LC3-II) by
western blot. Moreover, LC3 flux was analyzed by using chloroquine
as a lysosome inhibitor and by using the RFP-GFP-LC3 plasmid to
quantify autophagosome maturation. Mitochondrial morphology was
analyzed using a pDsRed2-Mito plasmid and by electron microscopy.
ROS levels were measured using 2’7’-dichlorodihydrofluorescein
(DCFH). Lipid peroxidation products were analyzed by ELISA.
4-HNE aggregates were localized with immunocytochemistry.
Results: A significant reduction of MTT occurred first at 600
mM EtOH, after 24 h. EtOH exposure induced LC3 synthesis in
a concentration dependent-manner (starting 80 mM). Moreover,
EtOH also promoted a concentration-dependent increase in
autophagosome-lysosome fusion and mitochondrial fragmentation.
Obvious ultraestructural morphological changes were observed at
every EtOH concentration used. Furthermore, EtOH increased DCFH
fluorescence (ROS) levels in treated cells compared to non-treated.
4-HNE aggregates increased after EtOH exposure, forming large ballshaped aggregates in the vicinity of the nucleus.
Conclusions: Alcohol exposure produces ROS and mitochondrial
fragmentation. Furthermore, EtOH induces 4-HNE labelled protein
aggregates. In order to eliminate damaged organelles, autophagy
is activated. In summary, EtOH induces all neurodegenerative
hallmarks in RPE cells such as mitochondrial damage, protein
aggregation, and autophagy activation.
Commercial Relationships: Luis Bonet, None; Miguel FloresBellver, None; Sara Saez-Atienzar, None; Natalia Martínez, None;
Javier Sancho-Pelluz, None; Jorge M. Barcia, None; Joaquin
Jordan, None; Maria F. Galindo, None; Francisco J. Romero,
None
Support: Ministerio de Ciencias e Innovacion (SAF2010-21317)
Program Number: 1744 Poster Board Number: A0113
Presentation Time: 11:00 AM–12:45 PM
Hypoxia activates calpain in the nerve fiber layer of monkey
retinal explants
Masayuki Hirata1, 3, Emi Nakajima2, 3, Thomas R. Shearer3,
Mitsuyoshi Azuma1, 3. 1Senju Laboratory of Ocular Sciences, Senju
Pharmaceutical Co.,Ltd, Portland, OR; 2Senju Laboratory of Ocular
Sciences, Senju Pharmaceutical Co.,Ltd, Kobe, Japan; 3Integrative
Biosciences, Oregon Health & Science University, Portland, OR.
Purpose: The vascular ischemic theory of retinal nerve damage
assumes that decreased blood flow in the ophthalmic artery reduces
oxygenation and thereby damages neurons. Optic nerve damage from
hypoxia in glaucoma includes ischemia, impaired axonal transport,
and free radical formation. Hypoxia also leads to retinal cell death by
activating calpain enzyme. Data from our cultured monkey retinas
show that: 1) Hypoxia induces retinal cell death. 2) An active form
of calpain is observed in retina during hypoxia. 3) Calpain substrates
are proteolyzed. 4) All such changes are ameliorated by calpain
inhibitor SNJ1945. However, we still do not know in which specific
retinal layer(s) calpains are activated. The purposes of the present
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
experiment were to: 1) determine where calpain is activated in retina,
and 2) improve the methodology for culture of monkey retina.
Methods: Equal-sized pieces of retinas were dissected from
enucleated monkey eyes and cultured with the RGC side facing up
on micro porous membranes in 6-well culture plates. After 3 hrs of
normoxia, the retinal explants were incubated under hypoxia for 16
hrs in medium with 0.5 mM glucose, and then reoxygenated for 8
hrs in medium with 5.5 mM glucose. When used, calpain inhibitor
SNJ-1945 was added 1 hr before hypoxia treatment. Formalin-fixed
paraffin embedded sections were subjected to TUNEL staining and
immunohistochemistory for α-spectrin.
Results: Under improved culture conditions, TUNEL positive cells
were minimal under normoxia and indicated that proper culture time
was somewhat less than 2 days. During hypoxia/reoxygenation,
the 150 kDa calpain-specific, α-spectrin breakdown product first
accumulated in the nerve fiber layer, and SNJ-1945 inhibited it’s
formation. TUNEL staining then increased in the ganglion cell layer.
Conclusions: During hypoxia/reoxygenation, calpain is activated
in the nerve fiber layer, the first point of injury. This is followed by
death of the parent retinal ganglion cells. These observations suggest
that calpain may be involved in the degeneration of retinal nerve
fibers during hypoxia in glaucoma.
Dr. Shearer receives a research contract and consulting fees from,
and Drs. Hirata, Nakajima and Azuma are employees of, Senju
Pharmaceutical Co. Ltd.
Commercial Relationships: Masayuki Hirata, Senju
Pharmaceutical Co.,Ltd (E); Emi Nakajima, Senju Pharmaceutical
Co.,Ltd (E); Thomas R. Shearer, Senju Pharmaceutical Co.,Ltd (C);
Mitsuyoshi Azuma, Senju Pharmaceutical Co.,Ltd (E)
274 Glia, Diabetic Retinopathy, and Retinal Angiogenesis
Monday, May 05, 2014 3:45 PM–5:30 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 2241–2277/A0145–A0181
Organizing Section: Retinal Cell Biology
Program Number: 2241 Poster Board Number: A0145
Presentation Time: 3:45 PM–5:30 PM
PLVAP Modulates Angiogenesis By Tuning VEGF Signaling In
Endothelial Cells
Joanna Wisniewska-Kruk1, 2, Ingeborg Klaassen1, 2, Ilse M. Vogels2,
Cornelis J. Van Noorden2, Reinier O. Schlingemann1, 3. 1Department
of Ophthalmology, Academic Medical Center, Amsterdam,
Netherlands; 2Department of Cell Biology and Histology, Academic
Medical Center, Amsterdam, Netherlands; 3Department of Clinical
and Molecular Ophthalmogenetics, Netherlands Institute for
Neuroscience, Royal Netherlands Academy of Arts and Science
(KNAW), Amsterdam, Netherlands.
Purpose: Plasmalemma vesicle associated protein (PLVAP) is an
endothelial cell-specific protein, detected by the PAL-E antibody that
has been used for decades as endothelial marker. PLVAP is expressed
in a large part of the vascular system, but is absent from blood
vessels with an intact blood-retinal barrier. However, in pathological
conditions associated with neovascularization in the central nervous
system, as occurs in diabetic retinopathy, PLVAP expression
significantly increases. In this study, we investigated the role of
PLVAP in angiogenesis and the potential molecular mechanisms that
underlie its pro-angiogenic activity.
Methods: To elucidate the role of PLVAP in new blood vessel
formation in vivo, we used the oxygen-induced retinopathy mouse
model and injected siRNA intraocularly. Five days after injection,
retinas were flat-mounted and the vasculature was analyzed.
Furthermore, the possible pro-angiogenic function of PLVAP was
investigated in in vitro models, including the aortic ring assay,
the endothelial spheroid-based assay and migration assays. The
silencing efficiency of siRNA and lentiviral delivered shRNA in
all experiments was confirmed on mRNA and protein levels. The
expression of VEGF receptors and its co-receptors after Plvap
silencing in endothelial cells was investigated using western blot,
immunohistochemistry and flow cytometry analysis.
Results: Silencing of Plvap in the oxygen-induced retinopathy model
resulted in decreased vascular and neovascular areas, and increased
avascular areas in the retinas. Plvap inhibition in cultured endothelial
cells exhibited decreased sprout formation, reduced numbers of
endothelial tip cells and diminished cell migration. However, no
effect on cell viability or cell proliferation was observed after Plvap
silencing in these cells. Reduced angiogenic capacity of cells that
lack PLVAP expression was linked to reduced VEGFR2 protein
levels, selective downregulation of VEGF co-receptors and VEGFR2
down-stream signaling kinases.
Conclusions: Our data highlight PLVAP as an important
endothelium-specific cofactor for physiological and pathological
angiogenesis through a VEGFR2 dependent mechanism. Therefore,
PLVAP may serve as a potential therapeutic target in proliferative
vascular eye disorders, such as diabetic retinopathy and retinopathy
of prematurity.
Commercial Relationships: Joanna Wisniewska-Kruk, None;
Ingeborg Klaassen, None; Ilse M. Vogels, None; Cornelis J. Van
Noorden, None; Reinier O. Schlingemann, None
Program Number: 2242 Poster Board Number: A0146
Presentation Time: 3:45 PM–5:30 PM
Angiogenin is a potent modulator of neovascularization and
inflammation that is upregulated in various human retinal
diseases
Carli M. Wittgrove1, Liliana P. Paris1, Yoshihiko Usui1, 2,
Yoshihiro Wakabayashi2, Edith Aguilar1, Daniel Feitelberg1, Peter
D. Westenskow1, Leah C. Byrne3, John G. Flannery3, Martin
Friedlander1. 1Cell and Molecular Biology, The Scripps Research
Institute, La Jolla, CA; 2Ophthalmology, Tokyo Medical University,
Tokyo, Japan; 3Helen Wills Neuroscience, University of California at
Berkeley, Berkeley, CA.
Purpose: Our lab is interested in identifying common stress-response
pathways that may contribute to the initiation or progression of
multiple retinal diseases. Diabetic retinopathy (DR), age-related
macular degeneration (AMD), retinitis pigmentosa (RP), and uveitis
are diseases with neurodegenerative, inflammatory, and neovascular
features observed with variable frequency. Current treatment
strategies, including laser photocoagulation and anti-inflammatory
and angiostatic agents, can be insufficient. Identifying a pathological
feature common to these diseases could lead to the development
of a therapeutic agent effective in slowing or preventing their
pathogeneses.
Methods: Cytokine levels in human aqueous and vitreous
samples from patients with various retinal diseases were analyzed.
Angiogenin was found to be dysregulated. Its expression was
examined in four murine models of ocular neurodegeneration and/or
neovascularization (RD1, RD10, RDS, Oxygen-induced retinopathy)
using immunofluorescence. Angiogenin was overexpressed in
mouse Müller glia using ShH10 virus (ShH10-Ang) and the retinas
were monitored in vivo for phenotypic and functional changes
using optical coherence tomography, ICG angiography, and
electroretinography. Immunofluorescence was also employed on flatmounted retinas and cryopreserved sectioned tissues.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: We detected significantly elevated levels of angiogenin
in aqueous and vitreous samples from human patients with DR,
AMD, RP, and uveitis. Similarly, in murine disease models we
detected elevated levels of angiogenin in Müller glia. Angiogenin
was experimentally potentiated in the Müller glia of wild type
mice using ShH10-Ang. Three days post injection, pronounced
neovascularization and clear signs of inflammation were observed
both in vivo and in histological preparations. Electroretinography
revealed neuronal dysfunction as early as eight days post injection.
Conclusions: Angiogenin is dysregulated in a number of human
ocular diseases and murine models, suggesting its upregulation may
be a common stress-activated response. Elucidating its beneficial
and pathological functions could provide insights into retinal disease
progression and provide a potential novel target for therapeutic
intervention.
Commercial Relationships: Carli M. Wittgrove, None; Liliana
P. Paris, None; Yoshihiko Usui, None; Yoshihiro Wakabayashi,
None; Edith Aguilar, None; Daniel Feitelberg, None; Peter D.
Westenskow, None; Leah C. Byrne, None; John G. Flannery,
None; Martin Friedlander, None
Support: EY11254, EY22025, The Lowy Medical Research Institute
Program Number: 2243 Poster Board Number: A0147
Presentation Time: 3:45 PM–5:30 PM
Relationship between vascular endothelial growth factor
(VEGFA), biomarkers of inflammation and dysglycemia
Katrin Engelmann1, 2, Dieter Appelt4, Frank Pistrosch4, Dirk
Sandner5, Elena Henkel4, Carsta Koehler3, M. Hanefeld4.
1
Ophthalmology, Klinikum Chemnitz gGmbH, Chemnitz, Germany;
2
CRTD Center for Regenerative Therapies Dresden - DFG Cluster
of Excellence, Dresden, Germany; 3Medical Consulting, GWT-TUD
GmbH, Dresden, Germany; 4Endocrinology and Metabolic Disorders,
GWT-TUD GmbH, Dresden, Germany; 5Klinik für Augenheilkunde,
Universitätsklinikum Carl-Gustav Carus, Dresden, Germany.
Purpose: Abnormal VEGF plays a pivotal role in the pathogenesis of
diabetic maculopathy and retinopathy. Vice versa low systemic VEGF
levels seem to be associated with impaired angiogenesis in patients
with type 2 diabetes and cardiovascular disease. Recently long lasting
decrease in systemic VEGF levels was reported after intraocular antiVEGF treatment of diabetic macular edema. So far little is known on
relationship between VEGF, activation of the inflammation cascade
and other cardiovascular risk factors in different categories of glucose
intolerance. This proatherogenic cascade may be a link between
systemic effects of intraocular anti-VEGF therapy and vascular
complications.
In addition to previous studies with prediabetes we therefore analysed
the relationship between VEGF-A, biomarkers of inflammation (BI),
other cardiovascular risk factors (RF) and HbA1c in advanced type 2
diabetes.
Methods: 251 subjects matched for BMI and age: 50 with normal
glucose tolerance (NGT), 50 with pre-diabetes (PRE-DM), 100 with
early diabetes (E-DM) and 51 with advanced A-DM were analysed
for VEGF-A, BI and HbA1c.
Statistics: t-test with CI, Anova, univariate correlation and stepwise
regression analysis with VEGF-A as dependent variable.
Results: Patients were well matched for age, BMI and sex. As shown
in tab. A-DM shows a significant increase in hsCRP and VEGF-A.
In univariate analysis we found a significant correlation of VEGF-A
with hsCRP and HbA1c in the overall population. However, VEGF-A
was inversely correlated to age. In A-DM age (R=-0,37) and hsCRP
(R= 0,36) were significantly correlated to VEGF-A. In multivariate
analysis only the parameter hsCRP and adiponectin have an
independent association to VEGF-A.
Conclusions: Our analysis reveals a robust correlation between
VEGF-A and hsCRP in the overall population and in addition for
HbA1c and VEGF-A in patients with poorly controlled diabetes. A
DM represents a Bermuda triangle of high HbA1c, elevated VEGF-A
and activated inflammation. These data suggest that hsCRP as gold
standard of low grade inflammation may be used as risk marker
also for follow-up of anti-VEGF-A therapy. Prospective studies
are needed to validate cardiovascular risk associated with systemic
effects of anti-VEGF-therapy.
Commercial Relationships: Katrin Engelmann, None; Dieter
Appelt, None; Frank Pistrosch, None; Dirk Sandner, None; Elena
Henkel, None; Carsta Koehler, None; M. Hanefeld, None
Program Number: 2244 Poster Board Number: A0148
Presentation Time: 3:45 PM–5:30 PM
Caspase-14 Expression Impairs Retinal Pigment Epithelial Cell
Barrier Function
Selina Beasley1, 2, Mohamed Elsherbiny1, 2, Sylvia Megyerdi1, 2, Sally
El-shafey1, 2, Nader Sheibani3, Mohamed Al-Shabrawey1, 2. 1Oral
Biology/Anatomy, Georgia Regents University, Augusta, GA; 2Vision
Discovery Institute and Department of Ophthalmology, Georgia
Regents University, Augusta, GA; 3Ophthalmology and Visual
Sciences, University of Wisconsin, Madison, WI.
Purpose: We recently showed that caspase-14 is a novel molecule
expressed in the retina with potential role in accelerated vascular
cell death during diabetic retinopathy (DR). Here we evaluated the
impact of caspase-14 expression on apoptosis, barrier properties, and
phagocytic activity of retinal pigment epithelial (RPE) cell.
Methods: Human RPE (ARPE-19) cells were transfected with
caspase-14 cDNA or control vector, followed by studying the
barrier and phagocytic function, and the changes in the activity of
other caspases (caspase1, 3, 5, 8, and 9). RPE cell permeability was
evaluated by FITC-Dextran Flux assay. The levels and distribution of
actin stress fibers (F-actin) were examined by Immunofluorescence
staining. The effect of caspase14 expression on the activity of other
caspases was determined by using a specific caspase activity assay
kit. We also tested the expression of caspase 4 by Western blotting.
Phagocytic activity was examined using a phagocytic activity kit.
Moreover we tested the impact of high glucose treatment (30 mM) on
the levels of caspase14 expression in RPE cells compared to normal
glucose (5 mM) or osmotic control (Mannitol (25 mM) + glucose
(5mM)).
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: We found that caspase-14 expression promoted FITCdextran leakage through the ARPE-19 confluent monolayer. This
was associated with a significant increase in the expression and
disorganization of F-actin stress fibers and the activity of caspase-3
and caspase-9 (p<0.05). There was also significant increase in the
levels of caspase 4 in caspase 14 expressing ARPE-19 cells. There
was no significant difference in the phagocytic activity between
caspase 14 expressing RPE and control cells. HG treatment
significantly increased caspase-14 expression in RPE cells compared
to NG treatment or osmotic control (P<0.05).
Conclusions: Our findings suggest that caspase-14 expression and/
or activity may compromise RPE barrier function disrupting outer
retinal barrier during diabetes. The effect of caspase 14 on RPE
barrier function is probably mediated through enhancement of
apoptotic pathways.
Commercial Relationships: Selina Beasley, None; Mohamed
Elsherbiny, None; Sylvia Megyerdi, None; Sally El-shafey, None;
Nader Sheibani, None; Mohamed Al-Shabrawey, None
Support: 1R01EY023315-01 and QNRF (NPRP4-1046-3-284)
Program Number: 2245 Poster Board Number: A0149
Presentation Time: 3:45 PM–5:30 PM
Vascular endothelial growth factor (VEGF)-induced retinal
vascular leakage in African green monkeys
Wenzheng Hu, Vernard Woodley, Rohn Brookes, Mike Struharik,
Steve Whittaker, Steve Henry, Robin J. Goody, Matthew S. Lawrence.
RxGen Inc, Hamden, CT.
Purpose: To characterize the time course of pathologic features such
as retinal vascular leakage following intravitreal (IVT) injection of
recombinant human VEGF165 (rhVEGF) in African green monkeys
(AGMs), and to evaluate the effect of anti-VEGF therapy in reducing
the pathogenic changes elicited in this model.
Methods: Eight adult male and female AGMs received repeat
bilateral IVT injections of 1 or 2 mg of rhVEGF. Eyes were examined
by color fundus photography, fluorescence angiography and
optical coherence tomography. Lucentis 0.25 mg was administered
intravitreally 2 days after the second dose of rhVEGF.
Results: A minimum of two serial IVT rhVEGF injections were
necessary to induce significant retinal vascular leakage in AGMs.
Following initial dosing with 2 mg rhVEGF, the retinal appearance
was similar to that observed in eyes receiving a first dose of 1 mg
rhVEGF. Slight vascular dilation occurred in all eyes at day 2 with
a minor retinal hemorrhage, and resolved by day 3. Two days after
a second 2 mg IVT dose of rhVEGF, there was moderate dilation
and tortuosity of retinal vessels, and minor vascular sheathing in the
posterior retina. Changes peaked at day 3 with small patches of intraretinal hemorrhage occurring in the posterior region. Six days after
the second rhVEGF dose, moderate dilation and tortuosity of retinal
vessels, and multiple intra-retinal hemorrhages were seen in both
the posterior and nasal retina. Vascular dilation and tortuosity fully
resolved within 11 days of the second dose. Intra-retinal hemorrhages
also resolved but abundant hard exudates were deposited in the
posterior retina. Lucentis-treated eyes demonstrated no fluorescein
leakage in the retina although the fluorescein signal was still evident
in the optic nerve head 1 day post-Lucentis treatment. No retinal
vascular pathology was observed 4 days post-Lucentis injection and
thereafter.
Conclusions: Intravitreal injections of rhVEGF produced retinal
vascular leakage and other pathological changes including vascular
dilation, tortuosity and sheathing, intra-retinal hemorrhage, exudates
and non-perfusion regions. Pathological features were similar to
those seen in diabetic retinopathy. Lucentis effectively inhibited
rhVEGF-induced vascular leakage and other pathological changes,
thus supporting its use as a positive control for testing anti-vascular
leakage compounds in the rhVEGF model.
Commercial Relationships: Wenzheng Hu, RxGen Inc. (E);
Vernard Woodley, RxGen Inc. (E); Rohn Brookes, RxGen Inc. (E);
Mike Struharik, RxGen Inc. (E); Steve Whittaker, RxGen Inc. (E);
Steve Henry, RxGen Inc. (E); Robin J. Goody, RxGen Inc. (E);
Matthew S. Lawrence, RxGen Inc. (E)
Program Number: 2246 Poster Board Number: A0150
Presentation Time: 3:45 PM–5:30 PM
Serum amyloid A supports RF/6A cells under chemical hypoxia
and high glucose
Jing Feng, Yanrong Jiang. Peking Univ People’s Hospital, Beijing,
China.
Purpose: We investigated the expression of serum amyloid A (SAA)
in the eyes with diabetic retinopathy. We searched for the effects of
high glucose and hypoxia on the expression of SAA in RF/6A cells
in vitro, and for the effects of exogenous SAA on the proliferation,
migration and apoptosis of RF/6A cells.
Methods: A total of 26 eyes of 26 patients with DR, and 10 eyes
of 10 patients with non-diabetic ocular disease were enrolled.
Multiplex bead assay was used to examine the serum amyloid A
level in aqueous humor.Localization of SAA was detected using
immunofluorescence. The mRNA and protein of SAA in the RF/6A
cells were measured using real-time PCR and western blot. CCK-8
assay was performed to examine the proliferative ability, transwell
chamber assay and Annexin/PI staining were used to detect cell
migration and apoptosis.
Results: Significantly higher concentrations of SAA were found in
the aqueous humor of DR patients than those in the aqueous humor
of the control patients (p<0.01). Immunofluorescence analysis
showed SAA were expressed in RF/6A cells in vitro. The expression
of SAA mRNA and protein were increased induced by hypoxia and
high concentration glucose (p<0.01). By CCK-8 assay, after 24h,
the values were significant different with those of control groups
either (p<0.01). Results of apoptosis confirmed the apoptosis rates of
cells exposed to SAA for 24h were higher than those exposed to its
control.
Conclusions: The expression of SAA was higher in DR patients.
SAA was unregulated by high glucose concentration and hypoxia,
which promoted cell proliferation, migration and inhibited apoptosis.
Commercial Relationships: Jing Feng, None; Yanrong Jiang,
None
Program Number: 2247 Poster Board Number: A0151
Presentation Time: 3:45 PM–5:30 PM
Isoform-specific inhibition of NFATc1 and NFATc2 reduces
VEGF- and TNFa-induced inflammation in retinal endothelial
cells
Colin A. Bretz1, 3, Sara R. Savage2, 3, John S. Penn3, 1. 1Cell &
Developmental Biology, Vanderbilt University Medical Center,
Nashville, TN; 2Pharmacology, Vanderbilt University Medical Center,
Nashville, TN; 3Vanderbilt Eye Institute, Vanderbilt University
Medical Center, Nashville, TN.
Purpose: Retinal levels of vascular endothelial growth factor
(VEGF) and tumor necrosis factor alpha (TNFa) are known to be
elevated at an early disease stage in diabetic patients. In retinal
endothelial cells (RMEC), VEGF and TNFa induce expression
of leukocyte adhesion proteins and inflammatory cytokines that
contribute to leukostasis, a complication of diabetic retinopathy (DR).
The nuclear factor of activated T-cells (NFAT) family of transcription
factors are also known to upregulate inflammatory proteins, and can
act downstream of VEGF and TNFa in this context. We hypothesize
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
that NFAT family isoforms play distinct roles in the expression of
inflammatory proteins in response to diabetes-relevant stimuli, and
the present study aimed to evaluate the roles of NFATc1 and NFATc2
under VEGF and TNFa treatment conditions.
Methods: RMEC were transfected with isoform-specific siRNA
or a scrambled sequence and collected 4hrs post VEGF (25ng/ml)
or TNFa (10ng/ml) treatment. Quantitative real-time RT-PCR was
performed by co-amplification of VCAM1 and ICAM1 vs. β-actin.
For parallel plate flow chamber (PPFC) assays, cells were grown on
glass slides and transfected prior to TNFa (10ng/ml) treatment. 4hrs
later, slides were placed in PPFC and peripheral blood mononuclear
cells (PBMC) were flowed over the monolayer. Flow was stopped,
non-adherent cells were washed off, and remaining cells were
counted.
Results: Both VEGF and TNFa stimulated increased expression of
VCAM1 and ICAM1 in treated human RMEC. NFATc1 knockdown
caused a 65% decrease in VEGF induced VCAM1 expression
(P<0.03), while NFATc2 knockdown caused a 69% and 51%
decrease in TNFa induced VCAM1 and ICAM1 expression (P<0.01
and P<0.05), respectively. TNFa stimulation of PPFC slides
caused increased PBMC adhesion, which is abrogated by NFATc2
knockdown.
Conclusions: Here we provide evidence that two distinct NFAT
isoforms play a role in the downstream signaling of VEGF and
TNFa. NFATc1 modulates VEGF-induced VCAM1 expression,
while NFATc2 modulates both TNFa-induced VCAM1 and ICAM1
expression. The latter effect underlies the reduction seen in adherent
PBMCs when NFATc2 knockdown is combined with TNFa
treatment. Together, these findings suggest that NFAT signaling may
be a valuable target for DR treatment, since multiple NFAT isoforms
contribute to the signaling of two distinct and pathologically relevant
peptides.
Commercial Relationships: Colin A. Bretz, None; Sara R. Savage,
None; John S. Penn, None
Support: NIH Grant EY 07533, an Unrestricted Grant from Research
to Prevent Blindness, The Reeves Foundation, OneSight Foundation
week 14. Oral CLT-005 protected against this decline in a dosedependent manner, and the 500 mg/kg dose significantly rescued
losses in spatial frequency threshold and contrast sensitivity at weeks
10, 12, and 14 (p<0.01, ANOVA). A similar rescue was observed
in insulin-implanted animals. Incidence and grade of cataracts was
also significantly reduced in the 125, 250, and 500 mg/kg treatment
groups at week 14. The blood glucose levels in CLT-005 treated
animals were notably lower than vehicle controls; however, they were
still elevated as compared to naïve or insulin-treated STZ-diabetic
rats. Oral administration of CLT-005 did not produce any gross
pathological findings or changes in body weight during the entire
study duration.
Conclusions: Oral delivery of CLT-005 has a profound effect to
prevent loss of spatial frequency threshold and contrast sensitivity in
the STZ-model of type 1 diabetes. Chronic delivery over 14 weeks
was well tolerated with no observable adverse effects. These data
support future clinical studies of CLT-005 to prevent and ameliorate
inflammatory and neovascular insults that lead to decreased visual
acuity in patients with Diabetic Retinopathy and other diabetic eye
diseases.
Program Number: 2248 Poster Board Number: A0152
Presentation Time: 3:45 PM–5:30 PM
Oral delivery of CLT-005, a Stat3 inhibitor, prevents visual acuity
decline in an animal model of type 1 diabetes
Rafal Farjo, Phillip Vanlandingham, Alexander Quiambao, Jodi
Green, Didier J. Nuno, Fadee Mondalek, Drew Wassel. Charlesson
LLC, Oklahoma City, OK.
Purpose: Activation of Stat3 in the posterior segment is associated
with early events that lead to inflammation and neovascularization.
We have previously demonstrated that topical delivery of CLT-005,
a small molecule inhibitor of Stat3, achieved therapeutic levels in
the posterior segment and was efficacious in multiple animal models
of Wet AMD and Dry AMD. We sought to assess the efficacy,
safety, and ocular distribution of CLT-005 following chronic oral
administration over 14 weeks to streptozotocin (STZ) diabetic rats.
Methods: Type 1 diabetes was induced in Brown Norway rats by
intraperioteneal administration of STZ to rats on Day 1. Following
confirmation of elevated blood glucose on Day 4, rats were treated
with daily gavage of vehicle or CLT-005 at 125, 250, and 500 mg/
kg for 14 weeks. In a positive control group, an insulin pellet was
subcutaneously administered. Optokinetic tracking (OKT) was
performed at regular intervals to quantify spatial frequency threshold
and contrast threshold.
Results: A decline in spatial frequency threshold and contrast
threshold was observed in STZ-diabetic rats beginning at 8-10 weeks
following STZ induction, which progressively worsened through
Commercial Relationships: Rafal Farjo, Charlesson LLC
(E); Phillip Vanlandingham, Charlesson LLC (E); Alexander
Quiambao, Charlesson LLC (E); Jodi Green, Charlesson LLC (E);
Didier J. Nuno, Charlesson LLC (E); Fadee Mondalek, Charlesson
LLC (E); Drew Wassel, Charlesson LLC (E)
Support: NIH EY018989
Program Number: 2249 Poster Board Number: A0153
Presentation Time: 3:45 PM–5:30 PM
Retinas from STZ-induced diabetic rats present angiographic
and focal ERG alterations as well as anti-angiogenic factors
inhibition
Anna Salas Torras1, Marina Riera1, Miguel A. Zapata1, Ibane
Abasolo2, Simo Schwartz2, Jose Garcia-Arumi1. 1Ophthalmology, Vall
Hebron Hospital, Research Institute, Barcelona, Spain; 2CIBBIMNanomedicine, Vall Hebron Hospital, Research Institute, Barcelona,
Spain.
Purpose: The aim of this study is to characterize the ocular
alterations of the STZ-induced diabetes model through fluorescence
angiography (FA), focal electroretinogram (fERG), and angiogenesisrelated factors study by immunohistochemistry and gene expression.
Methods: 6-week-old Long Evans male rats were randomly assigned
to treated (65 mg/kg STZ) and control groups (1 ml/kg citrate buffer)
and divided into four time-points (1, 2, 4 and 8 weeks of diabetes).
Two days after STZ administration, glucose levels were measured to
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
assess the hyperglycemia in the STZ-treated animals. Inner and outer
retinal functions were measured by fERG every week, comparing
central and peripheral retina. Once a week, animals were injected
with fluorescein and FA were taken to study vascular abnormalities.
After each time-point animals were euthanized and eyes were
enucleated. Left eyes were fixed with 4% paraformaldehide and
frozen on TFM for cryosection and retinas from right eyes were
isolated and frozen for RNA extraction and real time PCR. Gene and
protein expression of the main angiogenesis-related factors (VEGF as
the principal angiogenesis promoter and PEDF and SST as two of the
most important anti-angiogenic factors) was determined.
Results: fERG measurements showed a significant reduction of
the oscillatory potentials after 8 weeks of diabetes, being higher in
central retina, demonstrating inner-retinal dysfunction. Fluorescence
angiographies showed increased vessel tortuosity and vascular
leakage starting at week 2 after treatment, evidencing diabetesdriven retinal vascular abnormalities. Post-mortem analysis showed
increased VEGF levels, glial activation and apoptosis in retinas of
STZ rats compared with controls, as well as lower PEDF and SST
levels.
Conclusions: Taken together, these data give new evidences on the
ocular alterations in the STZ-induced diabetes model, suggesting
that this is a good aproach for the study of proliferative diabetic
retinopathy, highlighting the neural and vascular abnormalities
as well as proposing it for the study of neuroprotective and antiangiogenic agents.
Commercial Relationships: Anna Salas Torras, None; Marina
Riera, None; Miguel A. Zapata, None; Ibane Abasolo, None; Simo
Schwartz, None; Jose Garcia-Arumi, None
Support: FIS grant FI11/706. ISCIIII.
Program Number: 2250 Poster Board Number: A0154
Presentation Time: 3:45 PM–5:30 PM
Alterations of retinal vasculature in cystathionine-betasynthase heterozygous mice, a model of mild-moderate
hyperhomocysteinemia (HHcy)
Amany M. Tawfik1, 2, Sally Elshafey2, 3, Arul Shanmugam1, 2, Shanu
Markand1, 2, Kartik Angara1, 2, Mohamed Al-Shabrawey2, 3, Vadivel
Ganapathy2, 4, Sylvia B. Smith1, 2. 1Cellular Biology & Anatomy,
Georgia Regents University, Augusta, GA; 2Culver Vision Discovery
Institute, Georgia Regents University, Augusta, GA; 3Oral Biology /
Anatomy, Georgia Regents University, Augusta, GA; 4Biochemistry/
Molecular Biology, Georgia Regents University, Augusta, GA.
Purpose: HHcy is implicated in neurovascular diseases including
certain retinal diseases such as diabetic retinopathy and age-related
macular degeneration. Recently, we analyzed the retinal vasculature
of mice completely lacking cystathionine β−synthase (CBS), which
have 40-fold increase in homocysteine (Hcy), and observed severely
altered retinal vasculature mimicking ischemic retinopathy (Tawfik et
al, IOVS, 2013). The purpose of the current study was to assess the
retinal vascular phenotype in mice with mild-moderate HHcy, which
is much more prevalent in the human population.
Methods: HHcy mice deficient in the cbs gene (cbs+/-) were used to
evaluate retinal vascular integrity. Cbs+/+ (n=56) and cbs +/- (n-47)
mice (age: 16 - 52 wks) were subjected to fluorescein angiography
(FA) and optical coherence tomography (OCT) to assess retinal
vasculature in vivo. Retinas harvested for cryosectioning or flatmount
preparations were subjected to immunofluorescence microscopy
(IMF) to detect blood vessels (isolectin-B4), angiogenesis (antiVEGF, anti-CD105), gliosis (anti-GFAP), blood-retinal barrier
integrity (anti-ZO-1, anti-occludin) and hypoxia (anti-HP-1).
Levels of VEGF, GFAP, ZO-1 and occludin were determined by
immunoblotting.
Results: FA revealed neovascularization and vascular leakage in
cbs+/- mice that was progressive (mild at 16 wks, marked by 52 wks);
OCT confirmed new vessels in the vitreous chamber by 1 yr. IMF
demonstrated vascular patterns consistent with ischemia including
a capillary-free zone centrally and new vessels with capillary tufts
mid-peripherally in 36 and 52 wk mice. This was associated with
increased VEGF, CD105 and GFAP and decreased ZO-1/occludin
levels in the cbs+/- retinas. Western blotting showed a 2-fold increase
in VEGF and GFAP. Retinal vein occlusion was observed in some
cbs+/- mouse retinas.
Conclusions: Our earlier studies clearly showed a link between
severe HHcy and retinal vascular disease. The current studies provide
strong evidence that even mild-moderate elevation of Hcy as seen
in cbs+/- mice is accompanied by progressive alterations in retinal
vasculature including ischemia, neovascularization, incompetent
blood-retinal barrier and vascular occlusion. Studies are underway
to determine mechanisms by which HHcy triggers retinal vascular
alterations.
Commercial Relationships: Amany M. Tawfik, None; Sally
Elshafey, None; Arul Shanmugam, None; Shanu Markand, None;
Kartik Angara, None; Mohamed Al-Shabrawey, None; Vadivel
Ganapathy, None; Sylvia B. Smith, None
Support: NIH Grant EY012830
Program Number: 2251 Poster Board Number: A0155
Presentation Time: 3:45 PM–5:30 PM
Role of fractalkine receptor (CX3CR1) in microglial mediated
inflammation in the Diabetic Retina
Andrew S. Mendiola, Sandra M. Cardona, Andrew T. Tsin, Astrid
E. Cardona. Biology, The University of Texas at San Antonio, San
Antonio, TX.
Purpose: Diabetic retinopathy (DR) is the leading cause of blindness
in working Americans and a common complication of diabetes
mellitus. Resident microglia can play detrimental and protective roles
during CNS inflammation. Previous data from our laboratory showed
that in the normal CNS the fractalkine receptor (CX3CR1) inhibits
microglia activation and neurotoxicity. Although it is reported that
microglia increase in number and release TNF-α and VEGF in the
diabetic retina, the contribution of CX3CR1 in microglial activation
and neuronal cell loss during DR is unclear. We hypothesize that
CX3CR1 deficiency in resident microglia cells exacerbates activation
signaling and promotes an inflammatory milieu that leads to neuronal
and vascular damage associated with early stage DR.
Methods: Ins2Akita mice have a point mutation in the insulin2 gene
causing protein misfolding and loss of pancreatic β-cells, which
results in hyperglycemia by 4 weeks of age. These mice were
crossed with CX3CR1-deficient mice on a C57BL/6 background to
generate diabetic wildtype (Ins2Akita-WT) and CX3CR1 deficientmice (Ins2Akita-KO). Whole retinas isolated from 10-week old diabetic
and non-diabetic mice were assayed for: fractalkine production,
immunohistochemical analysis to evaluate microglia phenotype and
retina pathology using confocal microscopy, and in vitro production
of nitric oxide levels upon different stimuli utilizing the Griess assay.
Results: Our data showed a significant increase in the number of
retinal microglia cells in diabetic and non-diabetic CX3CR1 deficient
mice (CX3CR1-KO) in comparison to age-matched CX3CR1
wild type mice. Conversely, fractalkine levels were decreased in
the diabetic mouse retina. Our in vitro studies revealed that retinal
microglia are responsive to LPS, IFN-γ and PolyIC stimulation. In
addition, CX3CR1-deficient mice showed significantly increased
microglia cell roundness, suggesting an activated state. Whereas
nitric oxide levels in the culture media were decreased in the retina of
CX3CR1 heterozygotes.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Conclusions: These data suggest that the absence of CX3CR1
in hyperglycemia promotes an environment that leads to the
proliferation and activation of resident microglia, which may
contribute to the pathology of early DR.
Commercial Relationships: Andrew S. Mendiola, None; Sandra
M. Cardona, None; Andrew T. Tsin, None; Astrid E. Cardona,
None
Support: This project was supported by grants from the National
Institute on Minority Health and Health Disparities (G12MD007591)
and (1SC1GM095426 to AEC) from the National Institutes of Health
and The San Antonio Area Foundation.
Program Number: 2252 Poster Board Number: A0156
Presentation Time: 3:45 PM–5:30 PM
Effect of Intravitreal Anti-VEGF Injections on Retinal Nerve
Fiber Layer
Julio Chora, Victor H. Gonzalez. Retina, Valley Retina Institute,
McAllen, TX.
Purpose: To assess the effect of repeated anti-VEGF intravitreal
injections on the retinal nerve fiber layer thickness (RNFL).
Methods: This was a retrospective chart review of thirty-three
consecutive patients who received multiple intravitreal injections
of anti-VEGF medications in one eye. The fellow eye was used as a
control. RNFL thickness was measured using spectral domain OCT
(Spectralis SD-OCT, Heidelberg, Germany) with a minimum followup of 12 months.
Results: 60% of the patients were female, and 40% were male with
an average age of 71.6 years. 45% of the patients were being treated
for exudative age-related macular degeneration, 30% for diabetic
macular edema, 20% for proliferative diabetic retinopathy, and 5%
for venous occlusions. 50% received ranibizumab, 30% pegaptanib,
15% bevacizumab and 5% aflibercept.
At the 12 month follow up, the mean number of injections received
were 7.4 and the mean RNFL thickness was 100.5 microns in the
treated eyes versus 102 microns in the fellow eye (P=0.186).
Conclusions: There was a tendency toward thinner RNFL readings
in the anti-VEGF treated eyes versus their controls. No statistical
significance was observed.
Commercial Relationships: Julio Chora, None; Victor H.
Gonzalez, None
Program Number: 2253 Poster Board Number: A0157
Presentation Time: 3:45 PM–5:30 PM
X-box Binding Protein 1 (XBP1) is a Crucial Regulator of
Endothelial Tight Junction and Protects the Blood-retinal barrier
in Diabetic Retinopathy
Jacey H. Ma1, 3, Jingming Li2, Joshua J. Wang1, Sarah X. Zhang1, 2.
1
Department of Ophthalmology & Ross Eye Institute and Department
of Biochemistry, SUNY-Buffalo and SUNY Eye Institute, Buffalo,
NY; 2Department of Medicine and Endocrinology, OUHSC,
Oklahoma, OK; 3State Key Laboratory of Ophthalmology, Zhongshan
Ophthalmic Center, Sun Yat-sen University, Guanzhou, China.
Purpose: Breakdown of the blood-retinal barrier (BRB) is one of the
most important features of diabetic retinopathy (DR) and is largely
attributed to disruption of endothelial tight junction. The aim of our
study is to decipher the role of X-box binding protein 1 (XBP1), a
major transcription factor activated by ER stress, in regulation of
endothelial tight junction and BRB function in normal and diabetic
conditions.
Methods: Primary human retinal endothelial cells (HRECs), mouse
retinal endothelial cells (MRECs) and mouse brain endothelial cells
(MBECs) were used for in vitro study. Endothelial cell-specific
XBP1 knockout (XBP1EC-/-) mice were generated and crossed
with db/+ mice to produce a type 2 diabetic XBP1-KO mouse
model (XBP1EC-/-/db/db). A separate group of XBP1EC-/- mice
with controls received 5 consecutive intraperitoneal injections of
streptozotocin to induce type 1 diabetes.
Results: Treatment of HRECs with the ER stress inducer
tunicamycin and thapsigargin resulted in decreased protein levels of
ZO-1 and claudin-5, increased phosphorylation of occludin, disrupted
tight junction and reduced trans-endothelial electrical resistance
(TEER). These changes were exacerbated in cells pre-treated with
pharmacological inhibitor of XBP1 splicing. XBP1EC-/- mice exhibit
reduced ZO-1 and claudin 5 expressions, defective endothelial tight
junction, and increased vascular permeability in the retina. Diabetesinduced BRB breakdown and permeability increase were worsened
in XBP1EC-/-/db/db mice when compare to wild type. Similar results
were observed in STZ-induced type 1 diabetes model. Furthermore,
XBP1-deficient endothelial cells derived from XBP1EC-/- mice were
more sensitive to VEGF- and TNF-α-initiated disruption of tight
junction proteins.
Conclusions: Our findings provide the first evidence that XBP1
is important for maintaining endothelial barrier function, and that
activation of XBP1 protects against ER stress-induced tight junction
damage. Future studies will investigate whether overexpressing
active XBP1 could restore BRB function in diabetic retinopathy and
to identify potential pathways by which XBP1 regulates endothelial
tight junction.
Commercial Relationships: Jacey H. Ma, None; Jingming Li,
None; Joshua J. Wang, None; Sarah X. Zhang, None
Support: NIH grant EY019949, ADA Research Award 7-11-BS-182,
and Unrestricted Grants from Research to Prevent Blindness to the
Department of Ophthalmology of University at Buffalo
Program Number: 2254 Poster Board Number: A0158
Presentation Time: 3:45 PM–5:30 PM
Role of AKT-FOXO1-BIM axis in R28 retinal neurons response
to inflammatory stimuli
Edith Arnold, Lijie Gong, Patrice E. Fort, Thomas W. Gardner,
Steven F. Abcouwer. Ophthalmology and Visual Sciences, Kellogg
Eye Center, University of Michigan, Ann Arbor, MI.
Purpose: Diabetic retinopathy is a neurodegenerative
disease characterized by diminished retinal AKT activity and
neuroinflammation. AKT is crucial to neurotrophic signal
transduction. We hypothesized that AKT activity is a key determinant
of the response of retinal neurons to a pro-inflammatory stimuli, and
examined how manipulation of AKT affected the response of R28
cells to treatment with TNF-alpha.
Methods: R28 cells were cultured on laminin in the presence
of a cAMP analogue to induce neuronal differentiation. Serum
deprivation (SD) and pharmacological inhibitors were used to negate
AKT activity. IGF-1 treatment was used to acutely activate AKT.
AKT activation and AKT substrate phosphorylation was evaluated
by Western blotting with antibodies to phosphorylated and total
proteins. Apoptotic death was evaluated by caspase-3/7 activity, DNA
fragmentation ELISA and lactate dehydrogenase (LDH) release.
Messenger RNA levels were measured by qRT-PCR. Expression of
FOXO1 and BIM, a FOXO1-target gene, was knocked down using
RNA silencing.
Results: AKT activation coincided with phosphorylation of
several protein substrates of AKT kinase, with FOXO1 being most
prominent. SD resulted in dephosphorylation of FOXO1 at the
threonine 24 activation site, leading to nuclear localization of the
protein and increased expression of BIM. IGF-1 reversed these
effects. SD and inhibition of AKT activity caused a significant
increase TNF alpha-induced cell death response, coinciding with
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
increased BIM expression. BIM was the only member of the proapoptotic BH3-only family of genes that was responsive to AKT
deactivation and TNF-alpha treatment. Knockdown of FOXO1 and
BIM significantly diminished R28 cell death in response to SD and
TNF alpha-treatment.
Conclusions: In the absence of AKT activity R28 retinal neuronal
cells exhibited a greater apoptotic response to an inflammatory
stimuli, modeled by TNF-alpha treatment that coincided with
increased expression of BIM. Activation of FOXO1 and BIM
expression are partially responsible for R28 cell death in response to
TNF-alpha treatment in the absence of AKT activity.
Commercial Relationships: Edith Arnold, None; Lijie Gong,
None; Patrice E. Fort, None; Thomas W. Gardner, Aerpio (C),
Akebia (C), Kalvista (C), Penn State University (P); Steven F.
Abcouwer, None
Support: R01 EY20582, Taubman Institute, Research to Prevent
Blindness Physician-Scientist Award.
in the diabetic HYPO, as well as the increased Iba-1+ microglia in
diabetic retina could be mitigated by treatment with minocycline.
Conclusions: Taken together, our data leads us to suggest that loss of
the neuronal survival factors IGFBP-3 and IGF-1 result in neuronal
loss and shrinkage in HYPO and are accompanied by activation of
inflammatory factors such as Iba-1 and MMP-2. Agents that target
both brain and retinal inflammation may be more effective in the
treatment of DR than agents that only influence the retina.
Commercial Relationships: Ping Hu, None; Jeffery S.
Thinschmidt, None; Sergio Caballero, None; Robert Baxter,
None; Louise Cole, None; Samuel Adamson, None; Tailoi ChanLing, None; Maria Grant, None
Support: National Health & Medical Research Council of Australia
Project Grant #1005730, National Health & Medical Research
Council Principal Research Fellowship #571100, the Brian M. Kirby
Gift of Sight Initiative, the Baxter Charitable Foundation and the
Alma Hazel Eddy Trust.
Program Number: 2255 Poster Board Number: A0159
Presentation Time: 3:45 PM–5:30 PM
Loss of Survival Factors and Activation of Inflammatory
Cascades in the Autonomic Centers of the Brain: Implications for
the Pathogenesis of Diabetic Retinopathy (DR)
Ping Hu1, Jeffery S. Thinschmidt2, Sergio Caballero2, Robert Baxter3,
Louise Cole4, Samuel Adamson1, Tailoi Chan-Ling1, Maria Grant5.
1
Department of Anatomy, University of Sydney, Camperdown,
NSW, Australia; 2Department of Pharmacology & Therapeutics, The
University of Florida, Gainesville, FL; 3Kolling Institute of Medical
Research, Sydney, NSW, Australia; 4Advanced Imaging Facility,
Bosch Institute, The University of Sydney, Camperdown, NSW,
Australia; 5Department of Ophthalmology, The Eugene and Marilyn
Glick Eye Institute, Indiana University, Indianapolis, IN.
Purpose: Complex bidirectional interactions between cells of the
immune and nervous systems contribute additional regulatory
mechanisms that influence the cellular activities of both systems. We
asked if this immune-CNS interaction may influence the pathogenesis
of DR. To address this question, we investigated the roles of key
neuronal and immune modulators.
Methods: STZ-induced T1D mice, age-matched controls, and
T1D mice treated with minocycline (an agent that has marked
anti-inflammatory activity that crosses the BBB/BRB and has been
used to treat diabetic retinopathy) and 6 diabetic & 6 age-matched
control human eyes were used. Neuronal, astrocytic and microglial
changes were examined with multiple marker immunohistochemistry
using antibodies against insulin-like Growth Factor (IGF) -1,
insulin-like growth factor-binding protein (IGFBP)-3, MMP2, Iba-1
(for detection of activated microglia) and CD39 (ecto-ADPase -a
regulator of the purinergic system which plays a role in preventing
inflammation).
Results: In the diabetic hypothalamus (HYPO), neuronal soma
diameter was reduced by 20% (p<0.05) whilst neuronal expression of
IGFBP-3 and IGF-1 was reduced by 32% (p<0.05) and 15% (p<0.05)
respectively. Markers associated with inflammation, MMP2 and
Iba-1 were up regulated in astrocytes (46%, p<0.01) and microglia
(29%, p<0.05). The expression of CD39 was down-regulated by
27%, p<0.05) on the microglia and blood vessels. Consistent with
the changes in diabetic HYPO, CD39+ cell density was reduced by
18% (p<0.05), while Iba-1+ microglial density increased by 29%
(p<0.05) in the diabetic mouse retina. In human diabetic retina, CD39
expression on microglia and blood vessels was reduced by 35%
(p<0.05), while Iba-1+ cell density increased by 24% (p<0.05). The
increased Iba-1 and MMP2 and reduced CD39 expression observed
Program Number: 2256 Poster Board Number: A0160
Presentation Time: 3:45 PM–5:30 PM
Role of Akt in VEGF-, bradykinin-, and diabetes-induced retinal
dysfunction and edema
Ward Fickweiler1, 2, Allen C. Clermont1, 2, Nivetha Murugesan1, 2,
Edward P. Feener1, 2. 1Research Division, Joslin Diabetes Center,
Boston, MA; 2Department of Medicine, Harvard Medical School,
Boston, MA.
Purpose: The VEGF and kallikrein kinin systems have been
shown to mediate retinal dysfunction and edema. Both VEGF and
bradykinin (BK) activate the serine/threonine-protein kinase Akt,
which has been reported to mediate vascular hyperpermeability.
The allosteric Akt-inhibitor MK2206 is currently in multiple phase
II clinical trials in oncology. This study examines the effect of Akt
inhibition by MK2206 on VEGF-, BK- and diabetes- induced retinal
vascular permeability (RVP) and retinal thickness
Methods: Sprague-Dawley rats received intravitreal (IVT) injections
of 2 mM BK, 10 ng VEGF, and 5 mM MK2206. Streptozotocininduced diabetic rats received MK2206 for 2 weeks administered
systemically using subcutaneously-implanted osmotic pumps.
Retinal thickness, vessel tortuosity and vasodilation were measured
using spectral domain optical coherence tomography. RVP was
quantified using Evans-Blue dye permeation. Retinal proteomes were
characterized using tandem mass spectrometry. Effects of BK, VEGF,
and MK2206 on Akt phosphorylation were examined in primary
cultures of Bovine Retinal Endothelial Cells (BREC)
Results: BK and VEGF similarly increased retinal thickening by 46
mm (24.6%) vs. 39 mm (20.3%), respectively (p<0.01 vs. baseline)
at 24 hours post IVT injection, and retinal thicknesses normalized
at day 4 post-injection. Proteomic analysis identified a subset of
plasma proteins in the retina that correlated with BK-induced retinal
thickening, suggesting that plasma protein extravasation contributed
to retinal edema. Retinal vessel diameters and tortuosity were
significantly increased after IVT injections of BK or VEGF (p<0.05).
Intravitreal co-injection of MK2206 reduced BK- and VEGFinduced retinal thickness by 65.9% and 56.8% respectively (p<0.05)
and completely blocked BK-induced RVP (p<0.05). Systemically
administered MK2206 normalized diabetes-induced RVP (p<0.05),
without significantly affecting blood glucose, blood pressure, or body
weight in diabetic rats. In vitro studies showed that both BK and
VEGF increased Akt phosphorylation at Ser473 in BREC and these
effects were inhibited by MK2206
Conclusions: Our data shows that Akt inhibition using MK2206
ameliorates the effects of VEGF, BK, and diabetes on retinal vascular
leakage and retinal thickness in rats. These findings suggest that Akt
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
inhibition may provide a therapeutic opportunity to block multiple
pathways that may contribute to diabetic macular edema
Commercial Relationships: Ward Fickweiler, None; Allen C.
Clermont, None; Nivetha Murugesan, None; Edward P. Feener,
None
Support: NIH R01-EY019029, JDRF 17-2011-251, and Mass Lions
Eye Research Fund
Program Number: 2257 Poster Board Number: A0161
Presentation Time: 3:45 PM–5:30 PM
Retinal thinning in mice with streptozotocin-induced diabetes
mellitus
Murat Kucukevcilioglu1, 2, Woo-Jin Jeong1, 3, Kyung Moo Lee4, Mona
K. Garvin5, 7, Chunhua Jiao1, Allison Garmager1, Bhavna J. Antony5,
Michael D. Abramoff4, 6, Elliott H. Sohn1. 1Institute for Vision
Research, Ophthalmology and Visual Sciences, University of Iowa,
Iowa City, IA; 2Ophthalmology, Gulhane Military Medical School,
Ankara, Turkey; 3Ophthalmology, Dong-A University College of
Medicine and Medical Science Research Center, Busan, Republic of
Korea; 4Iowa Institute for Biomedical Imaging, University of Iowa,
Iowa City, IA; 5Electrical and Computer Engineering, University
of Iowa, Iowa City, IA; 6Ophthalmology, VA Medical Center, Iowa
City, IA; 7VA Center for the Prevention and Treatment of Visual Loss,
Iowa City VA Health Care System, Iowa City, IA.
Purpose: Inner retinal thinning in humans with type I diabetes
mellitus (DM) has been previously described by us and others in eyes
with no detectable diabetic retinopathy. We sought to compare retinal
topography in vivo of wild type mice to those with diabetes mellitus
using the Iowa Reference Algorithm for three-dimensional automated
layer segmentation of spectral domain optical coherence tomography
(OCT). We also wanted to determine whether similar findings could
be replicated with ex vivo histologic ganglion cell analysis.
Methods: C57BL/6 mice (3 months) were assigned into
streptozotocin-induced diabetes mellitus (DM; n=20) and agematched control cohorts (n=10). Retinal OCT imaging was performed
(Bioptigen, Morrisville, NC) at baseline, 6 weeks, and 20 weeks
after induction of diabetes. Using the Iowa Reference Algorithm,
mathematically enhanced for mouse OCT, average change in
thickness of the combined retinal nerve fiber layer and ganglion cell
layer (RNFL-GCL) and total retinal thickness were quantified and
compared using two-tailed paired t-test analysis at 0, 6 weeks, and
20 weeks for each group. To evaluate the histologic characteristics of
diabetic mouse retina, immunofluorescence staining were performed.
The number of ganglion cells in the RNFL and GCL was determined
and averaged in five peripheral regions of each section on either side
under light microscopy using 200 magnification.
Results: The mean thickness of RNFL-GCL layer in DM at 20 weeks
(6.24μm) was thinner (p<0.05) compared to baseline (8.85μm). The
mean total retinal thickness of the DM group at 20 weeks (216.5μm)
was similar to the control group (216.2μm). The mean number of
ganglion cell measured per ganglion cell layer length (count/μm) in
the DM group (0.0148/μm) was less than control group (0.0164/μm;
p=0.006).
Conclusions: Significant decrease in inner retinal layer thickness
(especially RNFL-GCL) is seen in mice after 6 and 20 weeks of
streptozocin-induced diabetes mellitus using both OCT and histologic
analysis. These findings suggest that neuroretinal alterations are
consistently seen in early and later stages of streptozotocin-induced
diabetes.
Commercial Relationships: Murat Kucukevcilioglu, None; WooJin Jeong, None; Kyung Moo Lee, None; Mona K. Garvin, None;
Chunhua Jiao, None; Allison Garmager, None; Bhavna J. Antony,
None; Michael D. Abramoff, None; Elliott H. Sohn, None
Program Number: 2258 Poster Board Number: A0162
Presentation Time: 3:45 PM–5:30 PM
Pro-inflammatory effect of 12/15-lipoxygenase derived lipid
metabolites in retina: Role of Pigment epithelium-derived factor
(PEDF) suppression
Ahmed S. Ibrahim1, 2, Amany M. Tawfik4, Sally Elshafey2, Elia J.
Duh3, Sylvia B. Smith4, Mohamed Al-Shabrawey2. 1Biochemistry,
Mansoura University-Faculty of Pharmacy, Mansoura, Egypt; 2Oral
Biology, Georgia Regents university, Augusta, GA; 3Ophthalmology,
The Johns Hopkins University School of Medicine, Baltimore,
MD; 4Cellular Biology and Anatomy, Georgia Regents University,
Augusta, GA.
Purpose: Retinal vascular homeostasis is regulated by a delicate
balance between the levels of VEGF and PEDF. We recently
demonstrated that 12/15-lipoxygenase (LOX)-derived eicosanoids
(12- and 15-hydroxyeicosatetraenoic acids or HETEs) contribute to
diabetic retinopathy (DR) via disrupting this balance. In addition,
NADPH oxidase (NOX), a major source of oxidative stress during
DR, is a downstream mediator from HETEs. The goal of this study
was to portray the causal relationship between LOX-NOX system
and PEDF during DR and to test whether PEDF restoration prevents
HETEs-mediated retinal vascular injury.
Methods: Fluorescein angiogram (FA) was used to characterize
the effect of intravitreal injection of 12- HETE (0.1μM) with or
without PEDF on retinal vascular function in normal wild type (Wt)
living mice. Retina, were then collected for further assessment of
inflammatory markers. Rat Muller cells (rMC1) were treated with
12-HETE in the presence or absence of PEDF followed by analysis
of pNFκB by Western blotting on cell lysates. Condition medium was
collected for ELISA measurement of TNFα and IL6. Experimental
diabetes was induced Wt and NOX2-deficient mice by streptozotocin.
One group of the wt diabetic mice received NADPH oxidase
inhibitor, apocynin (50mg/kg/day). Immunofluorescence and Western
blotting were used to assess the retinal expression of PEDF
Results: FA showed marked increase in the inflammatory response
to the intravitreal injection of 12-HETE into the Wt control mice
as evidenced by marked vascular leakage and increased VCAM-1
expression. Concurrent injection of PEDF showed less vascular
inflammatory response to 12-HETE injection. Furthermore, in-vitro
results showed that HETEs significantly suppressed PEDF expression
while increased levels of p-NFκB, TNFα and IL6 in rMC1. All these
effects were prevented in PEDF-treated cells. Since our previous
studies linked the pro-inflammatory effect of HETEs to the NADPH
oxidase activation, we also examined the impact of NADPH oxidase
inhibition or deletion of NOX2 on retinal expression of PEDF.
Retinal PEDF was preserved in diabetic mice treated with apocynin
or lacking NOX2
Conclusions: Our findings suggest that interfering with LOXNOX signaling system opens up an entirely new terrain for treating
DR by restoring the endogenous PEDF that carries out multilevel
neurovascular protective function.
Commercial Relationships: Ahmed S. Ibrahim, None; Amany M.
Tawfik, None; Sally Elshafey, None; Elia J. Duh, None; Sylvia B.
Smith, None; Mohamed Al-Shabrawey, None
Support: 1R01EY023315-01 and QNRF (NPRP4-1046-3-284)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 2259 Poster Board Number: A0163
Presentation Time: 3:45 PM–5:30 PM
Vasculogenic potential of Adipose stromal cells in hyperglycemic
environment
Gangaraju Rajashekhar1, 2, Stephanie Merfeld-Clauss3, 2, Clifford
Babbey3, 2, Dmitry Traktuev3, 2, Keith L. March3, 2. 1Ophthalmology, IU
School of Medicine, Indianapolis, IN; 2Vascular and Cardiac Center
for Adult Stem Cell Therapy, IU School of Medicine, Indianapolis,
IN; 3Medicine, IU School of Medicine, Indianapolis, IN.
Purpose: Adipose Stromal Cells (ASC) have been shown to play a
regenerative therapeutic role in early stage diabetic retinopathy (DR),
a leading cause of blindness in working-age adults. We have shown
that ASC in co-cultures with human retinal endothelial cells (HREC)
enhance endothelial survival and collaborate to form vascular
networks. In this study we evaluated the effects of hyperglycemia
on bioactivity of ASC and its ability to maintain sustained vascular
networks.
Methods: Human ASC expressing both pericyte and mesenchymal
cell surface markers were exposed to varying doses of glucose
(5.5mM to 100mM) for 7 days. For vascular network formation
(VNF) assay, ASC were co-cultured with HREC at a 6:1 ratio. At day
6 of co-cultivation vascular networks were visualized by staining the
cultures with Agglutinin I and anti-α-smooth muscle actin IgG. Total
tube length (TTL) of the networks was assessed by Angiogenesis
Tube formation assay module of MetaMorph software. Human cord
blood derived endothelial cells (HCBDEC) served as a non-retinal
control endothelial cell type. Accumulation of bioactive molecules
secreted into the media by ASC exposed to physiological and
hyperglycemic conditions was evaluated by ELISA and confirmed by
Western blotting.
Results: ASC cultured under chronic hyperglycemic state secreted
both VEGF and HGF at the same rates as those cells incubated with
physiological glucose level. While the densities of the vascular
networks at high glucose levels were the same as observed in
normal glucose cultures, co-cultures exposed only to 100mM
glucose demonstrated a significant decrease in TTL. Interestingly, a
similar observation were made when ASC were co-cultivated with
HCBDEC. Short term (6 days) pre-treatment of ASC monolayers
with 25mM glucose did not affect their vasculogenic potency when
evaluated in subsequent in vitro tests: 1) media collected from
ASC after exposure to hyperglycemia was able to promote HREC
survival to the same degree as media conditioned by ASC in control
environment; 2) ASC potency to promote HREC to organize into
vascular cords was independent from glucose level.
Conclusions: Our findings demonstrate that ASC have a high
tolerance to hyperglycemia, suggesting that ASC could be a potential
candidate for cell therapy in DR. In our future studies we will explore
the significance of specific biomolecules in this vascular stabilization.
Commercial Relationships: Gangaraju Rajashekhar, None;
Stephanie Merfeld-Clauss, None; Clifford Babbey, None; Dmitry
Traktuev, None; Keith L. March, None
Support: National Eye Institute EY023427
Program Number: 2260 Poster Board Number: A0164
Presentation Time: 3:45 PM–5:30 PM
Microglia dynamics in experimental branch vein occlusion in a
mouse model
Cavit Agca, Andreas Ebneter, Martin S. Zinkernagel. Ophthalmology,
Inselspital, Bern, Switzerland.
Purpose: Branch retinal vein occlusion (BRVO) is a common
vision-threatening disease in the adult population. Loss of vision
develops rapidly and mainly occurs due to formation of macular
edema as a result of occluded vein. BRVO can be induced by laser
photocoagulation in mouse and other animal models. In most
reports on experimental vein occlusion the majority of veins was
occluded, resulting in a phenotype resembling central vein occlusion
disease. We describe here a mouse model of BRVO using laser
photocoagulation to block a single vein and study retinal disease
mechanisms in this model. Additionally, we wanted to characterize
the microglia and Muller cell dynamics in this model.
Methods: Mice were injected with Bengal Rose dye through
tail vein. Laser burns were applied to the superior vein inducing
intravascular coagulation and blockage. Retinas were analyzed in
vivo by fundus imaging and fluorescein angiography. To determine
microglial response, vein occluded retinas were fixed and whole
mount immunohistochemistry was performed using F4/80 and Iba1
antibodies as well as isolectin for co-imaging of the vasculature. Vein
occluded retinas were also collected for RT-PCR analysis Vegf, Tnf,
Ccl5, Cntf and Gfap transcript levels were determined at day 7 and 14
after laser.
Results: We have successfully characterized a mouse model with
a single vein occlusion. Whereas ischemic areas surrounding the
lasered vein were evident at day 3, reperfusion was detectable at
day 7 and 14 by fluorescein angiography often in conjunction with
collaterals. Consistent with this finding, there was no significant
upregulation of Vegf levels at day 7 and 14 timepoints compared to
healthy controls. Immunohistochemical analysis using F4/80, Iba1
and isolectin identified a region of activated microglia/macrophages
that co-localized with the drainage area of the occluded vein.
This finding was also supported by increased levels of Ccl5 and
Tnf expression. Moreover, Gfap and Cntf, which are expressed in
activated Muller cells, are also upregulated.
Conclusions: These results strongly argue that BRVO of a single
vein is sufficient to induce activation of Muller cells and microglia in
mouse neuronal retina but does not upregulate Vegf. The experimental
vein occlusion model resembling BRVO may be a very valuable tool
to study disease mechanisms especially because specific questions
can be answered by using genetically modified mouse models.
Commercial Relationships: Cavit Agca, None; Andreas Ebneter,
None; Martin S. Zinkernagel, None
Program Number: 2261 Poster Board Number: A0165
Presentation Time: 3:45 PM–5:30 PM
Up-Regulation of VEGF by Hyaluronic Acid in Microglia
Bongsu Kim1, Rania Kusibati1, Elaine Binkley1, Mohamed H. AbdelRahman1, 2, Colleen M. Cebulla1. 1Department of Ophthalmology and
Visual Science, The Ohio State University, Columbus, OH; 2Division
of Human Genetics, The Ohio State University, Columbus, OH.
Purpose: Vascular endothelial growth factor (VEGF) expression is
critical to many diseases at the vitreoretinal interface. Inflammatory
cells, particularly macrophages and microglia, play an important role
in disease modulation and may be a source of VEGF. We investigated
the hypotheses that vitreous component hyaluronic acid (HA) and
sheared HA (HAS - low molecular weight) induce VEGF expression
in murine microglia and that AZD 6244, a MAPK/ERK kinase
(MEK) inhibitor down-regulates this VEGF expression.
Methods: Murine microglial BV-2 cells or primary microglia isolated
from CNS were treated with HA and HAS in serum free media
(SFM), with or without AZD6244 or DMSO vehicle. Total RNA, cell
lysate and culture supernatant were collected at 6 and 12 hours. Real
time q-PCR was performed to evaluate gene expression. Western blot
and ELISA assays were performed to evaluate the protein expression.
Results: At 12 hours, BALBC primary microglia show Vegf
expression increased by 1.56-fold in HAS but not HA treatment.
HA and HAS induced Vegf gene expression in BV-2 microglia by
1.27- and 1.44-fold, respectively. VEGF protein on Western blot was
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
increased 3.09-fold in HA and 2.22-fold in HAS compared to SFM.
HAS, but not HA, also increased VEGF expression 1.48-fold at 6hr.
The ELISA assay result shows that HA and HAS with DMSO induce
VEGF expression in cell culture supernatant by 4.01-fold and 3.97fold at 6hours and 1.51-fold and 3.13-fold at 12hours, respectively.
AZD6244 decreased VEGF supernatant concentrations in HA and
HAS to that in SFM with DMSO in all time points.
Conclusions: HA and HAS induced VEGF expression in murine
microglia. MEK inhibitor AZD6244 decreased VEGF supernatant
concentrations in BV-2 cells treated by HA and HAS.
Commercial Relationships: Bongsu Kim, None; Rania Kusibati,
None; Elaine Binkley, None; Mohamed H. Abdel-Rahman, None;
Colleen M. Cebulla, None
Support: Ohio Lions Eye Research Foundation, NIH
1K08EY022672-01, the Patti Blow Fund.
Program Number: 2262 Poster Board Number: A0166
Presentation Time: 3:45 PM–5:30 PM
Age-related Changes in Microglial Signaling in the Aging Retina:
Potential Role of CXCL13
Wenxin Ma1, Lian Zhao1, Robert N. Fariss2, Wai T. Wong1. 1UNGIRD,
National Eye Institute, Bethesda, MD; 2Biological Imaging Core,
National Eye Institute, Bethesda, MD.
Purpose: Age-related retinal diseases, including AMD and diabetic
retinopathy, involve neuroinflammation, indicating a role for immune
dysregulation in their pathogenesis. Retinal microglia, the resident
immune cell type, demonstrate multiple aging changes which may
relate to age-related immune dysfunction. We explore the potential
of CXCL13, an aging-regulated microglial cytokine, to drive
pathologically-significant cell-cell interactions in the aging retina.
Methods: CXCL13 mRNA expression was monitored by qPCR and
microarray analyses, while protein expression was measured with
ELISA. Localization of CXCL13 and CXCR5 in the aged retina
was analyzed by immunohistochemistry. A human RPE cell line
(ARPE19) was used to evaluate CXCL13-CXCR5 signaling in RPE
cells.
Results: Microarray gene profiling demonstrated that CXCL13
mRNA expression increased monotonically as a function of
advancing age in retinal microglia. Age-related increases in CXCL13
expression was verified on the mRNA level by qRT-PCR in retinal
microglia isolated ex vivo by flow-cytometry, and on the protein
level by ELISA in retinal cell lysates. Immunohistochemical
analyses demonstrated that while microglia in the young (2 monthold) retina were immunonegative for CXCL13, those in aged (20
month-old) retina, located in the OPL, IPL, and subretinal space,
were immunopositive. A small subpopulation of AP2α-positive
amacrine cells in the INL were also immunopositive in both young
and aged retina. Microglial expression of CXCL13 was found to
increase with (1) retinal inflammation, as induced by intravitreal
LPS, and (2) with retinal degeneration in 10-month old rd8/rd8 mice.
CXCR5, the cognate receptor of CXCL13, was found constitutively
expressed in RPE cells at the basal and lateral cell membranes and in
αSMA+ smooth muscle cells in retinal and choroidal blood vessels.
Exogeneous application of CXCL13 to ARPE19 cells increased
in ARPE19 cells the expression of pro-inflammatory mediators,
IL1β and TNFα, as well as markers of epithelial-to-mesenchymal
transformation (EMT), snail, twist, and αSMA.
Conclusions: CXCL13 is a microglial-derived cytokine whose
expression increases in an aging-dependent manner and which can
mediate abnormal microglia-RPE signaling in the aged retina. These
interactions may induce changes in RPE cells and alterations in the
retinal immune environment in ways relevant to AMD pathogenesis.
Commercial Relationships: Wenxin Ma, None; Lian Zhao, None;
Robert N. Fariss, None; Wai T. Wong, None
Support: NEI Intramural Research
Program Number: 2263 Poster Board Number: A0167
Presentation Time: 3:45 PM–5:30 PM
The morphology of microglia in physiological and pathological
retinal neovascularization
Akito Shimouchi1, Harumasa Yokota1, Chiemi Matsumoto1, Taiji
Nagaoka1, S. Priya Narayanan3, Shoji Kimura2, Hiroya Kobayashi2,
Ruth B. Caldwell3, Akitoshi Yoshida1. 1Ophthalmology, Asahikawa
Medical University, Asahikawa, Japan; 2Pathology, Division of
Immune Pathology, Asahikawa Medical University, Asahikawa,
Japan; 3Vascular Biology Center, Georgia Regents University,
Augusta, Georgia.
Purpose: Microglial form and function have been shown to be
inextricably linked in neurodegenerative conditions. Quantitative
analysis of microglial morphology can provide an index of retinal
inflammation. This study is the first to quantify microglial shape
changes during retinal neovascularization.
Methods: Oxygen-induced retinopathy (OIR) was induced by
exposing C57BL/6J mice to 75% oxygen from the postnatal day
(P)7 to P12 and then returning them to room air until P17. Control
mice were kept in room air. Retinal vessels were labeled with lectin
and microglia were labeled with Iba1. Confocal images of retinal
microglia were prepared and processed with Image J to calculate
fractal dimension (DF, structural complexity), form factor (FF,
roundness), branching density (BD), convexity (CON) and solidity
(SOL, spatial density).
Results: Microglia were uniformly distributed throughout the
retinas of control mice on P5, P7and P17. During physiological
neovascularization, DF was significantly lower in both central (c)
and peripheral (p) retina as compared with the fully vascularized
P17 retina. Mean ± SD DF in P5c =1.35±0.02, P5p=1.35±0.10,
P7c=1.35±0.05, P7p=1.30±0.06, P17con=1.51±0.31 (p<0.01,
n=5). Comparison of the vascularized central retina and avascular
peripheral retina at P5 and P7 showed no differences in DF, FF, BD,
CON or SOL. Microglia were clustered around neovascular tufts in
the P17 OIR retina. Furthermore, DF was significantly lower in both
the avascular (1.39±0.06) and neovascular (1.38±0.03) areas of the
P17 OIR retina as compared with the P17 control retina (1.51±0.31,
p<0.05). Both FF and SOL were significantly higher in areas of
neovascularization in P17 OIR retina as compared with the avascular
areas (p<0.01). In addition, there was a statistically significant
decrease in FF for both P17 OIR and control retinas as compared
with the P5 and P7 retinas.
Conclusions: Computer-assisted morphometry demonstrated
significant changes in microglial shape during retinal
neovascularization. Microglia in areas of pathological
neovascularization of the OIR retina showed increased roundness
and spatial density as compared with those in the normal retina.
Microglial surface irregularity/complexity was reduced during both
physiological neovascularization and pathological neovascularization.
Studies using adaptive optics and microglia morphometry may offer a
biomarker for detecting early retinopathy in patients.
Commercial Relationships: Akito Shimouchi, None; Harumasa
Yokota, None; Chiemi Matsumoto, None; Taiji Nagaoka, None; S.
Priya Narayanan, None; Shoji Kimura, None; Hiroya Kobayashi,
None; Ruth B. Caldwell, None; Akitoshi Yoshida, None
Support: Grant-in-Aid for Young Scientists (B) 25861609, Tokyo,
Japan
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 2264 Poster Board Number: A0168
Presentation Time: 3:45 PM–5:30 PM
Microglial Depletion in the Adult Mouse Retina: Effects on
Retinal Cell Death and Inflammation
Xu Wang1, Lian Zhao1, Christopher Parkhurst3, Wen-biao Gan3,
Robert N. Fariss2, Wai T. Wong1. 1Unit on Neuron-Glia Interactions
in Retinal Disease, National Eye institute, Bethesda, MD; 2Biological
Imaging Core, National Eye institute, Bethesda, MD; 3Physiology
and Neuroscience, New York University School of Medicine, New
York, NY.
Purpose: In retinal pathologies, microglia have been implicated
in regulating trophic influences to retinal neurons and gliosis in
macroglia. However, whether microglia exert such regulatory
functions in a constitutive manner in the healthy adult retina is
unclear. We explore this question by examining the effects of
sustained microglial depletion in the adult mouse retina.
Methods: Transgenic mice on a C57Bl6 background in which the
Cx3cr1 gene was replaced by a sequence encoding a mutant Cre
protein with a tamoxifen (TAM)-dependent estrogen ligand-binding
domain (Cx3cr1CreER) were crossed with mice carrying lox-P-flanked
(‘floxed’) diphtheria toxin (DTA) to create Cx3cr1CreER/+ DTA/+ (TG)
mice. Depletion of retinal microglia in TG mice was induced by TAM
gavage and was sustained over 30 days by repeated administration.
Histological changes in the retina were monitored in retinal sections
and flat-mounted preparations.
Results: Following 30 days of sustained microglial depletion (<1%
of microglia remaining), histological analyses revealed that overall
retinal organization relative to untreated controls was maintained
without detectable changes in retinal lamination or thicknesses in
retinal cell layers. Apoptosis, as detected by TUNEL staining, was not
induced in any retinal layer. The structure and laminar organization
of retinal vasculature, which are normally in close proximity
to microglia, remained unaffected. The morphologies of retinal
astrocytes and Müller cells, as revealed by immunohistochemical
labeling for GFAP and glutamine synthetase respectively, were
similar to controls; signs of Müller cell gliosis, as evidenced by
process hypertrophy or GFAP upregulation, were also absent.
Conclusions: Microglial absence in the mouse retina for intervals
up to 1 month did not induce detectable neuronal cell death, vascular
change, or alterations in macroglial morphology and activation.
Retinal microglia do not appear necessary for retinal cell survival
or for the maintenance of astrocyte and Müller cell morphology
or physiology. Studies to discover any constitutive microglial
contribution to retinal cell function and retinal synaptic structure are
currently ongoing.
Commercial Relationships: Xu Wang, None; Lian Zhao, None;
Christopher Parkhurst, None; Wen-biao Gan, None; Robert N.
Fariss, None; Wai T. Wong, None
Program Number: 2265 Poster Board Number: A0169
Presentation Time: 3:45 PM–5:30 PM
Genetic model system for microglia depletion in the adult mouse
retina
Lian Zhao1, Xu Wang1, Christopher Parkhurst2, Wen-biao Gan2,
Robert N. Fariss3, Wai T. Wong1. 1Unit on Neuron-Glia Interactions in
Retinal Disease, National Eye Institute, Bethesda, MD; 2Department
of Physiology and Neuroscience, New York University School of
Medicine, New York, NY; 3Biological Imaging Core, National Eye
Institute, Bethesda, MD.
Purpose: Microglia have been implicated as a contributing factor to
retinal disease in humans and animal models. However, endogenous
functions of microglia in the healthy adult retina have not yet
been well-defined. We describe the use of a genetic model system
for depleting microglia in the retina that can be used to study the
constitutive contributions of retinal microglia to retinal function.
Methods: Transgenic mice on a C57Bl6 background in which the
Cx3cr1 gene was replaced by a sequence encoding a mutant Cre
protein with a tamoxifen (TAM)-dependent estrogen ligand-binding
domain (Cx3cr1CreER) were crossed with mice carrying lox-P-flanked
(‘floxed’) diphtheria toxin (DTA) to create Cx3cr1CreER/+DTA+/-(TG)
mice. TG mice were gavaged with TAM (500mg/kg) at different
time intervals and the density and distribution of retinal microglia
analyzed.
Results: Histological comparisons of retinas from young adult
(6-12 weeks old) wild type C57Bl6 mice (WT) and control TG
mice (not treated with TAM) demonstrated similarities with respect
to: 1) retinal anatomy (lamination and thickness of retinal layers),
and 2) retinal microglia (distribution, density and morphology).
Following gavage with TAM (2 cycles, 2 days apart), near-complete
depletion of microglia in all retinal locations and layers was achieved
2 days post-gavage (DPG) as evidenced by Iba1 and CD11b
immunohistochemical analyses. Beginning at around 9 DPG, isolated
Iba1+ cells with amoeboid to ramified morphologies appeared in the
IPL in the peripapillary retinal, spreading into the equatorial retina
by 16 DPG. Colonization of the retina from central to the peripheral
retina was relatively complete by 30 DPG. Near-complete microglia
depletion in the retina was sustained for up to 30 days by repeated
TAM gavage, maintaining total retinal microglia densities at ≈0.5%
of that in control TG retina.
Conclusions: In the absence of TAM exposure, TG mice
demonstrated normal retinal anatomy and retinal microglial
distribution, but become highly depleted of microglia following a
brief period of TAM exposure. Sustained and near-complete depletion
up to 30 days can be induced by maintaining TAM exposure. This
genetic system demonstrates utility in studies aimed at studying
the contributions of microglia to retinal structure, physiology, and
disease.
Commercial Relationships: Lian Zhao, None; Xu Wang, None;
Christopher Parkhurst, None; Wen-biao Gan, None; Robert N.
Fariss, None; Wai T. Wong, None
Support: National Eye Institute Intramural Research Program
Program Number: 2266 Poster Board Number: A0170
Presentation Time: 3:45 PM–5:30 PM
Lipopolysaccaride (LPS)-Induced Inflammation in the Retina:
Effects on Microglia Activation, Cytokine Expression Profile and
Retinal Ganglion Cell Death
Patrik Bauer1, Marina Castro Zalis2, Tomas Deierborg3, Fredrik
Johansson1, Ulrica Englund Johansson2. 1Dept Biology, Functional
Zoology, Lund University, Lund, Sweden; 2Inst Clin Sci, Dept
Ophthalmology, Lund University, Lund, Sweden; 3Inst Exp Med,
Experimental Neuroinflammation Laboratory, Lund University, Lund,
Sweden.
Purpose: Multiple factors play important roles in the development of
glaucoma, with retinal ganglion cell death (RGC) as a major hallmark
of the disease. In the central nervous system, microglia is activated
in response to neurodegenerative disease (e.g. Alzheimer’s disease)
as well as in glaucoma. Thus, it is suggested that inflammation can
contribute to disease progression. The purpose of the present project
was to study the temporal aspects of microglial activation, cytokinerelease profile and RGC death after lipopolysaccharide (LPS)induced inflammation in the mouse retina.
Methods: An organotypic culture system of the postnatal mouse
retina was used. The optic nerve (ON) axotomy, and the explantation
process results in RGC loss and causes microglia activation. LPS
(100 ng/ml) was added to cultures at 48 hours after dissection, and
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
at 24 h, 48 h and 5 days retinas were fixed. Microglia activation
is assessed by 1) numbers of proliferating microglia (Ki-67
immunostaining), 2) morphological changes using Iba-1 and ED1immunostaining and 3) cytokine release. Conditioned media were
collected and a multiplex immunoassay is used to determine cytokine
levels. Degeneration in the ganglion cell layer is examined by
quantification of number of nuclei (DAPI- staining), apoptotic cells
(TUNEL) staining) and RGCs (NeuN- staining).
Results: We demonstrate a 50% significant loss of NeuN-positive
RGCs by apoptosis within 48 hrs after axotomy of the ON. Thereafter
the numbers of DAPI- as well as NeuN-positive cells is stable up
to 14 days. Initial results show enhanced microglial activation after
LPS-treatment judged by a higher number of ED1-positive cells in
the inner retina at all time-points studied, suggesting more activated
microglia, but no significant difference in RGC numbers. Further
analysis is on-going.
Conclusions: Since inflammation in the central nervous system
may have a critical role in the neurodegenerative process in
many diseases, including glaucoma, appropriate models of
neuroinflammation are necessary for understanding the disease
progression. Here we propose a controlled system for studying the
effects of microglial activation on the degeneration in the retina, and
especially the RGCs, using an organotypic mouse retina model.
Commercial Relationships: Patrik Bauer, None; Marina Castro
Zalis, None; Tomas Deierborg, None; Fredrik Johansson, None;
Ulrica Englund Johansson, None
Support: Lund University, VELUX stiftung, Claes Chroschinsky-,
Olle Engqvist funds
Program Number: 2267 Poster Board Number: A0171
Presentation Time: 3:45 PM–5:30 PM
Microglial Activation and Translocator Protein Expression in a
Rat Optic Nerve Crush Model
Nobuharu Asai, Masayoshi Nakatani, Takao Nakamura, Kan Ohtsuki.
Bioengineering Institute R&D Division, NIDEK Co Ltd, Gamagori,
Japan.
Purpose: Microglia are located in central nerve system including
retina. In normal conditions, they have a ramified morphology.
After injury, microglia are activated and their morphology becomes
amoeboid. This study was undertaken to characterize the reaction of
microglia during degeneration of the optic nerve and retinal ganglion
cells (RGCs).
Methods: Optic nerve injury and RGC death were induced by
optic nerve crush (ONC) in adult male Brown Norway rats.
Retinal thicknesses were monitored using spectral-domain optical
coherence tomography. At 1 to 14 days after ONC, the retinas
were isolated, and cells positive for Brn-3a (a marker of RGCs)
were counted. Microglial morphology was observed by using
immunohistochemistry for Iba-1, a marker of microglia. Translocator
protein (TSPO) mRNA expression levels were assessed by using
semiquantitative PCR.
Results: The thickness of ganglion cell complex (GCC) 400 μm from
the optic nerve head (ONH) was 89.6 ± 1.6 mm in naive retinas. The
GCC thickness was visibly reduced 14 days after ONC (71.7 ± 0.6
μm). The number of Brn-3a positive RGCs decreased slowly over the
14 days (104% ± 2%, 90% ± 5%, 42% ± 2% and 7% ± 1% relative
to the numbers in naive retinas, at 1, 3, 7, and 14 days, respectively).
The pattern of microglial staining was altered around part of the
ONH 3 days after ONC. This alteration of microglia had spread
entirely around the ONH at 7 and 14 days after ONC. TSPO mRNA
expression was 50% higher than the naive level at 1 day after ONC,
and this high expression continued for at least 7 days.
Conclusions: In an ONC model, microglia were activated before
reductions were seen in the GCC thickness and number of RGCs.
These results suggest that microglia respond to RGC injury at an
early stage. Because microglial activation and up-regulation of TSPO
expression continued during the progression of tissue degeneration
and RGC death, these events might serve as biomarkers of retinal
diseases such as glaucoma.
Commercial Relationships: Nobuharu Asai, None; Masayoshi
Nakatani, None; Takao Nakamura, None; Kan Ohtsuki, None
Program Number: 2268 Poster Board Number: A0172
Presentation Time: 3:45 PM–5:30 PM
Mechanical Insult Abrogates the Anti-Apoptotic Role of PEA15
Rachel Exler1, 3, John G. Flanagan2, 3, Darren Chan3, Jeremy M.
Sivak2, 3. 1Laboratory Medicine and Pathobiology, University of
Toronto, Toronto, ON, Canada; 2Department of Opthamology
and Vision Science, University of Toronto, Toronto, ON, Canada;
3
Toronto Western Research Institute, University Health Network,
Toronto, ON, Canada.
Purpose: To determine the role of increased levels of astrocytic
PEA15, and it’s associated changes in phosphorylation and function
in the mechanism directing reactive gliosis. Phosphoprotein enriched
in astrocytes (PEA15) was found following proteomic analysis
on human optic nerve head astrocytes exposed to biomechanical
insult. PEA15 is a molecular adaptor implicated in cell proliferation,
apoptosis, and ECM remodelling, and depending on the
phosphorylation state, it can bind and alter the function of several
proapoptotic factors and ERK1/2.
Methods: Primary rat retinal astrocytes and a rat optic nerve
astrocyte cell line (A7 cells) were stretched to induce pathologically
relevant biomechanical insult. This was combined with PEA15
overexpression and knockdown techniques to better understand the
role of PEA15 in apoptosis and the regulation of MMP expression.
Levels of apoptosis were evaluated using flowing cytometry
and western blot, while levels of MMPs were determined via
zymography.
Results: Levels of PEA15 protein were seen to increase at 1, 6, and
12 hours following mechanical insult while ERK and Caspase 8
activation were seen to peak at 6 hours. PEA15 knockdown revealed
a two-fold increase in levels of apoptosis compared to control cells.
Conversely, transfected PEA15 caused a two-fold decrease in rate
of apoptosis. When mechanical insult was combined with PEA15
misexpression, the rate of apoptosis doubled compared to acquiescent
cells, however there was no difference in the level of apoptosis
between PEA15 overexpression, knockdown and mechanically
insulted controls. Zymography analysis of culture media following
mechanical insult revealed increased levels of MMPs 2 and 9 after 6
hours of insult. PEA15 knockdown also increased levels of MMP2
and MMP9, while PEA15 overexpression decreased MMP2 and
MMP9 compared to control.
Conclusions: PEA15 is antiapoptotic and regulates secreted levels
of MMPs 2 and 9 in quiescent astrocytes, however this activity
is abrogated when cells undergo mechanical insult. Additional
experiments are planned to investigate the mechanism for this effect.
Commercial Relationships: Rachel Exler, None; John G.
Flanagan, None; Darren Chan, None; Jeremy M. Sivak, None
Support: CIHR, Vision Science Research Award
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 2269 Poster Board Number: A0173
Presentation Time: 3:45 PM–5:30 PM
Granulocyte Colony Stimulation Factor (G-csf) Blocks Retinal
Gliosis in a Mouse Model of Retinitis Pigmentosa
Wei Wang1, Sang Joon Lee3, Guomin Jiang1, Yongqing Liu1, Ni Xu1,
Henry J. Kaplan1, Douglas C. Dean1, 2. 1Ophthalmology & Visual
Sciences, University of Louisville, Louisville, KY; 2Molecular
Targets Program, James Graham Brown Cancer Center, University
of Louisville, Louisville, KY; 3Department of Ophthalmology, Kosin
University, Kosin, Republic of Korea.
Purpose: The leading cause of blindness in western countries is
retinal degeneration. In lower vertebrates, Muller glia proliferate
in response to retinal damage and dedifferentiate to retinal
photoreceptor progenitors, which in turn replace lost photoreceptors.
But, in higher vertebrates Muller cells instead undergo a gliotic
response leading to scar formation. Such gliotic responses are
a hallmark of CNS injury where they not only inhibit tissue
regeneration but also serve as a barrier to stem cell transplantation
therapy. During photoreceptor degeneration, a glial scar forms
between photoreceptors and the RPE, which acts as a barrier
to subretinal transplantation of stem cell-derived photoreceptor
progenitors. Granulocyte Colony Stimulation Factor (G-csf) is
classically used to stimulate proliferation and survival of bone
marrow progenitors in patients undergoing chemotherapy. But, G-csf
also has well recognized antiapoptotic effects and it has been utilized
as a neuroptotective agent in the retina and to protect cardiomyocytes
in the heart. Here, we examined a potential role for G-csf on Müller
cells in culture and in a mouse model of retinitis pigmentosa.
Methods: Mouse Müller cells were cultured on Matrigel in DMEM
complete medium with 10% FBS. The cells were treated with
100 ng/mg G-csf for 5 or 10 days, then the cells were fixed and
immunostaining Gfap as a marker of gliosis. rd10 mice were injected
with G-csf and the effect on retinal gliosis was evaluated at various
times histologically.
Results: We found that Muller cells expressed the G-csf receptor,
and that G-csf repressed expression of the gliotic marker Gfap in
the cell in culture. In vivo, G-csf injection also blocked Muller
cell expression of Gfap, and it prevented gliotic scar formation as
photoreceptors degenerated in rd10 mice. .
Conclusions: Although glial cells are critical for neuronal survival,
following CNS injury in higher vertebrates they express Gfap and
produce a glial scar that constitutes a major block to tissue repair
and to stem cell transplantation therapy. Our results suggest that
G-csf represses Gfap and it inhibits retinal gliosis that occurs as
photoreceptor degeneration occurs. Thus, G-csf might be an effective
treatment to inhibit glial scarring and facilitate regeneration and stem
cell transplantation therapy not only in the retina but in other CNS
tissues damaged by injury or disease.
Commercial Relationships: Wei Wang, None; Sang Joon Lee,
None; Guomin Jiang, None; Yongqing Liu, None; Ni Xu, None;
Henry J. Kaplan, None; Douglas C. Dean, None
Support: Research to Prevent Blindness, American Health
Assistance Foundation, NIH Grants (P20 RR018733 and EY015636)
and The Commonwealth of Kentucky Research Challenge.
Program Number: 2270 Poster Board Number: A0174
Presentation Time: 3:45 PM–5:30 PM
The Protective Role of AMPK/SIRT1/PGC-1α Signalling in
Retinal Astrocytes
Qi Jiang1, 2, John G. Flanagan2, 3, Darren Chan2, Jeremy M. Sivak2,
3 1
. Laboratory Medicine and Pathobiology, University of Toronto,
Toronto, ON, Canada; 2Vision Science, Toronto Western Research
Institute, Toronto, ON, Canada; 3Ophthalmology and Vision Science,
University of Toronto, Toronto, ON, Canada.
Purpose: The transcriptional co-activator PGC-1α (peroxisome
proliferator-activated receptor gamma coactivator-1 alpha) is a key
regulator of energy metabolism and oxidative stress response. When
activated by upstream regulators such as AMP kinase (AMPK)
and sirtuin 1 (SIRT1), PGC-1α modulates the expression of genes
including those involved in antioxidant defense. We have previously
found that the loss of PGC-1α compromised retinal ganglion cell
(RGC) homeostasis and induced pathological astrocyte reactivity.
We aim to determine whether promoting activity through the AMPK/
SIRT1/PGC-1α pathway in astrocytes can increase their resistance
to oxidative stress and ultimately enhance their ability to support the
RGCs.
Methods: In vitro experiments were performed using primary rat
retinal astrocytes and an astrocytic cell line (A7). After exposure to
oxidative stress, the level or activation of AMPK, SIRT1, and PGC1α was determined by Western blot. To evaluate whether stimulating
the pathway offers protection, pharmacological agonists of AMPK
(AICA-Riboside, AICAR) and SIRT1 (SRT1720) were applied to
A7 cells under oxidative stress. Cell viability was then assessed by
a colorimetric XTT assay. Subsequently, the expression of several
PGC-1α-regulated genes was analyzed after treatment to determine
downstream targets that are significantly regulated.
Results: Following oxidative stress, increased phosphorylation
of AMPK and elevated nuclear levels of SIRT1 and PGC-1α
were evident. Treatment with AICAR and SRT1720 was able to
significantly improve cell viability in a dose dependent manner
(p<0.05). Combined treatment with both compounds further
increased viability compared to either alone. Lastly, analysis of gene
expression revealed the upregulation of antioxidant genes, including
a two- to three-fold increase in superoxide dismutase 1 (SOD1), in
response to AMPK and SIRT1 activation.
Conclusions: The AMPK/SIRT1/PGC-1α axis is stimulated
when astrocytes are challenged by oxidative stress. Through
pharmacological activation of AMPK and SIRT1, astrocytes exhibit
increased resistance against oxidative insult through the expression of
antioxidant defense genes.
Commercial Relationships: Qi Jiang, None; John G. Flanagan,
None; Darren Chan, None; Jeremy M. Sivak, None
Support: Canadian Institutes of Health Research Master’s Award,
Vision Science Research Program OSOTF Award (University of
Toronto, Toronto Western Research Institute)
Program Number: 2271 Poster Board Number: A0175
Presentation Time: 3:45 PM–5:30 PM
Deimination as a potential astrocytic activation marker for
retinal astrocytes following temperature incubation
Mabel E. Algeciras1, 2, Sanjoy K. Bhattacharya1, Horacio M.
Serra2. 1Ophthalmology, Univ of Miami/Bascom Palmer, Miami,
FL; 2Neuroscience, Universidad Nacional de Cordoba, Cordoba,
Argentina.
Purpose: To determine whether temperature fluctuation elicits an
increase in deimination (conversion of protein bound arginine into
citrulline) in retinal astrocytes, and whether elevated deimination
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
levels can serve as an astrocytic activation marker (along with other
activation markers) in the retina.
Methods: Isolated retinal astrocytes obtained from C57BL/6J mice
(about 1000 per plate) were cultured and subjected to different
incubation temperatures for one hour, followed by a stabilization
period of 23 hours at 37°C. The exposed and control astrocytes
were evaluated for deimination levels as well as for levels of other
established markers of astrocyte activation (for example, glial
fibrillary acidic protein: GFAP) using immunohistochemical, Western
blot and ELISA analyses.
Results: Optimal growth of retinal astrocytes occurred at
37°C. Increased levels of astrocyte activation markers such as
Aquaporin4, and Thrombospondin were found in astrocytes
subjected to hypothermia; whereas decreased GFAP, deimination and
peptidylarginine deiminase type 2 (PAD2) levels were found in cells
subjected to this condition. The astrocytes subjected to hyperthermia
showed an increase in deimination and PAD2 levels. Deimination
and PAD2 levels were higher for hyperthermia (approximately
1.5-fold) than for the cells subjected to hypothermia. Hyperthermia
was accompanied by an increase in some astrocytic markers
such as GFAP and aldehyde dehydrogenase 1 family member L1
(ALDH1L1).
Conclusions: The level of deimination undergoes a shift (change in
level compared to baseline) on either side of optimal temperature
incubation (37°C) for retinal astrocytes. The level of deimination
correlates with astrocyte activation markers in the retina when cells
are subjected to temperature treatment. These results agree with our
previous work performed on brain cortex astrocytes, also from the
C57BL/6J mouse strain, in which a similar pattern is observed.
Commercial Relationships: Mabel E. Algeciras, None; Sanjoy K.
Bhattacharya, None; Horacio M. Serra, None
Support: NIH grant P30EY014801
Program Number: 2272 Poster Board Number: A0176
Presentation Time: 3:45 PM–5:30 PM
Induction of Citrullination during Pathological Retinal Gliosis
John Wizeman1, Anthony P. Nicholas2, Royce Mohan1. 1Neuroscience,
University of Connecticut Health Center, West Hartford, CT;
2
Neurology, University of Alabama at Birmingham School of
Medicine, Birmingham, AL.
Purpose: Gliosis is an insidious process underlying a majority of
retinal diseases. While the upregulation of the type III intermediate
filaments (IFs) vimentin (vim) and the glial fibrillary acidic protein
(GFAP) are hallmark features of gliosis, the mechanisms of gliosis
are poorly characterized. IFs undergo several post-translational
modifications, including citrullination. Citrullination is increased in
glaucoma patients, but the direct impact of citrullination on gliosis is
unknown. Here we identify gliosis-related citrullination in the retina
and investigate the possible contribution of IFs to hypercitrullination.
Methods: The DBA/2J mouse model of glaucoma and control
C57BL/6 mice were used to investigate onset of citrullination
in glaucoma. Vim-deficient (Vim-KO) and WT 129SvEV were
subjected to a corneal injury with 1N NaOH. Injured and uninjured
eyes were enucleated after 3 to 7 days. Eyes were then frozen down
for analysis by immunohistochemistry with GFAP, vim and F95
(citrullinated proteins) antibodies, or posterior eye cups were put
into explant culture for 1 to 3 days as a novel method for inducing
hypercitrullination. Retinal proteins were extracted as soluble and
insoluble fractions. Samples were subjected to western blot analysis
for GFAP, vim and F95 expression.
Results: DBA/2J mice accumulate citrullinated proteins by 8 months
of age, while citrullination was not detected in age-matched C57BL/6
mice by western blot. Müller glia express citrullinated filaments in
DBA2/J mice. There was no difference in GFAP expression between
strains. Vim-KO mice exhibit a pattern of hypercitrullination at 1
year of age that was not found in young mice or in WT controls.
With injury, WT eyes displayed increased expression of citrullinated
proteins in the retina compared to uninjured retinas. Injured eyes
exhibit hypercitrullination and a corresponding GFAP overexpression
in explant cultures.
Conclusions: Our results suggest that citrullination is a pathological
process underlying gliosis in the retina. Significantly, increased
citrullination occurs in the DBA/2J model of glaucoma, suggesting
that it could be an early trigger for pathological gliosis. The injuryexplant model revealed a molecular switch from citrullination
to hypercitrullination. Hypercitrullination was also observed in
uninjured aged Vim-KO mice, suggesting that IFs may play a role in
the regulation of citrullination in the retina.
Commercial Relationships: John Wizeman, None; Anthony P.
Nicholas, None; Royce Mohan, None
Support: NIH R01 EY016782, John A. and Florence Mattern
Solomon Endowed Chair
Program Number: 2273 Poster Board Number: A0177
Presentation Time: 3:45 PM–5:30 PM
Increased expression of PSA-NCAM in the optic nerve of
experimental glaucoma
Jooseppi Puranen1, Anne-Mari Haapaniemi1, Sunna Lappalainen2,
Giedrius Kalesnykas1, 2. 1Department of Ophthalmology, University
of Eastern Finland, Kuopio, Finland; 2Experimentica Ltd., Kuopio,
Finland.
Purpose: To examine polysialic acid neural cell adhesion molecule
(PSA-NCAM) expression after microbead-induced ocular
hypertension in retinas and optic nerves of mice that lack brain
derived neurotrophic factor (BDNF+/-) and their wild type littermates
(WT).
Methods: Intraocular pressure (IOP) was elevated unilaterally by
injection of polystyrene microbeads into the anterior chamber to
occlude aqueous outflow. Retinas and optic nerves were harvested 1,
3 and 6- weeks after the treatment. Effects of the treatment on retinal
ganglion cell layer (RGCL) cells and axons of the optic nerve were
determined by stereological methods. The immunoreactive expression
of PSA-NCAM in the eye was detected by immunofluorescence and
electron microscopy.
Results: Mean IOP in the glaucoma-treated eyes was 10.8±1.2
mmHg in WT mice and 11.0±0.6 mmHg in BDNF+/- mice.
Contralateral control eyes had a steady IOP level that was
maintained at an average value of 7.7±0.2 mmHg. There was no
statistically significant RGCL cells loss or axon damage detected in
the glaucomatous eyes. PSA-NCAM expression was increased in
astrocytes in optic nerves and retinas with experimental glaucoma.
The lack of BDNF did not influence on the expression levels of PSANCAM as compared to WT littermates.
Conclusions: Our data indicate that partial loss of BDNF does
not induce higher glaucomatous damage in the retina and optic
nerve. Retinal and optic nerve glia respond to increased IOP by
overexpressing PSA-NCAM. However, we did not observe PSANCAM expression in RGCL neurons.
Commercial Relationships: Jooseppi Puranen, None; AnneMari Haapaniemi, None; Sunna Lappalainen, None; Giedrius
Kalesnykas, None
Support: Finnish Cultural Foundation
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 2274 Poster Board Number: A0178
Presentation Time: 3:45 PM–5:30 PM
Inositol 1, 4, 5-trisphosphate receptors and ryanodine receptors
control intracellular calcium signaling in adult rat optic nerve
head astrocytes
Peter Koulen, Andrew J. Payne, Krupa R. Patel, Yuliya Naumchuk,
Simon Kaja. Ophthal/Vision Research Ctr, University of MissouriKansas City, Kansas City, MO.
Purpose: In glaucomatous retinopathy, pathological changes to
optic nerve head astrocytes (ONHAs) include activation, migration,
extracellular matrix remodeling and altered gene and protein
expression. However, little is known regarding intracellular signaling
pathways in ONHAs. We conducted a detailed quantitative analysis
of intracellular Ca2+ signaling in ONHAs.
Methods: We optimized the culture of primary cultured adult rat
ONHAs and performed a detailed immunocytochemical analysis of
expression and distribution of intracellular Ca2+ channels. Optical
imaging of the intracellular Ca2+ concentration was used to determine
the ion channel-specific contributions to stimulus-induced Ca2+
release from intracellular stores.
Results: We identified strong immunoreactivities for type 1 and type
2 inositol 1, 4, 5-trisphosphate receptors (IP3Rs) in the endoplasmic
reticulum and the nuclear envelope, respectively. Immunoreactivity
for type 3 IP3R was not detected in primary cultured ONHAs. All
ryanodine receptor (RyR) subtypes showed strong immunoreactivity
in primary cultured ONHAs. Our functional analyses revealed
significant responses to pharmacological stimulus-induced
intracellular Ca2+ release from both IP3Rs and RyRs. Subcellular
quantification of intracellular Ca2+ transients showed differential
IP3R-mediated Ca2+ release in nuclear vs. cytosolic compartments
indicating a strong correlation with the differential subcellular
distribution of IP3R subtypes and type 2 IP3R as the major contributor
to intracellular Ca2+ release.
Conclusions: ONHAs utilize differentially distributed intracellular
Ca2+ channels to control their intracellular Ca2+ homeostasis. Our data
provides a critical foundation for future studies investigating potential
changes in Ca2+ signaling in ONHAs as a result of glaucomatous
retinopathy. Furthermore, our protocol for primary culture of adult
rat ONHAs provides new feasibility data for using ONHAs for
drug discovery research for glaucomatous retinopathy and related
disorders affecting the optic nerve and optic nerve head.
Commercial Relationships: Peter Koulen, None; Andrew J.
Payne, None; Krupa R. Patel, None; Yuliya Naumchuk, None;
Simon Kaja, None
Support: NIH grants EY014227, EY022774, AG010485, AG022550,
AG027956, RR022570, RR027093, Felix and Carmen Sabates
Missouri Endowed Chair in Vision Research, Challenge Grant from
Research to Prevent Blindness, Vision Research Foundation of
Kansas City
Program Number: 2275 Poster Board Number: A0179
Presentation Time: 3:45 PM–5:30 PM
βA3/A1-crystallin is a local mediator in astrocytes, regulating the
Notch/STAT3 signaling axis
Mallika Valapala1, Jian Fei Hu1, J S. Zigler1, Stacey L. Hose1, Eric F.
Wawrousek2, Jiang Qian1, Debasish Sinha1. 1Ophthalmology, Johns
Hopkins Univ Sch of Med, Baltimore, MD; 2National Eye Institute,
National Institutes of Health, Bethesda, MD.
Purpose: We have shown that decreased Notch signaling in Nuc1 (a
spontaneous mutation in the Cryba1 gene) astrocytes accounts for the
reduced promoter activity for glial fibrillary acidic protein (GFAP).
This has led us to explore the signaling mediators that regulate GFAP
expression. It is known that the signal transducer and activator of
transcription 3 (STAT3) operates downstream of Notch. The objective
of this study was to determine if βA3/A1-crystallin is required for
the phosphorylation of STAT3 and the subsequent dimerization and
translocation to the nucleus, activating transcription of Cryba1 and
concomitantly, GFAP.
Methods: Optic nerve astrocytes were isolated from postnatal
day 1-2 Cryba1-floxed mice. A lentiviral vector expressing Cre
recombinase was used to delete Cryba1. The activity of STAT3 was
upregulated by IL-6 treatment and inhibited by a STAT3-specific
inhibitor, Stattic. Immunoblotting and ELISA were used to quantify
the phosphorylation of STAT3. ENCODE and synteny mapping of
human to mouse genome were used to identify STAT3 binding sites
in the mouse Cryba1 gene. Quantitative real time PCR and luciferase
assays were used to study the expression of Cryba1 and to validate
the potential STAT3-binding sites in Cryba1.
Results: Our studies show that phosphorylation of STAT3 is inhibited
in the Cryba1-floxed cells upon lentiviral-mediated knockout of
Cryba1. Our data also show that STAT3 could be activated by
IL-6 and inhibited by Stattic. The expression of βA3/A1-crystallin
was elevated by IL-6 and inhibited upon treatment with Stattic.
Furthermore, ENCODE search for STAT3-binding sites in Cryba1,
suggested two potential STAT3-binding sites, one in the promoter
and another within the second intron. Our promoter-based luciferase
assay confirmed that Cryba1 possesses a functional STAT3-binding
site in its promoter. The Notch pathway inhibitor, DAPT also inhibits
STAT3 activity.
Conclusions: Our data suggest that in astrocytes, STAT3 and βA3/
A1-crystallin are co-regulated. This leads to a positive feedback
loop in astrocytes, with βA3/A1-crystallin participating in the
phosphorylation of STAT3 in the cytosol and in turn, STAT3
regulating the transcription of Cryba1 in the nucleus. βA3/A1crystallin modulates the Notch/STAT3 signaling axis in astrocytes
and is involved in regulating GFAP, which might potentiate the
abnormal astrocyte template formation in Nuc1.
Commercial Relationships: Mallika Valapala, None; Jian Fei
Hu, None; J S. Zigler, None; Stacey L. Hose, None; Eric F.
Wawrousek, None; Jiang Qian, None; Debasish Sinha, None
Support: NIH Grant R01EY019037 (to DS) and Research to Prevent
Blindness (unrestricted grant to The Wilmer Eye Institute)
Program Number: 2276 Poster Board Number: A0180
Presentation Time: 3:45 PM–5:30 PM
EXPRESSION OF AQUA/AQUAGLYCEROPORINS IN
MURINE OPTIC NERVE HEAD ASTROCYTES
Rumi Kawashima, Kenji Matsushita, Kohji Nishida. Ophthalmology,
Osaka University Hospital, Suita, Japan.
Purpose: Aqua/aquaglyceroporins (AQPs) absorb small metabolites
around the optic nerve head (ONH). We analyzed the expression of
AQPs in the ONH astrocytes.
Methods: We studied ONH astrocytes with high glial fibrillary
acidic protein (GFAP) positivity cultured from C57BL6 mice and
ONH tissue. The AQP (AQP1 to 12) expression levels in mRNAs
from cultured astrocytes were analyzed by reverse-transcription
polymerase chain reaction (RT-PCR). Expressed AQPs (AQP 1, 4, 5,
and 9) were immunostained in cultured astrocytes and ONH tissue.
Whole-mount immunostaining was performed for AQP4 and AQP9
to investigate the communication between vessels and glia cells.
LSM Software ZEN 2008 (Carl Zeiss MicroImaging Co., Ltd.) was
used for image analysis.
Results: RT-PCR showed cultured ONH astrocytes expressed
AQP1, 5 and AQP9 strongly and AQP4 weakly. In culture, the
immunostaining showed AQP1 and AQP5 in cytosol, the perinuclear
area, and cell membranes and AQP9 was on cell membranes; no
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
AQP4 was expressed. Immunostaining of tissue sections showed
mainly AQP9 in ONH astrocytes and little AQP4 at the edge of the
ONH on the vitreous surface and in the optic nerve astrocytes. In
whole mounts, within a 75-micron radius from the ONH center,
AQP9 was colocalized with GFAP; within that area AQP4 were not
colocalized with GFAP. AQP9 was expressed in the cell body and
processes of the astrocyte (cell body 100%; primary processes 100%;
branchings 42.1%; endfeet 0% [n=10]). AQP9s on the astrocytes
might have contact with surrounding nerve fiber layers. Astrocytes
had endfeet on the retinal large vessels (35.70±9.91 microns [n=10])
and small vessels (7.93±1.46 microns [n=15]). No AQP9 was
expressed at the endfeet of the astrocytes. AQP4 was immunostained
on the large and small retinal vessels, which might be expressed at
the endfeet of the retinal Müller cells.
Conclusions: Murine cultured ONH astrocytes expressed aquaporin
and aquaglyceroporin. Some AQPs were expressed in tissue and
distributed in different ways . Thus, the various distributions of
each AQP might have a role in controlling homeostasis of the small
metabolites in neural tissue.
Commercial Relationships: Rumi Kawashima, None; Kenji
Matsushita, None; Kohji Nishida, None
Program Number: 2277 Poster Board Number: A0181
Presentation Time: 3:45 PM–5:30 PM
Effect of glucocorticoids on neuronal and vascular pathology in a
transgenic model of selective Müller cell ablation
Weiyong Shen1, So-Ra Lee1, Joana Araujo1, 2, Sook H. Chung1, Ling
Zhu1, Mark C. Gillies1. 1Clin Ophthal & Eye Health, University of
Sydney, Sydney, NSW, Australia; 2Department of Physical Chemistry,
Institute of Nanoscience and Nanotechnology, Faculty of Pharmacy,
University of Barcelona, Barcelona, Spain.
Purpose: Many retinal diseases exhibit pathological processes that
affect both neurons and blood vessels. Treatments that address both at
the same time might have advantages over more specific approaches,
such as vascular endothelial growth factor (VEGF) inhibitors which
are used to treat vascular leak but are suspected to have a neurotoxic
effect. The aim of this study was to evaluate the effects of an
intravitreal injection of triamcinolone acetonide (TA) in a transgenic
model in which patchy Müller cell ablation leads to photoreceptor
degeneration, vascular leak and intraretinal neovascularization.
Methods: TA was injected 4 days before induced Müller cell
ablation. Changes in photoreceptors, microglia and Müller cells,
differential expression of P75 neurotrophin receptor (p75NTR),
tumor necrosis factor-α (TNFα), the precursor and mature forms of
neurotrophin 3 (pro-NT3 and NT3) and activation of the p53 and p38
stress-activated protein kinase (p38/SAPK) signalling pathways were
examined. We also evaluated the effects of TA on retinal vascular
pathology.
Results: We found that TA prevented photoreceptor degeneration and
inhibited reactive activation of microglial and Müller cells. TA also
attenuated Müller cell loss and inhibited overexpression of P75NTR,
TNFα, pro-NT and the activation of p53 and p38/SAPK signalling
pathways. TA not only prevented the development of retinal vascular
lesions but also inhibited fluorescein leakage from established
vascular lesions. TA inhibited overexpression of VEGF in transgenic
mice but without affecting its basal level expression in the normal
retina.
Conclusions: Our data suggest that glucocorticoid treatment may be
beneficial for treatment of retinal diseases that affect both neurons
and the vasculature.
Intravitreal injection of triamcinolone acetonide protected
photoreceptors concurrently with inhibition of microglial activation.
Double labelling was conducted on retinal wholemounts using
an antibody against ionized calcium binding adaptor molecule 1
(Iba-1) for microglia (green) and peanut-agglutinin (PNA) for cone
photoreceptor outer segments (red) in a transgenic model of selective
Muller cell ablation.
Commercial Relationships: Weiyong Shen, None; So-Ra Lee,
None; Joana Araujo, None; Sook H. Chung, None; Ling Zhu,
None; Mark C. Gillies, None
Support: This study was supported by grants from Lowy Medical
Research Institute, National Health and Medical Research Council
(APP1028393, APP1050373) and Ophthalmic Research Institute
of Australia. Mark Gillies is a fellow of Sydney Medical School
Foundation and is supported by a National Health and Medical
Research Council (Australia) Practitioner Fellowship.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
306 Stem Cells: Biology and Therapeutic Applications
Tuesday, May 06, 2014 8:30 AM–10:15 AM
S 310E-H Paper Session
Program #/Board # Range: 2671–2677
Organizing Section: Retinal Cell Biology
Program Number: 2671
Presentation Time: 8:30 AM–8:45 AM
Differentiation of Human Stem Cells to Retinal Ganglion-like
Cells using a CRISPR/Cas9 Engineered Reporter Line
Donald J. Zack, Valentin Sluch, Vinod Ranganathan, Cynthia
Berlinicke. Ophthalmology, Wilmer Eye Inst, Johns Hopkins Univ,
Baltimore, MD.
Purpose: A number of diseases lead to retinal ganglion cell (RGC)
death and vision loss, the most common of which is glaucoma. Once
the optic nerve is damaged, few avenues of intervention exist since
the mammalian optic nerve does not regenerate. One ambitious
goal is to use cell replacement therapy to restore vision. Human
pluripotent stem cell (hPSC) differentiation could, in theory, provide
the necessary RGCs for this strategy. In addition, human stem
cell-derived RGCs could make possible an approach for medically
relevant drug screening that would have advantages over the current
use of rodent RGCs. In addition, such cells could provide insights
into human RGC development, gene regulation, and neuronal
biology. Here, we describe a protocol for differentiation of hPSCs to
RGC-like cells that express a variety of RGC-enriched markers and
exhibit spontaneous transient calcium activity typical of neurons.
Methods: H7 human embryonic stem cells were genetically
engineered using the CRISPR/Cas9 nuclease system to contain a
membrane Cherry fluorescent reporter downstream of BRN3B, an
RGC-enriched transcription factor. This augmented cell line was
differentiated in N2B27 neuronal medium in the presence of 2%
matrigel. Following differentiation, we were able to use fluorescent
activated cell sorting (FACS) to isolate the RGC-like cells. We
analyzed the cells using qPCR, immunostaining, and calcium
imaging.
Results: By day 30 of differentiation, BRN3B reporter expression
was evident and the cells displayed long neurite projections. The
cultures were enriched for BRN3B, BRN3A, SNCG, NEFH, NRN1,
and RBPMS expression by qPCR. Additionally, we were able to
detect low levels of melanopsin expression. Calcium imaging of day
35 cultures showed spontaneous calcium activity, typical of neuronal
electrical activity. Immunostaining confirmed the presence of cells
double-labeled for MAP2 and BRN3A, neuronal and RGC-enriched
markers, respectively.
Conclusions: We were able to differentiate hPSCs to RGC-like cells.
Additionally, through the use of CRISPR technology, we generated a
stem cell line containing a human RGC reporter. These cells can be
FACS-sorted to obtain isolated RGCs. We believe that our protocol
lays the groundwork for further experiments on stem cell-derived
human RGCs which can be used for drug screening, developmental
and biological studies, as well as cell replacement experiments.
Commercial Relationships: Donald J. Zack, None; Valentin
Sluch, None; Vinod Ranganathan, None; Cynthia Berlinicke,
None
Support: NIH
Program Number: 2672
Presentation Time: 8:45 AM–9:00 AM
A High Throughput Screen for small molecules that promote
stem cell differentiation into Retinal Pigmented Epithelium
Julien Maruotti1, John Fuller1, Karl Wahlin1, Valentin Sluch1,
Catherine Kim1, Jun Wan1, Kapil Bharti2, Janine Davis2, Sheldon S.
Miller2, Donald J. Zack1. 1Ophthalmology, Johns Hopkins University,
Baltimore, MD; 2National Eye Institute, Bethesda, MD.
Purpose: Age-related macular degeneration (AMD) affects the
retinal pigment epithelium (RPE), the layer of cells that surrounds
and nourishes the neurosensory retina. RPE dysfunction can lead to
photoreceptor death, and subsequent vision loss.
Cell-based transplantation strategies offer the promise of being able
to restore RPE cells. Human pluripotent stem cells (hPSCs) may
prove suitable for this purpose: significant advances have recently
been made in inducing the differentiation of hPSCs toward an
RPE-like cell fate. Nevertheless, the length and efficiency of RPE
generation from hPSCs are still not optimal. We therefore sought to
develop a high-throughput screen aimed at finding small molecules
that could improve RPE differentiation in terms of efficiency and
time course.
Methods: hPSC were cultured until confluence in 384 well plate
format. Thereafter, they were treated with a focused library of
over 300 compounds for 10 days and analyzed by high-throughput
quantitative real-time PCR for the expression of the following key
RPE markers: MITF, OTX2 and PMEL17. Bioinformatic analysis
was performed to identify primary hits. For hit confirmation, doseresponse experiments were done folllowed by qPCR for an extended
panel of RPE markers. To further assess the small molecule efficiency
at generating RPE, the area of pigmented cells was measured after
compound treatment.
Results: After bioinformatic anlysis, 4 compounds were found
to up-regulate both MITF and OTX2 above the 3-fold threshold,
while a single compound was able to increase all 3 markers. qPCR
dose-response assays confirmed that 2/5 primary hits significantly
up-regulated RPE markers compared to vehicle conditions. These two
compounds were BIO and Chetomin. Next we checked the ability
of these small molecules to induce RPE differentiation from hPSC:
after 50 days of differentiation, the area of pigmented cells following
BIO or chetomin treatment was 10 and 15 times larger, respectively,
compared to vehicle treatment.
Conclusions: In conclusion, we have developed a successful
HTS qPCR approach to identify molecules that promote RPE
differentiation from hPSC. We are currently studying how
combinations of these new compounds may help set up faster and
more efficient ways to produce RPE from hPSC.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
After 50 days of differentiation, area of RPE pigmented colonies is
increased following Chetomin or BIO treatment.
Commercial Relationships: Julien Maruotti, None; John Fuller,
None; Karl Wahlin, None; Valentin Sluch, None; Catherine Kim,
None; Jun Wan, None; Kapil Bharti, None; Janine Davis, None;
Sheldon S. Miller, None; Donald J. Zack, None
Support: NIH Grant R21
EY023812
Program Number: 2673
Presentation Time: 9:00 AM–9:15 AM
Induced Pluripotent Stem Cell-derived Tissues Elicit Selective
Immunogenicity
Daniel Feitelberg1, Peter D. Westenskow1, Stephen Bravo1, Tongbiao
Zhao2, Zhili Rong2, Carli M. Wittgrove1, Liliana P. Paris1, Dennis O.
Clegg3, Yang Xu2, Martin Friedlander1. 1Cell and Molecular Biology,
The Scripps Research Institute, La Jolla, CA; 2Molecular Biology,
University of California, San Diego, La Jolla, CA; 3Molecular, Cell
and Developmental Biology, University of California, Santa Barbara,
Santa Barbara, CA.
Purpose: Age-Related Macular Degeneration (AMD) is the leading
cause of vision loss in the elderly. While no cure exists, evidence
suggests that stem cell-derived retinal pigment epithelium (RPE)
grafts can prevent photoreceptor degeneration in atrophic retinas.
Clinical trials utilizing embryonic stem (ES) cell-derived RPE are
ongoing, but require immune suppression; this may be unnecessary
if autologous induced pluripotent stem cells (iPS) are employed.
However, some studies suggest that reprogramming may actually
make naïve iPS cells more immunoreactive.
Methods: Mice with humanized immune systems were generated
and fibroblasts were collected from the same human source. iPSCs
were generated using episomal vectors and were either directly
injected into the host to generate teratomas, or converted into
RPE or smooth muscle cells (SMCs) and characterized. These
iPSCs were then transplanted directly into the muscle or eyes of
humanized mice (HuSCID) as “autografts.” (RPE were injected in
muscle since eyes are relatively immune privileged). ES-RPE and
ES-SMCs were generated as allograft controls. The grafted regions
of the muscle and eyes were examined for T-cell infiltration using
immunohistochemistry.
Results: After injecting naïve autologous iPSCs into HuSCID mice,
unusually small teratomas formed which lacked specific cell types
like smooth muscle, but contained RPE. iPS-RPE and iPS-SMCs that
strongly resemble their primary counterparts (based on expression of
terminal differentiation genes and functional assays) were injected as
autologous grafts. While significant T-cell infiltration was observed
around allografts, none of the iPS-RPE lines elicited obvious T-cell
infiltration. However, all of the autologous iPS-derived SMC lines
examined had significant T-cell infiltration in HuSCID mice. No
T-cell infiltration was observed in eyes injected with iPS- or ES-RPE.
Conclusions: The presence of small teratomas lacking smooth
muscle and other cell types (but containing RPE) in HuSCID
mice suggested that cells derived from iPS may elicit selective
immunogenicity. This was confirmed by injecting autologous RPE
cells and SMC cells and observing pronounced differences in T-cell
infiltration. While this represents a promising outcome for RPE cell
transplantation studies, it also serves as a warning against using other
iPS-derived cells without first doing adequate pre-screening to ensure
that they are not immunoreactive.
Commercial Relationships: Daniel Feitelberg, None; Peter D.
Westenskow, None; Stephen Bravo, None; Tongbiao Zhao,
None; Zhili Rong, None; Carli M. Wittgrove, None; Liliana P.
Paris, None; Dennis O. Clegg, None; Yang Xu, None; Martin
Friedlander, None
Support: CIRM grant TR1-01219 and EY11254
Program Number: 2674
Presentation Time: 9:15 AM–9:30 AM
Safety outcome of subretinal human embryonic stem cell-derived
pigment epithelium (hESC-RPE) transplantation in Yucatan
mini-pigs with oral or intravenous immunosupression.
Paulo Falabella1, 2, Michael J. Koss1, Francisco R. Stefanini1, Marcel
Pfister1, Gerald J. Chader2, Biju B. Thomas2, Padmaja Thomas1,
Dennis O. Clegg3, David R. Hinton2, Mark S. Humayun2. 1Doheny
Eye Institute, Los Angeles, CA; 2Department of Ophthalmology
- Keck School of Medicine, University of Southern California,
Los Angeles, CA; 3University of California, Santa Barbara, Santa
Barbara, CA.
Purpose: To evaluate retinal morphology after subretinal
transplantation of a human embryonic stem cell-derived
retinal pigment epithelium (hESC-RPE) monolayer in Yucatan
mini-pig eyes, dependent on oral or intravenous tacrolimus
immunosuppression regimen as a safety model for future human trials
of dry AMD treatments.
Methods: Five Yucatan mini-pigs underwent vitrectomy with the
creation of a localized subretinal saline solution bleb. A limited
peripheral retinotomy was performed and the transplant consisting
of hESC-RPE polarized cells cultured on parylene membranes
was placed into the subretinal space using a specially designed
inserter. Animals received tacrolimus during the 1 month followup either orally (group 1; n=2) or intravenously, through a vascular
access port and a continuous infusion pump (group 2; n=3).
Examinations included in vivo spectral domain optical coherence
tomography (SD-OCT) and fluorescein angiography (FA). Ex vivo
histological analyses included hematoxylin and eosin (H&E) with
immunohistochemical anti-RPE65 (RPE cell marker), rhodopsin and
TRA-1-85 (human cell marker) antibody evaluation.
Results: All animals reached the 1 month follow-up with thorough in
vivo and ex vivo documentation. Both groups achieved blood levels
of tacrolimus within the therapeutic range in humans (4-10ng/mL).
No animal had retinal detachment or evidence of unduly increased
intraocular inflammation. SD-OCT showed excellent placement for
the majority of the implant with only rare small areas of folds in the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
parylene substrate. FA similarly demonstrated no anterior or posterior
segment leakage. Immunohistochemical analysis showed hESC-RPE
cell survival in both groups.
Conclusions: In this animal model, pars plan vitrectomy followed by
subretinal RPE-parylene sheet implantation was very well tolerated.
The immunosuppression delivered either via intravenous or oral was
effective in reducing the inflammation induced by the xenograft. The
optimum immunosuppression to be used in patients as this approach
is advanced will require clinical evaluation.
Commercial Relationships: Paulo Falabella, None; Michael
J. Koss, None; Francisco R. Stefanini, None; Marcel Pfister,
None; Gerald J. Chader, None; Biju B. Thomas, None; Padmaja
Thomas, None; Dennis O. Clegg, None; David R. Hinton, None;
Mark S. Humayun, None
Support: California Institute for Regenerative Medicine (CIRM)
grant DRI-01444, the National Eye Institute (NEI Core Grant
#EY03040), the German Research Society (DFG KO4294/1-1) and in
part by a grant from Research to Prevent Blindness, Inc.
Program Number: 2675
Presentation Time: 9:30 AM–9:45 AM
Using patient-derived iPSCs to identify new drug treatments for
JNCL
Luke A. Wiley1, Kristin Anfinson1, Emily E. Kaalberg1, Suruchi
Shrestha1, Svetha Swaminathan1, Arlene V. Drack1, Robert Mullins1,
Edwin M. Stone1, 2, Budd A. Tucker1. 1Stephen A. Wynn Institute
for Vision Research, Department of Opthalmology & Visual
Sciences, University of Iowa, Iowa City, IA; 2Howard Hughes
Medical Institute, Department of Ophthalmology & Visual Sciences,
University of Iowa, Iowa City, IA.
Purpose: Juvenile neuronal ceroid lipofuscinosis (JNCL) is
an autosomal recessive lysosomal storage disorder that causes
irreversible blindness, epilepsy, cognitive deficits and premature
death. The most common mutation that causes JNCL is a onekilobase genomic deletion in the gene CLN3. There is no cure for
JNCL and treatments that are effective in slowing disease progression
are limited. The purpose of this study was to generate patient-specific
iPSC-derived retinal neurons stably expressing a caspase-sensitive
fluorescent apoptosis reporter construct, Apoliner, for the purpose of
high-throughput chemical screening and identification of therapeutic
targets.
Methods: Skin biopsies were obtained from patients with
molecularly confirmed CLN3-associated JNCL and from Cln3associated mice (both Cln3ΔlacZ and Cln3Δex7-9). Pluripotent iPSCs
were generated from cultured fibroblasts via viral transduction of the
transcription factors OCT4, SOX2, KLF4 and c-Myc. To monitor the
fate of diseased cells, iPSCs that stably-express the caspase-sensitive
fluorescent apoptosis reporter, Apoliner, were engineered. iPSCs were
subjected to retinal differentiation protocols to obtain retinal neurons.
RT-PCR, Western blotting, and immunocytochemical analysis
were performed to confirm presence of JNCL phenotype(s) and the
function of the Apoliner construct.
Results: We have successfully generated iPSCs from patients
harboring the most common genetic mutation in the gene CLN3 and
from 2 separate mouse models of JNCL. As JNCL retinal neurons
prematurely succumb to apoptotic cell death, the caspase-sensitive
apoptosis fluorescent reporter, Apoliner (Bardet et.al., 2006, PNAS),
was adopted for high-throughput screening purposes. The Apoliner
construct was cloned into a lentiviral vector upstream of an antibiotic
resistance cassette (LV-APO). Each of the above described iPSC
lines were transduced with LV-APO and at 1-week post-transduction
antibiotic resistant stable Apoliner-iPSC lines were clonally
expanded. Stable lines were subjected to our previously developed
stepwise differentiation protocol. Functionality of the Apoliner
construct was confirmed in JNCL-specific neurons via confocal
microscopy.
Conclusions: The iPSC reporter lines generated in this study
provide the basis for future high-throughput small molecule/
chemical screening studies focused on finding compounds capable of
mitigating the JNCL phenotype.
Commercial Relationships: Luke A. Wiley, None; Kristin
Anfinson, None; Emily E. Kaalberg, None; Suruchi Shrestha,
None; Svetha Swaminathan, None; Arlene V. Drack, None; Robert
Mullins, None; Edwin M. Stone, None; Budd A. Tucker, None
Support: NIH Grant 1-DP2-OD007483-01, NEI Grant EY017451,
Howard Hughes Medical Institute, Foundation Fighting Blindness,
Stephen A. Wynn Foundation, and Leo, Jacques & Marion Hauser
Family Vision Restoration Fund
Program Number: 2676
Presentation Time: 9:45 AM–10:00 AM
Gene Therapy for MAK-associated RP
Edwin M. Stone1, 2, Arlene V. Drack1, Rebecca M. Johnston1, Heather
T. Daggett1, Jeremy M. Hoffmann1, Christine M. Hass1, Jessica A.
Penticoff1, Malia M. Collins1, Robert Mullins1, Budd A. Tucker1.
1
Stephen A Wynn Institute for Vision Research, Department of
Ophthalmology and Visual Sciences, University of Iowa, Iowa
City, IA; 2Howard Hughes Medical Institute, Department of
Ophthalmology and Visual Sciences, University of Iowa, Iowa City,
IA.
Purpose: One in 55 people with Ashkenazi Jewish ancestry harbor an
Alu repeat in exon 9 of the MAK gene. Homozygosity of this founder
mutation results in a loss of normal MAK transcript, abnormal
elongation of the photoreceptor connecting cilia and development of
autosomal recessive retinitis pigmentosa (RP). The purpose of this
study was to develop a viral gene transfer approach to treat MAKassociated RP.
Methods: Dermal fibroblasts were obtained from patients with
MAK-associated RP. These cells were used to both investigate the
pathophysiology of the disease and as a cell source for derivation of
patient specific induced pluripotent stem cells (iPSCs). These iPSCs
were differentiated into photoreceptor precursor cells using our
previously published protocol consisting of a stepwise addition of
neurotrophic growth factors, and inhibitors of Wnt, Notch and BMP.
Patient specific cell lines carrying the MAK mutation and Mak-/mice were used as gene transfer recipients. A combination of rt-PCR,
western blotting, immunocytochemistry, confocal microscopy,
electron microscopy, fundus photography, OCT and ERG were used
to evaluate the efficacy of the gene transfer approach.
Results: Full-length retinal MAK cDNA, generated via rt-PCR
amplification from total human retinal RNA, was TA cloned and
subsequently ligated into both lentiviral and AAV vectors for in
vitro and in vivo gene transfer experiments respectively. One week
following transduction of patient specific fibroblasts and iPSC
derived photoreceptor precursor cells, full length MAK could be
detected via rt-PCR and western blotting. Confocal microscopy
revealed that restoration of wild type MAK protein resulted in a
significant shortening of primary cilia. Subretinal injection of AAVMAK into Mak-/- mice resulted in a significant slowing of disease
progression.
Conclusions: We have demonstrated that viral-mediated gene
replacement of MAK can mitigate the pathogenic effects of diseasecausing mutations in this gene in mice and in patient-specific human
cell lines. This is an important step toward a clinical trial of gene
replacement therapy for patients affected with MAK-associated RP.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Edwin M. Stone, None; Arlene V.
Drack, None; Rebecca M. Johnston, None; Heather T. Daggett,
None; Jeremy M. Hoffmann, None; Christine M. Hass, None;
Jessica A. Penticoff, None; Malia M. Collins, None; Robert
Mullins, None; Budd A. Tucker, None
Support: NIH DP2OD007483, Stephen A Wynn Foundation,
Foundation Fighting Blindness
Program Number: 2677
Presentation Time: 10:00 AM–10:15 AM
Targeting protein degradation pathways in macular degeneration
using a human iPS cell model of Best disease
Ruchira Singh1, 3, David Kuai1, Jackie Meyer1, Molly Smith1, Kyle
Wallace1, Amelia Verhoeven1, David M. Gamm2, 3. 1Waisman Center,
University of Wisconsin, Madison, WI; 2Dept. of Ophthalmology and
Visual Sciences, University of Wisconsin, Madison, WI; 3McPherson
Eye Research Institute, University of Wisconsin, Madison, WI.
Purpose: Retinal pigment epithelium (RPE) dysfunction in
degrading photoreceptor outer segments (POS) is linked to increased
accumulation of autofluorescent material in several maculopathies,
including Best disease (BD). Similarly, we previously demonstrated
that human induced pluripotent stem cell (hiPSC)-derived RPE
from two patients with BD (BD-hiPSC-RPE) possessed delayed
degradation of POS and increased autofluorescence accumulation
after chronic POS feeding. In the present study, we used this model
system to determine 1) if alterations in specific protein degradation
pathways are associated with delayed digestion of POS and 2)
whether we could pharmacologically target autofluorescent material
accumulation
Methods: hiPSC-RPE monolayers were derived from two
BD patients and unaffected siblings (Ctrl). Western blotting,
immunocytochemistry, ELISA and fluorescence-based assays
were utilized to evaluate expression, localization and/or activity
of key enzymes involved in lysosomal, ubiquitin-proteasomal and
autophagy-mediated protein degradation before and after chronic
POS feeding. Exosomes secreted by RPE in the apical and basal
media were isolated by differential ultracentrifugation and probed for
protein content. Time course experiments were used to evaluate the
effect of drugs that selectively targeted protein degradation pathways.
This analysis included rate of POS degradation and amount of
autofluorescent material accumulation in RPE cells after chronic POS
feeding.
Results: Differences in the expression and/or activity of specific
proteins/enzymes involved in intracellular protein degradation was
observed 1) between BD and Ctrl hiPSC-RPE and 2) before and after
POS feeding. Differences were also seen in the amount of exosome
released from BD vs. Ctrl hiPSC-RPE both prior to and after chronic
POS feeding. Application of selected drugs increased the rate of
POS degradation in BD hiPSC-RPE. Furthermore, continuous drugtreatment decreased the amount of autofluorescent material after
chronic POS feeding in this BD hiPSC-RPE model.
Conclusions: Our results show that we can treat a key mechanistic
defect in BD, delayed POS degradation, by targeting protein
degradation pathways. Furthermore, by modulating the rate of
POS digestion in this BD hiPSC-RPE model, we can reduce the
accumulation of autofluorescent material in RPE, a pathological
manifestation of various macular dystrophies.
Commercial Relationships: Ruchira Singh, None; David Kuai,
None; Jackie Meyer, None; Molly Smith, None; Kyle Wallace,
None; Amelia Verhoeven, None; David M. Gamm, None
Support: Foundation Fighting Blindness Wynn-Gund Translational
Research Award, Macula Vision Research Foundation, Retina
Research Foundation
321 RPE/Retina Cell Biology and Degeneration, II
Tuesday, May 06, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 2950–2985/D0001–D0036
Organizing Section: Retinal Cell Biology
Program Number: 2950 Poster Board Number: D0001
Presentation Time: 8:30 AM–10:15 AM
Expression of HLA-G in the retinal pigment epithelial cell line,
ARPE-19
Signe G. Svendsen1, Ching-Lien Wu2, Helene B. Juel1, Carsten
Faber3, Edgardo D. Carosella2, Joel LeMaoult2, Mogens H.
Nissen1. 1Int. Health, Immunology and Microbiology, University
of Copenhagen, Copenhagen, Denmark; 2Hemato-Immunology
Research, CEA, Hospital Saint Louis, Paris, France; 3Ophthalmology,
Glostrup Hospital, Glostrup, Denmark.
Purpose: The nonclassical human leukocyte antigen (HLA)
molecule, HLA-G, regulates the immune system and is important
for immunological tolerance during pregnancy. HLA-G mediates the
inhibition of the maternal immune response, which is instrumental
in protecting the semi-allogeneic fetus, thus allowing a successful
pregnancy. Furthermore, HLA-G inhibits NK and T cell cytotoxicity,
T cell proliferation, and antigen presentation, and is involved
in cancer, transplantation and a number of autoimmune and
inflammatory diseases.
NK cells have been described in the subretinal lesions of patients
with age-related macular degeneration (AMD) and are believed to
contribute to the pathogenesis of AMD. Since HLA-G inhibits NK
cells, its expression by RPE cells might be of significance to AMD.
The purpose of this study is to investigate the expression of HLA-G
in RPE cells in vitro and to determine its regulation in response to
proinflammatory cytokines.
Methods: As an inflammatory model, ARPE -19 cells were cultured
and stimulated with IFN-γ and/or TNF-α.
Gene expression analysis was performed by whole-transcriptome
microarray. Protein expression was quantified by western blot and
immunocytochemistry.
Results: Gene expression analyses of RPE cells show that HLA-G is
constitutively expressed at a high level. Furthermore, the expression
is up regulated when the cells are stimulated with IFN-γ and/or
TNF-α. The results from protein analyses confirm these results on the
protein level.
Conclusions: HLA-G is constitutively expressed in RPE cells in
vitro. This expression is up regulated by proinflammatory cytokines
and may be a part of the immune privilege of the posterior segment of
the eye. HLA-G can inhibit NK cell and T cell mediated cytotoxicity,
and can furthermore turn these effector cells into regulatory
cells. Thus this molecule is an important immune modulator, and
expression by the RPE cells may indicate a role for HLA-G in ocular
diseases mediated by inflammation.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Microarray data from the RPE cell line, ARPE-19, show that HLA-G
is constitutively expressed at a high level. The expression is increased
when the cells are stimulated with IFN-γ and/or TNF-α.
A) Western Blot showing HLA-G protein expression.
Expression is up regulated after stimulation with INF-γ and /or
TNF-α.
B) Protein expression is confirmed by immunofluorescence confocal
microscopy.
Commercial Relationships: Signe G. Svendsen, None; Ching-Lien
Wu, None; Helene B. Juel, None; Carsten Faber, None; Edgardo
D. Carosella, None; Joel LeMaoult, None; Mogens H. Nissen,
None
Support: Faculty PhD Scolarship from the Faculty of Health and
Medical Sciences, University of Copenhagen
Program Number: 2951 Poster Board Number: D0002
Presentation Time: 8:30 AM–10:15 AM
Oxytocinergic Signaling via GPCR in a Single HEK293 Cell with
Stable Expression of the Oxytocin Receptor
De-Ann M. Pillers1, 2, Michelle Chiu1, Patrick Halbach1, Nathan
York1, Bikash R. Pattnaik1, 3. 1Pediatrics, University of WisconsinMadison, Madison, WI; 2McPherson Eye Research Institute,
University of Wisconsin-Madison, Madison, WI; 3Ophthalmology &
Visual Sciences, University of Wisconsin-Madison, Madison, WI.
Purpose: We have shown that oxytocin (OXT) localizes to the
cone photoreceptor outer segment whereas the oxytocin receptor
(OXTR) localizes to the retinal pigment epithelium (RPE). OXT
is a neuropeptide hormone traditionally recognized for its role in
parturition. A rise in intracellular calcium ([Ca2+]i) occurs as a result
of OXT binding to the oxytocin receptor (OXTR), most likely
through G-protein coupled receptor (GPCR) mediated activation. We
used molecular and live-cell imaging techniques to visualize OXTOXTR signaling by real-time fluorescence.
Methods: We generated Human Embryonic Kidney (HEK293)
cells stably expressing human OXTR (hOXTR-HEK). Intracellular
changes in [Ca2+] in response to OXT and ATP were measured using
a FURA-2AM ratiometric assay. We used a live-cell fluorescent
marker (pleckstrin homology domain-fused GFP or PH-GFP) for
the detection of membrane phosphatidylinositol 4,5-biphosphate
(PIP2). In the hypothesized rhodopsin-type class I GPCR pathway,
upon agonist binding, membrane PIP2 is hydrolyzed to inositol
1, 4, 5-trisphosphate (IP3) and diacylglycerol (DAG) catalyzed
by phospholipase C (PLC). hOXTR-HEK cells were transiently
transfected with PH-GFP using TransIT-HEK (MirusBio, Madison,
WI). ATP binding was used as a positive control. GFP positive cells
were imaged within 24-72 hr post transfection. Fluorescence images
were acquired every 10 sec.
Results: In response to OXT, hOXTR-HEK cells demonstrated an
average ratiometric increase of 0.0738 ± 0.0028 units (P<0.005)
corresponding to an increase in ~ 75 nM of free [Ca2+]. The
response to ATP was not significantly different, consistent with the
involvement of a GPCR mechanism. PH-GFP has a high affinity
for PIP2 but when PIP2 hydrolyzes, PH-GFP translocates with IP3
to the cytoplasm. In our live-cell imaging experiments, resting cells
expressing GFP in the sub-membrane domain showed translocation
of GFP fluorescence from the membrane to the cytoplasm (an
average increase in cytoplasmic pixel density of 512.09 ± 54.27 units,
P<0.005) when the cells were exposed to OXT. The time course of
GFP translocation correlated with the increase in intracellular [Ca2+].
Conclusions: In a HEK293 OXTR expression cell model, we have
shown that OXT-OXTR signaling uses a GPCR mechanism to
mobilize intracellular [Ca2+]. These results suggest that intercellular
communication may occur in the eye via OXT-OXTR mediated
GPCR signaling.
Commercial Relationships: De-Ann M. Pillers, None; Michelle
Chiu, None; Patrick Halbach, None; Nathan York, None; Bikash
R. Pattnaik, None
Support: Meriter Hospital Foundation, Department of Pediatrics of
UW-Madison, UW-Madison Graduate School
Program Number: 2952 Poster Board Number: D0003
Presentation Time: 8:30 AM–10:15 AM
Increased Wnt Inhibitory Factor 1 in Light and Oxidative Stressinduced Retinal Degeneration
Ae Jin Choi1, Jeehyun Yoon1, Hyunjung J. Lim2, Hyewon Chung1.
1
Ophthalmology, Konkuk University School of Medicine, Seoul,
Republic of Korea; 2Biomedical Science & Technology, Konkuk
University, Seoul, Republic of Korea.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Oxidative stress has been documented as the pathogenesis
of degenerative disease of the retinal pigment epithelium (RPE) and
retina, such as AMD. However, details on the protective mechanism
of the retina in oxidative environment are not well known. We
recently found that the increased level of Wnt inhibitory factor
1 (WIF-1) in the aqueous humor of AMD patients by proteomic
analysis. Here we investigate the potential correlation between the
increase of WIF-1 and cell survival mechanism in light and oxidative
stress-induced retinal degeneration.
Methods: Primary retinal cell and primary Muller cell cultures were
generated from the retinas of newborn or one-week-old Sprague–
Dawley rats as described previously. Cell viability and death were
monitored using CCK-8 assay. Western blot analysis of WIF-1, DKK3, and β-catenin was performed in the retinal and Muller cells and
their conditioned medium. SB216763 was used as a Wnt activator
in cell cultures. Adult C57BL6 mice were exposed to bright light of
20,000 lux for 2h, or injected with NaIO3 into tail vein. Eyes were
collected 2h, 1, 5, and 7 days after exposure and prepared for western
blotting and immunofluorescence (IF) of the RPE and retina.
Results: Exposure to H2O2 (50 mM, 24h) did not cause apoptosis or
cell death in retinal cell cultures. Expression of WIF-1, DKK-3, and
β-catenin were increased in retinal cells and conditioned medium
by exposure to oxidative stress. Exposure of retinal cell cultures to
2 - 4 mM SB216763 with 50 mM H2O2 showed increased expression
of WIF-1 and decreased cell death. Among mixed cells in retinal
cell cultures, Muller cells were markedly expressed WIF-1 and
DKK-3 proteins by IF. Prominent increase of above proteins upon
exposure to oxidative stress was found in Muller cell cultures and
its conditioned medium. Exposure to bright light or NaIO3 resulted
in a marked increase in WIF-1 expression, along with β-catenin
until 5 days after exposure. Increased expression of WIF-1 was colocalized with increased expression of Muller cell marker, glutamine
synthetase.
Conclusions: Increased Wnt signaling in the retina exposed to
light and oxidative stress is protective response against cell death.
Increased WIF-1 produced by Muller cells might be protective
mechanism against light and oxidative stress-induced retinal
degeneration.
Commercial Relationships: Ae Jin Choi, None; Jeehyun Yoon,
None; Hyunjung J. Lim, None; Hyewon Chung, None
Support: National Research Foundation of Korea funded by the
Ministry of Science, ICT & Future Planning (2012M3A9B2028333
and 2012R1A1A11012171)
Program Number: 2953 Poster Board Number: D0004
Presentation Time: 8:30 AM–10:15 AM
Expression of Piwi/piRNA in human ocular tissues: Role in
maintaining functional integrity of retinal pigment epithelial cells
and implication in proliferative diabetic retinopathy
Subbulakshmi Chidambaram1, Suganya Sivagurunathan1, Karthikka
Palanisamy1, Sulochana Natarajan1, Pukhraj Rishi2, Jayamuruga
Pandian Arunachalam1. 1Vision Research Foundation, Chennai,
India; 2Medical Research Foundation, Sankara Nethralaya, Chennai,
India.
Purpose: Piwi clade comprises of 4 proteins namely Piwil1-4 which
specifically bind a novel group of non coding small RNAs, Piwiinteracting RNA (piRNA). Expression of Piwi/piRNA is believed to
be confined to germline and stem cells. Serendipitously we found the
presence of Piwil1 in vitreous from donor eye ball during MALDI
ToF analysis. Intrigued by the result we set out to examine the
presence of Piwi/piRNA in ocular tissues and their possible role.
Methods: Human primary cells were isolated from donor eye balls.
NGS was done in Illumina GAIIX. VEGF, PEDF and adiponectin
cDNA were cloned in pACGFPC1 vector. Panaroma protein antibody
cell signalling array (Sigma) was scanned in Perkin Elmer system.
The study was done with approval from Institution Ethics Board.
Results: RT-PCR and qPCR showed that the transcripts of piwil1-4
were expressed in ocular tissues and cells. Western blot for Piwil1-4
showed stronger expression in retina followed by RPE/choroid.
In addition, IHC of human retinal section confirmed the presence
of Piwi proteins in RPE and RGC layer. Piwil1-4 localized in the
cytoplasm of hRPE cells as shown by IF. The presence of piRNA in
human retina was identified by NGS and validated by RIP and qPCR.
Piwil4 was knocked down in hRPE using DsiRNA and expression
of around 37 genes, involved in the vital functions of RPE was
screened. Decreased levels of tight junction proteins (ZO1, CLDN1
and OCLN), Phagocytosis and trafficking proteins (Syn1A, Syn16,
Rab5 and Vamp8) were observed in Piwil4 knockdown. Interestingly
TGFβ and VEGF levels were also decreased. When VEGF was
overexpressed in hRPE, Piwil4 was upregulated. In contrast,
Adiponectin and PEDF downregulated Piwil4. Protein antibody
array was used to analyse the changes in the proteome of Piwil4
knockdown cells. Further, western blot of vitreous from normal donor
eye ball (n=6) and PDR (n=3) showed reduced level of Piwil4 in
PDR.
Conclusions: The novelty of current finding is the demonstration
of widespread presence of Piwi/piRNA in human somatic (ocular)
tissue. Transdifferentiation of RPE into fibroblast like cells leads to
pathological fibrosis in PDR. Our preliminary results suggest that
Piwil4/piRNA pathway may have a significant role in regulating the
epithelial mesenchymal transition of RPE.
Commercial Relationships: Subbulakshmi Chidambaram, None;
Suganya Sivagurunathan, None; Karthikka Palanisamy, None;
Sulochana Natarajan, None; Pukhraj Rishi, None; Jayamuruga
Pandian Arunachalam, None
Support: Department of Biotechnology BT/PR055/
MED/30/761/2012, India; Department of Science and Technology
SERB/F/1495/2012-13, India
Program Number: 2954 Poster Board Number: D0005
Presentation Time: 8:30 AM–10:15 AM
Expression of the lactate receptor Gpr81 in mouse retina and its
regulation in mouse models of hemochromatosis and diabetes
Vadivel Ganapathy1, Pachiappan Arjunan1, Jaya P GnanaPrakasam1, Sudha Ananth1, Pamela M. Martin1, 2, Sylvia B. Smith2,
3 1
. Biochemistry & Molecular Biology, Georgia Regents University,
Augusta, GA; 2Ophthalmology, Georgia Regents University, Augusta,
GA; 3Cellular Biology and Anatomy, Georgia Regents University,
Augusta, GA.
Purpose: Lactate is an important metabolite that is a critical energy
source for retinal neurons. Extracellular lactate increases retinal blood
flow via angiogenesis and vasodilation. Lactate shuttle is also critical
for retinal function: glucose is metabolized into lactate in Muller
cells and the Muller-cell-derived lactate serves as the energy source
for retinal neurons. Recently, a cell-surface receptor for lactate has
been described in non-retinal tissues. The receptor, GPR81, is linked
to G-proteins Gi/Go, eliciting intracellular signaling with a decrease
in cAMP and/or an increase in calcium. Given the critical role for
lactate in retina, we examined the expression and regulation of Gpr81
in mouse retina to determine the relevance of the receptor to retinal
lactate biology.
Methods: Gpr81 expression was examined by qPCR and
immunofluorescence (IF). Neuronal localization of the receptor was
determined by assessing co-localization with the neuronal marker
NeuN. Polarized distribution of Gpr81 in RPE was assessed using
the monocarboxylate transporter MCT1 as a marker for RPE apical
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
membrane. Relative expression of Gpr81 in primary cultures of
mouse RPE, Muller cells, and retinal ganglion cells was also studied.
Retinal Gpr81 expression was investigated in mouse models of
hemochromatosis and streptozotocin (STZ)-induced diabetes.
Results: Gpr81 is expressed in mouse retina in all cell types. The
expression is comparable in RPE and ganglion cells but lower in
Muller cells. The expression co-localizes with NeuN in ganglion
cells. In RPE, the expression is restricted to the apical membrane.
Gpr81 expression is increased in retina in two different mouse
models of hemochromatosis: Hfe-/- mouse and Hjv-/- mouse, and the
increase is higher in Hjv-/- mouse retina than in Hfe-/- mouse retina.
STZ-induced diabetes is also associated with a marked upregulation
of Gpr81 in the retina.
Conclusions: Gpr81 is expressed widely in murine retina, suggesting
extracellular actions of lactate in target cells through a cell-surface
receptor. The apical membrane localization in RPE suggests that it
is the retinal lactate, not the circulating lactate, that is relevant to
Gpr81 signaling in this cell. The upregulation in hemochromatosis
and diabetes indicates that lactate/Gpr81 signaling is likely a critical
determinant in the retinal pathology of iron overload and diabetes.
Commercial Relationships: Vadivel Ganapathy, None;
Pachiappan Arjunan, None; Jaya P Gnana-Prakasam, None;
Sudha Ananth, None; Pamela M. Martin, None; Sylvia B. Smith,
None
Support: EY19672
Program Number: 2955 Poster Board Number: D0006
Presentation Time: 8:30 AM–10:15 AM
NADPH Oxidase4-derived H2O2 Promotes Aberrant Retinal
Neovascularization via Activation of VEGF Receptor 2 Pathway
in Oxygen-induced Retinopathy
Jingming Li1, 2, Joshua J. Wang1, 3, Sarah X. Zhang1, 3. 1Department
of Medicine and Endocrinology, OUHSC, OKLAHOMA CITY, OK;
2
Department of Ophthalmology, Affiliated Eye Hospital of Nanchang
University, Nanchang, China; 3Departments of Ophthalmology &
Biochemistry, SUNY-Buffalo and SUNY Eye Institute, Buffalo, NY.
Purpose: Oxidative stress plays an important role in the pathogenesis
of neovascular retinal diseases, such as retinopathy of prematurity
(ROP) and diabetic retinopathy (DR). Our previous study
demonstrates that NADPH oxidase 4 (Nox4), a major isoform of
NADPH oxidase in retinal endothelial cells, is responsible for retinal
vascular leakage in type 2 diabetes. However, the role of Nox4
in retinal neovascularization (NV) remains largely unknown. The
purpose of this study is to investigate the function and mechanisms of
Nox4 in the development of retinal NV.
Methods: A mouse model of oxygen-induced retinopathy (OIR) was
used to study retina NV. Nox4 expression was examined by westernblot analysis, real-time RT-PCR and immunohistochemistry in normal
and OIR retinas. Cultured human retinal microvascular endothelial
cells (HREC) were used for in vitro study. Over-expression or
knockdown of Nox4 expression in mouse retinas or HREC was
achieved by adenoviral infection. Generation of intracellular and
extracellular H2O2 was measured by DCF assay and Amplex red
assay respectively. Matrigel angiogenesis assay and transwell
migration assay or wound healing assay were used to evaluate
endothelial angiogenic capacity and migration. Phosphorylation of
VEGF receptor2 (VEGFR2) and ERK were also determined.
Results: Nox4 was mainly localized in the vasculature of mouse
retina and its expression was markedly increased in OIR, in parallel
with enhanced phosphorylation of ERK. Over-expression of Nox4
by adenoviral transduction of wild-type Nox4 gene significantly
increased extracellular H2O2 generation, potentiated VEGFstimulated VEGFR2 activation, and promoted endothelial tube
formation. Conversely, knockdown of Nox4 by siRNA or scavenging
H2O2 by overexpression of catalase inhibited endothelial migration,
VEGF-induced VEGFR2 phosphorylation and tube formation.
Importantly, reducing retinal Nox4 expression by siRNA suppressed
ERK phosphorylation and remarkably attenuated retinal NV
formation in OIR.
Conclusions: Upregulation of Nox4 contributes to retinal NV
formation in OIR. Modulation of retinal Nox4 expression may
present a promising therapeutic approach for neovascular retinal
diseases.
Commercial Relationships: Jingming Li, None; Joshua J. Wang,
None; Sarah X. Zhang, None
Support: NIH grant EY019949; Research Award from American
Diabetes Association 7-11-BS-182; Research Grant HR 10-060 from
Oklahoma Center for the Advancement of Science and Technology.
Program Number: 2956 Poster Board Number: D0007
Presentation Time: 8:30 AM–10:15 AM
Absence of the anti-inflammatory receptor GPR109A is
associated with compromised outer blood-retinal barrier integrity
Pamela M. Martin1, 2, Deeksha Gambhir1, Wanwisa Promsote1,
Rajalakshmi Veeranan-Karmegam1. 1Biochemistry and Molecular
Biology, Georgia Regents University, Augusta, GA; 2Ophthalmology,
Georgia Regents University, Augusta, GA.
Purpose: Diabetic retinopathy (DR) is a leading cause of blindness
worldwide. Alterations of blood-retinal barrier (BRB) integrity
contribute principally to cellular dysfunction and vision loss in the
disease. Much attention has been given to factors and mechanisms
regulating the maintenance of inner-BRB integrity however, the
outer-BRB, of which RPE is a major component, has received
considerably less attention. We demonstrated previously, the
expression of the Gi-linked protein coupled receptor, GPR109A, in
retina, a tissue in which the receptor localizes largely to the RPE
basolateral membrane. Subsequent in vitro and in vivo studies of
GPR109A function in this cell type revealed a role for the receptor
in the regulation of inflammatory signaling. Increased inflammation
and BRB breakdown go hand-in-hand. Therefore, here we examined
the relevance of GPR109A expression to factors relevant to the
preservation of outer-BRB integrity in Gpr109a+/+ and Gpr109a-/mouse retina.
Methods: Micron III technology was used to obtain fundoscopic
images of Gpr109a+/+ (wild type, WT) and Gpr109a-/- (knockout,
KO) mouse eyes at various ages. ZO-1 and occludin expression
was examined in WT and KO RPE flatmount preparations by
immunofluorescence. qPCR and Western blot analyses of these
proteins in RNA and protein samples obtained from WT and KO
RPE/eyecup tissues were also performed. Primary RPE cell cultures
were established from additional WT and KO mouse eyes and
cultured on permeable supports for a period of 6 weeks. These
polarized cells were then used for in vitro permeability assays.
Results: Disruptions in RPE morphology were readily apparent upon
fundoscopic imaging of KO mouse eyes and immunofluorescence
analysis of ZO-1 and occludin expression in corresponding RPE
flatmount preparations. Additionally, levels of ZO-1 and occludin
mRNA and protein were significantly reduced. Altered RPE
junctional protein expression/compromised barrier function was
confirmed by in vitro permeability assays which revealed increased
apical to basolateral leakage of FITC-Dextran dye in KO RPE.
Conclusions: GPR109A expression is important to the maintenance
of outer-BRB integrity. Strategies to augment GPR109A expression
in pathologic conditions such as diabetes may be useful to the
prevention and treatment of outer-BRB breakdown and in turn to the
prevention of vision loss and blindness.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Pamela M. Martin, None; Deeksha
Gambhir, None; Wanwisa Promsote, None; Rajalakshmi
Veeranan-Karmegam, None
Program Number: 2957 Poster Board Number: D0008
Presentation Time: 8:30 AM–10:15 AM
Age-dependent increases in lysosomal pH, lysosomal gene
expression and autofluorescence of mouse RPE cells; parallels
with the ABCA4-/- mice suggest causal factors in age-dependent
pathophysiology
Jason Lim1, Wennan Lu1, Alan M. Laties2, Claire H. Mitchell1,
3 1
. Anatomy and Cell Biology, University of Pennsylvania,
Philadelphia, PA; 2Ophthalmology, University of Pennsylvania,
Philadelphia, PA; 3Physiology, University of Pennsylvania,
Philadelphia, PA.
Purpose: RPE cells change in many ways as they age, but identifying
which changes have a causal impact on the development of diseases
like age-related macular degeneration is difficult. Parallels between
aging mice and genetic disease models may identify key pathological
changes, and thus possible targets for intervention. As lysosomes
are emerging as an important factor in RPE pathophysiology, we
compared changes in RPE lysosomes of aged mice and the ABCA4-/model of Stargardt’s early-onset retinal degeneration.
Methods: Lysosomal pH was measured directly from 6 and 24
month old mice using the Lysosensor assay. Gene expression was
determined using qPCR. RPE autofluorescence was measured from
sections using the spectral detector function of the Nikon A1 confocal
microscope using 406, 488 and 561 nm lasers.
Results: As ABCA4-/- mice have an elevated lysosomal pH, this
pH was determined in young and old control mice. Lysosomal pH
was elevated in older mice and the rise in pH was proportional to
mouse age. RPE cells from aged mice had a reduced expression of
genes for the vesicular proton pump vHATPase and of the lysosome/
autophagy transcription factor TFEB, consistent with the reduction
in lysosomal pH. As lysosomal alkalinization is predicted to impair
enzymatic degradation and lead to accumulation of partially digested
lipids and proteins, levels of lipofuscin-like autofluorescence in aged
RPE cells were determined. Spectral analysis of the autofluorescence
of aged RPE cells showed a difference in emission at 650-660 nm
as compared to younger mice. The emission for ABCA4-/- mice
was also increased at this wavelength, but the magnitude was much
greater than that measured from aged control mice.
Conclusions: In summary, aging of mouse RPE cells induces
changes in lysosomal pH and autofluorescence similar to those found
in ABCA4-/- mice. Whether treatment to restore lysosomal pH found
effective in RPE cells from ABCA4-/- mice prevents age-dependent
pathological changes remains to be determined.
Commercial Relationships: Jason Lim, None; Wennan Lu, None;
Alan M. Laties, None; Claire H. Mitchell, None
Support: NIH Grant EY013434
Program Number: 2958 Poster Board Number: D0009
Presentation Time: 8:30 AM–10:15 AM
Outer retinal parameters studied with Artificial Intelligence
methods predict teleost predatory behavior
Joaquin De Juan1, Noemi Martinez-Ruiz1, Jose L. Girela1, Bassima
Boughlala1, David Gil2. 1Biotecnologia, University of Alicante,
Alicante, Spain; 2Computer Technology, University of Alicante,
Alicante, Spain.
Purpose: Teleots is a successful vertebrate group, constituting more
than half of vertebrate species. Its retinal structure is determined
more by ecological and ethological factors, imposed for the visual
system, than for belonged to a given taxonomic group. Previously
we observed that telosts species with the most retinal spinules were
also the most predatory and vice versa. The aim of this work was to
compare several morphometrics retinal parameters with trophic and
predatory behavior, in twelve species of teleosts.
Methods: The study was performed on twelve species of teleosts
from different habitats. Light-adapted fishes were sacrificed and their
retinas processed for optical and transmission electron microscopy
studies. Thickness (mm) of Outer Nuclear Layer (ONL), Inner
Nuclear Layer (INL), Outer Plexiform Layer (OPL), Inner Plexiform
Laye (IPL), and density of nuclei (N/100 mm2) in ONL and INL were
measured in semithin vertical sections. The number of spinules (SpN)
and synaptic ribbons (SRN) per cone pedicle were counted, using
thin vertical sections. Finally, species were classified into four groups
according to their trophic and piscivorous levels from FishBase data,
and others references. The relationship between retinal parameters
and trophic and predatory levels, were studied using Factorial
Analyses and Decision Tree, one powerful Artificial Intelligence
method for classification and prediction.
Results: The SpN per cone pedicle varies in a wide range between
<4 and >8. Fishes with higher spinule number were also the more
predatory and vice versa. A factorial analyses groups in the first
factor the following parameters: SpN and SRN, trophic level values,
thickness of OPL and nuclei density in ONL. Decision Tree method
has been carried out using the following attributes: thickness of OPL,
ONL, INL, IPL, and SpN and SRN per pedicle. The results showed
that thickness of OPL and ONL were attributes that participated in the
first level in the classification process with accuracy ranging between
75% and 98%.
Conclusions: (1) The amount of spinules per pedicle, synaptic
ribbons, thickness of OPL and ONL correlates positively with
carnivore and predatory behavior. (2) Decision Tree method
accurately predicts the trophic and piscivorous behavior of telosts.
(3) In sum, higher morphometric parameters of the outer retina mean
higher piscivorous behaviors.
Commercial Relationships: Joaquin De Juan, None; Noemi
Martinez-Ruiz, None; Jose L. Girela, None; Bassima Boughlala,
None; David Gil, None
Support: Vicerrectorado de Investigación, University of Alicante,
Spain (Vigrob-137)
Program Number: 2959 Poster Board Number: D0010
Presentation Time: 8:30 AM–10:15 AM
Olfactomedin 1 may suppress APP cleavage through its
interaction with BACE1
Shokichi Takahama, Naoki Nakaya, Stanislav I. Tomarev. National
Eye Institute / National Institutes of Health, Bethesda, MD.
Purpose: Death of retinal ganglion cells (RGCs) is one of the key
pathogenic features of glaucoma. The accumulation of amyloid beta
(Aβ) may contribute to the RGC death. Olfactomedin 1 (Olfm1),
also known as noelin and pancortin, is a secreted glycoprotein
highly conserved in vertebrates. Olfm1 belongs to the family of
olfactomedin domain-containing proteins, and is expressed both in
the retina and brain. Available data suggest that Olfm1 may interact
with amyloid precursor protein (APP), suppress its cleavage, and
inhibit the subsequent production of Aβ. The mechanisms of APP
cleavage inhibition by Olfm1 are unknown. Here, we investigated
these mechanisms.
Methods: Primary RGS cultures were established using an
immunopanning method from 1 to 5 day-old mice. Interactions
between Olfm1 and putative Olfm1 binding proteins were
investigated with an alkaline phosphatase fusion protein assay, as
well as a co-immunoprecipitation assay using lysates of HEK293
cells transfected with corresponding expression constructs.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Localization of Olfm1 and candidate proteins was investigated by
immunofluorescence microscopy.
Results: Analysis of the proteins interacting with Olfm1 confirmed
binding with APP and identified beta-site amyloid precursor proteinconverting enzyme (BACE1) as a novel Olfm1-binding protein.
The 136 amino acid N-terminal region of Olfm1 was sufficient for
its interaction with both BACE1 and APP. Olfm1 and APP were
preferentially expressed in the RGC layer, while BACE1 was
also expressed in the inner nuclear layer. These proteins were also
detected in primary RGC cultures. Available data suggest that Olfm1BACE1 interaction may affect the BACE1 cleavage activity with
APP as a substrate.
Conclusions: Modulation of Olfm1-BACE1 interaction may result in
the accumulation of Aβ in RGCs and brain. The N-terminal portion
of Olfm1 might be considered as a potential neuroprotective agent in
glaucoma and Alzheimer’s disease.
Commercial Relationships: Shokichi Takahama, None; Naoki
Nakaya, None; Stanislav I. Tomarev, None
Support: The Intramural Research Program of the National Eye
Institute, NIH
Program Number: 2960 Poster Board Number: D0011
Presentation Time: 8:30 AM–10:15 AM
Activation of inflammatory signaling in the T17M RHO mice
Tapasi Rana, Marina S. Gorbatyuk. Vision Sciences, University of
Alabama, Birmingham, AL.
Purpose: Previously conducted studies have demonstrated a
contribution of inflammatory response in the pathogenesis of
retinal degenerative disorders. Results from our laboratory have
also indicated that the level of the TNFα marker is significantly
up-regulated in the mouse retina expressing the human T17M
Rhodopsin leading to sever autosomal dominant retinitis pigmentosa.
Therefore, the goal of the study is to identify whether the pro and
anti-inflammatory signaling is involved in the progression of ADRP
in T17M RHO mice.
Methods: RNA was extracted from T17M RHO retinas at P15, P30,
P45 and P60 and QPCR was performed to evaluate the expression
of 22 inflammatory markers associated with pro- (CCL2, CX3CR1,
CXCR1, CXCL1, TNFα, TNFR1, TNFR2, IL-1b and IL-6) and anti(IL-10, NFKB1 and NFKB2) inflammatory responses.
Results: We demonstrated increased expression of pro-inflammatory
chemokines such as CX3CR1, CCL2 and CXCL1 by 3-, 8- and
3-fold, respectively at P15; CX3CR1, CXCR1 and CXCL1 by 2-,
2- and 5-fold, respectively at P30; and CX3CR1, CCL2 and CXCL1
by 2-, 6- and 5-fold, respectively at P45. Moreover, expression of
the pro-inflammatory cytokines TNFα, TNFR2, IL-1b and the proangiogenic cytokine IL-6 were also increased by 4-, 2- and 9- and
4- fold, respectively, at P15. In addition, the TNFα, IRAK1, TNFR2
and TNFR1 expressions were upregulated in P45 ADRP retina by 4-,
2-, 2- and 2- fold respectively. The anti-inflammatory response in the
ADRP mouse retina was detected as well. Expression of the IL-10,
NFKB1 and NFKB2 genes by 7-, 3- and 6-fold, respectively was
observed in P30 ADRP retinas, and by 3-, 2- and 3-fold in P45 ADRP
retinas. In addition, upregulation of the Iba1 expression by 4-fold
was found at all-time points suggesting the activation of microglial
response.
Conclusions: Our study indicated that the progression of ADRP is
associated with a strong immune response .Activation of both the
pro-inflammatory and the anti-inflammatory signaling occur in ADRP
mouse retina not concomitantly.
Commercial Relationships: Tapasi Rana, None; Marina S.
Gorbatyuk, None
Support: NIH Grant RO1EY020905
Program Number: 2961 Poster Board Number: D0012
Presentation Time: 8:30 AM–10:15 AM
ARL13b and CCDC41 is a key molecules for docking and
tethering of a primary ciliary vesicle in human retinal pigment
epithelial cells
Kwangsic Joo1, Jongshin Kim1, Seok Hyun Lee2, Joon Kim1.
1
Graduate school of medical science and engineering, KAIST,
Daejeon, Republic of Korea; 2Ophthalmology, Incheon Metropolitan
Medical Center, Incheon, Republic of Korea.
Purpose: Dysfunction of primary cilium, also known as a ciliopathy,
causes diverse hereditary retinal degenerations such as X-linked
retinitis pigmentosa and Leber’s congenital amaurosis. Recent
reports show that human ciliopathy-related genes, such as CEP164
and CCDC41, are related to the centriole-vesicular docking step.
However, the process of centriole-vesicular docking was not clearly
defined. Here, we show the correlation and interaction between key
molecules of ciliogenesis, ARL13b, CCDC41, CEP164 and CP110 in
the centriole-vesicular docking step.
Methods: We used human RPE1 cellline stably expressing EGFPtagged smoothened (Smo-GFP) was established as previously
reported. Cells were transfected with 5-10nM siRNAsand human
ARL13b and CCDC41 cDNAs cloned into plasmid vectors using
Lipofectamine. For indirect immunofluorescence, cells were fixed in
paraformaldehyde 8 minutes at room temperature and then methanol
for 2 minutes at -20 ‘C. Primary antibodies and secondary antibodies
(Alexa 488-, 594- or 647-conjugated) were applied for 1 h at room
temperature. Immunoprecipitation were performed with Anti-FLAG
M2 affinity gel.
Results: ARL13b colocalizes with single Smo-GFP dot representing
a primary ciliary vesicle and does not overlap with centriole
marker γ-Tubulin and distal appendage marker CEP164. ARL13b
depletion inhibits ciliogenesis and accumulates Smo-GFP vesicles,
illustrating that ARL13b play a key role in formation of the primary
ciliary vesicle. Moreover, immunofluorescence staining and coimmunoprecipitation reveals an overlap and interaction between
exogenously expressed EGFP-tagged ARL13b and FLAG-tagged
CCDC41, whereas mutant forms of FLAG-CCDC41 cDNAs do not
overlap and interact with EGFP-ARL13b cDNAs.
Conclusions: The recruitment of ARL13b to the primary ciliary
vesicle is indispensable for the selective formation of the primary
ciliary vesicle in human RPE1 cells. And, CCDC41 is a key tether for
maintaining ARL13b into the primary ciliary vesicle.
Commercial Relationships: Kwangsic Joo, None; Jongshin Kim,
None; Seok Hyun Lee, None; Joon Kim, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 2962 Poster Board Number: D0013
Presentation Time: 8:30 AM–10:15 AM
Calcium-Induced Apoptosis in retinal degeneration of
S334terRho and P23H Rho Rats
Vishal M. Shinde, Austin Lenox, Marina S. Gorbatyuk. Vision
Science, University of alabama at Birmingham, Birmingham, AL.
Purpose: The S334ter-4 rhodopsin (Rho) and the P23H-3 Rho rats
bear mutant rhodopsin transgenes with an early termination codon at
residue 334 and a point mutation substituting Proline to Histidene at
position 23, correspondingly. Expression of S334ter RHO (Class 1)
and P23H RHO (Class II) is known to activate the Unfolded Protein
Response (UPR) and to trigger the photoreceptor cell death through
apoptosis. However, the ADRP progression in the S334ter RHO rats
is more rapid compared to P23H RHO. It is generally known that
the persistent UPR is capable of provoking a cytosolic Ca+2 release
from the Endoplasmic Reticulum (ER), thus inducing a cell death.
Therefore, the goal of this study is to investigate whether the ADRP
photoreceptors have a raise in the cytosolic Ca2+ and whether the
progression of retinal degeneration in ADRP retina is associated with
Ca2+-induced apoptosis.
Methods: The RNA and protein extracts were obtained from ADRP
and Sprague Dawley (SD) retinas at different time points: P13, P21,
P30, P41, and P60. QRT-PCR and western blotting were performed
to analyze expression of Ca2+-sensing genes and proteins from
different cellular compartments including the ER, cytosol and the
mitochondria.
Results: We found that expression levels of the ER residents, BAX
Inhibitor-1 and calreticulin proteins was steady increased during
the ADRP progression in both rat models starting at P21. However,
this increase was more prominent in P23H RHO retina. Both ADRP
models demonstrated an elevated expression of the ER Ca2+releasing
channel, the IP3R mRNA starting at P13 and a transient up-regulation
of the SERCA2b mRNA in P21 retina. The level of the cytosolic
calpastatin mRNA was significantly higher in both ADRP models
compared to SD but the expression was higher in the P23H RHO than
in the S334ter retina. Western blotting analysis confirmed the upregulation of the IP3R protein and revealed no changes in the SERCA
2b protein in both ADRP retinas.
Conclusions: Results of the study indicate a potential rise in the
intracellular Ca2+ concentration and suggest that Ca2+ signaling
might be involved in the ADRP photoreceptor cell death
Commercial Relationships: Vishal M. Shinde, None; Austin
Lenox, None; Marina S. Gorbatyuk, None
Support: R01EYO20905, TA-GT-0409-0606-UAB.
Program Number: 2963 Poster Board Number: D0014
Presentation Time: 8:30 AM–10:15 AM
Mouse Prpf3, 8 and 31 mutants show altered rhythms of retinal
phagocytosis and adhesion
Deborah Lew1, 2, Michael H. Farkas3, Kinga M. Bujakowska1, 3,
Jonathan Chatagnon1, 2, Shomi S. Bhattacharya4, 5, Eric Pierce3,
Emeline F. Nandrot1, 2. 1Therapeutics, INSERM, U968, UPMC Univ
Paris 06, UMR_S 968, Paris, France; 2CNRS, UMR_7210, Institut
de la Vision, Paris, France; 3Ophthalmology, Ocular Genomics
Institute, Massachusetts Eye and Ear Infirmary, Harvard Medical
School, Boston, MA; 4Institute of Ophthalmology, University College
London, London, United Kingdom; 5Centro Andaluz de Biología
Molecular y Medicina Regenerativa, Andalusian Center of Molecular
Biology and Regenerative Medicine, Sevilla, Spain.
Purpose: Mutations in the Pre-mRNA Processing Factors 3, 8 and
31 (PRPF3, 8 and 31) cause autosomal dominant retinitis pigmentosa
(adRP). We previously showed that gene-targeted mice for these
3 factors display late-onset morphological changes in the retinal
pigment epithelium (RPE). Since the RPE is critically important
for the overall health of the retina, we wanted to determine which
abnormalities precede the morphological changes observed in
these mice. We have focused our investigation on two of the most
important RPE functions that follow a daily rhythm: phagocytosis of
photoreceptor outer segment (POS) and retinal adhesion.
Methods: Phagocytosis was analyzed in vitro and in vivo on
Prpf-mutant and wild-type mice. In vitro phagocytosis assays
were performed on primary RPE cell cultures from 9-10 day-old
mice fed with FITC-labeled POS. Ratios of bound and ingested
phagosomes versus cell numbers were assessed on a fluorescent
microscope. For in vivo phagocytosis assays, mice were euthanized
at different times of the day, eyes processed for electron microscopy
or paraffin sectioning and phagosome numbers were counted on
electron micrographs or paraffin section lengths labeled with an antirhodopsin antibody. Strength of retinal adhesion was evaluated by
quantification of RPE-specific melanin and RPE65 proteins present
on peeled retinas. Expression of phagocytosis receptors and ligands
were analyzed by immunohistological labelings.
Results: Primary Prpf-mutants RPE cells show a 40% decrease
in total phagocytosis. We found that POS binding is reduced by
around 53% in Prpf31-mutant cells, whereas POS internalization
remained similar to wild-type cells. In vivo, we observed the normal
daily phagocytosis peak 2 hours after light-onset in wild-type
controls, while it was greatly reduced in all three mutants. Similarly,
retinal adhesion was reduced at the peak adhesion time in mutants,
while being normal at other times. Some of the main phagocytic
proteins showed altered localization in RPE cells and the POS/
interphotoreceptor matrix area.
Conclusions: Our results suggest that early-onset defects in the
synchronized rhythmicity of retinal phagocytosis and adhesion
could explain the morphological changes observed in aging mutant
RPE cells. Studies are underway to identify the exact cellular
modifications created by altered splicing in these animals.
Commercial Relationships: Deborah Lew, None; Michael
H. Farkas, None; Kinga M. Bujakowska, None; Jonathan
Chatagnon, None; Shomi S. Bhattacharya, None; Eric Pierce,
None; Emeline F. Nandrot, None
Support: This study is supported by NEI-NIH grants #EY020902
and #F32-EY020747.
Program Number: 2964 Poster Board Number: D0015
Presentation Time: 8:30 AM–10:15 AM
Role of claudin-19 and claudin-3 on the barrier function of
human retinal pigment epithelium (RPE)
Shaomin Peng1, 2, Peter Y. Zhao1, Ron A. Adelman3, Lawrence J.
Rizzolo1. 1Surgery/Ophthalmology, Yale University, New Haven, CT;
2
Ophthalmology, 2nd Hospital of Harbin Medical University, Harbin,
China; 3Ophthalmology, Yale University, New Haven, CT.
Purpose: Human RPE uses principally claudin-3 and claudin-19
to form its tight junctions. The ion selectivity of these claudins is
not well known. Although newly confluent ARPE-19 expresses
many tight junction proteins, the expression of claudins is low and
non-uniform. Consequently the tight junctions are rudimentary and
non-functional. This circumstance gives a unique opportunity to
characterize individual claudins in a background that essentially lacks
other claudins, but contains other tight junction proteins.
Methods: ARPE-19 cells were seeded on Snapwell filters and
infected with adenoviral vectors that express either GFP, claudin-3
or claudin-19. The expression of claudin and various RPE genes or
proteins was monitored by quantitative RT-PCR, immunoblotting,
and confocal immunofluorescence microscopy. An Ussing chamber
was used to measure the transepithelial electrical resistance (TER),
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
transepithelial electrical potential (TEP), sodium dilution potential,
and sodium-potassium bi-ionic potential. From these data, we
estimated the permeability of sodium, potassium, and chloride. To
examine cell proliferation and migration, the cultures were scratched
and subsequently examined using phalloidin and Ki-67 to monitor
filamentous actin and cell proliferation, respectively.
Results: Over-expression of either claudin increased the expression
of bestrophin 1, PEDF and TRPC4 more than 2-fold. However,
only claudin-19 increased the expression of the EGF receptor.
Each claudin converted the morphology of cells from squamous to
cuboidal. Cell proliferation and the presence of actin stress fibers
were reduced. Wounds were healed principally by cell spreading.
Each claudin increased the TER and decreased the permeability
of sodium, potassium, and chloride. Both claudins were slightly
cation-specific, and claudin-3 had a slight preference for sodium over
potassium relative to claudin-19.
Conclusions: The study demonstrates that besides regulating
paracellular permeability, claudin-3 and claudin-19 regulate gene
expression. Both up-regulate the expression of several RPE-specific
genes, but only claudin-19 up-regulates the EGF receptor. Claudin-3
and claudin-19 formed general permeability barriers with only slight
ion-selectivity relative to the reports of other claudins. The functions
of claudin-3 and claudin-19 appear to be redundant except for their
regulation of gene expression.
Commercial Relationships: Shaomin Peng, None; Peter Y. Zhao,
None; Ron A. Adelman, None; Lawrence J. Rizzolo, None
Support: the Research to Prevent Blindness (Yale Department of
Ophthalmology), the International Retinal Research Foundation
(L.J.R.), Connecticut Stem Cell Research Fund 10SBC02 (L.J.R.),
the Leir Foundation (R.A.A.), the Newman’s Own Foundation
(R.A.A.), the National Natural Science Foundation of China NO:
30772381 (S.P.), and Connecticut Stem Cell Research Fund Core
Grant 08SCD-004 (Yale University). NIH CTSA-TL1 TR000141-08
Program Number: 2965 Poster Board Number: D0016
Presentation Time: 8:30 AM–10:15 AM
Investigation of new receptors of the scavenger family for the
phagocytosis of spent photoreceptor outer segments by RPE cells
Quentin Rieu1, 2, Jonathan Chatagnon1, 2, Emeline F. Nandrot1, 2.
1
Therapeutics, INSERM, U968, UPMC Univ Paris 06, UMR_S
968, Paris, France; 2CNRS, UMR_7210, Institut de la Vision, Paris,
France.
Purpose: Daily clearance of aged photoreceptor outer segment
(POS) tips by retinal pigment epithelial (RPE) cells is crucial for
retinal health and function. 2 main receptors have been identified,
alphavbeta5 integrin and MerTK, that rhythmically tether and
internalize POS fragments to be eliminated, respectively. However,
many other receptors are present at the RPE cell surface, among
which the scavenger receptor family. The only family member
explored so far, CD36, seems to intervene in the speed rate of
phagocytosis. Other receptors from this family have been suggested
to play a role in POS phagocytosis by RPE cells or in macrophage
elimination of apoptotic cells. Thus, we investigated the potential
implication of these receptors in POS phagocytosis in vitro.
Methods: The candidates investigated are class A scavenger
receptors SR-A and MARCO, and class B receptor SR-BI. We
analyzed the expression of the candidates before and during POS
challenge using immunoblots. We tested their co-localization with
POS at different times of phagocytosis using immunofluorescence
assays. We inhibited expression of the candidates in RPE-J using
siRNA or blocked the proteins function using antibodies, and then
analyzed the potential impact on the cells phagocytic capabilities
using in vitro phagocytosis assays.
Results: All receptors are expressed by RPE-J cells, and their total
cellular expression levels do not seem to vary extensively with POS
inbubation. SR-A and MARCO receptors co-localized partially with
POS, while the association was less observed for SR-BI after 1.5
hours of POS challenge. Inhibition of the candidates’ function using
siRNAs showed an overall decrease in POS phagocytosis that was
more marked after 3 hours of POS challenge. The decrease was more
pronounced for the binding step of phagocytosis for SR-BI, while
SR-A inhibition affected mostly the internalization step. Blocking
the candidates’ function using specific antibodies confirmed the
decreased phagocytic capabilities of RPE cells.
Conclusions: These results indicate that scavenger receptors might
participate in POS clearance by RPE cells, possibly in different steps
of the phagocytic process. Studies are under investigation in order to
identify how they intervene in POS phagocytosis, either directly or
by signaling to the cognate receptors alphavbeta5 integrin or MerTK.
Commercial Relationships: Quentin Rieu, None; Jonathan
Chatagnon, None; Emeline F. Nandrot, None
Support: This study is supported by a project grant from Lifesenses
Labex.
Program Number: 2966 Poster Board Number: D0017
Presentation Time: 8:30 AM–10:15 AM
Effect Of Low Oxygen Culture On Secretion Of Trophic And
Angiogenic Factors By RPE Cells
Jean-Michel Bourget1, 2, Véronique Beaulieu Leclerc1, 2, Olivier
Rochette-Drouin1, 2, Solange Landreville1, 2, Stephanie Proulx1, 2.
1
CUO-Recherche, Centre LOEX de l’Université Laval, Centre de
recherche du CHU de Québec, axe médecine régénératrice, Québec,
QC, Canada; 2Département d’ophtalmologie et d’ORL, Faculté de
médecine, Université Laval, Québec, QC, Canada.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of blindness affecting people over 65 years old. The “dry”
form results from atrophy of the retinal pigment epithelium (RPE).
Regenerative medicine aims at restoring this layer using in vitro cell
culture. In vivo, the RPE is exposed to 4.5-11% of oxygen. However,
RPE cell culture is usually performed under atmospheric conditions
(21% O2). This study was undertaken in order to evaluate the
influence of low oxygen level on the expression of secreted trophic
and angiogenic factors by RPE cells.
Methods: Primary cultures of RPE cells were established from
human donor globes and cultured in DMEM-Ham’s F12 (3:1)
medium supplemented with 10% serum and antibiotics under
physiological (<5% O2) or atmospheric (21% O2) oxygen conditions.
When the cells reached confluency, they were grown without serum
and the conditioned medium was collected after 48h. Trophic factors
secreted by RPE cells were analyzed using proteome profiler array
kits (human cytokines and angiogenesis arrays). Scanned films were
analyzed using ImageJ software.
Results: A higher secretion of IL-6, IL-8/CXCL8, C5/C5a, slCAM-1/
CD54, INF-gamma, IL-23, MCP1/CCL2, TSP-1, IGFBP-3, PAI-1,
PEDF and VEGF was observed in RPE cells cultured at low oxygen
level. Inversely, this condition resulted in lower protein expression of
CXCL16, Endothelin-1, uPA and PDGF-AA.
Conclusions: This study showed a different secretome in RPE cells
grown under physiological oxygen conditions. Further analysis
will be necessary to understand the extent of those changes on RPE
phenotype as well as on other retinal cell types in order to develop a
regenerative therapy for dry AMD.
Commercial Relationships: Jean-Michel Bourget, None;
Véronique Beaulieu Leclerc, None; Olivier Rochette-Drouin,
None; Solange Landreville, None; Stephanie Proulx, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Support: FRQS, FRQS Vision Health Research Network, FRQS
TheCell Network
Program Number: 2967 Poster Board Number: D0018
Presentation Time: 8:30 AM–10:15 AM
Three-dimensional analysis of transparent optic nerves reveals
enhanced regeneration and reduced branching of axons in EphA4
KO mice after traumatic lesion
Vincent Pernet, Sandrine Joly, Noémie Jordi, Martin E. Schwab.
Brain Research Institute, ETH/Univ Zurich, Zurich, Switzerland.
Purpose: In the present study, we investigated the influence of the
guidance molecules EphA4 and ephrinA3 on retinal ganglion cell
(RGC) survival and the regeneration of injured optic nerve axons in
adult mice.
Methods: Axonal regeneration was examined in 3D in the injured
mouse optic nerves of wild-type (WT), EphA4 knock-out (KO) and
ephrinA3 KO C57BL/6 mice. Intraorbital traumatic lesion of the
optic nerve was performed with a 9.0 suture. Thirteen days later,
optic nerve axons were anterogradely traced by injecting cholera
toxin β subunit coupled to alexa 594 (CTb-594) in the vitreous
space. The day after, animals were intracardially perfused with 4%
of paraformaldehyde, optic nerves and retinal flat-mounts were
processed for axonal growth and RGC survival analysis respectively.
To allow 3D analysis of labeled axons, optic nerves were dehydrated
in ethanol and cleared in a solution of benzylbenzoate/benzyl alcohol.
CTb-594-stained axons were imaged by confocal microscopy and
reconstructed in 3D with the Imaris software (Bitplane). The number
of regenerating axons, the directionality and branching of individual
axons were examined. The density of surviving RGCs was calculated
on retinal flat-mounts after immunostaining for β3tubulin.
Results: The number of regenerating axons was markedly increased
in EphA4 KO optic nerve compared with WT controls, at 100,
200, 300, 400, 500 μm from the lesion site. In contrast, ephrinA3
KO mice did not show more growing fibers than WT littermates.
The frequency of axonal U-turns was not different between the 3
experimental groups. However, the number of branched axons was
reduced in EphA4 KO relative to WT optic nerves. The measurement
of growth-associated protein mRNA (Sprr1a, Gap-43 and ATF3) by
qRT-PCR did not show stronger growth program activation in retinal
lysates from injured EphA4 KO mice. In EphA4 optic nerves, glial
fibrillary acidic protein (GFAP)-positive astrocytes retracted from the
injury site at 5 days post-lesion, suggesting that decreased gliosis in
the optic nerve may enhance RGC axon sprouting after trauma. The
survival of RGCs was not significantly affected in animals deprived
of EphA4 or ephrinA3.
Conclusions: Our results suggest that EphA4 negatively regulates
axonal regeneration in a mouse model of optic nerve trauma,
presumably by reducing the glial scar formation.
Commercial Relationships: Vincent Pernet, None; Sandrine Joly,
None; Noémie Jordi, None; Martin E. Schwab, None
Support: Velux Stiftung #817, SNF (31003A-149315-1),
Forschungskredit UZH #54150602
Program Number: 2968 Poster Board Number: D0019
Presentation Time: 8:30 AM–10:15 AM
Sildenafil Treatment Inhibits Zebrafish Rod Photoreceptor Outer
Segment Shedding
Leah J. Campbell, Abbie M. Jensen. Biology Department, University
of Massachusetts Amherst, Amherst, MA.
Purpose: The rod photoreceptor outer segment (ROS) is a modified
cilium composed of stacked, membranous, rhodopsin-filled discs.
New discs are added at the base and old discs are shed from the tip
of the ROS on a daily basis. The latter part of this renewal process
is linked to the light cycle, such that shedding occurs following
illumination. Given that phosphodiesterase (PDE) activity, a
key component of phototransduction, is also stimulated by light,
we sought to investigate whether PDE inhibition by sildenafil
(commercially sold as Viagra) would mimic the dark state and inhibit
shedding in zebrafish larval rods.
Methods: Transgenic zebrafish that expressed EGFP driven by the
Xenopus rhodopsin promoter and a transmembrane-tagged mCherry
protein under the control of a heat shock promoter were heat
shocked at 5 days post fertilization (dpf) and treated with sildenafil
until 8 dpf, 3 days post heat shock (dpHS). Retinal sections were
imaged using confocal microscopy to obtain optical z-stacks of the
photoreceptor layer. Images were analyzed by measuring the distance
from the mCherry stripe to the tip of the ROS (DS) of individual rod
photoreceptors (Fig. 1). Measurements of rods from sildenafil-treated
larvae were compared to rods from vehicle-treated control larvae.
Results: Sildenafil treatment inhibited ROS shedding. Drug-treated
larvae had a significantly longer DS measurement as compared to
vehicle-treated control larvae and similar to that of dark-reared
larvae. The growth distance (DG) from the base of the ROS to the
mCherry stripe was not affected by sildenafil treatment.
Conclusions: To our knowledge, this is the first demonstration of
a drug that inhibits ROS shedding in an in vivo system. Further
investigation of sildenafil treatment on models of human retinal
degeneration disease may provide a basis for potential treatment
of blinding disorders that are characterized by photoreceptor
degeneration.
Fig. 1. Genetically encoded marker of ROS renewal. (A) Construct
for generation of heat shock promoter-driven (hsp70), HA-tagged,
transmembrane (TM)-bound, mCherry transgenic fish. SP, signal
peptide. (B) Heat shock induction and displacement of mCherry
over time. (C) Confocal z-projection of rods from an 8 dpf, 3 dpHS
Tg(Xop:EGFP; hsp70:HA-mCherryTM); alb-/- larva. (D) Diagram of
ROS growth (DG), shedding (DS), and total (DT) distances.
Commercial Relationships: Leah J. Campbell, None; Abbie M.
Jensen, Pfizer (F)
Support: NIH Grant EY015420
Program Number: 2969 Poster Board Number: D0020
Presentation Time: 8:30 AM–10:15 AM
Myocilin is constitutively released with exosomes from RPE in
situ
Christina Locke1, Nicole R. Congrove1, W Daniel Stamer2, Brian S.
McKay1. 1Ophthalmology and Vision Science, University of Arizona,
Tucson, AZ; 2Ophthalmology, Duke University, Durham, NC.
Purpose: Myocilin is a ubiquitous protein that is found both soluble
in the cytoplasm and associated with cytoplasmic vesicles. We have
recently shown that myocilin enters the endocytic compartment
of cells during receptor-mediated endocytosis and is later released
from cells on the surface of exosomes. In this study, we tested the
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
hypothesis that myocilin recruitment and release from RPE cells in
situ is regulated by GPCR (GPR143) activation.
Methods: Using posterior segment prepared from fresh human
donor eyes containing RPE with the retina removed, GPR143 (OA1)
was stimulated with its endogenous ligand, L-DOPA and myocilinassociated exosome release was monitored over time. In a paired
donor eyes, one was stimulated with 5 μM L-DOPA in DMEM
while the contralateral eye was maintained in DMEM. The exosomes
released into the media from the RPE were purified by differential
ultracentrifugation. In parallel experiments, biotinylation was used
to label surface membrane proteins prior to receptor stimulation to
investigate whether myocilin was recruited to activated GPCRs in
situ. We harvested total RPE protein, captured biotinylated proteins
with bound cytoplasmic proteins using streptavidin conjugated beads.
Samples were analyzed for myocilin content by western blot.
Results: RPE in situ constitutively releases myocilin-associated
exosomes. During a 25-minute period of media conditioning, 54%
of total myocilin is released by the unstimulated RPE (n=4). Total
is defined as myocilin in both the cellular and media compartments.
Constitutive release of myocilin-associated exosomes was
dramatically halted following activation of GPR143, with just 10%
of t asotal of myocilin being released after stimulation. Similar to in
vitro models, myocilin was recruited to the membrane compartment
of RPE in situ after GPR143 activation (n=5).
Conclusions: Results show that myocilin-associated exosomes
are continually produced by RPE cells in situ. GPR143 activation
prompted two signal transduction-dependent outcomes in RPE;
trafficking of myocilin to the endosomal compartment, and inhibition
of myocilin-associated exosome release.
Commercial Relationships: Christina Locke, None; Nicole R.
Congrove, None; W Daniel Stamer, None; Brian S. McKay, None
Support: RPB: Center Grant
Program Number: 2970 Poster Board Number: D0021
Presentation Time: 8:30 AM–10:15 AM
RPE dedifferentiation can be inhibited by small molecules
Enrique Salero1, James Moroney1, Jeffrey L. Goldberg2.
1
Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;
2
Ophthalmology, UC San Diego, Shiley Eye Center, San Diego, CA.
Purpose: Retinal degeneration is characterized for progressive loss
of photoreceptor cells that largely are responsible for vision loss.
The etiology is attributed primarily to retinal pigment epithelium
(RPE) cells that are a monolayer of pigmented cells underlying
and supporting the neural retina. We have described that under
some conditions RPE cells undergo EMT and exhibit mesenchymal
properties. Ensuring that RPE cells are maintained as a cobblestone
and do not undergo EMT is critical for photoreceptor survival. Small
molecule has an effect on protein function and can inhibit a specific
function of a multifunctional protein and disrupt protein-protein
interactions. The purpose of this work is to study whether RPE
dedifferentiation can be inhibited or reverse through the use of small
molecules that allow us to identify the molecular pathways involve in
such mechanisms.
Methods: Human RPE cells from cadaveric donors were isolated
and cultured. To activate EMT process, we use different cell passages
(P0-P14) of hRPE cells in culture and test with EMT markers, then
we added individually or cocktail of small molecules that inhibit
the signaling pathways (TGF-β, Wnt/β-catenin, FGFR, MEK1/2)
involve in EMT process. Transepithelial electrical resistance (TEER)
was performed to analyze tight junction before and after treatment.
Western blot and immunostaining were performed before and after
treatments. Total RNA was extracted from hRPE at different passages
before and after treatment. Gene expression assay was carried out by
qRT-PCR analysis.
Results: Our results demonstrate that hRPE cells decrease the
expression of RPE markers and EMT markers activates over the
passages in culture. Our results suggest that inhibition by specific
small molecules targeting TGFβ, Wnt/β-catenin signaling pathway
decrease the expression of EMT markers at early passages rather
than late passages in RPE cells. Immunostaining corroborates the
observation made from gene expression and protein analysis. We
observe morphological changes in all RPE passages after one week of
treatment, cells become more epithelial.
Conclusions: The protection of RPE cells against EMT process is
afforded by the inhibition of TGFβ and Wnt/β-catenin pathways to
prevent dedifferentiation of RPE in early passages, however in late
passages other signaling pathway could be involve and need to be
study. The use of small molecules may be a novel therapeutic target
in retinal degenerative diseases.
Commercial Relationships: Enrique Salero, None; James
Moroney, None; Jeffrey L. Goldberg, None
Support: EY014801
Program Number: 2971 Poster Board Number: D0022
Presentation Time: 8:30 AM–10:15 AM
Disease-causing mutations associated with four
bestrophinopathies exhibit disparate effects on the localization,
but not the oligomerization, of Bestrophin-1
Adiv A. Johnson1, 2, Yong S. Lee2, Lihua Y. Marmorstein2, Alan D.
Marmorstein2. 1Physiological Sciences Graduate Interdisciplinary
Program, University of Arizona, Tucson, AZ; 2Ophthalmology, Mayo
Clinic, Rochester, MN.
Purpose: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause
five clinically distinct retinal degenerative diseases, including adultonset vitelliform macular dystrophy (AVMD), autosomal recessive
bestrophinopathy (ARB), retinitis pigmentosa (RP), and autosomal
dominant vitreoretinochoroidopathy (ADVIRC). Best1 is localized to
the basolateral plasma membrane of the RPE, where it forms homooligomeric anion channels and regulates intracellular Ca2+ signaling.
To gain insight into the pathogenesis of these diseases, we screened
28 Best1 mutants associated with AVMD, ARB, RP, and ADVIRC for
defects in localization and oligomerization.
Methods: Using confocal microscopy and immunofluorescence, we
assessed Best1 localization after expressing YFP-tagged Best1 in
highly confluent, polarized MDCK cells via adenovirus-mediated
gene transfer. Oligomerization was evaluated by live-cell, confocal
fluorescence resonance energy transfer (FRET) between WT Best1CFP- and WT or mutant Best1-YFP. FRET data were confirmed
by reciprocal co-immunoprecipitation as well as co-localization
experiments between WT Best1-c-myc- and WT or mutant Best1YFP.
Results: Many, but not all, AVMD- and ARB-associated mutants
were mislocalized to intracellular compartments. In contrast, all
RP- and ADVIRC-associated mutants were properly localized
to the basolateral plasma membrane. When mislocalized AVMD
and ARB mutants were co-expressed with WT Best1, all mutants
predominantly co-localized with WT Best1 in intracellular
compartments. All 28 mutants exhibited comparable FRET
efficiencies to and reciprocally co-immunoprecipitated with WT
Best1, indicative of unimpaired oligomerization.
Conclusions: All 28 mutants associated with AVMD, ARB, RP,
and ADVIRC formed oligomers with WT Best1, suggesting that
oligomeric defects are not associated with the bestrophinopathies.
Our data also show that, although other pathogenic mechanisms
besides mislocalization are involved for RP and ADVIRC,
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
mislocalization alone is insufficient to distinguish between different
disease phenotypes. Moreover, several recessive ARB mutants
mislocalized WT Best1 when co-expressed together. That a recessive
mutant would mislocalize WT Best1, yet not be pathogenic, indicates
that mislocalization on its own cannot cause disease, and that the
absence of Best1 at the plasma membrane is well tolerated.
Commercial Relationships: Adiv A. Johnson, None; Yong S. Lee,
None; Lihua Y. Marmorstein, None; Alan D. Marmorstein, None
Support: EY13160
Program Number: 2972 Poster Board Number: D0023
Presentation Time: 8:30 AM–10:15 AM
Isoform Switch of RPGR During Photoreceptor Development
Xun Sun1, Oleg V. Bulgakov1, Michael Adamian2, Zhijian Wu1,
Tiansen Li1. 1Neurobiology, Neurodegeneration & Repair Laboratory
(NNRL), National Eye Institute, Bethesda, MD; 2The Berman-Gund
Laboratory for the Study of Retinal Degenerations, Harvard Medical
School, Boston, MA.
Purpose: Mutations in the Retinitis Pigmentosa GTPase Regulator
(RPGR) gene leads to one of the most severe forms of retinal
degenerations and accounts for up to 80% cases of XLRP and 20% of
all RP. RPGR undergoes complex splicing and the ORF15 isoform,
considered to be photoreceptor specific, contains a glutamic acid-rich
domain and is a mutational hotspot. There is a current discrepancy
in subcellular localization of RPGR between rodent and primate
photoreceptors, which raises questions about their differential
functions among species and the use of rodent models to test gene
replacement therapies. This study aims to determine subcellular
localization of RPGR in human retinas and to explore a cellular and
biochemical basis for the genetic finding that all disease-causing
mutations in RP3 exclusively affect ORF15 transcripts only.
Methods: We generated and validated a battery of isoform-specific
antibodies against both human and mouse RPGR. We performed
Western blot, fluorescence microscopy, immuno-EM, and AAVmediated gene delivery in RPGR knockout mice.
Results: We found that native human RPGR predominantly localized
at the connecting cilia of human rod and cone photoreceptors.
Recombinant human RPGR transgene products, upon transduction
with AAV vectors, were able to localize at the connecting cilia
of mouse photoreceptors and interact with mouse RPGRIP1. The
ORF15 isoform of the protein is the predominant form in mature
photoreceptors of both humans and mice, of which the most
abundant version was found to lack the c-terminus. RPGR expression
undergoes an isoform switch during postnatal photoreceptor
development. The default RPGR isoform is detected during retinal
progenitor development, which is replaced by the ORF15 isoform
as the predominant variant beginning at postnatal day 7 and plateaus
when photoreceptors mature.
Conclusions: RPGR localize at the connecting cilia regardless of
species of origin. The RPGR default isoform may play a role in early
stages of ciliogenesis common to all ciliated cell types, whereas
the ORF15 isoform functions specifically in mature photoreceptor
cells where it is presumed to be involved in ciliary trafficking. This
explains why ORF15 is the only transcript clinically known to cause
retinal degeneration. Finally our unexpected findings that the bulk
of ORF15 in vivo variably lacks the C-terminal sequence may have
implications for understanding the normal functions of RPGR and
future therapeutic studies.
Commercial Relationships: Xun Sun, None; Oleg V. Bulgakov,
None; Michael Adamian, None; Zhijian Wu, None; Tiansen Li,
None
Program Number: 2973 Poster Board Number: D0024
Presentation Time: 8:30 AM–10:15 AM
Deletion of EFEMP1 is protective against the development of
basal deposits in mouse eyes
Youwen Zhang1, James B. Stanton2, Yong S. Lee1, Alan D.
Marmorstein1, Lihua Marmortein1. 1Ophthalmology, Mayo Clinic,
Rochester, MN; 2Surgery, University of Arizona, Tucson, AZ.
Purpose: EFEMP1 (fibulin-3) is mutated in Malattia Leventinese/
Doyne’s honeycomb retinal dystrophy (ML/DHRD), an autosomal
dominantly inherited macular degenerative disease with strong
similarities to age-related macular degeneration (AMD). Both ML/
DHRD and AMD are characterized by basal deposits beneath the
retinal pigment epithelium (RPE). While basal deposits develop in
knock-in mice carrying the diseasing-causing mutation R345W in
Efemp1, they are not observed in knock-out mice lacking Efemp1. A
number of experimentally applied stress conditions have been shown
to cause the formation of basal deposits in wild-type mice, including
combinational treatments of high-fat diet (HFD) and cigarette smoke,
or HFD and laser photochemical injury. This study is to test whether
basal deposits develop in Efemp1 knock-out mice exposed to HFD/
cigarette smoke or HFD/laser injury.
Methods: Efemp1 knock-out mice and wild-type littermates (control)
were fed with a synthetic HFD. Mice of the same genotype at the
same age were fed with standard laboratory rodent diet as controls.
Beginning one month after starting the HFD, one group of mice
under each condition was exposed to cigarette smoke daily for one
month using a Teague Enterprises mouse smoking system (TE-10z),
and another group of mice was subjected to photochemical injury
every other day for two weeks from a 488 nm blue argon laser.
Following the stress treatments, ocular phenotype analysis was
performed to assess whether basal deposits developed in any of the
mouse eyes.
Results: In wild type mice, basal laminar deposits (BLD) were
observed in the 18 month age group after exposure to HFD and
cigarette smoke or laser injury. No BLD or other deposit was
observed in younger age groups of wild type mice after the exposure.
BLD was observed in 24 month old wild type mice with or without
exposure to HFD/cigarette smoke or HFD/laser. In Efemp1 knock-out
mice, BLD was not observed in any age groups with or without the
exposure.
Conclusions: Mice lacking Efemp1 do not develop basal deposits.
Environmental oxidative stressors (HFD/cigarette smoke or HFD/
laser) known to cause BLD formation in wild type mice failed to
induce BLD formation in Efemp1 knock-out mice. These results
suggest that EFEMP1 is a central player in the development of BLD,
and deletion of EFEMP1 is protective against the development of
BLD.
Commercial Relationships: Youwen Zhang, None; James B.
Stanton, None; Yong S. Lee, None; Alan D. Marmorstein, None;
Lihua Marmortein, None
Support: NIH Grant EY013847 (to LM), EY013160 (to ADM), and
EY021153 (to ADM), and an unrestricted grant from Research to
Prevent Blindness.
Program Number: 2974 Poster Board Number: D0025
Presentation Time: 8:30 AM–10:15 AM
Diffusion across and proteoglycan content in Bruch’s membrane
are altered in mice carrying an Efemp1 mutation
Samuel D. Cross1, Astrid Zayas2, James B. Stanton3, Alan D.
Marmorstein1, Lihua Marmortein1. 1Ophthalmology, Mayo Clinic,
Rochester, MN; 2Pathology and Laboratory Medicine, Universidad
Central Del Caribe, Bayamón; 3Surgery, University of Arizona,
Tucson, AZ.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: A missense mutation R345W in EFEMP1 (fibulin-3) causes
Malattia Leventinese/Doyne’s honeycomb retinal dystrophy (ML/
DHRD), an autosomal dominant macular degenerative disease with
strong similarities to age-related macular degeneration (AMD).
Both ML/DHRD and AMD are characterized by sub-retinal pigment
epithelium (RPE) deposits. Impairment of Bruch’s membrane’s
diffusion properties is thought to contribute to the deposit formation.
This study was to determine whether the diffusion across and
proteoglycan content in Bruch’s membrane are altered using a mouse
knock-in model carrying the R345W mutation in Efemp1.
Methods: Cupromeronic Blue coupled with chondroitinase ABC
and nitrous acid treatment was used to determine the distribution
patterns of proteoglycans in mouse Bruch’s membrane of wild
type, knock-in carrying the R345W mutation, or knock-out lacking
Efemp1. Cupromeronic Blue bound proteoglycans were visualized
under electron microscopy as filaments with different sizes reflecting
different types of proteoglycans. Isolated mouse Bruch’s membrane/
chroid was mounted in a modified Ussing chamber with a small
exposed surface area. A solution containing 4 different tracers
was placed in one compartment of the Ussing chamber. Samples
were collected from both chambers at various time points and the
amount of each tracer determined using quantitative gel exclusion
chromatography.
Results: Two main types of filaments were found in mouse
Bruch’s membrane: small rod filaments primarily in the basement
membranes of the RPE and choroidal endothelium, and longer
filaments in the collagenous/elastic layers. The longer filaments,
which were eliminated by chondroitinase ABC, were chondroitin
and dermatan sulfate proteoglycans. The small rod filaments, which
were eliminated by nitrous acid treatment, were heparan sulfate
proteoglycans. In Efemp1 knock-out mice, the quantity of longer
filaments was similar to that of wild-type mice, but the small rod
filaments decreased. In Efemp1 knock-in mice, there were higher
amounts of both types of filaments. The diffusion of tracers was
reduced across Bruch’s membrane of Efemp1 knock-in mice.
Conclusions: EFEMP1 affects the distribution of different
proteoglycans. The R345W mutation causes an increase of both
heparan and chondroitin/dermatan proteoglycans in Bruch’s
membrane, and a decrease in permeability of Bruch’s membrane in
mice.
Commercial Relationships: Samuel D. Cross, None; Astrid Zayas,
None; James B. Stanton, None; Alan D. Marmorstein, None;
Lihua Marmortein, None
Support: NH Grant EY013847 (to LM), EY013160 (to ADM),
R01EY021153 (to ADM), and an unrestricted grant from Research to
Prevent Blindness.
Program Number: 2975 Poster Board Number: D0026
Presentation Time: 8:30 AM–10:15 AM
Activation of the TGF-beta and IGF signaling pathway during
retina regeneration in adult zebrafish
Markus Tschopp1, 2, Christoph Tappeiner1, Ellinor Maurer1, Kaspar
Schürch1, Pauline Sallin3, Anna Jazwinska3, Volker Enzmann1.
1
Department of Ophthalmology, University of Bern, Bern,
Switzerland; 2Department of Ophthalmology, University of Basel,
Basel, Switzerland; 3Department of Biology, University of Fribourg,
Fribourg, Switzerland.
Purpose: Zebrafish (Danio rerio) is an important model organism in
eye research, amongst others due to its cone rich retina. In contrast to
mammals, the retina regenerates even after severe damage. Analyzing
the underlying signaling pathways may elucidate why regeneration
occurs in the adult zebrafish. It has been shown that the TGF-beta and
the IGF signaling pathway are necessary for heart or fin regeneration.
In this study we investigate the activation of these two pathways
during retina degeneration and subsequent regeneration.
Methods: Retina degeneration was induced by placing adult
zebrafish in water containing 150 mg/l N-Methyl-N-Nitrosourea
for one hour. Thereafter, the time course of the activation of
the TGF-beta and the IGF signaling pathway was monitored by
immunohistological staining. Double staining was performed to
identify the activated cell types. The involved signaling molecules
were analyzed by in situ hybridization.
Results: Immunohistological staining for the TGF-beta and the IGF
signaling pathway was elevated during retina regeneration, starting
at day three. Staining showed a maximum in the inner nuclear layer
(INL) and co-localized with staining for Müller glial cells. In situ
hybridization revealed a marked increase of TGF-beta 3 and IGF 2b
expression in the INL and outer nuclear layer.
Conclusions: Both, the TGF-beta and the IGF signaling pathway
are activated during retina regeneration in the adult zebrafish. This
activation is also observed in the regeneration of the heart and fin
and may imply a shared mechanism in the regeneration of these three
organs.
Commercial Relationships: Markus Tschopp, None; Christoph
Tappeiner, None; Ellinor Maurer, None; Kaspar Schürch, None;
Pauline Sallin, None; Anna Jazwinska, None; Volker Enzmann,
None
Support: This research was supported by an unrestricted grant from
the Berne University Research Foundation and the Peter Mayor
Gedenkstiftung.
Program Number: 2976 Poster Board Number: D0027
Presentation Time: 8:30 AM–10:15 AM
Outer segment targeting of the CNG Channel in Xenopus rod
photoreceptors
Jillian N. Pearring, Vadim Y. Arshavsky. Duke Eye Center, AERI,
Duke University, Durham, NC.
Purpose: We aim to identify the elements that ensure proper
subcellular targeting of outer segment resident proteins. Cyclic
nucleotide gated (CNG) channels are located in the plasma membrane
of photoreceptor outer segments and mediate the electrical response
to light. The rod CNG-channel is composed of CNGα1 and CNGβ1
subunits that co-assemble to form a heterotetrameric channel.
A previous study identified a C-terminal RVXP sequence that is
required for ciliary localization of the olfactory CNGβ1b subunit.
In this report, we identify a different CNGβ1 targeting sequence
sufficient for its outer segment delivery.
Methods: DNA constructs expressed in Xenopus utilized a dual
promoter strategy; Xenopus opsin promoter used to express MYCtagged human CNGβ1 constructs in rods as well as a γ-crystallin
promoter to drive GFP expression in the lens. Transgenic Xenopus
tadpoles were produced using standard techniques. At developmental
stage 45, immunohistochemical staining was performed and
subcellular localization of each construct analyzed by confocal
microscopy. Primary mouse anti-MYC 9E10 (Santa Cruz) or antiCNGβ1 (GARP 8G8) antibodies and goat secondary antibodies
conjugated to Alexa Fluor 488 or 568 (Invitrogen).
Results: In order to identify a targeting signal in the CNGβ1
subunit, we transgenically expressed membrane-bound and cytosolic
fragments of the human CNGβ1 into Xenopus rods. Initially, we
examined the C-terminal of CNGβ1, including the predicted RVXP
motif, but determined that this sequence is not involved in outer
segment localization of the CNGβ1 channel. Instead we identified
a short domain on the cytoplasmic N-terminal that is sufficient for
outer segment delivery of the channel. Interestingly, this targeting
domain of CNGβ1 is separate from its N-terminal peripherin binding
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
site. However, peripherin binding is capable of bringing cytoplasmic
fragments of CNGβ1 to the outer segment, most likely through
protein-protein interactions.
Conclusions: These findings indicate that the rod CNGβ1 channel
does not contain a C-terminal RVXP targeting sequence (as reported
for olfactory CNGβ1b), but instead contains a targeting domain
present in the N-terminal. This targeting domain is separate from the
peripherin binding site and is sufficient for CNGβ1’s outer segment
delivery.
Commercial Relationships: Jillian N. Pearring, None; Vadim Y.
Arshavsky, None
Support: NIH Grants EY22508 (JNP), EY12859 (VYA), the NIH
Core Grant for Vision research EY5722, and an unrestricted grant
from Research to Prevent Blindness to Duke University.
Program Number: 2977 Poster Board Number: D0028
Presentation Time: 8:30 AM–10:15 AM
Apical localization of the CLC-2 chloride channel in polarized
RPE cells
Guillermo Lehmann-Mantaras1, Ignacio Benedicto1, Erwin
de la Fuente2, Diego Gravotta1, Enrique Rodriguez-Boulan1.
1
Ophthalmology, Well Cornell Medical College, New York, NY;
2
Facultad de Medicina, Universidad Catolica del Norte, Coquimbo,
Chile.
Purpose: The chloride channel 2 (ClC-2) is a polytopic membrane
protein ubiquitously expressed in the human body that plays
important physiological roles in cell volume regulation, ion transport
and acid-base balance. Knock-out of this channel in mice causes
retinal degeneration, suggesting that ClC-2 is involved in retinal
homeostasis. It is known that ClC-2 localizes to the basolateral
surface of most epithelia such as duodenum, colon and MDCK cells.
There is functional evidence based on pharmacologic manipulation
of Cl- conductance that suggests that ClC-2 is basolaterally localized
in RPE. However, the localization of ClC-2 in RPE has not been
addressed yet. Here we present a morphological analysis of the
subcellular localization of hClC-2 in RPE cells.
Methods: We performed confocal fluorescence microscopy in highly
polarized ARPE-19 and hfRPE cells grown on Transwell filters. To
explore the localization of endogenous hClC-2 we used an affinitypurified anti-ClC-2 antibody. We also studied the localization of
exogenous ClC-2 by transduction of hClC-2-GFP and hClC-2-HA
via liposomes, electroporation and lentivirus vectors.. To examine
the localization of the channel in RPE in situ, we performed in vivo
subretinal injection of a plasmid encoding hClC-2-GFP in wild type
C57BL/6 mice.
Results: To our surprise, endogenous hClC-2 was predominantly
localized to the apical membrane of polarized hfRPE cells.
Independently of the gene delivery method used, both hClC-2-GFP
and hClC-2-HA were found highly enriched at the apical domain in
ARPE-19 and hfRPE cells. The apical localization was confirmed
by colocalization of hClC-2-HA with the apical marker MCT1 in
non-permeabilized hfRPE monolayers. Finally, we analyzed the ClC2 localization in situ on mouse RPE transfected in vivo. Confocal
analysis of whole mount RPE sheets and transverse retinal vibratome
sections showed that hClC-2 was highly enriched at apical RPE
microvilli.
Conclusions: We provide for the first time a morphological analysis
of ClC-2 localization in RPE cells. To our surprise we found by
different methods that that hClC-2 is predominantly localized to the
apical plasma membrane in RPE cells.
Commercial Relationships: Guillermo Lehmann-Mantaras,
None; Ignacio Benedicto, None; Erwin de la Fuente, None; Diego
Gravotta, None; Enrique Rodriguez-Boulan, None
Support: NIH/NEI Grant EY08538
Program Number: 2978 Poster Board Number: D0029
Presentation Time: 8:30 AM–10:15 AM
miR-302 regulates retinal epithelial cell fate; New insights into
TGFβ signalling reveal a role for for the polycomb protein EZH2.
Darrell Andrews1, Noel Faherty1, Giorgio Oliverio1, Colm J.
O’Brien2, Gerard Cagney1, John Crean1. 1School of Biomolecular
and Biomedical Science, University College Dublin, Dublin, Ireland;
2
School of Medicine and Medical Science, University College
Dublin, Dublin, Ireland.
Purpose: Diabetic Retinopathy (DR), manifests as retinal
microvascular defects and neuroretinal dysfunction. The progression
of the fibroproliferative element of the disease is accepted to feature
growth factor mediated epithelial differentiation in a process
analogous to cell fate determination. Manipulating cell differentiation
in the context of retinal fibrosis represents a novel approach to
targeting fate transitions. We identified a signalling nexus comprising
TGFβ, miR-302 and the TGFβ type II receptor (TβRII) that
regulates retinal epithelial cell fate. We describe a novel interaction
between smad3 and polycomb repressive complexes in miR-302
overexpressing cells that regulate phenotypic responses of retinal
epithelial cells to TGFβ.
Methods: TβRll was validated as miR-302d target by 3’ UTR
reporter assay, qRT-PCR and Western blot. A polycistronic lentiviral
vector was generated for stable expression of miR-302. ARPE19
cells differentiated with TGFβ treatment were transduced with
lentivirus and the capacity of the virus to initiate epithelial cell fate
specification was assessed by Western blot and ICC using known
markers of epithelial and fibroblast fates including e-cadherin,
ZO-1, n-cadherin, vimentin and α-smooth muscle actin and their
transcriptional regulators (slug, snail). We assessed the expression
markers of induced pluipotency (Oct 4, Sox 2 and nanog). Analysis
of the Smad 3 interactome was undertaken by mass spectrometry and
immunopreciptation studies.
Results: Silencing of TβRll expression and downstream
TGFβ canonical signalling was evidenced by decreased smad
phosphorylation. Decreased mesenchymal marker expression and
increased in epithelial fate markers suggests a role for miR-302d
in regulating epithelial fate. Analysis of the Smad 3 interactome in
these cells identified a novel interaction with the polycomb repressive
complex EZH2. This interaction was confirmed by immunoprecipitation studies. Treatment of cells with an EZH2 inhibitor
recapitulated aspects of epithelial de-differentation.
Conclusions: These findings suggest polycomb mediated repression
is a feature of epithelial differentiation and may be manipulated to
restore epithelial phenotype and function in diabetic retinopathy with
the possibility of cellular reprogramming. This represents a novel
approach to targeting cell fate decisions in disease.
Commercial Relationships: Darrell Andrews, None; Noel Faherty,
None; Giorgio Oliverio, None; Colm J. O’Brien, None; Gerard
Cagney, None; John Crean, None
Program Number: 2979 Poster Board Number: D0030
Presentation Time: 8:30 AM–10:15 AM
RPE tight junctions are regulated by endothelial cells
Ignacio Benedicto1, 3, Guillermo Lehmann-Mantaras1, 3, Shahin
Rafii2, 4, Enrique Rodriguez-Boulan1, 3. 1Ophtalmology, Weill Cornell
Medical College, New York, NY; 2Genetic Medicine, Weill Cornell
Medical College, New York, NY; 3Margaret Dyson Vision Research
Institute, Weill Cornell Medical College, New York, NY; 4Ansary
Stem Cell Institute, Weill Cornell Medical College, New York, NY.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: The retinal pigment epithelium (RPE) separates the neural
retina from the fenestrated choroid endothelium. RPE tight junctions
(TJs) are key for the correct maintenance of the outer blood-retinal
barrier. It has been shown that RPE TJs are affected by factors
secreted by the neural retina. However, it is not known whether RPE
TJs are also regulated by choroid endothelial cells. Recent studies
have demonstrated that endothelial cells play key instructive roles in
the differentiation and maintenance of parenchymal cells in several
body organs. Thus, we studied whether endothelial cells regulate RPE
TJ function.
Methods: We performed co-culture experiments on Transwell
inserts using human fetal RPE (hfRPE) and human umbilical vein
endothelial cells expressing the adenoviral protein E4 (E4-HUVECs).
E4-HUVECs are able to survive in the absence of endothelial factors
or serum. hfRPE were seeded on top of the filter and E4-HUVECs
were seeded either on the other side of the filter or on the bottom
chamber of the insert. Transepithelial electrical resistance (TER)
was measured at different time points in the absence or presence
of E4-HUVECs. As controls, several cell lines were used instead
of E4-HUVECs. Alternatively, co-cultures were carried out with
E4-HUVECs that had been previously exposed to polarized hfRPE
conditioned medium. Expression levels of the TJ-associated proteins
claudin-1, -2, -3, -9, -10b, -12, -15, -16, -19 and occludin were
analyzed in hfRPE at different time points by real time PCR.
Results: hfRPE co-cultured with E4-HUVECs presented a
significantly increased TER both in the presence or absence of serum.
This effect was was not detected when other cell types were used
instead of endothelial cells. The increase in TER was faster when E4HUVECs had been previously primed with hfRPE. We also observed
that the presence of endothelial cells induced a specific decrease in
hfRPE claudin-2 mRNA levels.
Conclusions: Endothelial cells altered both TER and claudin-2
expression in hfRPE in vitro, suggesting that choroidal endothelium
may modulate RPE TJ function. In addition, the fact that the effect
on TER was faster when endothelial cells were previously primed
with hfRPE suggests a bidirectional communication model in which
endothelial cells receive a signal from RPE which in turn allows
them to modulate RPE tight junctions. This model fits well with
the observed synchronized development of RPE and choroidal
endothelium.
Commercial Relationships: Ignacio Benedicto, None; Guillermo
Lehmann-Mantaras, None; Shahin Rafii, None; Enrique
Rodriguez-Boulan, None
Support: Tri-Institutional Stem Cell Initiative (Tri-SCI), The Starr
Foundation.
Program Number: 2980 Poster Board Number: D0031
Presentation Time: 8:30 AM–10:15 AM
Neuroprotectin D1 is Synthesized during Moderate Light
Preconditioning
Eric J. Knott, William C. Gordon, Nicolas G. Bazan. Neuroscience
Center, Louisiana State Univ Hlth Sci Ctr, New Orleans, LA.
Purpose: NPD1, a lipid mediator made on demand is biosynthesized
from DHA. This bioactive mediator attenuates CNV, protects
neurons during ischemia reperfusion, and RPE cells from oxidative
stress. Bright light induces oxidative stress and other homeostatic
disruptions that results in photoreceptor degeneration; however
a moderate light (preconditioning) stimulus prior to bright light
treatment ameliorates photoreceptor cell death. It has been reported
that ischemic preconditioning releases DHA and AA, but the lipid/
docosanoid signaling during light induced degeneration has yet to be
investigated. Thus, the purpose of this study was to determine if DHA
was released and the docosanoids 14-HDHA, 17-HDHA, and NPD1
were synthesized during moderate light preconditioning.
Methods: Male Sprague Dawley rats (150-175g) Charles River,
were acclimated to facilities for 7-14 days (cage light 40-60 lux)
followed by stimulation with moderate light (1200 lux) prior to bright
light (18kLux) treatment. Photoreceptor survival was analyzed via
SD-OCT and outer retinal thickness (photoreceptor/RPE) reported.
Retinas were collected -1h, +1, +4, +8, +11h post preconditioning
light onset, lipids extracted, and analyzed via LC-ESI /MS/MS.
Results: Rats exposed to bright light displayed a 50% reduction in
superior outer retinal thickness, while rats given moderate light prior
to bright light treatment presented with an 8% reduction in outer
retinal thickness. Retinas of preconditioned rats showed increased
DHA release and synthesis of 17 HDHA, 14 HDHA, and NDP1
throughout the day, as compared to controls.
Conclusions: This data suggests that moderate light preconditioning
is neuroprotective through DHA release and synthesis of the
docosanoid NPD1. This supports the idea that DHA/NPD1 could
prevent or attenuate pathological conditions during early stages of
retinal degenerative diseases.
Commercial Relationships: Eric J. Knott, None; William C.
Gordon, None; Nicolas G. Bazan, None
Support: NIH/NEI EY005121, Research to Prevent Blindness, Inc
(NGB)
Program Number: 2981 Poster Board Number: D0032
Presentation Time: 8:30 AM–10:15 AM
Long term organotypic culture of the human retina
Arnold Szabo1, Anna Enzsoly2, Klaudia Szabo1, Agoston Szel1,
Ákos Lukáts1. 1Dept. Human Morphology and Developmental
Biology, Semmelweis University, Budapest, Hungary; 2Dept. of
Ophthalmology, Semmelweis University, Budapest, Hungary.
Purpose: Corneal transplantation has become a routine procedure in
ophthalmology nowadays. The anterior segment with the cornea of
the donor eyeball is removed shortly before transplantation, and the
otherwise intact posterior segment containing the retina that could
be used for scientific investigations is usually disposed of. Here we
present a potent in vitro application of these retinas that allows the
long term survival of retinal cells with maintained retinal integrity.
Methods: Approximately 5x5 mm pieces of central retina were
harvested from donor adult eyes 2-4 hours after enucleation. The
pieces of the neural retina without the pigment epithelium were
placed onto a nitrocellulose membrane and kept in culture using
serum-free medium for up to three weeks. The cultures were fixed at
different time points, processed and analyzed as histological sections
or whole mounts by immunohistochemistry using cell type-specific
antibodies.
Results: Although slight degenerative signs were present, the overall
retinal architecture was well preserved even after 21 days in culture
and the retinal thickness was comparable to that of in vivo controls.
As expected, we detected signs of glial activation with only moderate
GFAP immunoreactivity after 7 days in culture which gradually
increased thereafter. The most prominent alterations occurred in the
outer and inner plexiform layers, where reduction of thickness and
minor synaptic disorganization was observed. The cones showed
a near normal morphology with occasional swelling of the outer
segments. The rod outer segments gave a strong anti-rhodopsin
immunoreaction, but they were shorter than in in vivo controls. Some
picnotic nuclei in the outer and inner nuclear layers were detectable
after prolonged culturing, but no severe decrease in the number of
bipolar, amacrine and horizontal cells was found.
Conclusions: Our results indicate that the adult human retina can
be maintained in an appropriate culture system for at least three
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
weeks. Despite the minor changes detected, the retina shows normal
lamination, the retinal cells preserve their normal morphology
and staining characteristics. The presented culture method can be
reliably reproduced and could be adapted to pharmacological and
toxicological applications.
Commercial Relationships: Arnold Szabo, None; Anna Enzsoly,
None; Klaudia Szabo, None; Agoston Szel, None; Ákos Lukáts,
None
Support: Hungarian Scientific Research Fund (OTKA#73000),
TÁMOP-4.2.1.B-09/1/KMR-2010-0001
Program Number: 2982 Poster Board Number: D0033
Presentation Time: 8:30 AM–10:15 AM
Exogenous COL18A1 Restores Retinal Function in a Patient
Specific Model of Knobloch Syndrome
Huy V. Nguyen1, Yao Li2, Irene H. Maumenee3, Stephen H. Tsang4.
1
Columbia University College of Physicians and Surgeons, New
York, NY; 2Ophthalmology, Columbia University, New York, NY;
3
Illinois Eye and Ear Infirmary, University of Illinois at Chicago,
Chicago, NY; 4Pathology and Cell Biology, Columbia University,
New York, NY.
Purpose: Knobloch Syndrome (KNO, OMIM: 267750) is an
autosomal recessive disorder characterized by high myopia,
vitreoretinal degeneration with recurrent retinal detachment, and
congenital encephalocele. The confirmed genetic mutation of the
COL18A1 gene on 21q22.3 is responsible for the characteristic
dysfunctional retinal pigment epithelium (RPE) cells. There is
currently no approved treatment for KNO. In the current study, we
aim to rescue KNO RPE function with exogenous COL18A1 using
human induced pluripotent stem (iPS) cell technology.
Methods: Stem cells were generated from skin fibroblast from a
patient with KNO. Antibodies against standard pluripotency markers
Oct-4, Sox-2, TRA-1-60, SSEA4, and NANOG were applied to
characterize the iPS cells reprogrammed from this sample. Stem
cells were differentiated into morphological and functional RPE
cells, as shown by immunohistochemical staining, transmission
electron microscopy, and measurement of transepithelial resistance
(TER). iPS-derived RPE were seeded onto Transwell membranes and
grown on Matrigel matrix. Exogenous COL18A1 was applied to the
Matrigel matrix and TER of experimental RPE was compared to that
of untreated KNO-iPS-derived RPE and wildtype iPS-derived RPE.
Results: KNO-iPS-derived RPE lacking COL18A1 is
dysmorphologic. Application of exogenous COL18A1 to the Matrigel
matrix restored KNO-iPS-derived RPE morphology to that of
wildtype iPS-derived RPE in a dose-dependent manner. TER levels
of untreated KNO-iPS-derived RPE were extinguished while TER
levels of COL18A1-treated KNO-iPS-derived RPE approached that
of control RPE.
Conclusions: This is the first report of human iPS-derived RPE
being successfully used to model this disease phenotype. The results
indicate that exogenous COL18A1 can successfully be applied to
restore morphology and function of KNO-iPS-derived RPE.
Commercial Relationships: Huy V. Nguyen, None; Yao Li, None;
Irene H. Maumenee, None; Stephen H. Tsang, None
Support: Research to Prevent Blindness Medical Student Eye
Research Fellowship
Program Number: 2983 Poster Board Number: D0034
Presentation Time: 8:30 AM–10:15 AM
Functional Investigation of the Role of Prickle 2 in Retinal
Photoreceptors
Sameila Okpodu1, 2, Chunqiao Liu2, Helen May-Simera2,
Werner Graf1, Anand Swaroop2, Tiansen Li2. 1Physiology and
Biophysics, Howard University, Washington, DC; 2Neurobiology
Neurodegeneration & Repair Laboratory (N-NRL), National Eye
Institute/ National Institutes of Health, Bethesda, MD.
Purpose: Prickle 2, a cytoplasmic protein, is a core component of the
planar cell polarity (PCP) signaling pathway. PCP signaling is critical
for many aspects of development in numerous organ systems. Neural
Retinal Leucine Zipper (Nrl) is a transcription factor responsible
for rod photoreceptor fate determination. In the absence of Nrl, rod
precursors fail to develop mature rods resulting in a cone dominated
retina. In Nrl knockout retina, Prickle 2 (Pk2) expression is
upregulated. We posit that Pk2 may play a role in the determination,
differentiation, and/or maturation of cone photoreceptors. To
investigate the plausibility of this hypothesis we examined expression
and function of Pk2 in the mammalian retina.
Methods: In situ hybridization was used to identify cell type specific
localization of Pk2 expression in the retina.Immunoblotting,using
polyclonal antibodies against Pk2,and RNA sequencing were used
to study temporal expression of Pk2 during retinal development.
Outer retinal function of Pk2 knockout mice was assessed by
electroretinography (ERG).Quantification of retinal integrity was
accomplished through spectral domain-optical coherence tomography
(SD-OCT) and histological analysis of age-matched Pk2 wild-type
and knockout adult mice.
Results: In situ hybridization at postnatal day 21 confirms Pk2
expression in the Inner Nuclear Layer and the Ganglion Cell Layer
of the retina. Immunoblotting using wild-type retina shows Pk2 is
expressed after P10 and continues to be expressed in the adult retina;
time points that do not coincide with photoreceptor development. In
adult retina, RNA sequencing data shows relatively low expression
levels of Pk2 in the photoreceptor cells. ERG recordings show no
statistical difference between the a-wave of adult wild-type and
knockout retinas, suggesting no major deficit in photoreceptor
function. SD-OCT and histological analyses reflect overall retention
of retinal layer integrity and cell specification, however, the central
retina is thinner in the mutants.
Conclusions: Analysis of the effect of Pk2 in the retina suggests
that this gene is likely not playing a major, non-redundant, role in
photoreceptor development and/or function. While Pk2 is markedly
expressed in the inner retina, lack of an overt phenotype may be
explained by possible functional redundancy with Pk1. Further work
is in progress that will investigate the retinal phenotype of a Pk1 and
Pk2 double knockout.
Commercial Relationships: Sameila Okpodu, None; Chunqiao
Liu, None; Helen May-Simera, None; Werner Graf, None; Anand
Swaroop, None; Tiansen Li, None
Program Number: 2984 Poster Board Number: D0035
Presentation Time: 8:30 AM–10:15 AM
Evaluation of Human Embryonic Stem Cell-Derived Retina as a
Potential Retinoblastoma Model
Victor Liao, Narine Harutyunyan, Jennifer Aparicio, David Cobrinik,
Thomas C. Lee. Opthamology, Children Hospital Los Angeles, Los
Angeles, CA.
Purpose: Retinoblastoma is a childhood malignancy initiated by the
loss of Rb function during human retinal development. Mouse models
do not accurately mimic retinoblastoma since Rb1 loss alone does
not induce retinal tumors, and retinal tumors generated by combined
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
loss of Rb and Rb-related proteins have a distinct phenotype. Human
embryonic stem cells (hESCs) are a potential source of immature
retinal tissue that could be used to model retinoblastoma if it exhibits
developmental properties similar to human fetal retina. Our objective
is to characterize hESC-derived retina, particularly with respect
to Rb expression, to ascertain its potential to accurately model
retinoblastoma pathogenesis.
Methods: hESC-derived retinas were grown in 3-dimensional
cultures initiated by reaggregation of dissociated hESC colonies
into spheres which were differentiated using small molecules and
matrigel as described (Nakano et al 2012). hESC-derived retinas
were analyzed by immunofluorescence staining of cryosections after
various times of differentiation.
Results: Analogous to retinal tissue developing in vivo, retinal
progenitor cells (RPCs) of the hESC-derived retinas were
Chx10+,Ki67+, and Pax6+, underwent interkinetic nuclear migration,
and prominently expressed Rb. The RPCs also differentiated into
retinal cells that were appropriately positioned within stratified
retinal layers and were produced in the characteristic sequence seen
in vivo. For example, detection of Brn3+ ganglion cell precursors
preceded that of CRX+ photoreceptor precursors. Likewise, cone
markers (cone arrestion, L/M opsin) emerged prior to rod markers
(NRL). Although CRX+ cone precursors expressed L/M opsin and
cone arrestin, and exhibited structures resembling inner segments and
rudimentary outer segments, Rb was not detected in these cells, at
least up to 120 days in culture, the latest age examined. In contrast,
maturing human fetal cones express high levels of Rb.
Conclusions: hESC-derived retinas recapitulated many processes
of normal retinal development, including production of RPCs and
differentiated retinal cells in stratified layers. RPCs in hESC-derived
retinas exhibited high levels of Rb similar to what is seen in human
fetal retina. However, the retinas failed to induce Rb expression
in maturing cone precursors, bringing into question whether
they sufficiently simulate in vivo retinal maturation to serve as a
retinoblastoma model.
Commercial Relationships: Victor Liao, None; Narine
Harutyunyan, None; Jennifer Aparicio, None; David Cobrinik,
None; Thomas C. Lee, None
as well as preserved more RGCs compared to retinal treated with
AAV5-LacZ.
Conclusions: HO-1 might be a potential therapeutic target for
treatment of traumatic optic neuropathy.
Program Number: 2985 Poster Board Number: D0036
Presentation Time: 8:30 AM–10:15 AM
HO-1 gene therapy for protection of retinal ganglion cells after
optic nerve crush injury in rat
Ming-Hui Sun1, Chi-Chin Sun2, Kuan-Jen Chen1, Yeou-Ping Tsao3.
1
Ophthalmology, Chang Gung Memorial Hospital -LinKou, KweiShan, Tao-Yuan, Taiwan; 2opthalmology, Chang Gung Memorial
Hospital, KeeLong, Taiwan; 3ophthalmology, Mackay Memorial
Hospital, Taipei, Taiwan.
Purpose: to evaluate the protective effect of HO-1 on retinal
ganglion cells (RGCs) through gene delivery after optic nerve crush
injury
Methods: AAV5-HO-1 was injected intravitreally in left eye of rats
3 weeks before optic nerve (ON) crush injury in one group. AAV5LacZ injected intravitreally in left eye of rats was served as control
group for comparison. ON crush injuries were induced by applying a
60-g microvascular clip to the ON at a distance of 2 mm posterior to
the globe for 2 minutes. The protective effect of HO-1 on RGCs was
evaluated by TUNEL labelling and RGC counting through retrograde
labeling by fluoroGold and through immunofluorescence by Brn 3a
staining.
Results: Intraviteal injection of AAV5-HO-1 transduced the
expression of HO-1 in RGCs. Retina treated with AAV5-HO-1
attenuated the apoptosis in RGCs 2 weeks after ON crush injury
328 Mechanisms in retinal angiogenesis and retinopathy
Tuesday, May 06, 2014 11:00 AM–12:45 PM
S 310E-H Paper Session
Program #/Board # Range: 3014–3020
Organizing Section: Retinal Cell Biology
transgene expression of HO-1 in retina
Apoptosis in RGCs in retina treated with AAV-HO-1
Commercial Relationships: Ming-Hui Sun, None; Chi-Chin Sun,
None; Kuan-Jen Chen, None; Yeou-Ping Tsao, None
Program Number: 3014
Presentation Time: 11:00 AM–11:15 AM
Changes in retinal vessel caliber with flicker light stimulation in
eyes with diabetic retinopathy
Laurence S. Lim1, 2, Peng Guan Ong1, E SHyong Tai1, 2, Gemmy C.
Cheung1, Wallace S. Foulds1, Tien Y. Wong1, 2. 1Ophthalmology,
Singapore National Eye Center, Singapore, Singapore;
2
Ophthalmology, Yong Loo Lin School of Medicine, National
University of Singapore, Singapore, Singapore.
Purpose: Changes in retinal vessel calibre in response to flickering
light are believed to be mediated by nitric oxide release from the
retinal microvascular endothelium. This study investigated the
responses of retinal vessels to flickering light in diabetic patients with
various grades of diabetic retinopathy(DR).
Methods: This cross-sectional observational study evaluated adult
subjects with diabetes mellitus. The Dynamic Vessel Analyser (DVA)
was used to measure retinal vascular responses to diffuse illuminance
flicker. DR was graded from retinal photography. Each eye was
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
assigned a retinopathy severity score according to the modified
Airlie House classification system, and categorized as minimal
nonproliferative diabetic retinopathy (NPDR), mild NPDR, moderate
NPDR, severe NPDR, or proliferative retinopathy. Eyes were also
classified as having any DR (minimal NPDR or worse), moderate DR
(moderate NPDR or worse), or vision-threatening DR (severe NPDR
or worse, or clinically significant macular edema) according to the
Eye Diseases Prevalence Research Group definitions.
Results: There were 279 subjects in total, with a mean age of
59.9±9.2 years. The majority were male (73%) and the mean HbA1c
level and mean duration of diabetes were 7.7±1.4% and 13.9±10.4
years respectively. After adjustments for age, sex, smoking, duration
of diabetes, HbA1c, hypertension and hyperlipidemia, retinal
arteriolar and venular dilation responses to flicker stimulation
decreased continuously with increasing severity of diabetic
retinopathy.(p = 0.008 and <0.001 respectively). Subjects with
reduced arteriolar dilation responses were more likely to have any
DR [odds ratio (OR) 1.20 (95% confidence interval 1.01 – 1.45) per
standard deviation (SD) decrease, p=0.045]. Subjects with reduced
venular dilation responses were more likely to have any DR [OR
1.27(1.04 – 1.53) per SD decrease, p=0.02], moderate DR [OR 1.27
(1.06 – 1.49) per SD decrease, p = 0.007] and vision-threatening DR
[OR 1.51(1.14 – 1.50) per SD decrease, p = 0.002].
Conclusions: Retinal arteriolar and venular dilation responses to
flickering light are diminished in subjects with DR, and decrease
progressively with more severe stages of DR. Our findings suggest
that the severity of DR is correlated with measurable differences in
retinal microvascular endothelial function, supporting a role for the
latter in the pathogenesis of DR.
Commercial Relationships: Laurence S. Lim, None; Peng Guan
Ong, None; E SHyong Tai, None; Gemmy C. Cheung, None;
Wallace S. Foulds, None; Tien Y. Wong, None
Support: NMRC grant number R710/60/2009
Program Number: 3015
Presentation Time: 11:15 AM–11:30 AM
Amacrine cell-derived VEGF is required for development and
maintenance of the retinal vasculature in mice
Yoshihiko Usui, Toshihide Kurihara, Peter D. Westenskow, Edith
Aguilar, Liliana P. Paris, Stacey K. Moreno, Carli M. Wittgrove,
Daniel Feitelberg, Martin Friedlander. Cell Biology, The Scripps
Research Institute, San Diego, CA.
Purpose: The retinal vasculature of many organisms including
humans and mice consists of three distinct plexus layers. While it is
clear that the inner retinal vascular layer develops over a pre-existing
astrocytic network and that development of vascular and neuronal
networks are co-dependent, it is unclear how the outer retinal
vascular networks form. As retinal neurons populate the retina and
mature, oxygen demands change and activation of the oxygen sensing
VHL/HIF-α/VEGF pathway in maturing neurons may be a strong
driving force for development and maintenance of the outer plexus
layers. In this study, we examined the contribution of amacrine and
horizontal cells due to their close proximity to the intermediate and
outer retinal vascular layers.
Methods: Transgenic mice expressing Cre recombinase specifically
in amacrine and horizontal cells (Ptf1a-Cre mice) were mated
with floxed VHL, HIF-1α, HIF-2α and/or VEGF mice to generate
conditional knockouts. Amacrine and horizontal cells were
genetically ablated using Ptf1a-Cre and forced expression of
diphtheria toxin (DT) receptors.
Results: We show that amacrine and horizontal cell processes tightly
associate with intermediate and outer plexus retinal capillaries.
Pseudo-hypoxia in Ptf1a-Cre; VHL mutants induces formation of a
dense intermediate plexus compared to controls, while a dramatically
attenuated intermediate plexus is observed in Ptf1a-Cre; VEGF
and Ptf1a-Cre; HIF-1α mutants. Co-deletion of HIF-1α, but not
HIF-2α, rescued the vascular phenotypes of Ptf1a-Cre; VHL KO
mice. Amacrine and horizontal cell ablation by DT injection also
suppressed the formation of the intermediate plexus and DT injection
after the retinal vasculature had developed resulted in attenuation of
the vasculature. In all of these genetic manipulations the deep plexus
was less affected.
Conclusions: Dysregulated VEGF release from amacrine and
horizontal cells results in formation of a very dense intermediate
vascular plexus, while elimination of VEGF (or of amacrine and
horizontal cells themselves) prevents its formation. These data
demonstrate a novel function of amacrine cells, directing formation
of the intermediate plexus layer. Horizontal cells, on the other hand,
are likely strictly dependent on the vasculature, but do not determine
its formation or maintenance.
Commercial Relationships: Yoshihiko Usui, None; Toshihide
Kurihara, None; Peter D. Westenskow, None; Edith Aguilar,
None; Liliana P. Paris, None; Stacey K. Moreno, None; Carli M.
Wittgrove, None; Daniel Feitelberg, None; Martin Friedlander,
None
Support: The Lowy Medical Research Institute and EY11254
Program Number: 3016
Presentation Time: 11:30 AM–11:45 AM
Ataxia telangiectasia mutated (ATM) dysregulation precipitates
in diabetic retinopathy
Ashay D. Bhatwadekar1, 2, Maria Korah3, Sergio Caballero3,
Justin Baas1, Maria Grant1, 3. 1Ophthalmology, Indiana University,
Indianapolis, IN; 2Pharmacology and Toxicology, Indiana University,
Indianapolis, IN; 3Pharmacology and Therapeutics, University of
Florida, Gainesville, FL.
Purpose: Diabetic retinopathy (DR) is a vasodegenerative condition
with apoptosis of endothelial cells and pericytes resulting in
widespread areas of ischemia. Contrary to the belief that duration
of diabetes is one of the strongest predictors for development of
DR, we identified a unique cohort of patients who in spite of longstanding (>40 yrs) poorly controlled diabetes, remained free of DR.
We reasoned that hematopoietic stem cells (HSCs) and vascular
progenitors were unique in this group of patients. Microarray analysis
of HSCs from this ‘protected cohort’ revealed upregulation of
tumor suppressor protein ataxia telangiectasia mutated (ATM). We
hypothesized that the loss of ATM in bone marrow HSCs could result
in inadequate retinal repair and accelerated development of DR.
Methods: We developed gender mismatched mouse chimeras in
which hematopoietic tissue of wild type mice was replaced with
that from ATM-/- mice, WT. ATM-/- chimera. These chimeras were
sacrificed 6 months post induction of diabetes with streptozotocin and
tissues were harvested for further analysis. Long-term repopulating
(LTR) and short-term repopulating (STR)-HSCs were evaluated as
lin-Sca1+c-kit+CD34- and lin-Sca1+c-kit+CD34+ cells respectively
using flow cytometry. Retinas were processed for trypsin digestion to
evaluate the degree of DR and femurs were embedded to quantify the
numbers of LTR and STR-HSCs.
Results: We observed a 50% decrease (p<0.05) in the number of
LTR-HSCs in diabetic mice. This decrease in LTR-HSCs was further
accelerated in diabetic WT.ATM -/- chimeras (p<0.05). The diabetic
WT.ATM -/- also showed a tendency of myeloid bias and a 2-fold
increase (p<0.05) in STR-HSCs was observed compared to nondiabetic WT.ATM -/- chimeras. Cell cycle analysis further revealed
a profound decrease (p<0.05) in quiescent (G0 phase) LTR-HSCs in
control and diabetic WT.ATM-/- chimeras. Also, a substantial portion
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
of STR-HSCs was in either G1 or G2 phase, suggesting a complete
decline of quiescent HSCs in. WT.ATM-/-chimeras. Quantification
of retinas in diabetic WT.ATM-/- chimeras showed an accelerated
increase in the number of acellular capillaries.
Conclusions: In conclusion, our study suggests the critical role of
ATM in protecting bone marrow HSCs from diabetic stress and
highlights the importance of maintaining long-term repopulating
ability of HSCs to participate in retinal vascular repair in DR.
Commercial Relationships: Ashay D. Bhatwadekar, None; Maria
Korah, None; Sergio Caballero, None; Justin Baas, None; Maria
Grant, None
Support: Thomas H Maren Junior Investigator Award
Program Number: 3017
Presentation Time: 11:45 AM–12:00 PM
The role of 53bp1 and of the endothelial DNA repair cascade in
the vasoprolifarative retinopathies
Matina Economopoulou1, 2, Ria Zengler1, 2, Lutz E. Pillunat1,
Andre Nussenzweig3, Triantafyllos Chavakis2. 1Ophthalmology,
University Clinic Dresden, Dresden, Germany; 2Institute for clinical
pathobiochemistry, University clinic, TU, Dresden, Germany;
3
Laboratory of Genome Integrity, CCR, NCI, NIH, Bethesda, MD.
Purpose: Hypoxia is central in the vasoprolifearative retinopathies
(DR, ROP) as it is the driving force for the endothelial cell (EC)
proliferation. On the other hand hypoxia and reoxygeneation lead to
stalled replication forks and activation the DNA repair machinery
leading to replication arrest and apoptosis. The DNA is repaired
either by homologous recombination (HR) or non homologous
end-joining (NHEJ). During the S Phase of the cell cycle, HR is the
main mode of DNA repair. p53 binding protein1 (53bp1) is a DNA
repair factor that promotes NHEJ and competes with BRCA1 (that
promotes HR) for the DNA repair mode. Given our previous finding
that H2Ax and endothelial DNA repair are critical for hypoxia-driven
angiogenesis in retinopathy (Economopoulou et al., Nat Med 2009),
we here explored the role of 53bp1 in ROP.
Methods: We subjected ECs to hypoxia/ reoxygenation (H/Reox)
conditions and studied the phosphorylation of 53bp1 (p-53bp1)
by IF and WB. In vivo we subjected 53bp1-/- and +/+ mice to the
ROP model. Their retinas were analysed for neovascularisation, EC
proliferation and apoptosis and WB quantification of the DNA repair
factors BRCA1 and Rad51.
Results: The exposure of ECs to H/Reox conditions resulted in an
increase in p-53bp1. Furthermore, H/Reox upregulated other DNA
repair factors, mostly involved in HR, like RAD51. The retinas of
53bp1-/- ROP mice showed significantly higher neovascularisation
compared to their wt littermates. This was due to higher EC
proliferation and lower apoptosis in the retinas of 53bp1-/- mice.
Furthermore BRCA1 and Rad51 were significanty upregulated in the
retinas of 53bp1-/- ROP mice compared to 53bp1+/+ suggesting a
higher rate of HR in the 53bp1-/- retinas.
Conclusions: Our study shows a novel role of 53bp1 in
vasoproliferative retinopathies. The absence of 53bp1 results in
increased neovascularisation due to enhanced EC proliferation and
decreased apoptosis in the retinas of ROP mice. We propose that
the increase in BRCA1 and Rad51 in the retinas of 53bp1-/- mice
lead to a more efficient EC proliferation under H/Reox conditions
in the ischemic retina. Intriguingly, our results show an opposite
role of 53bp1 to the function of histone H2AX in the ROP. Overall,
our study strengthens the notion that the DNA Repair cascade is
important in vasoproliferative retinopathies and identifies the variable
effects of different DNA repair proteins in this context.
Commercial Relationships: Matina Economopoulou, None; Ria
Zengler, None; Lutz E. Pillunat, None; Andre Nussenzweig, None;
Triantafyllos Chavakis, None
Support: Else Kröner-Fresenius-Stiftung Grant
Program Number: 3018
Presentation Time: 12:00 PM–12:15 PM
Laminin β2 and γ3 chains regulate microglial activation and the
downstream effects of microglia on retinal vascular development
Saptarshi Biswas1, 2, Julianne Chu1, 2, Galina Bachay1, 2, Dale D.
Hunter1, 2, William J. Brunken1, 2. 1Ophthalmology, SUNY Downstate
Medical Center, Brooklyn, NY; 2SUNY Eye Institute, Brooklyn, NY.
Purpose: Microglia play important roles in vascular plexus formation
both by mediating vascular anastomosis and secreting pro- or antiangiogenic cytokines. Here, we investigated the role of laminin
β2 and γ3 chains in recruiting retinal microglia to the developing
vascular plexus, subsequent activation of microglia, and the effect of
microglial activation states on retinal vascular development.
Methods: Global and activated microglia density in different regions
of the retina, their association with vascular branch points, and
mitotic and apoptotic endothelial cell quantification were analyzed
using immunohistochemistry and 3D reconstruction.
Results: Previously, we showed that there is an increase in the
number of microglia associated with the vascular plexus in the
laminin γ3-/- retina. Here, we show that the laminin γ3-/- retina has
increased microglia density in the ganglion cell layer (GCL) both
centrally and peripherally compared to wild type retina. Moreover,
consistent with th in the laminin γ3-/- retina there is an increase in
both the vascular branch points and microglia associated with them at
the nascent plexus. In the laminin β2-/- retina, the normally orderly
arrangement of microglia in the GCL is disrupted, and microglia
aggregate around the persistent hyaloid vessels and malformed retinal
vasculature. In the laminin γ3-/- retina, more activated microglia
are present around the vascular plexus than in the wild type. The
increased number of vascular branch points associated microglia in
the laminin γ3-/- retina suggests more anastomotic events, leading to
a denser plexus. In contrast, in the laminin β2-/- retina, the number
of activated microglia remains the same. The developing vascular
plexus of the laminin γ3-/- retina also has increased endothelial cell
proliferation compared to the wild type, whereas the number of
apoptotic endothelial cells remains unchanged in the laminin γ3-/retina compared to the wild type.
Conclusions: Our results suggest that laminins containing the β2
and γ3 chains differentially regulate distribution of microglia in
the retina as well as the recruitment of microglia to the developing
vascular plexus with β2 laminins having pro-angiogenic effects and
γ3 laminins having anti-angiogenic effects.
Commercial Relationships: Saptarshi Biswas, None; Julianne
Chu, None; Galina Bachay, None; Dale D. Hunter, None; William
J. Brunken, None
Support: NEI Grant EY12676, RPB Challenge Grant to Department
of Ophthalmology
Program Number: 3019
Presentation Time: 12:15 PM–12:30 PM
Targeting of calcium/calmodulin-dependent protein kinase II
delta and gamma isoforms inhibits growth factor-induced retinal
angiogenesis in vitro
Sadaf Ashraf, Hannah McCauley, Alan W. Stitt, Graham J.
McGeown, Tim M. Curtis. Centre for Experimental Medicine, Queens
University Belfast, Belfast, United Kingdom.
Purpose: Previous studies from our group have shown that calcium/
calmodulin-dependent protein kinase II (CaMKII) plays a critical
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
role in VEGF-induced retinal angiogenic signalling. In the present
study, we have extended this work to examine the wider contribution
of CAMKII signalling to growth factor (GF)-induced retinal
angiogenesis in vitro and the specific involvement of the γ and δ
isoforms of CAMKII in this process.
Methods: Human retinal microvascular endothelial cells (hRMECs)
were cultured and stimulated over a 24h period with a range of
GFs (vascular endothelial growth factor [VEGF], fibroblast growth
factor [FGF], insulin-like growth factor [IGF], hepatocyte growth
factor [HGF], placental growth factor [PlGF] and platelet derived
growth factor [PDGF]) at 50ng/ml. Total and phosphorylated
protein levels of CaMKII were detected using western blotting.
The effects of CaMKII inhibition using 10μM KN93 (CaMKII
inhibitor) and its inactive analogue KN92 on GF-induced sprouting
angiogenesis in vitro were evaluated. Small interference RNA
(siRNA; 50nM) mediated knockdown of CaMKIIγ and δ isoforms
was used to investigate the relevance of these isoforms in GF-induced
retinal endothelial cell migration, tube formation and sprouting
angiogenesis.
Results: Exposing hRMECS to VEGF, FGF, HGF, IGF and PlGF
triggered a time-dependent increase in total and phospho-CAMKII
protein levels. In contrast, PDGF had no effect on CAMKII
phosphorylation or total CAMKII levels. All GFs with the exception
of PlGF and PDGF stimulated sprout formation compared with
controls. VEGF, FGF, HGF and IGF also stimulated hRMEC
migration and tube formation. KN93 reduced GF-induced sprout
formation to control levels, whereas KN92 (inactive analogue) had no
effect. siRNA knockdown of CaMKIIδ isoform significantly reduced
GF-induced sprouting angiogenesis, migration and tube formation
to control levels, whereas siRNA targeting of CaMKIIγ had only a
partial effect.
Conclusions: These results suggest that both CaMKIIδ and γ
isoforms are involved in mediating GF-induced angiogenic activity.
CaMKII is thus an important regulator of GF-induced retinal
angiogenesis and treatments targeting the γ and δ isoforms of this
protein have the potential to reduce abnormal angiogenesis in ocular
diseases.
Commercial Relationships: Sadaf Ashraf, None; Hannah
McCauley, None; Alan W. Stitt, None; Graham J. McGeown,
None; Tim M. Curtis, None
Support: British Heart Foundation R2828CVS
Program Number: 3020
Presentation Time: 12:30 PM–12:45 PM
Deletion of Thioredoxin Interacting Protein (TXNIP) Augments
Hyperoxia-induced Vaso-obliteration in Ischemic Retinopathy
Azza B. El-Remessy1, 2, Mohammed A. Abdelsaid1, 3, Adviye Ergul1,
3
, Suraporn Matragoon1, 2. 1Clin & Experimental Therapeutics,
University of Georgia, Augusta, GA; 2Culver VDI, Georgia Regents
University, Augusta, GA; 3Physiology, Georgia Regents University,
Augusta, GA.
Purpose: We have recently shown that thioredoxin interacting
protein (TXNIP) is required for VEGF-mediated VEGFR2 receptor
activation and angiogenic signal. Retinas from TXNIP knockout
mice (TKO) exhibited higher cellular antioxidant defense compared
to wild type (WT). The current study was undertaken to examine the
impact of TXNIP deletion on hyperoxia-induced vaso-obliteration in
ischemic retinopathy model.
Methods: TKO and WT pups were subjected to oxygen-induced
retinopathy model. Retinal central capillary dropout was measured
at p12. Retinal redox and nitrative state were assessed by reducedglutathione (GSH), thioredoxin reductase activity and nitrotyrosine
formation. Western blot and QT-PCR were used to assess VEGF,
VEGFR-2, Akt, iNOS and eNOS, thioredoxin expression, ASK-1
activation and downstream cleaved caspase-3 and PARP in retinal
lysates.
Results: Retinas from TKO mice exposed to hyperoxia showed
significant increases (1.5-fold) in vaso-obliteration as indicated by
central capillary drop out area compared to WT. Retinas from TKO
showed minimal nitrotyrosine levels (10% of WT) with no change in
eNOS or iNOS mRNA expression. There was no change in levels of
VEGF or activation of VEGFR2 and its downstream Akt in retinas
from TKO and WT. In comparison to WT, retinas from TKO showed
significantly higher level of GSH and thioredoxin reductase activity
in normoxia but comparable levels under hyperoxia. Exposure
of TKO to hyperoxia significantly decreased the anti-apoptotic
thioredoxin protein (~50%) level compared with WT. This effect was
associated with a significant increase in activation of the apoptotic
ASK-1, PARP and caspase-3 pathway.
Conclusions: Our results showed that despite comparable VEGF
level and signal in TKO, exposure to hyperoxia significantly
decreased Trx expression compared to WT. This effect resulted
in liberation and activation of the apoptotic ASK-1 signal. These
findings suggest that TXNIP is required for endothelial cell survival
and homeostasis especially under stress conditions including
hyperoxia.
Commercial Relationships: Azza B. El-Remessy, None;
Mohammed A. Abdelsaid, None; Adviye Ergul, None; Suraporn
Matragoon, None
Support: Support: CDA-JDRF, RO1EY042208, VDI grant
346 AMD: Mechanism and Protection
Tuesday, May 06, 2014 11:00 AM–12:45 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 3432–3465/C0280–C0313
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 3432 Poster Board Number: C0280
Presentation Time: 11:00 AM–12:45 PM
Characterization of the Stoichiometry of Human Complement C5
Binding to LFG316
Ana Carrion, Bijan Etemad-Gilbertson, Jing Zhou, Aditi Soni,
Michael Roguska. Novartis Institutes for BioMedical Research,
Cambridge, MA.
Purpose: LFG316 is a fully-human therapeutic IgG that targets
complement C5. The goal of this study was to characterize the
binding stoichiometry of LFG316 to human C5.
Methods: Three methods were used to assess binding stoichiometry;
surface plasmon resonance (Biacore), isothermal titration calorimetry
(ITC) and ELISA. The Biacore method comprised comparison
C5 binding to LFG316 IgG or LFG316 Fab in parallel flow cells
under conditions which allowed for sequential binding by the IgG
to C5. ITC was performed by titration of LFG316 against C5 and
a binding ratio calculated from the exothermic reaction. Finally, a
non-competing anti-C5 antibody was used as a capture and detection
reagent in a sandwich ELISA format in which binding to C5 was
compared for LFG316 IgG and LFG316 Fab antibodies.
Results: In the Biacore analysis, LFG316 that was bound to C5
ligand captured on a biocap chip was able to bind sequentially to C5
analyte, whereas the LFG316 Fab could only bind to the C5 ligand
on the chip, indicating 1:2 binding of the IgG. A heat plot generated
from the ITC assay yielded a binding ratio of 1:2. In the ELISA,
LFG316 IgG bound to two bound C5 molecules could be detected,
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
whereas as expected only a single bound C5 was detected with the
LFG316 Fab, also indicating a 1:2 binding ratio.
Conclusions: Using three different analytical methods, Biacore,
ELISA, and ITC, we observed a 1:2 binding stoichiometry between
LFG316 and human complement C5.
Commercial Relationships: Ana Carrion, Novartis Institutes for
BioMedical Research (E); Bijan Etemad-Gilbertson, Novartis
Institutes for BioMedical Research (E); Jing Zhou, Novartis
Institutes for BioMedical Research (E); Aditi Soni, Novartis
Institutes for BioMedical Research (E); Michael Roguska, Novartis
Institutes for BioMedical Research (E)
Program Number: 3433 Poster Board Number: C0281
Presentation Time: 11:00 AM–12:45 PM
Generation and Characterization of LFG316, A Fully-Human
Anti-C5 Antibody for the Treatment of Age-Related Macular
Degeneration
Michael Roguska1, Igor Splawski1, Beate Diefenbach-Streiber3,
Elizabeth Dolan1, Bijan Etemad-Gilbertson1, Jean-Michel Rondeau2,
Mark Keating1. 1Novartis Institutes for BioMedical Research,
Cambridge, MA; 2Novartis Institutes for BioMedical Research, Basel,
Switzerland; 3MorphoSys AG, Martinsried, Germany.
Purpose: Polymorphisms in the alternative pathway of the
complement system correlate with risk of AMD. Cleavage of C5
generates C5a and the membrane attack complex (MAC), key
mediators of the terminal complement pathway and complement
activation. We designed LFG316 to test whether inhibition of C5
cleavage may prevent AMD or slow the rate of progression.
Methods: LFG316, a fully-human antibody, was generated using
phage-display technology. Antibodies were identified using the
HuCAL GOLD library by selections on human and cynomolgus
C5 protein. The selection criteria of the final candidate antibody
was based on assessments of potency in hemolytic assays, affinity,
expression and biophysical behaviour.
Results: LFG316 is an IgG1 antibody with modifications to the
Fc region to attenuate immune effector activity. LFG316 binds to
the human and cynomolgus C5 with affinities of 12.1 pM and 74.1
pM, respectively. In an alternative pathway hemolytic assay the
antibody has an IC50 of 23.9 nM in 10% human serum and an IC50
of 37.3 nM in 10% cynomolgus monkey serum. LFG316 blocks the
generation of C5a during the hemolysis reaction with an IC50 of 25.4
nM. These results indicate that LFG316 binds to C5 and prevents
cleavage by the C5 convertase to C5a and C5b. LFG316 also blocks
C5 cleavage due to complement classical pathway activation with an
IC50 of 20.1 nM in the presence of 10% human serum.
Conclusions: LFG316 is a fully-human, high affinity antibody
that prevents the cleavage of C5 via the complement classical or
alternative pathways. Inhibition of C5 with LFG316 is currently
being tested as a therapy for the treatment of geographic atrophy.
Commercial Relationships: Michael Roguska, Novartis Institutes
for BioMedical Research (E); Igor Splawski, Novartis Institutes for
BioMedical Research (E); Beate Diefenbach-Streiber, Morphosys
AG (E); Elizabeth Dolan, Novartis Institutes for BioMedical
Research (E); Bijan Etemad-Gilbertson, Novartis Institutes for
BioMedical Research (E); Jean-Michel Rondeau, Novartis Institutes
for BioMedical Research (E); Mark Keating, Novartis Institutes for
BioMedical Research (E)
Program Number: 3434 Poster Board Number: C0282
Presentation Time: 11:00 AM–12:45 PM
Smoke-exposure causes endoplasmic reticulum stress and lipid
accumulation in retinal pigment epithelium through oxidative
stress and complement activation
Baerbel Rohrer1, 2, Carl Atkinson3, Kannan Kunchithapautham1.
1
Ophthalmology, Med Univ of South Carolina, Charleston, SC;
2
Research Service, Ralph H. Johnson VA Medical Center, Charleston,
SC; 3Microbiology and Immunology, Med Univ of South Carolina,
Charleston, SC.
Purpose: Age-related macular degeneration is a complex disease
that is caused by genetic and environmental factors, including
genetic variants in complement components and smoking. Smoke
exposure has been shown individually to lead to oxidative stress,
complement activation, endoplasmatic reticulum (ER) stress and lipid
dysregulation, which have all been proposed to be associated with
the pathogenesis of AMD. Here we examine the effects of smoke
exposure on retinal pigment epithelium (RPE) of mice, and cigarette
smoke extract (CSE) on immortalized human RPE cells (ARPE-19).
Methods: Mice were exposed to cigarette smoke or filtered air for
6 months. RPE cells grown as stable monolayers were exposed to
5% smoke extract. Barrier function of monolayers was determined
by transepithelial resistance (TER) measurements; and effects of
smoke were determined by biochemical, molecular and histological
measures. Effects of the alternative pathway (AP) of complement
as well as anaphylatoxin receptor signaling were analyzed using
knockout mice or specific inhibitors.
Results: ER stress markers were elevated in the RPE of smoke
exposed mice, which could be prevented by eliminating the AP. To
further examine this mechanism, RPE monolayers were exposed
to 5% CSE, a concentration which did not alter TER. Short-term
smoke exposure resulted in epithelial C3 release, generation of
the complement anaphylatoxins, C3a and C5a, oxidative stress,
complement activation on the cell membrane, and ER stress. In
addition, long-term exposure resulted in lipid accumulation and
secretion. All measured outcomes were significantly reduced by
blocking anaphylatoxin receptor function, and AP signaling. Lipid
deposition induced by tunicamycin-mediated ER stress, however was
not susceptible to AP inhibition.
Conclusions: Our results provide clear evidence that smoke exposure
results in oxidative stress and complement activation involving
the AP, resulting in ER stress-mediated lipid accumulation. Taken
together our data suggests that smoking and complement activation
act synergistically in the disease process.
Commercial Relationships: Baerbel Rohrer, Alexion Therapeutics
(P); Carl Atkinson, None; Kannan Kunchithapautham, None
Support: NIH grant EY019320, NHLBI091944, a Department
for Veterans Affairs merit award RX000444, Foundation Fighting
Blindness, the Beckman Initiative for Macular Research, and
Research to Prevent Blindness.
Program Number: 3435 Poster Board Number: C0283
Presentation Time: 11:00 AM–12:45 PM
The Role of Nrf-2 in Alternative Pathway (AP) Complement
and Hydroquinone (HQ)-mediated Heme Oxygenase-1 (HO-1)
Expression in Human RPE (hRPE) Cells
Michelle Bao1, Zhe Ma1, Ping Yang1, Peter Baciu2, Glenn J. Jaffe1.
1
Ophthalmology, Duke University Eye Center, Durham, NC;
2
Biology, Allergan Inc, Irvine, CA.
Purpose: Oxidative stress and complement activation are implicated
in age-related macular degeneration (AMD). HO-1 is a stressinducible protein with antioxidant and potential anti-inflammatory
effects. We previously reported that complement activation through
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
the AP attenuates HQ-mediated HO-1 mRNA induction. Multiple
signaling pathways, including Nrf-2, NF-κB, AP-1, and MAPK,
modulate HO-1 expression in non-ocular cells, and Nrf-2 is thought
to play an important role. To better understand complement-mediated
attenuation of HO-1, we investigated the role of Nrf-2 signaling on
HO-1 expression in response to HQ alone, or in combination with AP
complement.
Methods: Cultured hRPE were treated with 125 uM HQ for 1.5
hours, primed with sheep anti-ARPE-19 antibody for 30 minutes
and incubated in C1q-depleted human serum (C1q-Dep) for 3 hours.
To study the role of Nrf-2 in HQ-mediated HO-1 induction, 80%
confluent hRPE were transfected with 30 nM of small interfering
RNA (siRNA) specific for Nrf-2. Once confluent, cells were exposed
to 125 uM HQ for 1.5 hours and incubated in serum-free media
for 3.5 hours. Cytoplasmic and nuclear protein was harvested, and
protein levels were determined by Western blot. Confluent hRPE
were treated with 40 ng/ml TNF-α, various doses of HQ, or AP
complement at multiple time points. NF-κB translocation was
examined with immunofluorescent staining and western blots of
cytoplasmic and nuclear extracts.
Results: AP complement activation attenuated HQ-induced HO-1
protein; the magnitude of this effect varied among experiments.
Increased nuclear Nrf-2 accompanied HQ-mediated HO-1 induction,
and AP complement attack had minimal effect on HQ-induced Nrf-2
protein. HQ-induced Nrf-2 protein was knocked down by Nrf-2
siRNA, but Nrf-2 inhibition had minimal effect on HQ-induced HO1. TNF-α caused NF-κB nuclear translocation at 40 and 90 minutes,
but HQ and AP complement alone did not cause NF-κB nuclear
translocation at any dose or time point.
Conclusions: AP complement activation attenuated HQ-mediated upregulation of HO-1 protein expression. These phenomena appeared
largely Nrf-2 and NF-κB independent. These data suggest that other
transcription factors may play more significant HO-1 regulatory roles
and represent further targets of study to understand the synergistic
effects of oxidative stress and AP activation on RPE cell function.
Commercial Relationships: Michelle Bao, None; Zhe Ma, None;
Ping Yang, None; Peter Baciu, Allergan Inc (E); Glenn J. Jaffe,
None
Support: NIH P30 EY-005722 (Core grant), Research to Prevent
Blindness, Inc. (RPB)
Program Number: 3436 Poster Board Number: C0284
Presentation Time: 11:00 AM–12:45 PM
Upregulation of complement components and chemokines
by interferon gamma (IFN-γ) stimulation of retinal pigment
epithelium (RPE) in vitro
Sijia Cao, Aikun Wang, Jing Z. Cui, Joanne A. Matsubara. Ophthal
& Visual Sciences, University of British Columbia, Vancouver, BC,
Canada.
Purpose: Drusen are hallmark deposits of age-related macular
degeneration (AMD) and are hypothesized to mediate RPE
dysfunction. Our previous studies demonstrated that two drusen
components, advanced glycation end products and amyloid-beta,
upregulated IFN-γ and IFN-γ signaling pathways in RPE cells in
vitro. IFN-γ is a soluble cytokine associated with innate and adaptive
immunity, and recent studies point towards an emerging relationship
between IFN-γ and mechanisms underlying the pathogenesis of
AMD. Here we focus on understanding effects of IFN-γ on RPE gene
expression.
Methods: Primary fetal RPE cells were exposed to 12.5 or 25ng/
ml IFN-γ in serum-free DMEM culture medium for 12h and 24h,
respectively. The IFN-γ induced cytotoxicity was evaluated by lactate
dehydrogenase (LDH) assay. Cells were lysed to collect mRNA
and the RNA expression of complement components, inflammatory
cytokines and chemokines were analyzed by quantitative PCR
(q-PCR).
Results: Cell death was unchanged for both 12.5 and 25ng/ml IFN-γ
at 24h compared to untreated cells. IFN-γ treatment upregulated
complement factor H (CFH), complement factor B (CFB) and C3
(fold change > 2, p < 0.05), but did not affect the expression level
of complement regulators CD46/55/59, complement factor I (CFI),
complement factor D (CFD) or C5. IFN-γ also upregulated CX3CL1
(fractalkine) (>10) and MCP-1 (>2) in RPE cells at 12h and 24h (p <
0.01). IFN-γ at the tested concentration did not change the expression
level of IL-1β, IL-18, IL-6, IL-8, TNF-α, IL-1ra and VEGF-A.
Conclusions: IFN-γ at the concentration tested does not have an
adverse effect on survival of RPE cells, but it can affect the gene
expression of RPE related to pro-inflammatory mediators. Our data
demonstrated that several complement components are upregulated
by IFN-γ. In addition, IFN-γ stimulation promoted chemokine
upregulation, especially CX3CL1 from RPE cells. The significant
upregulation of fractalkine, CX3CL1, a membrane-bound chemokine,
may play an important role in the early AMD by promoting immune
cell chemotaxis in outer retina in response to drusen components.
Therefore, targeting the release of cleaved CX3CL1 from RPE origin
may be an important strategy to suppress immune cell chemotaxis
into outer retina.
Commercial Relationships: Sijia Cao, None; Aikun Wang, None;
Jing Z. Cui, None; Joanne A. Matsubara, None
Support: CIHR MOP 97806 and VGH+UBC Hospital Foundation
Program Number: 3437 Poster Board Number: C0285
Presentation Time: 11:00 AM–12:45 PM
Proteomics Analysis of Protein Expression in Human RPE Cells
Exposed to Oxidant and Complement Attack through Alternative
Pathway
Ping Yang1, Nikolai P. Skiba1, Michelle Bao1, Angel Long1, Peter
Baciu2, Glenn J. Jaffe1. 1Ophthalmology, Duke University Eye
Center, Durham, NC; 2Biology, Allergan, Inc, Irvine, CA.
Purpose: Complement activation and oxidative stress has been
increasingly implicated in the pathogenesis of AMD. We previously
reported a new model whereby oxidative stress sensitizes RPE
cells to complement attack by the alternative complement pathway.
Herein, we pursued proteomic analyses to screen potential candidate
molecules/pathways to better understand the mechanisms of RPE
injury in this model.
Methods: Cultured hRPE cells were stimulated with or without
hydroquinone (HQ) for 1.5 hours, primed with an anti-ARPE-19
antibody for 30 minutes followed by incubation for 6 hours with
either 6% C1q-depleted human serum (C1q-Dep) to initiate
complement attack, or 6% heat inactive C1q-Dep. Quantified proteins
were electrophoresed. Stained bands were excised and subjected
to in-gel tryptic digestion. The peptide mixes were subjected to
a LC-MS/MS. Three independent protein quantifications were
conducted by three spectra counting methods: the normalized
spectral abundance factor, the normalized weighted spectra, and
the exponentially modified protein abundance index. Percentage of
CV (SD/average) per protein of 4 treatments greater than 100% (≥2
average of CV%) from three methods were considered as detected
altered proteins.
Results: Over 1000 proteins were compared. As expected, and
as controls for the model, complement components such as C3,
C5 and C9 were deposited in cells incubated with complement or
HQ+complement, but not in control- or HQ-treated cells. Heme
oxygenase-1 (HO-1) and hexokinase-1 were elevated in cells
stimulated with HQ and HQ+complement when compared to controls
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
and complement alone. HO-2 levels were not altered by any of the
treatments. Detected proteins altered by complement alone were
predominantly involved in calcium homeostasis, regulation of cell
signaling processes and energy metabolism. Proteins altered by HQ
alone were involved in cellular protein metabolic process, apoptosis
and cellular iron ion homeostasis. Proteins altered by the combination
of complement and HQ were involved signal transduction, gene
expression and response to stress.
Conclusions: Complement and oxidative stress altered several
proteins that are involved in key cellular processes. These results may
help to improve our understanding of complement- and oxidative
stress-mediated RPE injury, and provide insight into potential
therapeutic targets.
Commercial Relationships: Ping Yang, None; Nikolai P. Skiba,
None; Michelle Bao, None; Angel Long, None; Peter Baciu,
Allergan, Inc (E); Glenn J. Jaffe, None
Support: NIH P30 EY-005722 (Core grant), Research to Prevent
Blindness, Inc. (RPB)
Program Number: 3438 Poster Board Number: C0286
Presentation Time: 11:00 AM–12:45 PM
Peroxisome proliferator-activated receptor (PPAR) β/δ expression
and activation in retinal pigment epithelial and choroidal
endothelial cells: implication in age-related macular degeneration
Mayur Choudhary, Goldis Malek. Opthalmology, Duke Eye Center,
Durham, NC.
Purpose: PPARs are members of a superfamily of nuclear receptors
that play pivotal roles in the regulation of lipid homeostasis,
energy metabolism, inflammation, extracellular matrix remodeling,
cellular differentiation, proliferation and apoptosis, events also
important in initiation and progression of age-related macular
degeneration (AMD). Among the three isoforms identified, the
physiological function of the PPARβ/δ [NR1C2] isoform, remains
relatively unknown. It is expressed in a variety of cells and its
activation has been postulated to exert anti-atherosclerotic effects by
modulating lipoprotein metabolism and increasing the availability
of inflammatory suppressors. Here, we investigated the expression
and activity of the PPARβ/δ in retinal pigment epithelial (RPE) and
choroidal endothelial cells, cells affected in AMD, and its effect on
disease related pathogenic pathways.
Methods: PPARβ/δ expression and activity were determined
in ARPE19 and RF/6A endothelial cells treated with synthetic
agonists and antagonists of the receptor using qPCR, western blot
and luciferase assays. Expression of inflammatory, extracellular
matrix (ECM) molecules, and growth factors involved in fibrosis
were measured in cells following knock-down of the receptor, and/
or inflammatory stimulation and oxidative stress. RPE cells were
also treated with native and oxidized lipids to validate them as
endogenous ligands.
Results: The PPARβ/δ pathway is active in ARPE19 and RF/6A cells
as shown by transcriptional activity assays and target gene expression
in response to agonists. The induction of the PPAR reporter activity
and target genes in response to dietary lipids confirmed their role
as endogenous ligands. Agonist treatment caused an upregulation
of TGFβ mRNA, and collagenIV and fibronectin secretion, which
was inhibited by the antagonist. PPARβ/δ knockdown resulted in an
increase in collagen1A1, IV, and vitronectin expression, underlining
the involvement of the PPAR signaling pathway in ECM regulation.
Conclusions: Since homeostasis of the ECM and apoptosis of RPE
and endothelial cells are potentially vital mechanisms in AMD
pathogenesis and specifically deposit formation and fibrosis, our
results support the hypothesis that the PPARβ/δ may be a regulatory
signaling pathway in AMD. Further investigation is ongoing.
Commercial Relationships: Mayur Choudhary, None; Goldis
Malek, None
Support: IRRF Postdoctoral award (MC), NIH Grant EY02868
(GM), P30 EY005722 (Duke Eye Center), Research to Prevent
Blindness Special Scholar Award (GM) and Research to Prevent
Blindness (Core to Duke University)
Program Number: 3439 Poster Board Number: C0287
Presentation Time: 11:00 AM–12:45 PM
Different Structures of Aβ1-40 Assemblies Induce Distinctive
Primary RPE Cell Responses in vitro
Jiangyuan Gao, Jing Z. Cui, Aikun Wang, Joanne A. Matsubara.
Ophthalmology and Visual Sciences, University of British Columbia,
Vancouver, BC, Canada.
Purpose: Aβ1-40 is a pathological drusen component whose role
in AMD etiology is under investigation. However, little is known
about its conformational changes and the associated effects on RPE
during AMD pathogenesis. This study aims to compare and elucidate
the differential impact of high and low molecular weight Aβ1-40
assemblies on RPE cells.
Methods: High molecular (>170 KDa, fibrillar) Aβ1-40 species was
collected after 1 week incubation in Tris-buffered saline (TBS) at
37 Celsius following initial suspension in sterile deionized water.
Similarly, low molecular (<15 KDa, oligomeric/monomeric) Aβ1-40
species was obtained after 24 hour incubation following initial
suspension in hexafluoroisopropanol. Both Aβ1-40 preparations were
subjected to electrophoresis separation and detected by immunoblot
to verify proper structures. Human primary RPE cells at passage 9
were treated with either form of Aβ1-40 at various concentrations for
MTT and LDH assays. Quantitative RT-PCR (qRT-PCR) was applied
to quantify gene expression in oxidative stress, for example, NAD(P)
H dehydrogenase, quinone 1 (NQO1) and in inflammatory responses,
such as complement factor H (CFH) and Caspase-1 (CASP1), after
Aβ1-40 treatment.
Results: At 24 hour, both forms of Aβ1-40 increased LDH release and
decreased MTT colorimetric reaction when compared to non-treated
RPE cells. At the same concentration, fibrillar Aβ1-40 presented
equivocal cytotoxicity and concomitantly a dose-dependent decrease
of intracellular reductants compared to oligomeric/monomeric Aβ1-40.
In qRT-PCR, 5.0 μM fibrillar Aβ1-40 downregulated CFH by 1.5 fold
compared to non-treated RPE cells. Both CASP1 and NQO1 were
decreased by 1.5 fold in 0.3 μM fibrillar Aβ1-40 treated cells compared
to 0.3 μM oligomeric/monomeric Aβ1-40 treated ones.
Conclusions: The current study addresses an important topic
that different forms of Aβ1-40 assemblies may affect primary RPE
differentially. Fibrillar Aβ1-40 seems more likely to cause oxidative
stress than its low molecular counterpart. High concentration of
fibrillar Aβ1-40 sequesters RPE’s self-protection from complement
activation and therefore may play a role in AMD pathogenesis. Given
the presence of both forms of Aβ1-40 in drusen deposits, understanding
their distinctive biological effects on RPE may provide insights
on AMD disease progression and also shed light on the future
development of focused treatment strategies.
Commercial Relationships: Jiangyuan Gao, None; Jing Z. Cui,
None; Aikun Wang, None; Joanne A. Matsubara, None
Support: CIHR MOP 97806 and VGH+UBC Hospital Foundation
Program Number: 3440 Poster Board Number: C0288
Presentation Time: 11:00 AM–12:45 PM
Exogenous regulation of the HIF pathway in RPE cells
Helder Andre, Malena Ekstrom, Anna Takei, Yu Ma, Anders P.
Kvanta. Clinical Neurosciences, St Erik Eye Hospital, Stockholm,
Sweden.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: In this study we investigate the effects of a series of
hypoxia-inducible factors (HIF)-regulating molecules (HRM) on the
hypoxia pathway in retinal pigment epithelial (RPE) cells, critically
involved in neovascular age-related macular degeneration (nAMD)
pathogenesis.
Methods: ARPE-19 were maintained at normoxia (21% O2), hypoxia
(1% O2), or in hypoxia-mimicking agents (CoCl2). HUVEC were
kept at normoxia.
ARPE-19 cells were transfected with plasmids encoding FLAGtagged HRM (PHD1, PHD2, PHD3, VHL, FIH-1), as well as HIF-1α
and HIF-2α, as needed.
Effects of HMR on ARPE-19 were analyzed by a luciferase reporter
assay (DLR), Western blot, and soluble vascular endothelial factor
(VEGF)-capture assay. Effects on endothelial cells (EC) were studied
by exposing HUVEC cells to ARPE-19 pre-conditioned media after
transfection with particular HRM.
Results: ARPE-19 cells displayed endogenous expression of
HIF-1α, but not -2α, at the protein level. Moreover, transfection
of all plasmids encoding FLAG-tagged HRM was normalized for
comparable levels of protein expression.
The DLR denoted a marked negative regulation of HIF-1α activity
in ARPE-19 in the presence of all exogenous PHDs. Analysis of
HIF-1α protein showed a very considerable decrease, particularly
in the presence of PHD2. Additionally, PHD2 also showed the most
dramatic reduction of HIF-1α protein life-time.
Following, ARPE-19 were transfected with PHD2-encoding plasmid,
and the level of secreted VEGF was analyzed by immunocapturing
assay using bevacizumab (anti-VEGF). The immunoprecipitated level
of VEGF from the media of ARPE-19 cells transfected with PHD2
was considerably lower than that of an empty control. Subsequently,
HUVEC were exposed to the media from these experiments, and EC
proliferation was decreased in pre-conditioned media from ARPE-19
transfected with PHD2.
Conclusions: In this study we have compared the effects of canonical
HMR on the hypoxia-mediated response in ARPE-19 cells. Our data
indicates that PHD2 seems to be the most potent negative-regulator
of the HIF pathway. Furthermore, the negative effects of PHD2 were
clearly associated with decrease in secreted VEGF. This decrease in
VEGF level displayed further effects in reducing EC proliferation.
These results may have implications for the clinical treatment of
patients with nAMD, particularly regarding the use of gene therapy to
negatively regulate the neoangiogenesis present in these patients.
Commercial Relationships: Helder Andre, None; Malena
Ekstrom, None; Anna Takei, None; Yu Ma, None; Anders P.
Kvanta, None
Support: KMA, TNS, KI
Program Number: 3441 Poster Board Number: C0289
Presentation Time: 11:00 AM–12:45 PM
Effect of Dexamethasone on Polarized H9RPE Cells
Danhong Zhu1, Jamie Hsiung1, David R. Hinton1, 2. 1Pthology,
University of Southern California, Los Angeles, CA;
2
Ophthalmology, University of Southern California, Los Angeles,
CA.
Purpose: The transplantation of embryonic stem cell-derived
polarized RPE monolayers to restore the lost or dysfunctional RPE
is a promising new treatment for age-related macular degeneration
(AMD), however, immune rejection remains to be the biggest
challenge for long term survival of those implanted allogeneic cells.
Dexamethasone (DEX), a widely used synthetic corticosteroid, is
known to inhibit allogeneic immune rejection, but also promote cell
death in several cell types. This study is to investigate the effect and
potential side-effect of DEX on polarized RPE cells derived from the
H9 embryonic stem cell line (H9-RPE).
Methods: RPE cells derived from H9 human embryonic stem
cells were cultured on substrates for 4 weeks to form a polarized
monolayer; the polarized RPE monolayer was then treated with
different concentrations (0, 10, 100 nM) of DEX for a month. Live/
dead cell staining, quantitative PCR, immunofluorescent staining, and
ELISA were used to examine the cell viability, the changes of mRNA
and protein expression levels of RPE signature genes and apoptosis
related genes.
Results: After DEX treatment, significantly increased cell death was
observed in non-polarized H9RPE cells, but not in polarized H9RPE
cells, as compared to non-DEX treated corresponding control cells.
The polarized RPE cells kept their hexagonal shape and integrity
of tight junctions after long term DEX treatment. The expression
level of RPE signature genes, such as RPE65, PMEL and PEDF had
no significant difference in DEX treated polarized H9RPE cells, as
compared to non-DEX treated control cells.
Conclusions: Although it may increase non-polarized RPE cell death,
DEX does not impose adverse effects on polarized RPE cells.
Commercial Relationships: Danhong Zhu, None; Jamie Hsiung,
None; David R. Hinton, None
Support: Grant From CIRM Disease Team Research Award, NIH
Core Grant EY 03040
Program Number: 3442 Poster Board Number: C0290
Presentation Time: 11:00 AM–12:45 PM
Permeability of ranibizumab and bevacizumab through the
polarized RPE cells in vitro
Hiroto Terasaki, Naoya Yoshihara, Makoto Shirasawa, Hiroki
Otsuka, Shozo Sonoda, Taiji Sakamoto. Ophthalmology, Kagoshima
University, Kagoshima, Japan.
Purpose: To investigate the penetration of ranibizumab and
bevacizumab through the polarized RPE cells in vitro and its
mechanism.
Methods: Highly polarized RPE cells were cultured in Boyden
chamber (Sonoda S, et al. Nat Protoc, 2009). Polarization was
confirmed by asymmetrical secretion of VEGF between apical and
basal side and significant expression of ZO-1. Ranibizumab (1-100
μg/ml) or bevacizumab (2.5-250 μg/ml) were added to the upper
chamber. After 3 hours, the medium in lower chamber was evaluated
by measuring the concentration of them using ELISA. The effect
of pharmacological inhibitors was evaluated to investigate the
mechanism of permeation of ranibizumab/bevacizumab.
Results: Ranibizumab was more permeable than bevacizumab
through RPE layer at any concentration (2.0-2.6 times greater
than bevacizumab, Mann-Whitney U, P<0.05). Light and electron
microscopy showed no cytotoxic changes in either ranibizumabor bevacizumab-treated RPE cells. Permeation of ranibizumab/
bevacizumab was significantly inhibited by protein kinase C inhibitor
in contrast to control.
Conclusions: Ranibizumab was more permeable than bevacizumab
through highly polarized RPE cells. And its permeation through
RPE cells was, at least in part, PKC-dependent. Considering
its similarity to RPE in vivo, ranibizumab might be more
effective than bevacizumab on the treatment for type 1 choroidal
neovascularization.
Commercial Relationships: Hiroto Terasaki, None; Naoya
Yoshihara, None; Makoto Shirasawa, None; Hiroki Otsuka,
None; Shozo Sonoda, None; Taiji Sakamoto, Novartis JapaNovartis
Pharmaceuticals Japan (F)
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 3443 Poster Board Number: C0291
Presentation Time: 11:00 AM–12:45 PM
Dissecting molecular pathways of mTORC1 signaling in the
retinal pigment epithelium
Bo Yu, Pei Xu, Zhen-Yang Zhao, Bo Long, Jiyang Cai, Yan Chen.
Ophthalmology, UTMB, Galveston, TX.
Purpose: Activity of the mechanistic target of rapamycin (mTOR)
is controlled by upstream small GTPase protein Rheb, as well as its
subcellular localization. Although a broad spectrum of downstream
effectors exist for mTOR complex 1 (mTORC1), activated mTORC1
regulates specific substrates to accommodate with tissue- and
cell-specific functions. The majority of literature studies on mTOR
used transformed cancer cell lines whose signaling networks can be
distinct from the retinal pigment epithelium (RPE). The purpose of
this study is to explore RPE-specific pathways of mTOR, both in
vitro and in vivo.
Methods: Major regulatory proteins of mTOR signaling, including
TSC1, TSC2, Rheb, Rag and Ragulator complex, were downregulated by specific siRNAs in cultured human fetal RPE cells or
143B osteosarcoma cells. Responses of mTORC1 and mTORC2
to nutrients and growth factors were monitored by measuring the
phosphorylation status of S6 and Ser473 of Akt, respectively. To
activate mTORC1 in vivo, mice were fed with a high fat, cholesterol
rich (HF-C) diet which contained 20% fat (w/w) and 1.5% cholesterol
for 1 month with or without administration of rapamycin. Activity of
mTORC1 and subcellular location of mTOR-related proteins were
analyzed in the isolated RPE/choroid tissue.
Results: mTORC1 in the RPE and 143B cells both responded to
nutrients and growth factors. However, regulation by the TSC1TSC2 complex was different. In RPE cells, TSC1-TSC2 did not exert
negative regulation on mTOR signaling. Instead, the presence of
TSC complex was required for maximum activation of mTORC1. In
vivo, HF-C diet selectively activated mTORC1 in the RPE, without
affecting the Akt phosphorylation. Moreover, rapamycin regulated
different sets of mTOR substrates in mice fed with HF-C diet as
compared to animals on regular diet.
Conclusions: In contrast to transformed cells with strong mitogenic
signaling, RPE cells have unique upstream regulatory mechanisms
of mTORC1. There is a positive association between TSC1TSC2 complex and mTORC1 activity in the RPE. How mTOR
contributes to HF-C diet-induced RPE degeneration warrants further
investigation.
Commercial Relationships: Bo Yu, None; Pei Xu, None; ZhenYang Zhao, None; Bo Long, None; Jiyang Cai, None; Yan Chen,
None
Support: NIH grants EY 019706, EY 021937, International Retinal
Research Foundation and Ted Nash Long Life Foundation
Program Number: 3444 Poster Board Number: C0292
Presentation Time: 11:00 AM–12:45 PM
Complement component C5a primes the NLRP3 inflammasome
in retinal pigment epithelial cells
Carolina Brandstetter, Frank G. Holz, Tim U. Krohne.
Ophthalmology, University of Bonn, Bonn, Germany.
Purpose: Photooxidative damage of the retinal pigment epithelium
(RPE) is associated with the pathogenesis of age-related macular
degeneration (AMD). In addition, involvement of a chronic
immune response in the sub-RPE space including activation of the
complement system has been demonstrated in AMD. To identify
a molecular link between these mechanisms we investigated the
capability of activated complement components to prime RPE
cells for activation of the NLRP3 inflammasome by lipofuscin
phototoxicity.
Methods: Lipofuscinogenesis was induced in primary human RPE
cells and ARPE-19 cells by incubation with isolated photoreceptor
outer segments following modification with lipid peroxidation
products. For inflammasome priming, lipofuscin-loaded cells
were incubated in serum-free media or media supplemented with
full human serum, C5-deficient serum, or isolated C5a. Specific
C5a receptor (CD88) antibodies were used to block C5a binding.
Control cells were primed with IL-1α. Following priming, cells were
irradiated with blue light for up to 6 hours. NLRP3 inflammasome
activation was assessed by measuring IL-1β and IL-18 secretion.
Pyroptotic cell death was analyzed using LDH release assay, TUNEL
staining, and DNA/histone-specific ELISA.
Results: Priming of RPE cells with full human serum or isolated
complement component C5a resulted in a lipofuscin load- and
light dose-dependent activation of the NLRP3 inflammasome with
secretion of IL-1β and IL-18. Complement heat-inactivation, C5
depletion, or C5a receptor inhibition suppressed the priming effect of
human serum. Specific inhibition of caspase-1 or cathepsin B, L, or
D likewise prevented NLRP3 activation. Inflammasome activation
was followed by RPE cell death by pyroptosis as identified by
morphological and molecular characteristics.
Conclusions: Complement component C5a is capable of providing
the priming signal for subsequent activation of the NLRP3
inflammasome by phototoxic effects of lipofuscin. This molecular
pathway may represent a functional link between hallmark features
of AMD such as lipofuscin accumulation, photooxidative damage,
chronic immune response, and progressive degeneration of the RPE
and may provide a novel target for therapeutic intervention in AMD.
Commercial Relationships: Carolina Brandstetter, None; Frank
G. Holz, None; Tim U. Krohne, None
Support: German Research Foundation (DFG, grant KR 2863/71); Pro Retina Research Foundation; German Ophthalmological
Society (DOG): University of Bonn BONFOR/SciMed Program; Dr.
Eberhard und Hilde Rüdiger Foundation
Program Number: 3445 Poster Board Number: C0293
Presentation Time: 11:00 AM–12:45 PM
Humanin protects RPE cells from oxidative stress induced cell
death
Parameswaran G. Sreekumar1, Keijiro Ishikawa1, 4, Christine Spee2,
Hemal Mehta3, Kelvin Yen3, Pinchas Cohen3, Ram Kannan1, David
R. Hinton2, 4. 1Ophthalmology, Doheny Eye Institute, Los Angeles,
CA; 2Ophthalmology, University of Southern California, Los
Angeles, CA; 3USC Davis School of Gerontology, Los Angeles, CA;
4
Pathology, University of Southern California, Los Angeles, CA.
Purpose: Humanin (HN) is a 24-amino acid peptide encoded from
the mitochondrial chromosome. Recent studies have reported a
neuroprotective role for HN in vitro and in various animal models.
Yet, the role of HN in age-related macular degeneration is hitherto
unknown. Hence, in this study, we investigated the protective role of
HN in human fetal RPE cells (hfRPE) and evaluated the participation
of mitochondrial respiration in this process.
Methods: To study the protective effect of HN, primary hfRPE cells
were co-treated with varying doses of HN (0.5-10ug/ml) and 150 mM
of tert-Butyl hydroperoxide (tBH) for 24 h in serum-free medium.
HN localization in RPE cells was assessed by confocal microscopy.
Mitochondrial respiration was measured in RPE cells grown on
96-well plates using XF96 analyzer (Seahorse Bioscience Inc, MA).
RPE cell death and caspase-3 activation induced by tBH were studied
by TUNEL staining and immunoblot analysis.
Results: Our studies showed a prominent expression of HN in
the cytoplasm and nucleus of hfRPE cells. In the cytoplasm, HN
co-localized with mitochondria. A similar pattern of expression
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
was found in human polarized RPE monolayers. Mitochondrial
respiration was significantly decreased with oxidative stress (p<0.05
vs control) while HN co-treatment upregulated mitochondrial
respiration significantly (p<0.01 vs tBH treated cells). HN protected
RPE cells from oxidative stress-induced cell death and prevented
caspase-3 activation.
Conclusions: HN protects RPE cells from oxidant injury by
attenuating cell death and restoring mitochondrial function as
evidenced by increased mitochondrial respiration. These data suggest
a potential role for humanin therapy in the prevention and treatment
of age-related macular degeneration.
Commercial Relationships: Parameswaran G. Sreekumar, None;
Keijiro Ishikawa, None; Christine Spee, None; Hemal Mehta,
None; Kelvin Yen, None; Pinchas Cohen, None; Ram Kannan,
None; David R. Hinton, None
Support: EY03040, EY01545, Arnold and Mabel Beckman
Foundation, Research to Prevent Blindness.
Program Number: 3446 Poster Board Number: C0294
Presentation Time: 11:00 AM–12:45 PM
Mitochondrial elongation in the retinal epithelium of aging
monkeys: evidence of metabolic stress
Peter Gouras1, L. Ivert2, Martha Neuringer3, Takayuki Nagasaki1.
1
Ophthalmology, Columbia University, New York, NY; 2St Erik’s
Hospital, Karolinska Institute, Stockholm, Sweden; 3Neuroscience,
Oregon National Primate Research Center, Beaverton, OR.
Purpose: Oxidative stress is suggested as a cause of age related
macular degeneration (AMD) but evidence that stress is occurring in
the major pathogenic target of AMD, the retinal pigment epithelium
(RPE), is lacking. Mitochondrial elongation is a morphological
change caused by metabolic stress. Mitochondria are dynamic
organelles continuously undergoing fusion and fission. The balance
between these opposing processes shapes the mitochondria and
reflects the metabolic status of a cell. With stress produced by
starvation and/or increases in reactive oxygen species (ROS),
mitochondria elongate, perhaps as an attempt to increase energy
production. We measured the number and length of mitochondria
in the macula RPE of young and old rhesus monkeys as a potential
marker of metabolic stress.
Methods: Eyes of seven monkeys (Macaca mulatta), 1, 2, 6.5, 24,
24, 26, and 35 years of age, were studied by electron microscopy.
The number and length of mitochondria were measured in macula
RPE. Measurements were assessed separately for the basal,
middle and apical regions of the RPE cell. Because mitochondria
are concentrated in the basal third of the cell, we use the basal
measurements for comparisons of mitochondrial number and length
versus age.
Results: Except for a high in the youngest and a low in the oldest,
mitochondrial number remained similar for most of the lifespan of
the monkeys, the latter similar to results of Feher et al. (2006) for
human RPE. An average length of mitochondria, however, increased
with age. Some of these mitochondria became extremely long, 4-5
microns in length, and tended to appear in clusters.
Conclusions: Elongation of mitochondria with age in the macular
RPE of monkeys suggests that metabolic stress occurs and is
increasing with age, consistent with the contribution of oxidative
stress to AMD. It would be interesting to know if similar changes
occur in non-macula RPE and if there is an association in monkeys
that develop AMD versus those that do not.
Commercial Relationships: Peter Gouras, None; L. Ivert, None;
Martha Neuringer, None; Takayuki Nagasaki, None
Support: NIH EY015293
Program Number: 3447 Poster Board Number: C0295
Presentation Time: 11:00 AM–12:45 PM
The role of IL-6 and TNF-alfa in generating the vicious
inflammatory cycle between macrophages and retinal pigment
epithelium in age-related macular degeneration
Jun Yamada1, 2, Kenichi Kimura2, Atsushi Mukai2, Junji Hamuro2,
Shigeru Kinoshita2. 1Ophthalmology, Meiji University of Integrative
Medicine, Kyoto, Japan; 2Ophthalmology, Kyoto Prefectural
University of Medicine, Kyoto, Japan.
Purpose: The interaction between macrophages (Mps) and retinal
pigment epithelium cells (RPE) reportedly plays an important
role in exacerbating age-related macular degeneration (AMD).
Oxidized (Ox) low-density lipoprotein (LDL) contributes to the
pathogenesis of AMD, and the presence of a high concentration of
transforming growth factor beta 2 (TGF-β 2) in the eye may enhance
the development of AMD. The purpose of this present study was to
determine the cytokine network comprising the vicious inflammatory
cycle in order to explore the possibility of developing new strategies
for the prevention of AMD.
Methods: C57BL6-derived primary RPE (mRPE) and adherent
peritoneal Mps (AdPC) were prepared. The mRPE and AdPC were
co-cultured, and the produced monocyte chemotactic protein-1
(MCP-1), interleukin-6 (IL-6), and vascular endothelial growth factor
(VEGF) in supernatants were analyzed by ELIZA assay. LDL (50 mg/
ml), ox-LDL (50 mg/ml), or TGF-β 2 (10 ng/ml) stimulation were
performed to determine the synergistic effect for enhancement of this
cycle. Tumor necrosis factor alpha (TNF-α) or IL-6 was neutralized
to determine the main factor composing their vicious cycle.
Results: The mRPE/AdPC co-culture significantly produced MCP-1
(16.2 ng/ml, > x100), IL-6 (3.1 ng/ml, > x100), and VEGF (1034
pg/ml, > x2) than solely mRPE or AdPC culture. The anti-TNF-α
Ab neutralizing significantly suppressed MCP-1 (7.8 ng/ml), IL-6
(0.51 ng/ml), and VEGF (420 pg/ml) production, and anti-IL-6 Ab,
and anti-gp130 Ab neutralizing significantly suppressed MCP-1
(10.9 and 10.1 ng/ml, respectively) and VEGF (399 and 451 pg/ml,
respectively) production. LDL stimulation did not enhance MCP-1
and VEGF, but did enhance IL-6 production. Ox-LDL enhanced
IL-6 and VEGF production much more than LDL stimulation, but
decreased MCP-1 production. TGF-β also had a synergistic effect on
the production of MCP-1, IL-6, and VEGF from mRPE.
Conclusions: The findings of this study show that TNF-α and IL-6
constitute the vicious inflammatory cycle between RPE and Mps.
Ox-LDL and TGF-β are able to enhance this cycle and might enhance
the development of the early stage of AMD. Since Mps are the main
source of TNF-α production, selective treatment of TNF-α produced
by Mps is a possible new strategy for preventing the development of
AMD.
Commercial Relationships: Jun Yamada, None; Kenichi Kimura,
None; Atsushi Mukai, None; Junji Hamuro, None; Shigeru
Kinoshita, None
Support: The Research Funding for Longevity Sciences (21-17)
(23-29)
Program Number: 3448 Poster Board Number: C0296
Presentation Time: 11:00 AM–12:45 PM
Age-dependent changes in clearance of phagocytosed
photoreceptor outer segments in the Retinal Pigment Epithelium
Pei Xu, Zhen-Yang Zhao, Bo Yu, Bo Long, Jiyang Cai, Yan Chen.
Ophthalmology, UTMB, Galveston, TX.
Purpose: Retinal pigment epithelial (RPE) cells are the most
active phagocytes that are responsible for the removal of daily shed
photoreceptor outer segments (POS). Incomplete degradation of
ingested POS results in gradual accumulation of lipofuscin in RPE
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
phagolysosomes. Increasing lipofuscin with age in RPE is considered
to be one of the causative factors for blindness in patients with agerelated macular degeneration (AMD). Emerging evidence suggests
that maturation of phagosomes depends on molecular components
of the autophagic pathway; and autophagy is closely related to aging
through its housekeeping function in the degradation and recycling
of cytoplasmic components and damaged organelles. The goal of the
study was to determine the functional relationship between autophagy
and phagocytosis; and how aging influences the trafficking and
lysosome-dependent turnover of POS in RPE.
Methods: Porcine POS were purified by sucrose gradient
centrifugation. Cultured human RPE cells were fed with POS for 16
hours and chased for 8 hours to monitor the turnover of phagocytosed
POS. The amount and subcellular localization of rhodopsin were
measured by Western blot and confocal microscopy. To inhibit
autophagic and endolysosomal pathways, cells were treated with
chemical inhibitors, 3-methyladenine (3-MA) or wortmannin, or
transfected with siRNA targeting Rab7, Atg3, Atg5, or LC3. RPE/
choroid flat-mounts from young and aged mice were co-stained with
rhodopsin and Rab7 or LAMP2 to determine the colocalization of
POS with endosome or lysosome, respectively.
Results: Inhibiting autophagy by 3-MA and wortmannin reduced
the degradation of POS. The remaining POS formed aggregates in
lysosomes. Similar effects were achieved by down-regulating Rab7,
Atg3, Atg5, or LC3. Phagocytosed POS were co-localized with the
late endosome marker Rab7 and were more quickly degraded by
young RPE than the aged one. Rab7 level was increased but Atg5 and
Atg3 levels were decreased in aged RPE.
Conclusions: Young RPE had higher capacity in clearance of POS
than aged RPE. Autophagy activity is likely decreased with aging.
Inhibiting the endolysosomal pathway led to reduced degradation
of POS. The results indicate that autophagy is essential for both
self-renewal and clearance of phagocytosed POS in the RPE. The
interactions among autophagy, phagocytosis and endocytic vesicular
trafficking are important for RPE function.
Commercial Relationships: Pei Xu, None; Zhen-Yang Zhao,
None; Bo Yu, None; Bo Long, None; Jiyang Cai, None; Yan Chen,
None
Support: NH Grants EY 019706, EY 021937, International Retinal
Research Foundation and Ted Nash Long Life Foundation
Program Number: 3449 Poster Board Number: C0297
Presentation Time: 11:00 AM–12:45 PM
Inhibition of peroxisome proliferator-activated receptor β/δ
(PPARβ/δ) reduces choroidal angiogenesis
Sara R. Savage, Laura L. Davia, John S. Penn. Pharmacology,
Vanderbilt Univ Medical Center, Nashville, TN.
Purpose: Age-related macular degeneration (AMD) is one
of the leading causes of blindness in the United States. AMD
is characterized by abnormal proliferation of the choroidal
vasculature into the sub-retinal space, hypothetically in response
to VEGF produced by hypoxic retinal pigmented epithelium
(RPE). Peroxisome proliferator-activated receptor β/δ (PPARβ/δ)
is a transcription factor that regulates energy homeostasis, lipid
catabolism, and glucose metabolism. Our lab recently reported
a potent inhibitory effect of PPARβ/δ antagonism on pre-retinal
neovascularization. In the present study, we sought to determine if
PPARβ/δ plays a similar role in sub-retinal neovascularization of the
sort occurring in AMD.
Methods: Primary mouse RPE and ARPE-19 were treated with
increasing concentrations of the PPARβ/δ antagonist GSK0660 in
hypoxia. Secreted VEGF was analyzed by ELISA. The angiogenic
behavior of human choroidal endothelial cells (HCEC) was assessed
by proliferation and tube formation assays in the presence of
GSK0660. Vascular sprouting from mouse choroid explants was also
analyzed in the presence of GSK0660.
Results: Treatment with GSK0660 reduced hypoxia-induced VEGF
production by both primary mouse RPE and ARPE-19 (p<0.025).
Proliferation of HCEC stimulated by both VEGF and 2% FBS
medium was reduced by GSK0660 (p<0.0001), as was tube formation
stimulated by VEGF (p=0.0007). GSK0660 reduced the area of
angiogenic sprouting of choroid explants by 30% (p=0.025).
Conclusions: PPARβ/δ plays a role both upstream and downstream
in choroidal angiogenesis. Inhibition of PPARβ/δ reduces VEGF
secretion from RPE while also inhibiting the angiogenic response of
HCEC to VEGF. Based on these findings, PPARβ/δ may constitute a
rational therapeutic target for treatment of neovascular AMD.
Commercial Relationships: Sara R. Savage, None; Laura L.
Davia, None; John S. Penn, None
Support: NIH EY07533, Unrestricted Grant from Research to
Prevent Blindness
Program Number: 3450 Poster Board Number: C0298
Presentation Time: 11:00 AM–12:45 PM
Comparative analysis of retinal phenotype in Cfh-/-, Cfb-/- and Cfh/Cfb-/- double knock-out mice
Jennifer A. Williams1, John Greenwood1, Judy Latcham2, Peter
S. Adamson2, Stephen E. Moss1. 1Cell Biology, Institute of
Ophthalmology, UCL, London, United Kingdom; 2Ophthiris
Discovery Performance Unit and Department of Laboratory Animal
Science, GlaxoSmithKline, Stevenage, United Kingdom.
Purpose: Single nucleotide polymorphisms in genes of the
alternative complement pathway are linked to susceptibility to
developing age-related macular degeneration. The Y402H mutation
in complement factor H (CFH) predisposes to the disease whereas
the R32Q mutation in complement factor B (CFB) is protective. The
purpose of this study was to use Cfh-/- and Cfb-/- mice, together with
the Cfh-/-Cfb-/- double knock-out mouse, to investigate the functional
interplay between CFH and CFB in the context of retinal phenotype.
Methods: Retinas were isolated from wild type (WT), Cfh-/-, Cfb-/-,
Cfh-/-Cfb-/- and Cfh-/-Cfb+/- mice. Retinal morphology was assessed
using toluidine blue stained semi-thin sections. Fixed frozen sections
were stained with antibodies to C3 and the C3 breakdown products
C3b, iC3b and C3c. Neuroretinal whole mounts were stained with
collagen IV to visualise the retinal vessels and F4/80 for microglia/
macrophages.
Results: WT mice showed C3 staining in the retinal vasculature and
along the basal surface of the retinal pigment epithelium (RPE) where
C3 breakdown products were also detected. Cfb-/- mice also exhibited
C3 staining in the retinal vasculature but less C3 staining along the
basal surface of the RPE compared to WT and no accumulation of C3
breakdown products. Loss of CFH caused secondary depletion of C3
from the serum, and in the retina staining for C3 and its breakdown
products was absent along the basal surface of the RPE. Deletion of
both Cfb and Cfh in Cfh-/-Cfb-/- double knock-out mice restored C3 to
levels similar to those observed in WT mice, however this reversal
of phenotype was not observed in Cfh-/-Cfb+/- mice. Cfh-/-Cfb-/- mice
differed from WT mice in having very low C3 breakdown products at
the basal surface of the RPE. Overall the retinal morphology, retinal
vasculature and number of infiltrating macrophages did not appear
different across the 5 different genotypes.
Conclusions: C3 accumulates at the basal surface of the RPE in WT,
Cfb-/- and Cfh-/-Cfb-/- mice, but is absent in Cfh-/- and Cfh-/-Cfb+/- mice,
consistent with its consumption in the serum of mice lacking CFH
when CFB is present. The partial or complete absence of CFB in Cfh/Cfb+/- and Cfb-/- mice was associated with a lack of C3 breakdown
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
products at the basal RPE suggesting that the C3b, iC3b and C3c
observed in WT mice are correlates of an active and functionally
normal alternative pathway.
Commercial Relationships: Jennifer A. Williams,
GlaxoSmithKline (F); John Greenwood, None; Judy Latcham,
GlaxoSmithKline (E); Peter S. Adamson, GlaxoSmithKline (E);
Stephen E. Moss, None
Support: Wellcome Trust Grant 090669
Program Number: 3451 Poster Board Number: C0299
Presentation Time: 11:00 AM–12:45 PM
Generation of iPSC derived vascular endothelial cells for the
treatment of AMD
Allison E. Songstad1, Cathyrn M. Cranston1, Miles J. Flamme-Wiese1,
Edwin M. Stone1, 2, Robert Mullins1, Budd A. Tucker1. 1Stephen A.
Wynn Institute for Vision Research, Carver College of Medicine,
Department of Ophthalmology and Visual Sciences, University of
Iowa, Iowa City, IA; 2Howard Hughes Medical Institute, University
of Iowa, Iowa City, IA.
Purpose: Age-related macular degeneration (AMD), the most
common cause of incurable blindness in the western world, is
characterized by the dysfunction and eventual death of choroidal
endothelial, retinal pigment epithelial (RPE), and photoreceptor
cells. Stem-cell based treatment strategies designed to replace both
photoreceptor and RPE cells are currently a major scientific focus,
and success of these approaches will undoubtedly also require
replacement of choroidal vasculature. The purpose of this study
was to generate a Tie2-GFP iPSC reporter line to develop efficient
vascular endothelial cell differentiation and transplantation protocols.
Methods: Dermal fibroblasts from the Tie2-GFP mouse (carrying
a GFP reporter gene under the control of the endothelial cellspecific Tie2 promoter) were isolated and reprogrammed into
induced pluripotent stem cells (iPSCs) via viral transduction of the
transcription factors Oct4, Sox2, Klf4, and c-Myc. iPSC potency was
characterized via RT-PCR, immunocytochemistry, western blotting
and teratoma assays. Tie2-GFP iPSCs were differentiated into
embryoid bodies using both a co-culture method as well as a modified
version of our previously developed step-wise differentiation
protocol. Translatability of developed protocols was tested using
human control iPSCs.
Results: Tie2-GFP iPSCs, generated from murine fibroblasts,
expressed the pluripotency markers Nanog, Oct4, Sox2, Klf4,
and c-Myc as determined by RT-PCR, western blot analysis and
immunocytochemistry. Following both embryoid body formation
and transplantation into immune compromised SCID mice,
undifferentiated cells formed tissues specific to each of the 3
embryonic germ layers. Pluripotent iPSCs subjected to co-culture
with the monkey choroidal endothelial cell line RF/6A differentiated
into vascular endothelial cells that expressed the choroidal endothelial
markers VE-cadherin and CD34 and were morphologically
indistinguishable from native choroidal endothelial cells.
Conclusions: We have successfully generated Tie2-GFP iPSCs
and used them to develop vascular endothelial cell differentiation
protocols. This work has set the stage for future studies focused
on investigation of disease pathophysiology and endothelial cell
replacement.
Commercial Relationships: Allison E. Songstad, None; Cathyrn
M. Cranston, None; Miles J. Flamme-Wiese, None; Edwin M.
Stone, None; Robert Mullins, None; Budd A. Tucker, None
Support: Elmer & Sylvia Sramek Charitable Foundation, NIH
DP2OD007483
Program Number: 3452 Poster Board Number: C0300
Presentation Time: 11:00 AM–12:45 PM
Induction of vascular endothelial growth factor and vascular
endothelial growth factor receptor after repeated bevicizumab
treatment in human umbilical vein endothelial cells
Ji Eun E. Lee1, 2, Hye Shin Jeon1, Jin Young Kim1, Jae Ho Jung1, 3,
Dong Hoon Shin4, Min Kyu Shin1. 1Ophthalmology, Pusan National
Univesity, Busan, Republic of Korea; 2Medical Research Institute,
Pusan National University Hospital, Busan, Republic of Korea;
3
Convergent Biomedical Research Institute, Yangsan Pusan National
University Hospital, Busan, Republic of Korea; 4Pathology, Pusan
National Univesity, Busan, Republic of Korea.
Purpose: To investigate expression of vascular endothelial growth
factor (VEGF) and VEGF receptor (VEGFR) in hypoxic human
umbilical vein endothelial cells (HUVECs) after repeated anti-VEGF
treatment.
Methods: HUVECs were incubated under the hypoxic environment
with single or repeated treatment of bevacizumab at 1.0 or 2.5 mg/
ml concentration. Treatment with identical volume of excipient was
served as control. Expression of VEGF and phosphorylated VEGFR2
were assessed by enzyme-linked immuno-sorbant assay (ELISA) and
western blot. Cytotoxicity and cell proliferation were also evaluated
by MTT assay and immunocytochemistry of Ki-67.
Results: VEGF secretion was significantly higher after the single
treatment than control and the repeated treatment groups (P < 0.05).
VEGFR2 expression increased significantly after both a single and
repeated bevacizumab treatments than control (P < 0.05). VEGFR2
expression was higher after repeated treatment than a single
treatment, and in lower concentration of bevacizumab than higher
concentration (P < 0.05). Cytotoxicity or inhibition of proliferation by
bevacizumab was not observed.
Conclusions: A single treatment of anti-VEGF induced secretion
of VEGF from HUVECs, whereas repeated treatments increased
expression of VEGFR2. Elevated receptor expression may increase
responsiveness to VEGF, and would be one of the mechanisms for
losing efficacy of anti-VEGF treatment in vivo.
Commercial Relationships: Ji Eun E. Lee, None; Hye Shin Jeon,
None; Jin Young Kim, None; Jae Ho Jung, None; Dong Hoon
Shin, None; Min Kyu Shin, None
Program Number: 3453 Poster Board Number: C0301
Presentation Time: 11:00 AM–12:45 PM
Regulation of Exosomes by Autophagy in Retinal Pigment
Epithelium: an Implication of the Formation of Drusen
Jeehyun Yoon1, Ae Jin Choi1, Hyunjung J. Lim2, Hyewon Chung1.
1
Ophthalmology, Konkuk University School of Medicine, Seoul,
Republic of Korea; 2Biomedical Science & Technology, Konkuk
University, Seoul, Republic of Korea.
Purpose: The formation of drusen has been considered a risk factor
for developing age-related macular degeneration (AMD). Although
some of the molecular components of drusen are known, there is
little understanding of the cell biology that leads to the formation
of drusen. Previous research found that drusen in AMD donor eyes
contain markers for autophagy and exosomes. Here we investigated
the relationship between the regulation of autophagy and the release
of exosomes in the retinal pigment epithelium (RPE).
Methods: ARPE-19 cells were transfected with GFP-LC3
plasmid DNA and labeled with DII (1,10-dioctadecyl-3,3,3’3’tetramethylindocarbocyanine perchlorate) dye (lipophilic tracers
for exosomes staining). ARPE-19 cells were exposed to oxidative
stress (H2O2, 200 mM) with co-treatment of 3-methyladenine (3ma) or siRNA against Atg5 as inhibition of autophagy. Rapamycin
or torin was used as stimulation of autophagy. Exosome pellets
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
from conditioned media (CM) from ARPE-19 cells exposed to 200
mM H2O2 for 24 h were obtained with ExoQuickTM Exosome
Precipitation Solution. Immunoblotting for multivesicular and late
endosomal membrane marker proteins (Tsg101 and CD63) were
performed. Atg7flox/flox;Best1-Cre mice were exposed to bright
light. Immunoblotting and immunoflourescence (IF) of the RPE and
retina were carried out.
Results: GFP-LC3 presented a punctuate distribution and partially
co-localized with DII labeled exosomes in ARPE-19 cells exposed
to H2O2. Inhibition of autophagy with 3-ma or siRNA against Atg5
increased the expression levels of Tsg101 and CD63 in exosomes
derived from ARPE-19 cells exposed to H2O2, whereas decreased
expression levels of these proteins were found in ARPE-19 cells
exposed to rapamycin or torin by immunoblotting and IF. Tsg101 and
CD63 expression were increased in the RPE and retina from bright
light-exposed Atg7flox/flox;Best1-Cre mice compared to control
mice by immunoblotting and IF.
Conclusions: The present study indicates that increasing autophagy
activity in a discretely controlled manner may decrease exocytotic
activity of the RPE, thus preventing the formation of drusen in
patients with AMD.
Commercial Relationships: Jeehyun Yoon, None; Ae Jin Choi,
None; Hyunjung J. Lim, None; Hyewon Chung, None
Support: National Research Foundation of Korea funded by the
Ministry of Science, ICT & Future Planning (2012M3A9B2028333
and 2012R1A1A11012171)
Program Number: 3454 Poster Board Number: C0302
Presentation Time: 11:00 AM–12:45 PM
Aberrant Cell and Basement Membrane Architecture Contribute
to Sidestream Smoke-induced Choroidal Endothelial Dysfunction
Xiao Yang, Harry Scott, Soroush Ardekani, Kaustabh Ghosh.
Bioengineering, University of California, Riverside, Riverside, CA.
Purpose: Environmental tobacco smoke (ETS) is widely regarded as
a major modifiable risk factor for age-related macular degeneration
(AMD). Yet, precisely how it exerts its pathological effects is poorly
understood. Since early-stage AMD is characterized by choroidal
capillary loss, this study examined the effect of sidestream smoke
(SS), the major component of ETS, on the viability of choroidal
endothelial cells (EC), with an emphasis on the role of aberrant cell
and basement membrane (BM) architecture in mediating SS-induced
response.
Methods: Chorioretinal ECs (RF/6A) were treated with SS and
cell viability and architecture were analyzed by colorimetric assay
and actin cytoskeletal organization, respectively. The structure of
RF/6A EC-secreted BM was examined by immunofluorescence for
collagen IV and immunoblotting for lysyl oxidase (LOX), a collagencrosslinking enzyme. Finally, fresh RF/6A ECs were cultured on
decellularized SS-treated BM to evaluate its active role in EC
dysfunction.
Results: RF/6A EC viability decreased progressively with increasing
SS dose, which correlated strongly with a significant decline in
actin cytoskeleton-dependent EC spreading. SS also caused marked
disruption of RF/6A EC-secreted BM that was accompanied by
suppression of LOX expression. Further, fresh, non SS-treated RF/6A
ECs exhibited a significant loss in viability and actin cytoskeletal
organization when cultured on SS-treated corrupt BM.
Conclusions: These findings indicate that aberrant physical cues in
the form of EC and BM architecture likely play an important role in
choriocapillaris dysfunction seen in SS-associated early AMD, and
implicate choroidal BM as a potential target for AMD management
strategies.
Commercial Relationships: Xiao Yang, None; Harry Scott, None;
Soroush Ardekani, None; Kaustabh Ghosh, None
Program Number: 3455 Poster Board Number: C0303
Presentation Time: 11:00 AM–12:45 PM
Non-vascular effects of VEGF in a model of age-related macular
degeneration
Michael R. Kozlowski. Optometry, Midwestern Univ/Arizona Coll of
Optom, Glendale, AZ.
Purpose: The purpose of this work is to assess whether vascular
endothelial growth factor (VEGF) has effects in addition to
promoting blood vessel growth that could contribute to the
development of age-related macular degeneration (AMD). Previous
studies have found that VEGF can weaken the connections between
retinal pigment epithelial (RPE) cells, in vitro. The potential
relevance of this phenomenon to the development of AMD is
partially addressed in this study.
Methods: ARPE-19 cells were grown in 6-well plates on membranecontaining inserts to model the native RPE cell layer. Cells from
subcultures at a lower population doubling level (PDL; 23 to 35) and
at a higher PDL (56 to 96) were compared. After being grown on the
inserts for two weeks, the trans-epithelial electrical resistance (TEER)
of the cell layers was measured. Once baseline measurements were
obtained, the cells were treated with either VEGF (20ng) or its
vehicle (PBS with 0.1% BSA), and TEER was again measured.
One week later, the cells, still growing on the inserts, were stained
for senescence-associated β-galactosidase (SABG) activity as an
indicator of cell senescence.
Results: The baseline TEER values did not differ significantly
between lower and higher PDL cells (35 ohms-cm2 and 34 ohmscm2, respectively; t-Test, p = .74). In contrast, the effect of VEGF
was much greater on the higher PDL cells than on the lower PDL
cells (22% decrease in TEER vs. 6% decrease; t-Test, p > .005).
SABG staining was present in both the high and low PDL ARPE-19
cells, but the pattern of the staining was more darkly mottled in the
higher PDL cells.
Conclusions: In addition to promoting vascular growth in the retina,
VEGF can also decrease the TEER of RPE cells. Since TEER is an
indicator of the quality of the tight junctions between the ARPE19 cells, this effect could interfere with the functioning of the RPE
layer in situ and might be permissive to the entry of choroidal
neovascularization into the retina. Here we confirm the disruptive
effect of VEGF on RPE cell layer integrity and show that it is greater
in higher PDL cells than in lower PDL cells. The pattern of SABG
staining of the higher PDL cells suggests that they are approaching
senescence. A greater disruptive effect of VEGF on senescent RPE
cells, together with the proposed occurrence of RPE senescence
in AMD, suggests that his additional action of VEGF could also
contribute to the pathology of AMD.
Commercial Relationships: Michael R. Kozlowski, None
Program Number: 3456 Poster Board Number: C0304
Presentation Time: 11:00 AM–12:45 PM
Increased expression of mitochondrial glutaredoxin 2 (Grx2)
protects human retinal pigment epithelial cells from oxidative
stress-induced cell death
Hongli Wu1, 2, Xiaobin Liu1, Jamieson Jann3. 1Pharmaceutical
Sciences, University of North Texas Health and Science Center, Fort
Worth, TX; 2Institute for Cancer Research, University of North Texas
Health and Science Center, Fort Worth, TX; 3University of Georgia,
Athens, GA.
Purpose: Glutaredoxin 2 (Grx2) is a mitochondrial isozyme of
thioltransferase in the oxidoreductase family that is a key regulator of
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
redox homeostasis through dethiolation of protein-glutathione mixed
disulfides (PSSG). Grx2 is expressed in various human and rodent
tissues, including the retina, where its functional role is unknown.
The purpose of this study is to investigate the presence of Grx2 in
human retinal pigment epithelial cells and evaluate its potential antiapoptotic function.
Methods: Human retinal pigment epithelial (ARPE-19) cells were
transfected with either a Grx2-containing plasmid or an empty
vector. Normal ARPE-19 cells and transfected cells were treated
with or without 200 mM H2O2for 24 h. Grx2 protein expression
was detected by Western blots. Grx2 enzyme activity was assayed
by spectrophotometric method in the mitochondrial fraction. Cell
viability was measured by a colorimetric assay with WST8. The
morphology of nuclear chromatin was assessed by staining with
Hoechst 33342. Apoptosis was quantitatively analyzed by flow
cytometry. Mitochondrial membrane potential was evaluated by using
JC-1 assay kit. The level of mitochondrial PSSG was measured by
immunoblotting using anti-PSSG antibody.
Results: Grx2 protein level and enzyme activity in Grx2 transfected
cells were significantly increased as compared to non-transfected
and vector transfected cells. Grx2 overexpression protected
ARPE-19 cells from H2O2-induced cell viability loss. Assessment
of apoptosis indicated that cells transfected with Grx2 were
relatively more resistant to H2O2 with fewer cells undergoing
apoptosis as compared to vector control or non-transfected cells.
Furthermore, Grx2 overexpressed cells were more resistant to the
loss of mitochondrial membrane potential induced by H2O2. PSSG
accumulation in mitochondria was also dramatically attenuated by
Grx2 overexpression.
Conclusions: Grx2 can protect human retinal pigment epithelial cells
against H2O2-induced cell death. The mechanism of this protection
is likely associated with its ability to prevent lethal accumulation of
PSSG in mitochondria.
Commercial Relationships: Hongli Wu, None; Xiaobin Liu, None;
Jamieson Jann, None
Support: UNTHSC Start-Up Grant; UNTHSC Intramural Grant:
RI6074 (ICR)
Program Number: 3457 Poster Board Number: C0305
Presentation Time: 11:00 AM–12:45 PM
Histone Deacetylase Expression and Inhibition in Age Related
Macular Degeneration
Mark E. Kleinman1, Andre Berner1, Kablian Mohan1, Ding-Yuan
Lou1, Jennifer Brown1, Justin West1, Rei Kono1, Ilene Sugino3, Marco
Zarbin3, Jayakrishna Ambati1, 2. 1Ophthalmology & Visual Sci, Univ
of Kentucky, Lexington, KY; 2Physiology, University of Kentucky,
Lexington, KY; 3Institute of Ophthalmology & Visual Science, New
Jersey Medical School, Newark, NJ.
Purpose: The role of histone deacetylases (HDACs) in the postnatal
control of cell survival and gene expression remains unclear. Reports
of improved retinal function in retinitis pigmentosa and age-related
macular degeneration (AMD) led us to investigate the effects of
specific HDAC inhibition on the retinal pigment epithelium (RPE) in
vitro and in vivo and analyze HDAC expression in situ.
Methods: Eyes with dry AMD and controls (n=3) were examined
by immunohistochemistry for HDAC expression. Total RNA
was harvested from RPE/choroid (n=3) and interrogated with
Nanostring™ and real-time PCR. Wild-type (Wt) C57BL/6J mouse
eyes (n=3) were evaluated with HDAC immunofluorescence. Mice
(n=6) were imaged by fundus photography 1 week after intravitreous
injection of HDAC inhibitors (suberoylanilide hydroxamic acid
(SAHA), trichostatin-A (TSA)) and vehicle (DMSO). ZO-1 immunostained RPE/choroid flatmounts (n=3) were analyzed by fluorescent
microscopy. RPE/choroid tissue (n=3) was harvested at 48 hours and
analyzed by targeted PCR arrays (Taqman®). Primary human RPE
(hRPE) isolates (n=6, individual donors) were treated with various
HDAC inhibitors. Short-interfering RNAs targeting HDACs were
also evaluated in vitro. Cell viability assays and targeted PCR arrays
were performed. Statistical analyses were performed with nonparametric tests (Mann-Whitney U) with significance determined at
P<0.05.
Results: HDAC-2 was down-regulated in eyes with AMD compared
to controls (-1.45±0.23, P<0.05) and expressed in mouse RPE and
in hRPE. Intravitreous injection of Class I/II HDAC inhibitors
led to focal atrophy of RPE as observed on fundus imaging and
ZO-1 immunofluorescence. HDAC inhibition in hRPE resulted in
upregulated cell death and wide-spread changes in the cytokine
expression profile. Similar disruptions in pro-inflammatory gene
expression were observed in the mouse model and with specific
targeting of HDAC-2 via RNA interference in vitro.
Conclusions: We found that Class I/II HDAC inhibition resulted
in increased RPE cell death and inflammatory cytokine expression.
Down-regulation of HDAC-2 was observed in RPE from eyes with
AMD suggesting a role for this important epigenetic mechanism in
the pathogenesis of this disease.
Commercial Relationships: Mark E. Kleinman, None; Andre
Berner, None; Kablian Mohan, None; Ding-Yuan Lou, None;
Jennifer Brown, None; Justin West, None; Rei Kono, None; Ilene
Sugino, None; Marco Zarbin, None; Jayakrishna Ambati, None
Support: National Eye Institute 1K08EY021757, Foundation
Fighting Blindness, Research to Prevent Blindness, and American
Federation for Aging Research
Program Number: 3458 Poster Board Number: C0306
Presentation Time: 11:00 AM–12:45 PM
Liver X Receptor signaling pathways and age-related macular
degeneration
Goldis Malek1, Mayur Choudhary2, Edwin Meade2, Erik Nelson3,
Donald McDonnell3. 1Ophthalmology and Pathology, Duke
University, Durham, NC; 2Ophthalmology, Duke University, Durham,
NC; 3Pharmacology and Cancer Biology, Duke University, Durham,
NC.
Purpose: Age-related macular degeneration (AMD) is the leading
cause of vision loss in the elderly. The early dry sub-type is
characterized by accumulation of cholesterol-rich deposits below the
retinal pigment epithelium (sub-RPE). Recent studies have identified
several genes associated with AMD risk, in the high-density
lipoprotein cholesterol pathway, including the ATP binding cassette
transporter 1 (ABCA1). Liver X receptors (LXRs) are nuclear
receptors that act as cholesterol sensors regulating not only lipidmetabolism and genes associated with reverse cholesterol transport
including ABCA1, but also inflammation. Given the importance of
lipid metabolism and inflammation in the pathogenesis of early dry
AMD, we investigated the impact of LXR activation in RPE and
choroidal endothelial cells, cells affected in AMD, and examined the
eye phenotype of aged LXR double knockout mice.
Methods: LXR expression and activity were determined in ARPE19
and RF/6A endothelial cells treated with endogenous lipids, synthetic
agonists and antagonists of the receptor using qPCR, western
blot and luciferase assays. Expression of inflammatory and lipidprocessing genes were measured in cells following treatment with
agonists and antagonists and following knockdown of the receptor.
In vivo examination of LXR knockout mice included evaluating the
morphology of retina/RPE/choroid and histochemical staining for
lipids.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: We found that the LXRs are present and the signaling
pathway can be activated using synthetic agonists in human RPE
and endothelial cells. Inflammatory genes including MCP-1, TLR3
and 4 and lipid processing genes ABCA1 and SREBP are regulated
by LXR. In vivo, the absence of LXR in 9 month old mice resulted
in accumulation of lipid-rich, oil red o positive, sub-RPE deposits
underlying approximately 80% the length of the RPE, along with
lipid droplets within the RPE and Bruch’s membrane.
Conclusions: These findings support the physiological importance of
LXR in lipid metabolism and inflammation in the RPE and choroidal
endothelial cells and indicate that the LXR signaling pathway may
be a potential therapeutic target against cholesterol-rich deposit
formation.
Commercial Relationships: Goldis Malek, None; Mayur
Choudhary, None; Edwin Meade, None; Erik Nelson, None;
Donald McDonnell, None
Support: NIH Grant EY02868, P30 EY005722, Research to Prevent
Blindness Special Scholar Award, Research to Prevent Blindness
Core Grant.
Program Number: 3459 Poster Board Number: C0307
Presentation Time: 11:00 AM–12:45 PM
AMD-compatible lysosomal changes in Rab38-deficient mouse
model
Tanya Tolmachova1, Diogo A. Feleciano1, Silene Wavre-Shapton1,
2
, Martin J. Evans1, Clare Futter2, Miguel C. Seabra1. 1Molecular
Medicine, NHLI, Imperial College London, London, United
Kingdom; 2Division of Cell Biology, Institute of Ophthalmology,
University College London, London, United Kingdom.
Purpose: Accumulation of lipofuscin deposits, lysosome
disregulation and inflammation are hallmarks of age-related macular
degeneration (AMD). Retinal pigment epithelium (RPE) plays a
major role in AMD pathology. Age-related changes in the normal
RPE and AMD mechanisms are not fully understood. Rab38 is a
small GTPase that is important for biogenesis of lysosome-related
organelles. The purpose of the study was to assess lysosomal
function in Rab38-deficient (chocolate) mouse, including expression
of lysosomal proteins LAMP-1 and cathepsins, accumulation of
autofluorescent deposits and inflammatory cytokine upregulation.
Methods: RPE cells were isolated from the eyes of young (3-4
months) and old (12-15 months) C57Bl6 and Rab38-deficient
(chocolate) mice. LAMP-1 and cathepsin gene and protein expression
were analysed in freshly isolated RPE by real-time PCR, FACS
analysis and Western blotting. Cathepsin activity was measured in
freshly isolated RPE cells and primary RPE cultures established from
3-week old mouse eyes. IL-6 and MCP-1 cytokine secretion was
measured by ELISA. Transmission electron microscopy was used.
Results: We detected significant downregulation of LAMP-1 gene
and protein expression in chocolate mice in comparison to C57Bl6
animals. While LAMP-1 expression significantly increased with
age in C57Bl6 animals, in chocolate mice there was no significant
difference between young and old age groups. Activity of cathepsin
H was increased in the lysates of freshly isolated chocolate RPE cells
as well as in chocolate RPE cultures. Activity of cathepsin D was
also increased in chocolate RPE cell culture lysates. Furthermore,
maturation of cathepsin D was delayed in chocolate RPE. We
detected accumulation of autofluorescent material in RPE cells
isolated from old chocolate mice in comparison to old C57Bl6
and young chocolate animals. Electron microscopy identified
abnormalities in old chocolate mice such as cytoplasmic deposits,
autophagosomes, elongated mitochondria. This was accompanied
by significant upregulation of IL-6 in freshly isolated chocolate RPE
cells and RPE cultures.
Conclusions: We identified lysosomal defects in Rab38deficient (chocolate) mice, which correlated with accumulation
of autofluorescent material with age and upregulation of
proinflammatory cytokine IL-6. Our results suggest that Rab38 is
important for lysosomal function in aged RPE and may play a role in
AMD pathogenesis.
Commercial Relationships: Tanya Tolmachova, None; Diogo A.
Feleciano, None; Silene Wavre-Shapton, None; Martin J. Evans,
None; Clare Futter, None; Miguel C. Seabra, None
Support: Wellcome Trust
Program Number: 3460 Poster Board Number: C0308
Presentation Time: 11:00 AM–12:45 PM
Expression of ADAMs (A Disintegrin and Metalloproteinase)
10 and 17 in Human Eyes and in Experimental Models of Age
Related Macular Degeneration (AMD)
Chris Or1, Jun Wang2, Luba Kojic1, William Jia3, Max S. Cynader1,
Jing Z. Cui1, Joanne A. Matsubara1. 1Department of Ophthalmology
& Visual Sciences, University of British Columbia, Vancouver,
BC, Canada; 2Department of Anatomy and Embryology, Peking
University Health Science Center, Beijing, China; 3Department of
Surgery, University of British Columbia, Vancouver, BC, Canada.
Purpose: ADAMs are crucial mediators in the proteolytic release
of extracellular domains from membrane bound precursors. Recent
studies support a role for ADAMs in mediating inflammation by
the shedding of pro-inflammatory mediators. Chronic inflammation
contributes to the development of AMD, but the role of ADAM10
and ADAM17 in this eye disease is unknown. We report the
expression of ADAM10&17 in post-mortem eyes and in experimental
models of AMD in which amyloid beta (Aβ), a drusen component,
promotes pro-inflammatory events.
Methods: Young (<56) and old (>71) postmortem eyes were
fixed and processed with standard methods. Long Evans rats
underwent intravitreal injections of Aβ (1-40, 1.4 μg/μL) and eyes
at Day 1, 4, 14 and 49 post-injection, were fixed and processed.
Antibodies against ADAM10&17 were used at 1:200 and their
immunohistochemical expression visualized. Next, mRNA
abundances of ADAM10&17 were analyzed in ARPE19 or fetal
hRPE by qRT-PCR. ADAM10&17 mRNA expression levels in fetal
hRPE cells were also analyzed using qRT-PCR following Aβ (1μM)
stimulation for 24h.
Results: Moderate expression of ADAM10&17 was detected in RPE
of old eyes and mild expression of ADAM 17 was noted in the RPE
of young eyes. Older eyes with drusen demonstrated expression of
ADAM10&17 within the drusenoid deposits. Choroidal macrophages
also expressed ADAM10 in the old eyes. In vivo studies demonstrated
that rodent eyes expressed ADAM10 at D1 and D4 and ADAM17 at
D1 post-intravitreal injection of Aβ. In vitro studies demonstrated that
transcriptional expression of ADAM17 was significantly increased by
20% (p=0.02), and a trend for increased ADAM10, in RPE following
Aβ stimulation, The ARPE19 cell line and primary hRPE cells
showed similar levels of mRNA abundance, with higher levels of
ADAM17 compared to ADAM10.
Conclusions: Older postmortem eyes expressed higher levels
of ADAM10&17, mostly in RPE and drusenoid deposits. The
significance of this is currently unknown but the link between
ADAMs and inflammation suggest an age-related relationship in
ADAM expression that may contribute to AMD pathogenesis.
Furthermore, the in vivo and in vitro results suggest that increased
ADAM expression could be a result of Aβ stimulation, given a
possible role of ADAMs in the removal of Aβ. Future work will
allow us to clarify the role of ADAMs in the outer retina.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Chris Or, None; Jun Wang, None;
Luba Kojic, None; William Jia, None; Max S. Cynader, None;
Jing Z. Cui, None; Joanne A. Matsubara, None
Support: CIHR MOP 97806, CIHR MOP-126195 and VGH+UBC
Hospital Foundation
Program Number: 3461 Poster Board Number: C0309
Presentation Time: 11:00 AM–12:45 PM
XIAP’s Association with NLRP3 Inflammasome in Human Donor
Eyes
Jing Z. Cui, Jiangyuan Gao, Eleanor To, Joanne A. Matsubara.
Ophthalmology & Visual Sciences, University of British Columbia,
Vancouver, BC, Canada.
Purpose: X-linked inhibitor of apoptosis (XIAP) is a potent antiapoptotic factor, whose neuroprotective role has been demonstrated
in several retinal degeneration animal models. However, a number
of recent studies have expanded our knowledge of XIAP’s biological
functions by showing its involvement in cell signaling and immune
response regulation, but not in retinal pigment epithelium (RPE)
cells. Previously, we showed an age-dependent decrease of
XIAP protein in RPE tissues from normal human donor eyes. We
also showed a surge of XIAP immunoreactivity in RPE tissues
from patients with geographic atrophy (GA) (Gao et al IOVS
2013;54;E-abstract 160). In this study, we aim to understand XIAP’s
potential role in regulating NLRP3 inflammasome activity, a key
immune response underlying GA.
Methods: The same set of donor eyes from previous XIAP study
were used here. The eyes were categorized into three groups, a
normal Young group (< 57 years of age), a normal Old group (>
70 years of age), and a GA group (averaging 83.7 years of age).
Antibodies against phosphorylated NF-κB p65 subunit, NLRP3
and cleaved Caspase-1 p10 subunit were applied to assess the
markers’ immunoreactivity in RPE layer. Nuclear immunolabeling
of phosphorylated NF-κB p65 in all groups were counted and
analyzed by One-Way ANOVA. Cytoplasmic labeling of NLRP3 and
cleaved Caspase-1 p10 were analyzed semi-quantitatively following
established protocols.
Results: In contrast to XIAP’s age-dependent decrease, the
immunoreactivity of phosphorylated NF-κB p65 and NLRP3 in
normal RPE tissues increased with age. Moreover, parallel to the
elevated XIAP level in the GA group, there was an augment in
cleaved Caspase-1 p10 subunit and NLRP3 immunoreactivity in the
GA group compared to the Old group. However, the phosphorylated
NF-κB p65 level was reduced in the GA group compared to the Old
group.
Conclusions: In this study, we correlated the changes in
immunoreactivity of XIAP and markers of the NLRP3 inflammasome
pathway using the same set of donor eyes. We showed an inverse
relationship between XIAP and NF-κB activation, an inflammasome
priming signal. Intriguingly, our findings also indicated a parallel
correlation between XIAP and two inflammasome components,
NLRP3 and Caspase-1 p10, suggesting XIAP’s potential involvement
in inflammasome regulation. Future experiments using cell culture
and animal models will provide mechanistic insights to fully
understand such interaction.
Commercial Relationships: Jing Z. Cui, None; Jiangyuan Gao,
None; Eleanor To, None; Joanne A. Matsubara, None
Support: CIHR MOP 97806 and VGH+UBC Hospital Foundation
Program Number: 3462 Poster Board Number: C0310
Presentation Time: 11:00 AM–12:45 PM
MOLECULAR AND FUNCTIONAL CHANGES IN THE
RETINA OF MICE DEFICIENT IN COMPLEMENT FACTOR
H AND APOLIPOPROTEIN E
Maria Hernandez1, Laura Garcia-Garcia1, Sergio Recalde1, Patricia
Fernandez1, Maite Moreno-Orduña1, Laura Fernandez Sanchez2,
Laura Ramirez3, Pedro de la Villa3, Nicolas Cuenca2, Alfredo GarciaLayana1, 4. 1University of Navarra, Pamplona, Spain; 2University
of Alicante, 33001, Spain; 3University of Alcala, 28871, Spain;
4
Opthalmology, Clinica Universidad de Navarra, Pamplona, Spain.
Purpose: The Apolipoprotein E deficient mouse (apoE-/-), an
experimental model of genetic hypercholesterolemia, shows some
retinal alterations. Complement factor H (cfh) is involved in
inflammatory processes associated with retinal degeneration such as
Age-related Macular Degeneration (AMD). We aimed to investigate
the effect of the absence of CFH and apoE genes in the expression
of specific molecular markers and to study the electrophysiological
activity in mouse retinas.
Methods: Twelve-month old wild type (WT), cfh-/-/apoE-/- (DKO)
and cfh+/-/apoE-/- (EKHH) mice were divided into 3-5 animals
per group. Electroretinograms (ERGs) were measured, the a-wave
was used as an index of outer retinal function, whereas the b-wave,
oscillatory potentials (OPs) and scotopic threshold response (STR)
were used as indices of inner retinal function. The animals were
euthanatized and enucleated. Retinal pigment epithelium (RPE) were
subjected to immunofluorescence using Zonula ocludens-1 (ZO-1)
antibody. Cryostat sections of the other eye were immunostaining to
detect outer and inner plexiform layer (OPL, IPL) (synaptophysin,
bassoon), horizontal cells (PKC, Calbindin) and inflammation-related
molecules (active caspase-1 and C5b9). Photographs were taken by
confocal microscopy.
Results: ZO-1 flat mount images showed alterations of intercellular
junctions between epithelial cells in the EKHH and DKO vs. WT
mice. The number of rows of photoreceptor nuclei were similar in
EKHH and WT retinas, although we observed in DKO a reduction
of the thickness in the outer nuclear layer. The dendrites of the
horizontal cells in EKHH retinas were numerous. Moreover, presence
of caspase-1 positive cells and C5b9 immunoreactivity were observed
in the ONL and INL in EKHH retinas. ERG recordings from DKO
mice showed their a-, b-wave and STR amplitudes were reduced
when compared to WT. There was no significant changes in the ERGs
of the EKHH mice.
Conclusions: ApoE and cfh genes seem to be related to the
maintenance of RPE cytoarchitecture. Moreover, they could be
involved in direct and indirect contact of photoreceptors with some
retinal cells. The morphological changes in DKO mice, correspond
to alteratios in functional retina. In addition, some inflammatory
pathways could be active in transgenic mice lacking apoE and cfh.
Commercial Relationships: Maria Hernandez, None; Laura
Garcia-Garcia, None; Sergio Recalde, None; Patricia Fernandez,
None; Maite Moreno-Orduña, None; Laura Fernandez Sanchez,
None; Laura Ramirez, None; Pedro de la Villa, None; Nicolas
Cuenca, None; Alfredo Garcia-Layana, None
Support: RETICS RD07/0062, RETIC RD12/0034/0010, Ministerio
de Ciencia e Innovación and RD12/0034, Ministerio de Economia y
Competitividad. Spain. LGG received a predoctoral grant from the
Asociación de Amigos de la Universidad de Navarra.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 3463 Poster Board Number: C0311
Presentation Time: 11:00 AM–12:45 PM
RNA sequencing reveals altered gene expression signatures in
complement challenged primate choroidal endothelial cells
S Scott Whitmore1, 2, Shemin Zeng1, 2, Megan J. Riker1, 2, Edwin M.
Stone1, 2, Budd A. Tucker1, 2, Todd E. Scheetz1, 2, Robert F. Mullins1,
2 1
. Stephen A. Wynn Institute for Vision Research, The University
of Iowa, Iowa City, IA; 2Ophthalmology & Visual Sciences, The
University of Iowa, Iowa City, IA.
Purpose: We hypothesized that activation of the complement
membrane attack complex (MAC) within the choroid may alter
homeostatic gene expression in endothelial cells, creating an
environment conducive to AMD. To test this hypothesis, we
challenged primate choroidal endothelial cells with MAC and
measured differential gene expression by RNA-Seq.
Methods: Rhesus macaque RF/6A choroidal endothelial cells
were grown in six-well culture plates and treated with either 50%
non-heated (n=3) or heat inactivated (n=3) human serum for 4
hours. Additional wells were used for MAC immunocytochemistry.
Following RNA extraction, samples were prepared by The University
of Iowa DNA Core Facility for sequencing on an Illumina HiSeq
2000. Sequence reads were mapped to the Ensembl macaque genome
build MMUL_1 using Tophat2 (ver. 2.0.6), transcript abundance was
estimated using Cufflinks (ver. 2.0.2), and differential expression was
assessed using CuffDiff (ver. 2.0.2) and cummeRbund (ver. 2.2.0).
Functional annotation clustering was performed with DAVID (ver.
6.7), using the MINT, UP_TISSUE, and UCSC_TFBS datasets in
addition to the default human annotations.
Results: Formation of the MAC was confirmed on cells exposed
to normal, but not heat inactivated, serum. RNA-Seq analysis
showed 850 differentially expressed genes (q-value <= 0.001 and
two fold expression up or down) of 11,732 reliably expressed genes
(FPKM >= 1 in all replicates of either treated or control cells). Of
differentially expressed genes, we submitted the top 200 genes
(ordered on q-value; ties ordered on decreasing magnitude of fold
change) to DAVID. Genes with increased expression in MAC-injured
endothelial cells included molecules (a) implicated in angiogenesis
and blood vessel development, (b) involved in TGF-beta or BMP
pathways, and (c) secreted as signaling proteins. Genes with
decreased expression in MAC-injured cells included those (a) bound
by thirteen transcription factors, (b) containing SH3-domains, and (c)
containing fibronectin-3 domains.
Conclusions: Complement activation of choroidal endothelial
cells alters a wide range of molecular pathways, including a robust
increase in genes associated with angiogenesis.
Commercial Relationships: S Scott Whitmore, None; Shemin
Zeng, None; Megan J. Riker, None; Edwin M. Stone, None; Budd
A. Tucker, None; Todd E. Scheetz, None; Robert F. Mullins, None
Support: NEI Grant EY016822; Elmer & Sylvia Sramek Charitable
Foundation; Hansjoerg E.J.W. Kolder, M.D., Ph.D., Professorship in
Best Disease Research
Program Number: 3464 Poster Board Number: C0312
Presentation Time: 11:00 AM–12:45 PM
The retina and the ascending visual pathway share common agerelated protein changes
Michael R. Boehm1, 2, Christina Nolte2, Arnd Heiligenhaus1, 2,
Solon Thanos1. 1Institute for Experimental Ophthalmology, School
of Med, WWU Muenster, Muenster, Germany; 2Department of
Ophthalmology, St. Franziskus Hospital, Münster, Germany.
Purpose: The visual consequences of age-related alterations in
the neural retina are well documented, but little is known about
the alterations along the visual pathway. We previously discovered
distinct proteins, e.g. PARK [Parkinson disease (autosomal recessive,
early onset)] 7/DJ-1 (DJ-1), stathmin (STMN), peroxiredoxin (Prx),
and beta-synuclein (SNCB) that are regulated in the aging retina.
Here, we performed a comparative analysis of these proteins along
the post-laminar visual pathway in rats, in order to unravel common
age-related changes.
Methods: Expression of DJ-1, STMN, Prx and SNCB were
compared in the newborn, juvenile, middle-aged, and aged parts
of the visual pathway including optic tract (OT), thalamus (TH),
superior colliculus (SC) and visual cortex (VC). The frontal
cortex (FC) served as non-visually associated area. Western-blot
(WB), quantitative reverse-transcriptase PCR (qRT-PCR), and
immunohistochemistry (IHC) analyses were employed to determine
whether changes identified by proteomics were verifiable at the
cellular and molecular level. Then we selected one of these proteins
(SNCB) to study its function in cultured neurons obtained from either
the postnatal retina or the cortex.
Results: Changes of the proteins were found throughout the life
of rats. The alterations were analogous to the retinal profiles. WB,
IHC and qRT-PCR analyses confirmed these findings. The proteins
were localized in certain cerebral areas within the visual pathway
and the frontal cortex, therefore assigning them a role within the
maturating visual pathway and brain. In-vitro studies with SNCB
showed changes in the pattern of cell differentiation and growth, thus
indicating its involvement in differentiation of cerebral and retinal
ganglion cells.
Conclusions: This study is the first to provide evidence that DJ-1,
STMN, Prx and SNCB are associated with aging within the visual
pathway. These changes occur in key functional pathways during the
processing of visual signals and may be involved in the development
of age-related pathologies. SNCB is strongly involved in key
pathways of cerebral and retinal ganglion cells. Most likely, SNCB
and perhaps the other proteins influence key pathways triggering
differentiation of visually relevant neuronal cells.
Supported by the DFG-Excellence Cluster “Cells in Motion, CiM”,
area C.4 and the DFG-grant Th386 20-1
Commercial Relationships: Michael R. Boehm, None; Christina
Nolte, None; Arnd Heiligenhaus, None; Solon Thanos, None
Support: Supported by the DFG-Excellence Cluster “Cells in
Motion, CiM”, area C.4 and the DFG-grant Th386 20-1
Program Number: 3465 Poster Board Number: C0313
Presentation Time: 11:00 AM–12:45 PM
Construction of Recombinant AAV-based Vectors bearing miR183 Cluster Genes
Maliheh Davari1, Hamid Ahmadieh2, Zahra-Soheila Soheili1,
Shahram Samiei1, Ehsan Ranaei1, Mozhgan Rezaei Kanavi2.
1
basic biotechnology, national institute of genetic engineering and
biotechnology, Tehran, Islamic Republic of Iran; 2Ophthalmic
Research Center, Shahid Beheshti University of Medical Sciences,
Tehran, Islamic Republic of Iran.
Purpose: miR-183 cluster as a sensory organ-enriched miRNA
cluster has been recognized to be an important class of retina-specific
miRNAs. Members of miR-183 cluster (miR-183, miR-96 and
miR-182) play a role in development and survival of the neuroretina particularly photoreceptors. This study aimed to construct
recombinant AAV-based vectors containing miR-183 cluster genes.
Methods: AAV Helper-Free System was purchased from Agilent
Technologies. miR-183, miR-96 and miR-182 complete sequences
were amplified from human genome through PCR using specific
linker primer pairs with suitable restriction sites at their 5’-ends.
Double-digested PCR products corresponding to each miRNA
sequence, as well as IRES-GFP fragment providing green
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
fluorescence gene as a reporter gene, were cloned into pAAV-MCS
vector. To check the accuracy of cloning of all three miRNA genes
into pAAV-MCS vectors, the resultant constructs were subjected to
digestion and sequencing experiments.
Results: Desired recombinant pAAV-MCS vector bearing all three
miR-183 cluster genes simultaneously followed by GFP gene was
constructed. On the other hand, recombinant pAAV-MCS vectors
bearing each of three miRNAs were individually constructed.
Conclusions: Given critical roles of miR-183 cluster in neuro-retina
maintenance and survival and high efficiency of AAV-based vectors
to provide high and stable gene expression levels in various tissues;
the resultant constructs would be applied to study the over-expression
effects of miR-183 cluster genes on different parts of the retina.
Commercial Relationships: Maliheh Davari, None; Hamid
Ahmadieh, None; Zahra-Soheila Soheili, None; Shahram Samiei,
None; Ehsan Ranaei, None; Mozhgan Rezaei Kanavi, None
369 Pathophysiology of AMD and retinopathies
Tuesday, May 06, 2014 3:45 PM–5:30 PM
S 310E-H Paper Session
Program #/Board # Range: 3553–3559
Organizing Section: Retinal Cell Biology
Program Number: 3553
Presentation Time: 3:45 PM–4:00 PM
IL18 is not therapeutic for neovascular age-related macular
degeneration
Bradley D. Gelfand1, 2, Yoshio Hirano1, Tetsuhiro Yasuma1,
Takeshi Mizutani1, Benjamin J. Fowler1, Miho Nozaki4, Hiroki
Kaneko5, Balamurali Ambati6, 7, David R. Hinton8, Jayakrishna
Ambati1, 3. 1Ophthalmology & Visual Sciences, University of
Kentucky, Lexington, KY; 2Biomedical Engineering, University of
Kentucky, Lexington, KY; 3Physiology, University of Kentucky,
Lexington, KY; 4Ophthalmology and Visual Science, Nagoya City
University Graduate School of Medical Sciences,, Nagoya, Japan;
5
Ophthalmology, Nagoya University Graduate School of Medicine,
Nagoya, Japan; 6Ophthalmology and Visual Sciences, Moran Eye
Center, University of Utah School of Medicine, Salt Lake City, UT;
7
Ophthalmology, Veterans Affairs Salt Lake City Healthcare System,
Salt Lake City, UT; 8Pathology and Ophthalmology, Keck School of
Medicine of the University of Southern California, Los Angeles, CA.
Purpose: Recent studies report apparently conflicting roles for
the inflammasome effector IL18 in the development of age-related
macular degeneration. Whereas IL18 has been demonstrated
to induce RPE degeneration in atrophic AMD, it has also been
promoted as a potential therapeutic to prevent neovascular AMD.
The purpose of this multi-centered study was to evaluate whether
IL18 possesses anti-angiogenic potential in preventing laser induced
choroidal neovascularization, and to evaluate the toxicity of IL18
administration.
Methods: Five independent laboratories administered a 4-log dose
range (1 to 1,000 ng) of recombinant mouse IL18 into the vitreous
humor of wild-type mice undergoing laser injury. Several anti-IL18
antibodies were tested for their ability to influence CNV volume in
wild-type and IL18-deficient mice. Seven and 14 days later, CNV
volumes were assessed. ERGs, RPE flat mount and histology were
utilized to examine retinal health after IL18 injection. Finally, mice
genetically or pharmacologically deficient in Nlrp3, Pycard, or
Caspase-1, which form the inflammasome complex that bioactivates
IL18, were analyzed for their angiogenic response.
Results: Of the five participating laboratories, none found that
administration of recombinant IL18 changed CNV volume. Further,
administration of recombinant IL18 caused RPE degeneration and
ERG a-wave and b-wave depression. Mice deficient in essential
components of the Nlrp3 inflammasome exhibited no significant
changes in CNV volume compared to control animals. The major
IL18 receptor, IL18R1 is abundantly expressed in normal healthy
human retina, which further supports the concept that IL18 treatment
is ill-advised in human patients. Surprisingly, in agreement with
a previous report, we found that one particular IL18 neutralizing
antibody increased CNV volume not only in wild-type but also in
IL18-deficient mice. However, we determined that this effect was
due to a pro-angiogenic effect of glycerol in the antibody preparation
mediated via aquaporin-1, rather than from target-specific IL18
blockade.
Conclusions: This study confirms that IL18 induces RPE
degeneration, argues against pursuing IL18 as a therapeutic agent
for neovascular age-related macular degeneration, and cautions
that antibody preparations containing glycerol must be carefully
monitored for potential confounding pro-angiogenic effects.
Commercial Relationships: Bradley D. Gelfand, None; Yoshio
Hirano, None; Tetsuhiro Yasuma, None; Takeshi Mizutani,
None; Benjamin J. Fowler, None; Miho Nozaki, None; Hiroki
Kaneko, None; Balamurali Ambati, None; David R. Hinton, None;
Jayakrishna Ambati, University of Kentucky (P)
Support: (BDG) AHA Scientist Development Grant, IRRF,
University of Kentucky CCTS Pilot Award; (JA) NEI/NIH
R01EY018350, R01EY018836, R01EY020672, and R01EY022238,
Doris Duke Distinguished Clinical Scientist Award, Burroughs
Wellcome Fund Clinical Scientist Award in Translational Research,
EMF Senior Scholar in Aging Award, Dr. E. Vernon Smith and Eloise
C. Smith Macular Degeneration Endowed Chair, FFB Individual
Investigator Research Award, Carl Reeves Foundation, Harrington
Discovery Institute Scholar-Innovator Award, Y.H. Alcon Japan
Research award; B.J.F. NIH T32HL091812 and UL1RR033173;
(BKA) NIH R01EY017182 and R01EY017950, VA Merit Award,
and Department of Defense; (DRH) by NIH P30EY003040 and
R01EY001545 and Arnold and Mabel Beckman Foundation; (U of
KY authors) RPB departmental unrestricted grant
Program Number: 3554
Presentation Time: 4:00 PM–4:15 PM
Evaluation of dosage dependent effects of subretinally
administered Sodium Iodate - a new model for dry AMD?
Michael J. Koss1, 2, Hossein Nazari Khanamiri1, 2, Douglas
Matsunaga2, Marcel Pfister1, Walid F. Abdallah1, 3, Yi Zhang1, 2, Mark
S. Humayun1, 2, David R. Hinton2. 1Ophthalmology, Doheny Eye,
Pasadena, CA; 2Ophthalmology, University of Southern California,
Los Angeles, CA; 3Ophthalmology, Zagazig University, Zagazig,
Egypt.
Purpose: Despite recent improvements in the treatment of exudative
age-related macular degeneration (AMD), few effective treatments
are available for non-exudative AMD, the most common subtype of
the disease. This is partly because of the lack of a suitable models
of non-exudative AMD and the most advanced from of this disease,
geographic atrophy. Systemic administration of Sodium iodate
(SoIod), that leads to a generalized toxic reaction in RPE cells have
been used as a model of RPE degeneration. But, this model creates
a generalized toxicity to ocular tissue that may interfere with studies
focused on the treatment of RPE degeneration. Here we describe
methods to create a localized area of RPE atrophy by subretinal
delivery of SoIod in rabbit eyes.
Methods: With a vitrectomy-like pars plana approach, a subretinal
bleb with SoIod solution wascreated in 14 pigmented rabbits with
different concentrations (10mmol, n=3;1mmol, n=7; 0.1mmol n=2).
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
In the control group (n=2) the bleb was created with BSS. The
bleb was left or drained after 10 min. The animals were studied at
four time points: 1 day, 4 days, 7 days and 14 days after surgery,
which included fluorescein angiography, infrared and red free
fundusphotography, spectral optical coherence tomography. RPE and
retina histologic changes were studied light and electron microscopy.
Results: Reproducible RPE and outer retina atrophy was induced
after localized exposure with SoIod by subretinal injection in all
animals without choroidal or retinal neovascularization as early as
4 days after surgery. Severe damage with complete retinal atrophy
was experienced in the 10mmol group. The effect of 0.1mmol
SoIod was not conclusive or reproducible. OCT measurements in
the 1mmol group demonstrated a decrease in retinal tissue/ RPEcomplex thickness in the targeted area from mean 148um to 107umm
(37.8%), which was clearly visible in vivo with infrared and red free
fundusphotography. Scanning and transmission electron microscopy
confirmed the corrupted retinal area indicating loss of micorvilli.
Conclusions: Our data indicate that subretinal application of 1mmol
SoIod in pigmented rabbits leads to a limited and circumscribed area
of atrophy of the RPE-outer retina complex similar to late stages
of dry AMD. This procedure introduces a new and reliable animal
model for geographic atrophy.
Commercial Relationships: Michael J. Koss, None; Hossein
Nazari Khanamiri, None; Douglas Matsunaga, None; Marcel
Pfister, None; Walid F. Abdallah, None; Yi Zhang, None; Mark S.
Humayun, None; David R. Hinton, None
Support: DFG KO 4294/1-1, EY01545
Program Number: 3555
Presentation Time: 4:15 PM–4:30 PM
Histological Evidence of Outer Retinal Atrophy Associated
with Geographic Atrophy Secondary to Age-related Macular
Degeneration
Richard F. Spaide1, 2, Christine A. Curcio2, Sotaro Ooto1, Mihoko
Suzuki1. 1Vitreous Retina Macula Consultants of New York, New
York, NY; 2Department of Ophthalmology, University of Alabama at
Birmingham, Birmingham, AL.
Purpose: To examine the histologic findings of eyes with geographic
atrophy (GA) associated with age-related macular degeneration for
potential retinal abnormalities that may impact perifoveal visual
function.
Methods: Macula-wide high-resolution sections were collected
starting at the superior edge of an 8-mm diameter full-thickness
punch, stained with toluidine blue, and examined by light
microscopy. GA was defined as discrete areas of loss of retinal
pigment epithelium (RPE) measuring at least 500 microns in
diameter.
Results: There were 13 eyes of 13 donors with GA. Within the zone
of confluent RPE loss there were thickened whorls of persistent basal
laminar deposit (BlamD). Although the layer of confluent RPE cells
usually ended co-terminously with the external limiting membrane
border, isolated RPE cells were found in the atrophic area. Overlying
neurosensory retina showed complete absence of photoreceptor
inner and outer segments and nearly complete outer nuclear layer
loss. Along the external border of confluent RPE BLamD was
abundant, and rounded RPE cells lacked obvious apical processes.
The superjacent retina at the ELM border showed a pronounced
depopulation of the outer nuclear layer, absence of outer segments,
and blunting or absence of inner segments. Surviving inner segments
showed retraction of the ellipsoid portion toward or even internal to
the internal limiting membrane. Extending radially away from the
area of RPE loss the inner and outer segments were compromised.
The number of nuclei in the outer nuclear layer returned to normal,
yet isolated nuclei were scattered as far inward as the photoreceptor
synaptic layer, signifying cellular stress. Subretinal drusenoid
deposits were frequently present.
Conclusions: Taken in aggregate the retinal changes can be termed
outer retinal atrophy and this abnormality extends well beyond the
area of RPE loss in GA eyes. Outer retinal atrophy has potential
negative impact on visual function of areas used for eccentric fixation
in low vision rehabilitation. Strategies currently employed to try to
halt the progression of GA need to consider the extent and severity of
concurrent outer retinal atrophy.
Restoration in inner and outer segment lengths and increasing nuclei
population in the outer nuclear layer progressing left to right away
from an area of GA. (Artifactual detachment in right panel.)
Commercial Relationships: Richard F. Spaide, Topcon (C),
Topcon (P); Christine A. Curcio, None; Sotaro Ooto, None;
Mihoko Suzuki, None
Support: Macula Foundation, NEI EY06109; Arnold and Mabel
Beckman Initiative in Macular Research; EyeSight Foundation of
Alabama; Research to Prevent Blindness
Program Number: 3556
Presentation Time: 4:30 PM–4:45 PM
Control of physiological and pathological angiogenesis in the
retina by the matricellular protein CCN1
Brahim Chaqour1, 2, Maria Grant3, Jinog Choi1, Izabella Krupska1,
Lulu Yan1, Hemabindu Chintala1. 1Cell Biology, SUNY Eye Institute
Downstate Medical Center, Brooklyn, NY; 2Ophthalmology, SUNY
Downstate Medical Center, Brooklyn, NY; 3Ophthalmology, Indiana
University, Indianapolis, IN.
Purpose: CCN1 is a non-structural bioactive extracellular matrixassociated heparin- and integrin-binding protein whose expression is
associated with sites of angiogenesis and tissue repair. In the retina,
CCN1 protein was prominently expressed at the leading front of
actively growing vessels. Conventional CCN1 gene deletion in mice
resulted in major vascular defects and embryonic lethality. Here we
used endothelial-specific inducible gene targeting strategy in mice
to define the function of CCN1 in retinal vascular development and
pathology.
Methods: Inducible loss-of-function of CCN1 in the endothelium
was carried out by crossing mice carrying floxed CCN1 alleles
with tamoxifen-activated Cdh5(PAC)-CreERT2 mice. Retinal
vascular alterations were characterized at postnatal day (P) 4 and
P7 upon injection of tamoxifen using immunohistochemical and
biochemical techniques. Effects of CCN1-loss-of-function on retinal
neovascularization were determined in the mouse model of oxygeninduced retinopathy (OIR).
Results: Endothelium-specific inactivation of CCN1 resulted in
a dense vascular network with increased endothelial cell (EC)
proliferation and apparent coalescence of retinal vessels leading
to loss of typical features of the retinal vascular architecture.
Endothelial tubes were devoid of pericyte coverage. Defects in
angiogenic sprouting were linked to increased activation of vascular
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
endothelial growth factor (VEGF) receptor 2 and altered downstream
Notch signaling activity at the distal growing end of new angiogenic
vessels. Subsequently, more ECs incorporate into the wall of forming
tubes rather than into new sprouts and branches. These changes were
directly coupled to enhanced VEGF signaling mechanisms required
for EC lumen and tube network formation. In the mouse model of
OIR, ectopic expression of CCN1 promoted normal vessel formation
while suppressing abnormal pathological neovascularization by
increasing resistance to vaso-obliteration and fine-tuning the
bioavailability of VEGF.
Conclusions: CCN1 is an angiogenic factor required for normal
retinal vessel morphogenesis and stabilization during normal
development as well as during progression of pathology. CCN1
activity may involve, at least in part, shaping gradients of VEGF
activity in areas of uniform ligand expression.
Commercial Relationships: Brahim Chaqour, None; Maria
Grant, None; Jinog Choi, None; Izabella Krupska, None; Lulu
Yan, None; Hemabindu Chintala, None
Support: NIH-NEI Grant EY022091-01
Program Number: 3557
Presentation Time: 4:45 PM–5:00 PM
Mechanistic role of arginase in inducing endothelial cell
senescence in diabetic retinopathy
Esraa Shosha1, Tahira Lemtalsi1, Zhimin Xu1, Robert William
Caldwell2, Ruth B. Caldwell1, 3, S. Priya Narayanan1. 1Vascular
Biology Center, Georgia Regents University, Augusta, GA;
2
Department of Pharmacology and Toxicology, Georgia Regents
University, Augusta, GA; 3VA Medical Center, Augusta, GA.
Purpose: Previous studies from our laboratory have shown
that diabetes-induced vascular inflammation and impairment of
endothelial-dependent vasorelaxation involve activation of the
urea/ornithine producing enzyme arginase. Recently, senescence
in endothelial cells has been considered as a cause of diabetes
related complications. We have shown that high glucose (HG)
accelerates retinal endothelial cell senescence and inhibiting arginase
activity blocks this effect. The present study investigated the
potential involvement of a novel cellular senescence marker DEC1
(differentiated embryo-chondrocyte expressed gene 1), a target of
the p53 family, the basic helix-loop-helix transcription factor, and an
upstream mediator, c-Jun NH2-terminal kinase (JNK), in this process.
Methods: Studies were performed using diabetic mice [Ins2 (Akita)
and streptozotocin (STZ)-diabetic], mice lacking one copy of
arginase 1 (A1 KO) and bovine retinal endothelial (BRE) cells treated
with high glucose. Protein extracts from BRE cells and fresh frozen
retinas were collected for Western blotting. RT-PCR was performed
using RNA isolated from fresh frozen retinal samples and isolated
retinal vessels. Retinal sections and fixed cells were evaluated for
senescence associated β-galactosidase activity assay. TUNEL assay
was used to determine cell death.
Results: Our studies showed that arginase expression/activity,
cellular senescence and cell death were increased in HG-treated BRE
cells as well as in diabetic retinas. These changes were associated
with activation of JNK in both HG treated BRE cells and diabetic
retinas. The JNK activation was significantly reduced by inhibiting
arginase activity with the pharmacological inhibitor ABH (amino-2borono-6-hexanoic acid) in BRE cells or by A1 KO in mice. RT-PCR
and Western blot analysis showed that the diabetes-induced activation
of JNK was associated with increased expression of DEC1.
Conclusions: This study shows for the first time that diabetes/
high glucose can induce vascular injury through arginase activation
resulting in cellular senescence and cell death through a pathway
involving JNK activation and upregulation of DEC1 expression.
Our data suggests that diabetes and hyperglycemia induce retinal
endothelial cell senescence and death by a mechanism involving
arginase-induced cell stress and DEC1 activation. Inhibiting arginase
can block this vascular injury.
Commercial Relationships: Esraa Shosha, None; Tahira Lemtalsi,
None; Zhimin Xu, None; Robert William Caldwell, None; Ruth B.
Caldwell, None; S. Priya Narayanan, None
Support: AHA-11SDG7440088, NEI-EY11766 and VA Merit Award
Program Number: 3558
Presentation Time: 5:00 PM–5:15 PM
Adiponectin mediates protective effect of omega-3 long-chain
polyunsaturated fatty acid in retinal neovascularization
Zhongjie Fu1, Chatarina Lofqvist2, Christian G. Hurst1, Zhenghao
Cui1, Lucy P. Evans1, Katherine T. Tian1, Hannah H. Bogardus1, Jing
Chen1, Ann Hellström2, Lois Smith1. 1Department of Ophthalmology,
Children’s Hosp Boston/Harvard Med Sch, Boston, MA; 2Department
of Ophthalmology, Sahlgrenska Academy at University of
Gothenburg, Gothenburg, Sweden.
Purpose: Retinopathy of prematurity (ROP) is a common blinding
disease in premature infants. It is caused by inadequate retinal
vascularization, resulting in retinal ischemia stimulating visionthreatening vaso-proliferative ROP. Lack of factors normally
available in the third trimester of pregnancy in utero, and lacking
after preterm birth such as omega-3 long-chain polyunsaturated fatty
acid (ω-3 LCPUFA), is associated with development of retinopathy.
Dietary supplement of ω-3 LCPUFA is protective in a mouse model
of ROP. Our preliminary data show that adiponectin (APN), an
adipocytokine abundantly expressed in white adipose tissue (WAT),
is significantly lower in serum from preterm infants with ROP versus
without ROP. We aimed to determine whether decreased APN levels
in ROP are associated with deficiency of ω-3 LCPUFA, and if APN
mediates in part the protective effect of ω-3 LCPUFA in ROP.
Methods: In the mouse model of oxygen-induced retinopathy
(OIR), APN-knockout (APN-/-) and wild-type (WT) mice were
fed isocaloric diets enriched with either ω-3 or ω-6 from postnatal
day (P)1. Pups were sacrificed at P17 for serum and WAT APN
levels, retinal vaso-obliteration and neovascularization. APN and its
receptors were examined with immunohistochemistry, endoplasmic
reticulum (ER) stress markers and ER proteins were examined in
WAT using Western Blot.
Results: In OIR, dietary ω-3 LCPUFA feed increased serum (ω-3:
5.8±1.4μg/ml vs. ω-6: 1.3±0.6μg/ml, P<0.05) and WAT APN level
(ω-3: 0.30±0.02ng/μg vs. ω-6: 0.23±0.02ng/μg, P<0.05) at P17
compared with ω-6 feed. Moreover, the vaso-protective effect of
ω-3 LCPUFA in OIR observed in WT retinae (neovascular area
ω-3/ω-6=0.3, P<0.001) versus ω-6 feed was abolished in APN/- retinae (neovascular area ω-3/ω-6=0.9, P<0.05). APN and its
receptors were localized in endothelial cells and macrophages in both
neovascular areas in OIR. ER stress (p-eIF2α/eIF2α: ω-3/ω-6=0.4,
P<0.05; CHOP: ω-3/ω-6=0.5, P<0.01) was attenuated and ER protein
was increased (Erp44: ω-3/ω-6=1.9, P<0.05) in WAT from ω-3
LCPUFA-fed mice versus ω-6 LCPUFA-fed mice
Conclusions: An increase in dietary ω-3 LCPUFA may regulate
production of APN through modulating ER stress in WAT and APN
in turn likely mediates in part the protective effects of ω-3 LCPUFA
in OIR
Commercial Relationships: Zhongjie Fu, None; Chatarina
Lofqvist, None; Christian G. Hurst, None; Zhenghao Cui, None;
Lucy P. Evans, None; Katherine T. Tian, None; Hannah H.
Bogardus, None; Jing Chen, None; Ann Hellström, None; Lois
Smith, None
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Support: NIH/ NEI (EY022275, EY017017, P01 HD18655, RPB Sr
Investigator Award, Lowy Medical Foundation, (LEHS)
Program Number: 3559
Presentation Time: 5:15 PM–5:30 PM
The role of IL-1β in progressive retinal degeneration associated
with retinopathy of prematurity
Tianwei (Ellen) Zhou1, Jose C. Rivera2, Isabelle Lahaie2, Zhuo Shao2,
Tang Zhu2, Baraa Noueihed1, Anna Polosa3, Allison L. Dorfman3,
Pierre Lachapelle3, Sylvain Chemtob2. 1Pharmacology and Medicine,
McGill University, Montreal, QC, Canada; 2Pediatrics, Ophtalmology
and Pharmacology, University of Montreal, Montreal, QC, Canada;
3
Neurosciences and Ophthalmology, McGill University, Montreal,
QC, Canada.
Purpose: Retinopathy of prematurity (ROP) is a serious complication
in premature infants. With the use of surfactant began in late 1980’s,
most premature neonates now survive and live into adulthood.
Besides immediate damages on the inner retina in ROP, progressive
photoreceptor malfunction is also observed, and appears to result
from a sustained involution of the choroid, the exclusive source of O2
and nutrients to the photoreceptors.
Sustained choroidal involution has been reported both in clinics
and in the established animal model of O2-induced retinopathy
(OIR). However, mechanisms for this choroidal involution remain
unexplained. While inflammation has often been proposed in
pathogenesis of ROP/OIR, the specific role of pro-inflammatory
cytokines in the neonatal sub-retina has yet to be explored. Therefore,
we investigated the role of the major pro-inflammatory cytokine,
notably IL-1β, in rats subjected to OIR.
Methods: Oxygen-induced retinopathy was achieved by exposing
rat pups to cycling O2 levels. Retinal and choroidal histology were
performed to study the thickness and integrity of microvasculature.
Expression of factors of interest was studied by immunohistology,
PCR, and Western blots. Finally, full-field and multifocal
electroretinograms (ERG) were performed to evaluate visual
function.
Results: Our longitudinal study revealed that OIR caused sustained
outer and sub-retinal hypoxia, and a progressive deterioration of the
sub-retina including an increase in abnormal mitochondria in RPE
cells, gradual degeneration of photoreceptor and ensuing decline in
visual function (ERG). These changes were associated with marked
increases in pro-inflammatory IL-1β, and drastic reductions in
photoreceptor response. Early neonatal treatment with IL-1 receptor
antagonist (IL-1ra [Kineret]) significantly decreased IL-1β levels and
attenuated choroidal involution. Moreover, IL-1ra effectively blunted
long-term photoreceptor loss induced by OIR. This amelioration in
outer retinal structure by IL-1ra was associated with improved retinal
function (ERG).
Conclusions: Our observations reveal a dominant role for IL-1β in
outer retinal damage associated with the ROP model. Our findings
set forth new mechanism notion of ROP and its long-term outcomes
in adults. IL-1 receptor blockers (administered early in the neonate)
may be protective to the retina and consequently limit progressive
deterioration initiated by ROP.
Commercial Relationships: Tianwei (Ellen) Zhou, None; Jose
C. Rivera, None; Isabelle Lahaie, None; Zhuo Shao, None; Tang
Zhu, None; Baraa Noueihed, None; Anna Polosa, None; Allison L.
Dorfman, None; Pierre Lachapelle, None; Sylvain Chemtob, None
384 Stem Cell II: Restoration of Photoreceptors
Tuesday, May 06, 2014 3:45 PM–5:30 PM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 3978–4006/D0037–D0065
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 3978 Poster Board Number: D0037
Presentation Time: 3:45 PM–5:30 PM
Human iPSC-derived neural progenitor cells preserve vision in a
rat retinal degeneration model
YuChun Tsai, Bin Lu, Sergey Girman, Benjamin Bakondi, Melissa
K. Jones, Anais Sahabian, Dhruv Sareen, Clive Svendsen, Shaomei
Wang. Cedars-Sinai Medical Center, Regenerative Medicine Institute,
Los Angeles, CA.
Purpose: Neural progenitor cells (NPCs) have been shown effective
in treating degenerative neurological disorders including retinal
degeneration. Induced pluripotent stem cell (iPSC) therapies are
gaining momentum for regenerative medicine. An ideal cell type to
treat retinal degeneration should be offering great preservation of
vision, renewable, no ethical concern, not causing immune rejection.
Here we study human NPC derived from iPSC (iNPCs) in preserving
vision after transplantation into the Royal College Surgeon (RCS) rat,
a well-established model for retinal degeneration.
Methods: Non-integrating human iPSCs were generated using
Yamanaka episomal plasmids from an Amaxa human dermal
fibroblast nucleofector kit. NPCs were derived from iPSC-EZ spheres
according to Sareen et al., 2013. iNPCs or medium were injected
into the subretinal space of RCS rats at 21 days postnatal (P21).
Visual functions were evaluated using Optokinetic response (OKR),
electroretinography (ERG), and luminance threshold recordings
(LTR) from superior colliculus. Histological examination of the
retinas was performed by light, confocal, and electron microscopy.
The in vitro phagocytosis assay of photoreceptor outer segments
(POS) by iNPCs was examined by RT-PCR, flow cytometry,
immunocytochemistry, and western blot after feeding naïve or FITC
labeled POS.
Results: At P90-100, iNPCs treated eyes sustained near normal
visual functions and retinal integrity compared with controls. Donor
cells showed extensive migration from the injection site, formation of
a continuous layer in the subretinal space, and limited distribution in
the inner retina. In cell treated eyes, the debris zone was significantly
diminished, suggesting the involvement of phagocytosis. iNPCs are
capable of phagocytosing POS in vitro in a time and dose dependent
manner, and express transcripts associated with RPE-specific
phagocytosis, including Mer Tyrosine Kinase (MERTK).
Conclusions: The results underscore the potential therapeutic
utility of iNPCs for age-related macular degeneration and other
degenerative retinal diseases.
Commercial Relationships: YuChun Tsai, None; Bin Lu, None;
Sergey Girman, None; Benjamin Bakondi, None; Melissa K.
Jones, None; Anais Sahabian, None; Dhruv Sareen, None; Clive
Svendsen, None; Shaomei Wang, None
Support: NIH R01 EY020488-02, W81XWH-DOD, FFB, Fund
from the Regenerative Medicine Institute at Cedars-Sinai Medical
Center
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Program Number: 3979 Poster Board Number: D0038
Presentation Time: 3:45 PM–5:30 PM
Functional consequences of the suppression of MHC-II
expression on human stem cell derived retinal pigment
epithelium (hES-RPE)
Hossein Nazari Khanamiri1, Keijiro Ishikawa1, Danhong Zhu1, 3,
Sherry T. Hikita4, Dennis O. Clegg4, David R. Hinton1, 3, Mark S.
Humayun1, 2. 1Ophthalmology, University of Southern California, Los
Angeles, CA; 2Biomedical Engineering and Cell and Neurobiology,
University of Southern California, Los Angeles, CA; 3Pathology,
University of Southern California, Los Angeles, CA; 4University of
California, Santa Barbara, Santa Barbara, CA.
Purpose: Subretinal implantation of human stem cell derived
retinal pigment epithelium cells (hES-RPE) is an emerging method
to replace degenerated RPE in age related macular degeneration.
But, immune reaction to the implanted cells, that necessitates
potentially harmful immunosuppressive agents, is a major obstacle
for the success of hESC-RPE implantation. Expression of Major
Histocompatibility Complex-II (MHC-II) molecules on RPE is the
key event for the recognition of allogeneic antigens of these cells
by the host immune system. We hypothesize that the suppression of
Class II Transactivator (CIITA), an essential regulator of MHC-II
expression, through RNA interference will down regulate MHCII expression on hESC-RPE cells, and thus, will reduce immune
recognition of these cells by host immune system.
Methods: The H9 human embryonic stem cell line was used for
derivation of RPE cells. hESC-RPE monolayer polarization (trans
epithelial resistance above 300 Ohm/cm2) was achieved with longterm growth on permeable inserts. A pool of constructs consisting
of three siRNA targeting CIITA were used to suppress CIITA
expression. Transfected cells and controls treated with scrambled
RNA were stimulated with interferon gamma to induce MHCII expression. Stimulated expression of CIITA and MHC-II was
quantified by qPCR and western blot techniques. Transfected cells
were co-cultured with naïve lymphocytes and reactive and regulatory
T cell differentiation was analyzed by flow cytometry.
Results: Polarized hES-RPE were successfully transfected with
anti-CIITA siRNA. Interferon gamma-stimulated CIITA and
MHC-II expression was knocked down with more than 95%
efficiency. CIITA-knocked down RPE’s ability to stimulate reactive
T-lymphocyte differentiation was limited.
Conclusions: RNA modulation of CIITA expression in polarized
hESC-RPE results in significant suppression of MHC-II expression
upon immune stimulation. Stable suppression of MHC-II expression
in differentiated hESC-RPE cells can render these cells less
immunogenic, potentially leading to improved survival of implanted
cells. Such treatment would have a major clinical impact if the need
for systemic use of immunosuppressive agents was eliminated.
Commercial Relationships: Hossein Nazari Khanamiri, None;
Keijiro Ishikawa, None; Danhong Zhu, None; Sherry T. Hikita,
None; Dennis O. Clegg, Regenerative Patch Technologies LLC (C);
David R. Hinton, Regenerative Patch Technologies LLC (C); Mark
S. Humayun, None
Support: CIRM Stem Cell Biology Training Program (TG2-01161)
Program Number: 3980 Poster Board Number: D0039
Presentation Time: 3:45 PM–5:30 PM
Induction of RPE-specific markers in human sweat gland-derived
stem cells by xenogeneic co-culture with porcine RPE cells
Mahdy Ranjbar1, Christine Örün1, Matthias Brandenburger2,
Charli Kruse2, Sandra Danner2, Salvatore Grisanti1. 1Department
of Ophthalmology, University of Lübeck, Lübeck, Germany;
2
Fraunhofer Research Institution for Marine Biotechnology, Lübeck,
Germany.
Purpose: There is an increasing interest in generating retinal pigment
epithelial (RPE) cells from stem cells for treatment of macular
degeneration. Human sweat glands are a rich source of nestinpositive stem cells. In this study, the possibility of inducing RPEspecific markers in human sweat gland-derived stem cells (SGSCs)
by xenogeneic co-culture with porcine RPE cells was investigated.
Methods: SGSCs were isolated out of adult human scalp skin,
purified and seeded on laminin-coated cover slips. Then they
were co-cultured with porcine RPE cells seeded on laminincoated transwell inserts or mono-cultured without RPE cells for
5 days. Afterwards they were washed, fixed, stained (Bestrophin,
MITF, PMEL, MERTK, CRALBP and RPE65) and analyzed by
fluorescence microscopy.
Results: SGSCs expressed Bestrophin and MITF on laminin-coated
cover slips even when mono-cultured without RPE cells whereas
PMEL, MERTK, CRALBP and RPE65 were only detectable after
co-culture with RPE cells.
Conclusions: Adult human sweat gland-derived stem cells can be
directed into expressing RPE-specific proteins even by xenogeneic
co-culture with porcine RPE cells. The presented system is an
effective tool to predict the behavior of SGSCs after transplantation
into the subretinal environment by mimicking the situation in vivo.
Commercial Relationships: Mahdy Ranjbar, None; Christine
Örün, None; Matthias Brandenburger, None; Charli Kruse,
None; Sandra Danner, None; Salvatore Grisanti, None
Program Number: 3981 Poster Board Number: D0040
Presentation Time: 3:45 PM–5:30 PM
Cone Transplantation
Sher A. Aslam2, 1, Alun R. Barnard2, Sumathi Sekaran2, Mandeep S.
Singh2, Robert E. MacLaren2, 1. 1Moorfields Eye Hospital, London,
United Kingdom; 2Nuffield Laboratory of Ophthalmology, University
of Oxford, Oxford, United Kingdom.
Purpose: Previous studies have shown that rod transplantation
is feasible. However, there are no reports that investigate the
transplantation of cones when isolated from rods. We show that cone
transplantation and restoration of cone function is possible using a
new approach to enrich cone photoreceptor donors.
Methods: Donor cells were derived from post-natal day (P)1
dissociated rd1, DsRed+, Opn1-EGFP+ retinas that exhibit ubiquitous
red fluorescence on an rd1 background and in which enhanced
green fluorescent protein (EGFP) expression is restricted to medium
wavelength-sensitive cones. These were transplanted into P1 hosts
of the following strains: C57BL/6 and Opn4-/-, Gnat1-/-, Cnga3-/[or TKO (triple knockout)]. The functional response of transplanted
cones in TKO mice was confirmed ex vivo by calcium imaging,
using the specific mGluR8 agonist, (S)-3,4-dicarboxyphenylglycine
(DCPG), in order to detect a similar response in donor to host cells,
the latter serving as positive controls. Behavioural light aversion
(BLA) was tested using a light-dark box.
Results: At three weeks after transplantation, cones integrated into
the host retina and displayed an atypical morphology (Figure 1).
Calcium imaging showed that the application of DCPG resulted in
an expected decrease in intracellular calcium concentration in 70.6 ±
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
1.4% of GFP+ (donor) cells vs. 70.2 ± 0.9% of control (TKO) cells
(t12 = 0.67, P = 0.52, N = 4, paired t-test). BLA testing showed that
treated TKO (N = 11) and wild type (N = 6) mice spent more time
(66.3 ± 5.0% and 79.0 ± 3.5% respectively) in the dark compartment
compared to sham-injected mice (N = 10) (53.7 ± 2.3%, F3,34 =
10.5, P = 0.031 and 0.0002 respectively, one-way ANOVA with
Dunnett’s post-hoc test).
Conclusions: These results demonstrate that cone transplantation
and restoration of cone-mediated visual behaviour is possible, with
calcium imaging demonstrating similar responses in transplanted to
host cells. Importantly, as opposed to the findings of previous studies
of photoreceptor integration, an atypical morphology excludes cell
fusion artefact.
Integration of cone photoreceptors. Three examples are shown of
integrated rd1.DsRed+, Opn1-EGFP+ cones, each with its cell body
within the subretinal space and a process extending towards the
outer plexiform layer. The identity of each cone is confirmed by the
presence of GFP and DsRed fluorescence (A-C) and by anti- arrestin
(D-F) and anti-recoverin staining (G-I). Scale bar: 20mm.
Commercial Relationships: Sher A. Aslam, None; Alun R.
Barnard, None; Sumathi Sekaran, None; Mandeep S. Singh,
None; Robert E. MacLaren, None
Program Number: 3982 Poster Board Number: D0041
Presentation Time: 3:45 PM–5:30 PM
INFLUENCE OF HMGA2 ON PHOTORECEPTOR
DIFFERENTIATION
Xiaohuan Xia, Sowmya Parameswaran, Iqbal Ahmad.
Ophthalmology and Visual Sciences, Univ of Neb Med Center,
Omaha, NE.
Purpose: The high-mobility group AT-hook 2 (Hmga2) protein
regulates transcription by modulating the chromatin structure. Hmga2
and its microRNA regulator Let7 are expressed in a reciprocal
manner in the developing retina; levels of Hmga2 transcripts
temporally decreases and Let7 microRNA increases with the
exhaustion of retinal progenitors as differentiation comes to an end.
Hmga2 is expressed in retinal progenitors and we demonstrated
earlier that it regulates the self-renewal of retinal progenitors
(Parameswaran et al., 2013, ARVO Abst). Here, we have examined
whether or not Hmga2 expression or the lack of it has any influence
on the differentiation potential of retinal progenitors during late
retinal histogenesis.
Methods: We carried out Hmga2 gain-of-function (GOF) and
loss-of-function (LOF) experiments using lentivirus-mediated
perturbation of expression in E18 retinal progenitor and E18 retinal
explant models of retinal differentiation. The perturbation of Hmga2
expression was verified by Q- PCR and immunofluorescence
analyses. The effects of the perturbations on the differentiation of
the late born cells, i.e., rod photoreceptors, bipolar cells, and Müller
glia were ascertained by examining the expression of regulators and
markers of the specific cell types using analyses mentioned above.
Results: We observed that both in retinal progenitor and retinal
explant models, levels of Hmga2 expression were decreased with
differentiation. When Hmga2 expression was maintained by Hmga2
lentivirus-mediated transduction, differentiation of rod photoreceptors
was negatively impacted, compared to controls. In contrast, when
Hmga2 expression was prematurely decreased by Hmga2 siRNA
lentivirus-mediated transduction a significantly higher number of rod
photoreceptors were observed, compared to controls. In both GOF
and LOF experiments, no significant difference in the number of
either bipolar cells or Müller glia was observed.
Conclusions: Our preliminary results suggest that Hmga2, which is
an intrinsic regulator of self-renewal of retinal progenitors, extends
its influence on rod photoreceptor differentiation.
Commercial Relationships: Xiaohuan Xia, None; Sowmya
Parameswaran, None; Iqbal Ahmad, None
Support: Pearson Foundation, Research to prevent blindness
Program Number: 3983 Poster Board Number: D0042
Presentation Time: 3:45 PM–5:30 PM
Human neural progenitor cells are not sufficient to support
degenerating photoreceptors in cultured porcine retina
Camilla Mohlin1, Tanzina Mollick2, Kjell Johansson1, 2. 1Natural
Sciences, Medicine and Optometry, Kalmar, Sweden; 2Örebro
University, School of Health and Medical Sciences, Örebro, Sweden.
Purpose: Neurodegenerative diseases like retinitis pigmentosa,
age-related macular degeneration and diabetic retinopathy commonly
cause photoreceptor degeneration over time, inducing secondary
injuries like gliosis and neuronal remodeling; such mechanisms
ultimately lead to impaired vision. Despite intense research, no
effective treatment has been found for these disorders. Human neural
progenitor cell (HNPC)-derived factors have been shown to protect
photoreceptor cells from apoptotic cell death. The current study was
undertaken to explore whether HNPC-derived factors could inhibit
the degenerative process of photoreceptors in adult porcine retinal
explants.
Methods: Adult porcine eyes were collected from a local abattoir.
The neural retina was gently detached from the pigmented epithelium
and peripheral sections were explanted onto a Millicell-PCF 0.4
μm culture plate insert with the vitreal side oriented upwards.
Adult porcine retinas were cultured with or without HNPCs as a
feeder layer in the bottom of the well. After co-culture for three
days the retinas were investigated for cell death by TUNEL assay.
Synaptic integrity was assessed by expression of the scaffold protein
postsynaptic density protein 95 (PSD95) and gliotic events were
detected by glial fibrillary acidic protein (GFAP).
Results: HNPCs were not able to inhibit photoreceptor cell death in
co-cultured retinal explants. However, the progenitor cells had the
ability to preserve synaptic integrity to some extent, as observed by
maintained PSD95 immunoreactivity in photoreceptor cell terminals.
Retinal co-cultures also showed reduced GFAP expression in Müller
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
cells which was interpreted as decreased gliosis. Milliplexed HNPCs
medium showed that several neuroprotective factors were secreted,
including FGF-2, G-CSF, GM-CSF and VEGF.
Conclusions: Our observations indicate that, although HNPC derived
factors did not have the capacity to protect photoreceptors from cell
death after three days of co-culture, maintenance of synaptic integrity
and decreased gliosis appeared to be favored.
Commercial Relationships: Camilla Mohlin, None; Tanzina
Mollick, None; Kjell Johansson, None
Program Number: 3984 Poster Board Number: D0043
Presentation Time: 3:45 PM–5:30 PM
The involvement of IL-17RC pathway in the inflammatory
stimuli of the multipotent retinal stem cells
Shida Chen1, 2, Defen Shen1, Nicholas Popp1, Jingsheng Tuo1,
Mones S. Abu-Asab1, Ting Xie3, Chi-Chao Chan1. 1National eye
institution, Bethesda, MD; 2Zhongshan Ophthalmic Center, Sun YatSen University, Guangzhou, China; 3Stowers Institute for Medical
Research, Kansas City, MO.
Purpose: Multipotent retinal stem cells (RSCs) from the adult
mouse retina are capable of producing functional photoreceptor
cells to replace degenerative and atrophic photoreceptor cells in
animal models. Inflammasome activation has been implicated in
the pathogenesis of age-related macular degeneration (AMD). The
downstream effectors of inflammasome activation are maturation of
IL-1β and IL-18, which can induce the expression of IL-17A in Th17
cells. IL-17A mediates signal transduction and pro-inflammatory
activities through its receptor, IL-17RC, which may contribute to
AMD pathogenesis. Additionally, IL-1β and IL-18 signals may lead
to apoptosis. This study aimed to determine whether IL-1β and IL-18
could induce expression of IL-17A and IL-17RC and to characterize
their effect on apoptosis in RSCs.
Methods: RSCs were cultured in culture medium for retinal stem
cells supplemented with 5% Knockout Serum Replacement, EGF
and FGF, and the cells doubled every 24 hours. The protein levels of
IL-17RC, cleaved caspase-3, and cleaved-caspase-9 were detected by
confocal immunofluorescent microscopy. Cells were treated with IL1β (1ng/ml, 10ng/ml, and 100ng/ml) or IL-18 (1ng/ml, 10ng/ml, and
100ng/ml) for 24 hours. Expression of IL-6, IL-17A, and IL-17RC
transcript was evaluated by quantitative RT-PCR.
Results: Naive RSCs expressed IL-17RC on the cell membrane and
in the cytoplasm. Expression of IL-17RC mRNA increased by 2-fold
when the cells were treated with the high dose of IL-1β (100ng/ml)
but was enhanced in a dose-dependent manner when treated with IL18. Treatment with IL-1β at 100ng/ml concentration induced a 3-fold
increase in IL-6 mRNA expression. IL-18 treatment led to increased
expression of IL-6 in a dose-dependent manner. IL-17A was not
induced by either IL-1β or IL-18 treatment. IL-1β (100ng/ml) or IL18 (10ng/ml) treatment induced expression of cleaved caspase-3 and
cleaved-caspase-9 in the RSCs.
Conclusions: RSCs express IL-17 receptor, which is upregulated
by IL-1β and IL-18. Neither IL-1β nor IL-18 treatment led to
measurable IL-17A expression in this cell line. However, both IL-1β
and IL-18, which are activated by inflammasome, can have proapoptotic effects on RSCs and may induce other pro-inflammatory
cytokine produced by these cells.
Commercial Relationships: Shida Chen, None; Defen Shen, None;
Nicholas Popp, None; Jingsheng Tuo, None; Mones S. Abu-Asab,
None; Ting Xie, None; Chi-Chao Chan, None
Support: NEI intrmural fund
Program Number: 3985 Poster Board Number: D0044
Presentation Time: 3:45 PM–5:30 PM
Activation of mTORC1 is sufficient for long-term cone survival in
Retinits Pigmentosa
Aditya Venkatesh, Shan Ma, Claudio Punzo. Ophthalmology and
Gene Therapy Center, University of Massachusetts Medical School,
Worcester, MA.
Purpose: The mechanism of cone death in Retinitis Pigmentosa
(RP) remains elusive. We previously proposed that cones are nutrient
deprived in RP based on gene expression changes seen during disease
in metabolic genes and genes of the insulin/mTOR pathway. These
findings led us to treat RP mice with insulin, which significantly
improved cone survival although only for a 4 week period. The
short-term effect was attributed to the feedback loop in the pathway.
To determine if insulin acted directly on cones, and to test the longterm therapeutic potential of the pathway, we have now genetically
activated the pathway directly in cones at two different junctions
downstream of the insulin receptor. This has been achieved by
conditional deletion of the phosphatase and tensin homolog (PTEN),
and separately by conditional ablation of the tuberous sclerosis
complex 1 (TSC1) in cones. In contrast to insulin, loss of PTEN or
TSC1 leads to constitutive activation of the pathway in a feedback
loop independent manner. Loss of PTEN activates the entire pathway
including both mTOR complexes and AKT thus promoting progrowth and pro-survival mechanisms, while loss of TSC1 activates
only mTOR complex 1 thereby promoting mainly pro-growth
mechanisms.
Methods: Mice carrying conditional knockout alleles for PTEN,
TSC1, raptor (loss of mTOR complex 1) and rictor (loss of mTOR
complex 2) were crossed to a cone-specific Cre recombinase line in a
retinal degeneration-1 (rd1) mutant background. Additionally, double
conditional alleles for PTEN & raptor, and TSC1 & raptor were
used to delineate the contribution of pro-growth and pro-survival
mechanisms. Retinal flat mounts were used to quantify surviving
cones.
Results: Activation of the pathway via removal of PTEN or TSC1
significantly improved cone survival for up to 8 months. The
combinatorial genetics showed that activation of mTORC1 is both
required and sufficient for long-term cone survival in RP. In fact,
upon loss of TSC1 at 2 months of age, the cone distribution in the
mutant background was similar to that of a wild-type retina.
Conclusions: Our results show that cell-autonomous activation of
the pathway is sufficient for long-term protection of cones in RP and
suggest that altering pro-growth mechanisms alone can promote cone
survival. The data suggests that mTORC1 targets are ideal candidates
to develop therapeutic strategies.
Commercial Relationships: Aditya Venkatesh, None; Shan Ma,
None; Claudio Punzo, None
Support: NIH/NEI grant R01 EY023570
Program Number: 3986 Poster Board Number: D0045
Presentation Time: 3:45 PM–5:30 PM
Using stem cells to develop gene therapy for Batten Disease.
Budd A. Tucker1, Luke A. Wiley1, Kristin Anfinson1, Dalyz Ochoa1,
Jeaneen Andorf1, Louisa M. Affatigato1, Arlene V. Drack1, Edwin M.
Stone1, 2. 1Stephen A. Wynn Institute for Vision Research, Department
of Ophthalmology and Visual Sciences, University of Iowa, Iowa
City, IA; 2Howard Hughes Medical Institute, Department of
Ophthalmology and Visual Sciences, University of Iowa, Iowa City,
IA.
Purpose: Batten Disease, or juvenile neuronal ceroid lipofuscinosis
(JNCL), is a devastating autosomal recessive lysosomal storage
disorder characterized by early onset retinal degenerative blindness,
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
epilepsy, cognitive decline and premature death. The most common
mutation that causes JNCL is a one-kilobase genomic deletion in the
gene CLN3. The purpose of this study is use patient-specific induced
pluripotent stem cells (iPSCs) as a model system to investigate the
pathophysiology of CLN3-associated JNCL and to develop gene and
autologous cell-based therapeutic approaches for the treatment of this
devastating disease.
Methods: iPSCs were generated via transduction of human dermal
fibroblasts obtained from patients with molecularly confirmed CLN3associated JNCL using the transcription factors Oct4, Sox2, c-Myc
and KLF4. iPSC potency was characterized via rt-PCR, Western
blotting, immunocytochemistry (ICC), and embryoid body formation.
Pluripotent iPSCs were differentiated into retinal neurons using our
previously published protocol consisting of the stepwise addition
of neurotrophic growth factors, and inhibitors of Wnt, Notch and
BMP. A combination of rt-PCR, Western blotting, ICC, and confocal
microscopy were used to evaluate disease phenotype and efficacy of
the developed gene therapeutic approach.
Results: Patient-specific iPSCs were generated from dermal
fibroblasts obtained from two separate patients with CLN3-associated
Batten disease. After 60 days of differentiation, retinal neurons were
identified via immunocytochemical analysis targeted against OTX2,
BRN3, TUJ1, NF200, MAP2, recoverin and cone opsins. As observed
in vivo, differentiated retinal neurons harbored the one-kilobase
deletion in CLN3 and loss of normal full-length transcript and
protein. Accumulation of autofluorescent lysosomal storage material
was accompanied by intense Lysosomal-Associated Membrane
Protein-1 (LAMP-1) expression. Full-length CLN3 was cloned and
packaged into lentiviral vectors and tested for the ability to drive
expression of full-length CLN3 and reversion of JNCL phenotype.
Conclusions: Retinal neurons generated from JNCL patients
recapitulate cardinal aspects of the disease in vitro. Genetic correction
of the disease phenotype in patient-specific iPSCs will pave the way
for combined gene and autologous cell replacement-based therapeutic
trials for the treatment of Batten disease.
Commercial Relationships: Budd A. Tucker, None; Luke A.
Wiley, None; Kristin Anfinson, None; Dalyz Ochoa, None;
Jeaneen Andorf, None; Louisa M. Affatigato, None; Arlene V.
Drack, None; Edwin M. Stone, None
Support: NIH DP2OD007483, Stephen A Wynn Foundation,
Foundation Fighting Blindness
Program Number: 3987 Poster Board Number: D0046
Presentation Time: 3:45 PM–5:30 PM
Engineering Isogenic Best Vitelliform Macular Dystrophy Patient
iPS cell lines using TALEN Technology
Ruchi Sharma, Vaishakh Rajan, Qin Wan, Kapil Bharti. NEI, NIH,
BETHESDA, MD.
Purpose: Best Vitelliform Macular Dystrophy (BVMD) is an
autosomal dominant form of retinal degeneration caused by the
mutations in VMD2 gene. The disease is initiated by functional
defects in Retinal Pigment Epithelium (RPE) leading to photoreceptor
degeneration and vision loss. The purpose of this study was to
generate patient specific induced pluripotent stem (iPS) cell lines,
genetically engineer them using gene-specific TALEN to generate
isogenic controls, and differentiate them into authenticated RPE to
identify mechanisms involved in disease onset and progression.
Methods: Patient specific iPS cell lines were generated using nonintegrating Sendai-Virus containing SOX2, OCT3/4, KLF4, and
c-MYC factors. The lines were authenticated using pluripotency
factor gene expression (qRT-PCR and immunofluorescence) and
three germ layer differentiation assay. Passage 15 iPS cells were
genetically engineered using VMD2 gene specific TALEN, which
disrupts the open reading frame of both VMD2 isoforms. iPS cells
were transfected with TALEN plasmids using Neon Transfection
system using 24 different transfection parameters. Mutated clones
were screened using a PCR based assay.
Results: BVMD patient (Trp 24 to Cys) iPS cell line showed strong
expression of several pluripotency markers and differentiated into all
three germ layers. Out of twenty-four transfection parameters tested,
ten parameters gave rise to healthy iPS cell colonies. Sequencing of
PCR fragments from genomic DNA and cDNA showed that TALEN
disrupted VMD2 open reading frame by non-homologous end joining
thus resulting in the complete knockout of one VMD2 allele. This
leads to two isogenic controls, one WT-heterozygous and the other
mutant-heterozygous. BVMD (Trp 24 to Cys) iPS cells have been
successfully differentiated into RPE at high efficiency. Currently,
these isogenic iPS cell lines derived RPE are being used to determine
BVMD disease mechanisms.
Conclusions: Isogenic iPS cells provide genetically matched controls
for patient-derived cells thus allowing the possibility to identify
disease cellular endophenotypes in the absence of genetic modifier
effects. TALEN technology is a viable approach to generate gene
knock-out using non-homologous end joining. BVMD isogenic
iPS cell lines generated in this study will help better understand the
dominant role played by VMD2 mutant allele.
Commercial Relationships: Ruchi Sharma, None; Vaishakh
Rajan, None; Qin Wan, None; Kapil Bharti, None
Program Number: 3988 Poster Board Number: D0047
Presentation Time: 3:45 PM–5:30 PM
TRANSPLANTED mESC-DERIVED RETINAL
PROGENITORS DIFFERENTIATE TO MATURE
PHOTORECEPTORS IN VIVO, MIGRATE AND INTEGRATE
IN THE MICE RETINA
Marcela Garita, Francisco Díaz-Corrales, Slaven Erceg, Shomi
Bhattacharya. CELL THERAPY AND REGENERATIVE
MEDICINE, CABIMER, Sevilla, Spain.
Purpose: Retinal dystrophies characterized by the progressive
degeneration of photoreceptors are the leading causes of incurable
blindness. Due to the limited capacity of the mammalian retina
to regenerate using embryonic stem cells (ESC) as an unlimited
source to replenish the lost cells has represented a main objective
for the scientific community. Despite great advances in the field of
differentiation of ESC towards photoreceptors in the recent years,
few drawbacks remain unresolved. Such as, efficiency, purity of the
population and the constant worry that once differentiated, cells could
not be able to integrate into the host retina
Methods: We optimized a new protocol to differentiate mouse
ESC (mESC), involving the control of the oxygen tension to mimic
the retinal niche conditions, as well as the manipulation of key
signaling pathways involved during normal retinal development.
The retinal progenitor cells generated were subretinally transplanted
in order to evaluate their migration and integration capacities by
immunohistochemistry analysis
Results: Hypoxia increases the efficiency of differentiation
towards photoreceptors, but as well it improves the modeling of
retinogenesis in vitro, by decreasing the time necessary to acquire
each specific phenotype. Transcription factors associated to each
stage of retinal differentiation such as eye field (Rax, Six3), optic cup
(Pax6, Mitf, Chx10), photoreceptor precursors (Nrl, Crx) and even
mature photoreceptors (Rhodopsin, Recoverin and Opsin-S) were
upregulated earlier and their levels of expression were significantly
higher than those reached under normoxic conditions as determined
by qPCR. Moreover, when photoreceptor precursors derived from
mESC cells following our protocol under hypoxia were transplanted
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
into the subretinal space of wild type mice they differentiated mainly
towards Recoverin/Rhodopsin positive cells, migrate and integrate
into the host retina and in some cases acquired the morphology of
mature rods with formation of structures suggesting outer segments
Conclusions: Our results demonstrate that hypoxia increases
the yield of retinal phenotypes from mESC, including mature
phenotypes. Moreover, in vivo experiments demonstrated that
hypoxia promotes the survival of the grafted cells, allowing the
retinal progenitors to differentiate towards photoreceptors, migrate
and integrate in the mice retina
Commercial Relationships: Marcela Garita, None; Francisco
Díaz-Corrales, None; Slaven Erceg, None; Shomi Bhattacharya,
None
Program Number: 3989 Poster Board Number: D0048
Presentation Time: 3:45 PM–5:30 PM
Cell fusion following photoreceptor transplantation into the nondegenerate retina
Mandeep S. Singh, Sher A. Aslam, Isabel L. Duncan, Alona O.
Cramer, Alun R. Barnard, Robert E. MacLaren. Nuffield Laboratory
of Ophthalmology, University of Oxford, Oxford, United Kingdom.
Purpose: Photoreceptor transplantation could be a future treatment
for retinal degenerations. In end-stage retinal degeneration,
when no photoreceptor cells remain, the goal is to repopulate the
retina with transplanted photoreceptors in order to restore vision.
However, in certain forms of congenital stationary night blindness
and Leber congenital amaurosis, there are vast numbers of intact
host photoreceptors in the outer nuclear layer (ONL) despite visual
dysfunction. Hence, in these conditions, photoreceptor transplantation
into the well-formed host ONL could lead to fusion between donor
and host cells that would result in the artefactual appearance of
integrated cells. We aimed to investigate if transplanted photoreceptor
precursors would fuse with coexisting photoreceptors in nondegenerate hosts.
Methods: Photoreceptor precursors were obtained from neonatal
donor mice, in which rod and/or cone photoreceptors were
fluorescent. Transplantation was performed into hosts with an intact
ONL. Hosts were selected on the basis of differential fluorescent
marker expression, in order to facilitate the accurate tracking of
donor and host cells after the procedure. Histological analysis was
conducted 2–3 weeks after transplantation.
Results: ONL cells were identified in which the fluorescent markers
of the donor and host photoreceptors were colocalised in the same
cell. In addition, fluorescence was detected in cells in which they
were not originally restricted, indicating transfer of fluorescent
proteins between cells. The arrangement of fluorescent photoreceptor
cell bodies in colour-concordant ONL columns suggested that
fluorescence had been transferred to the host from donor cells located
in the subretinal space, which were in intimate contact with the host
outer segments.
Conclusions: Donor photoreceptors, when transplanted into a nondegenerate host retina, fuse with host photoreceptors in the ONL.
This results in the transfer of proteins between the cells, including
fluorescent markers. The appearance of well-integrated transplanted
photoreceptors in non-degenerate hosts is the result of a fusion
artefact. This phenomenon should be considered when designing
clinical trials of stem cell transplantation in the future.
Commercial Relationships: Mandeep S. Singh, None; Sher A.
Aslam, None; Isabel L. Duncan, None; Alona O. Cramer, None;
Alun R. Barnard, None; Robert E. MacLaren, None
Support: NIHR Biomedical Research Centre, the Wellcome Trust,
Medical Research Council, UK Department of Health, Special
Trustees of Moorfields Eye Hospital, the Royal College of Surgeons
of Edinburgh, Singapore National Medical Research Council
Program Number: 3990 Poster Board Number: D0049
Presentation Time: 3:45 PM–5:30 PM
Subpopulations of bone marrow mesenchymal stem cells exhibit
differential effects on delaying retinal degeneration
Peng Li1, 2, Haibin Tian1, 2, Zongyi Li1, 2, Li Wang1, 2, Chunpin Lian1, 2,
Qingjian Ou1, 2, Lixia Lu1, 2, Weiye Li1, 3, Guo-Tong Xu1, 2. 1Department
of Ophthalmology of Shanghai Tenth Hospital, and Tongji Eye
institute,, Tongji University School of Medicine, Shanghai, China;
2
Department of Regenerative Medicine and Stem Cell Research
Center, Tongji University School of Medicine, Shanghai, China;
3
Department of Ophthalmology, Drexel University College of
Medicine, Philadelphia, PA.
Purpose: Bone marrow mesenchymal stem cells (BMSCs) have been
shown therapeutic functions on retinal degeneration (RD). However,
BMSCs are heterogenous, a new strategy for BMSCs treatment of
RD by choosing appropriate subpopulations was investigated.
Methods: Two subsets of rat BMSCs, termed as rBMSC1 and
rBMSC2, were obtained by cloning method. Specific surface
markers were analyzed by flow cytometry, proliferating rate and
gene expression were carried out by MTT and RNA sequencing
analysis. Their differentiation abilities were confirmed by culturing
rBMSCs in adipogenic, osteogenic and chondrogenic differentiation
media. rBMSC1 and rBMSC2 were transplanted into subretinal
space of RCS rats, their therapeutic functions were confirmed
by retinal nuclear layer thickness, apoptotic photoreceptors and
electroretinogram.
Results: Both subpopulations expressed MSC marker CD29 and
CD90 with null hemacyte antigen CD11b and CD45 expression. On
the other hand, rBMSC1 showed higher proliferating rate, stronger
colony forming capacity, and more adipogenic and chondrogenic
potential than rBMSC2, whereas the latter exhibited increased
osteogenic ability. RNA sequencing analysis further showed the
differential gene levels relative to proliferation, differentiation and
immunoregulation in rBMSC1 and rBMSC2. After transplanted into
subretinal space of RCS rats, rBMSC1 showed stronger vision rescue
function as compared to rBMSC2, in terms of increased b¬-wave
amplitude, restored retinal nuclear layer thickness, and decreased
apoptotic photoreceptors, whereas the rescue function of rBMSC2
was essentially no better than the control.
Conclusions: This study provides more information about the
heterogeneity of BMSCs and a new strategy for BMSCs treatment of
RD by choosing appropriate subpopulations.
Commercial Relationships: Peng Li, None; Haibin Tian, None;
Zongyi Li, None; Li Wang, None; Chunpin Lian, None; Qingjian
Ou, None; Lixia Lu, None; Weiye Li, None; Guo-Tong Xu, None
Program Number: 3991 Poster Board Number: D0050
Presentation Time: 3:45 PM–5:30 PM
Intervention of ADSC with modified medium on RCS rat
Chunpin Lian1, 2, Zongyi Li1, 2, Hui Lou1, 2, Li Wang1, 2, Peng Li1, 2,
Haibin Tian1, 2, Lixia Lu1, 2, Weiye Li1, 3, Guotong Xu1, 2. 1Department
of Ophthalmology of Shanghai Tenth People`s, Tongji Eye institute,
Tongji University School of Medicine(TUSM), Shanghai, China;
2
Department of Regenerative Medicine and Stem Cell Research
Center, Tongji University School of Medicine(TUSM), Shanghai,
China; 3Department of Ophthalmology, Drexel University College of
Medicine, Philadelphia, PA.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Retinal degeneration (RD) is one of major cause of
blindness in the world and no effective therapeutic strategies were
available nowadays. Stem cell-based therapy holds the promise of
curing retinal degenerative process. The protective effect of adiposederived stem cells (ADSCs) cultured in modified medium has been
investigated.
Methods: ADSCs (P1) were grown in modified medium and labeled
with lentivirus-GFP. The secretion of HGF and PEDF from ADSCs
was determined by ELISA kits. Proliferation ability was measured by
MTT method and colony-forming unit test. The therapeutic functions
of the modified-medium-cultured ADSCs (P4) were confirmed by
transplantation into subretinal space of Royal College of Surgeons
(RCS) rats at postnatal 3 weeks of age. RCS rats were divided in
three equal groups (n=6 per group), including ADSC (DMEM with
low glucose), ADSC (modified medium) and untreated control
groups. Q-PCR and TUNEL staining were performed to detect
neurotrophic factor gene expression and apoptotic photoreceptors.
ERG was performed to evaluate the improvement of visual function.
Results: Compared with DMEM-LG medium cultured ADSCs,
ADSCs cultured in modified medium possess higher proliferation
ability, and show an increased gene and protein expression levels
of HGF and PEDF. In the meantime, the inflammation factor IL-6
expression was significantly decreased. After transplantation into the
subretinal space of RCS rat, ADSCs cultured in modified medium
show an enhanced rescue effects compared to ADSCs cultured in
DMEM with low glucose medium, including increased b-wave
amplitude, restored retinal nuclear layer thickness, and decreased
apoptotic photoreceptors.
Conclusions: The gene expression and proliferation ability can be
affected by culture condition. ADSCs in modified medium show a
high proliferation ability, neurotrophic factor gene expression level
and decreased expression level of inflammation factor IL-6. The
enhanced rescue effects in modified-medium-cultured ADSC may
due to the stronger paracrine abilities. These data may provide useful
clues for further clinical intervention in RD patients.
Commercial Relationships: Chunpin Lian, None; Zongyi Li,
None; Hui Lou, None; Li Wang, None; Peng Li, None; Haibin
Tian, None; Lixia Lu, None; Weiye Li, None; Guotong Xu, None
Program Number: 3992 Poster Board Number: D0051
Presentation Time: 3:45 PM–5:30 PM
In-depth characterisation of retinal pigment epithelium (RPE)
cells derived from human induced pluripotent stem cells (iPSC)
Caroline Brandl1, 2, Stephanie Zimmermann2, Felix Grassmann2,
Vladimir Milenkovic3, Andrea Milenkovic2, Johanna Käsbauer2, Ute
Hehr2, Christian H. Wetzel3, Horst Helbig1, Bernhard H. Weber2.
1
Ophthalmology, University Hospital Regensburg, Regensburg,
Germany; 2Institute of Human Genetics, Regensburg, Germany;
3
Psychiatry and Psychotherapy, Molecular Neuroscience, University
of Regensburg, Regensburg, Germany.
Purpose: To establish and comprehensively characterise retinal
pigment epithelium (RPE) cells derived from adult human dermal
fibroblasts via induced pluripotent stem cell (iPSC) technology.
Methods: Adult human dermal fibroblast cultures were established
from skin biopsy material and subsequently reprogrammed following
polycistronic lentiviral transduction with OCT3/4, Sox2, Klf4 and
l-Myc. Chromosomal integrity was assessed by karyotyping. RPE
cell differentiation was achieved by induction with RPE medium
enriched for nicotinamide and Activin A. After 8 weeks, pigmented
clusters of RPE cells were manually excised and subcultured. Human
iPSCs were characterised by RT-PCR expression of specific stem
cell markers and immunofluorescence. IPSC-derived RPE cells
were characterised by RT-PCR expression of mature RPE markers,
confocal microscopy, scanning electron microscopy (SEM) and
functional analysis, the latter including feeding experiments with
porcine photoreceptor outer segments (POS) and measurements of
transepithelial resistance (TER).
Results: Fibroblast-derived human iPSCs showed typical
morphology and regular karyograms. Furthermore, they revealed
distinctive stem cell marker properties based on RNA- and
protein-expression profiling. Subsequently, human iPSCs were
differentiated into pigmented clusters reminiscent of RPE cells.
These cells maintained typical hexagonal RPE-morphology
during subcultivation. Starting at passage 6 replicative senescence
increased. RNA expression of mature PRE markers RPE65, RLBP
and BEST1 were found in comparison to human iPSCs. Confocal
microscopy demonstrated localisation of BEST1 at the basolateral
plasma membrane while SEM demonstrated typical microvilli at the
apical side of RPE cell. With regard to functional aspects, iPSCderived RPE cells phagocytosed and shredded POS. Finally, TER
measurements showed a significant increase and maintained high
levels of TER indicating functional formation of tight junctions.
Conclusions: Our data demonstrate the successful reprogramming
of human adult skin biopsy-derived fibroblasts to iPSCs and their
differentiation to RPE cells structurally and functionally indistinct
from native RPE cells.
Commercial Relationships: Caroline Brandl, None; Stephanie
Zimmermann, None; Felix Grassmann, None; Vladimir
Milenkovic, None; Andrea Milenkovic, None; Johanna Käsbauer,
None; Ute Hehr, None; Christian H. Wetzel, None; Horst Helbig,
Novartis (F); Bernhard H. Weber, Novartis (F)
Support: Novartis (PN3625340)
Program Number: 3993 Poster Board Number: D0052
Presentation Time: 3:45 PM–5:30 PM
Simple generation of self-forming neural retina and RPE cells
from confluent human iPS cells
Olivier Goureau1, Sacha Reichman1, Angélique Terray1, Amélie
Slembrouck1, Celine Nanteau1, Gael Orieux1, Christelle Monville3,
Jose A. Sahel1, 2. 1Institut de la Vision, UPMC Univ Paris 06, UMR_S
968; INSERM U968; CNRS UMR_7210, Paris, France; 2Centre
Hospitalier National d’Ophtalmologie des Quinze-Vingts, INSERMDHOS CIC 503, Paris, France; 3I-STEM, INSERM; UEVE U861,
Evry, France.
Purpose: For retinal cell therapy using human induced pluripotent
stem cells (hiPSCs), the current challenge is to improve the
generation of their therapeutic derivatives by eliminating time- and
labor-consuming manual steps to allow large-scale production
complying with certain criteria such as safety, efficiency,
reproducibility and low production cost. Here, we developed a new
retinal differentiation method using confluent hiPSCs bypassing
embryoid body EB formation and selection and the use of exogenous
molecules, coating or Matrigel.
Methods: Integration-free hiPSCs were generated via nucleofection
of adult human dermal fibroblasts, with plasmids coding for the
transcription factors OCT4, NANOG, SOX2, LIN28, KLF4 and
C-MYC. Confluent hiPSCs were directed toward a retinal lineage
in a serum free proneural medium containing N2 supplement.
Emergent neural retina (NR)-like structures were isolated and
cultured in floating conditions for their maturation. Capacity for
retinal differentiation was determined by immunohistochemistry and
qRT-PCR time course analysis triggering specific developmental and
mature retinal markers.
Results: In two weeks, confluent hiPSCs are able to generate
simultaneously RPE cells and self-forming NR-like structures
containing multipotent retinal progenitor cells (RPCs). Floating
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
cultures of isolated neuroretinal tissue enabled the differentiation of
RPCs into all types of retinal cells in a sequential manner consistent
with in vivo vertebrate retinogenesis. Indeed, early-born retinal cells
were identified as early as 21 days in culture (i.e. ganglion, amacrine
and horizontal cells), and late-born retinal cells appeared after 35
days (i.e. photoreceptor, Muller glial and bipolar cells). Interestingly,
early inhibition of the Notch pathway led to massive differentiation
of RPCs into photoreceptor precursors. hiPSC-derived RPE cells can
be amplified while retaining their phenotype close to their in vivo
state.
Conclusions: We thus propose an innovative process providing an
easy and scalable approach to generate large numbers of mitotic
RPCs and of both RPE cells and precursors of photoreceptors
needed for regenerative medicine or drug screening purposes. Our
retinal differentiation also provides an accessible in vitro model to
investigate mechanisms involved in human retinogenesis and retinal
diseases.
Commercial Relationships: Olivier Goureau, None; Sacha
Reichman, None; Angélique Terray, None; Amélie Slembrouck,
None; Celine Nanteau, None; Gael Orieux, None; Christelle
Monville, None; Jose A. Sahel, None
Support: ANR [GPiPS: ANR-2010-RFCS005], Investissements
d’Avenir programme [ANR-11-IDEX-0004-02] in the frame of the
LABEX LIFESENSES [ANR-10-LABX-65].
Program Number: 3994 Poster Board Number: D0053
Presentation Time: 3:45 PM–5:30 PM
A Stochastic Simulation to Determine Stem Cell Localization in
Retinal Vasculature
Howard C. Ray1, Bruce Corliss1, Stephen Cronk1, Paul A. Yates1, 2,
Shayn Peirce1, 2. 1Biomedical Engineering, University of Virginia,
Charlottesville, VA; 2Ophthalmology, University of Virginia,
Charlottesville, VA.
Purpose: The purpose of this work was to create an in silico Monte
Carlo model that predicts the expected association of intravitreally
injected adipose-derived stem cells (ASCs) with retinal vasculature,
assuming a random distribution of cells. These predictions were then
compared to experimental measurements of ASC associations with
in vivo retinal microvessels. This comparison allowed us to validate
this Monte Carlo method as a means for determining whether the
observed in vivo data is due entirely to random chance or might
suggest preferential adhesion or migration of injected ASCs towards
the retinal microvasculature.
Methods: Microvessel network architecture in the model is derived
from confocal images of excised murine retinae labeled with lectin
to visualize the vascular endothelium, as described in more detail
below. The simulation records the percent of ASCs that overlap
with vascular networks for each trial, then repeats the process and
displays the average percent of the ASC population that co-localizes
with the vasculature across all of the trials. A histogram is generated
to show the distance each ASC is away from the nearest vasculature
landmark.
Results: To determine the efficacy of the simulation, we used
fluorescent confocal images (20X magnification) of retinal
wholemounts. Eyes were previously injected intravitreally with DiI
labeled murine ASCs in eight week old Akimba or Akita mice. Eyes
were harvested 1 month later, retinal wholemounts prepared, and
retinal vasculature labeled with lectin. ASC association with the
vasculature was determined through blind counting the percentage
of murine ASCs contacting the vasculature. Using a Bland-Altman
analysis, we showed that there was no difference between the Monte
Carlo simulation and the blind counting method to measure the
percent of intravitreally murine ASCs contacting retinal vasculature
(Figure 1).
Conclusions: Our results suggest the Monte Carlo simulation
is a valuable stochastic simulation to determine the number of
intravitreally injected ASCs that make contact with retinal vasculature
purely by chance. The simulation can be extended to other cellular
applications in the eye to determine whether cell location and colocalization is caused by random chance or endogenous directional
signals.
Figure 1. Bland-Altman analysis between blind counting and the
Monte Carlo simulation in measuring percent of ASCs contacting
retinal vasculature.
Commercial Relationships: Howard C. Ray, None; Bruce Corliss,
None; Stephen Cronk, None; Paul A. Yates, Genentech/Roche
(C), RetiVue, LLC (I), RetoVue, LLC (E), U.S. Provisional Patent
Application Serial No. 61/684,375 (P); Shayn Peirce, None
Program Number: 3995 Poster Board Number: D0054
Presentation Time: 3:45 PM–5:30 PM
Co-culture of stem cell derived retinal progenitors and retinal
pigment epithelium promotes tissue maturity
Peter Y. Zhao1, Shaomin Peng1, Lilangi Ediriwickrema1, Caihong
Qiu2, Katherine J. Davis1, Ron A. Adelman3, Lawrence J. Rizzolo1.
1
Surgery/Ophthalmology, Yale University, New Haven, CT; 2Cell
Biology, Yale University, New Haven, CT; 3Ophthalmology & Visual
Science, Yale University, New Haven, CT.
Purpose: To restore vision, stem cell therapies for retinal
degenerations must address the loss of both photoreceptors and
retinal pigment epithelium. To explore the effects of each tissue layer
on the other’s maturation, we co-cultured human embryonic stem
cell derived retinal progenitor cells (hESC-RPC) with retinal pigment
epithelium (hESC-RPE).
Methods: Cultures were derived from the H9 human embryonic stem
cell line using modifications of previously published techniques. To
generate hESC-RPC, H9 were seeded as clusters to polycaprolactone
(PCL) scaffolds and cultured in retinal differentiation media.
hESC-RPE were expanded as monolayers on laminin-coated
Transwell filters in serum-free media, and adapted to the retinal
differentiation media for co-culture. In the co-culture group, hESCRPC cultures were placed on top of hESC-RPE monolayers during
the retinal differentiation protocol. For controls, cultures were
maintained separately in retinal differentiation media. Transepithelial
electrical resistance (TER) was monitored over time to assess
RPE integrity and function. After 2-4 weeks, co-cultured tissue
layers were separated and compared to controls by RT-PCR and
immunofluorescence.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Results: Neural retinal marker mRNAs were expressed by hESCRPC in both monoculture and co-culture. Co-cultures expressed
several of these markers at higher levels, including Crx and
Rhodopsin. Immunofluorescence revealed multilayered clusters
positive for markers including Otx2, Crx, and Recoverin. In cocultures, these markers localized to the surface opposing the RPE.
An RT-PCR array for monitoring RPE maturation showed that
co-cultured hESC-RPE was more mature. In addition, co-cultured
hESC-RPE maintained a high TER in retinal differentiation medium,
while in controls the TER decreased after 2-4 weeks.
Conclusions: Co-culture increases the maturation of both hESC-RPC
and hESC-RPE, underlining the interdependence of these tissues.
Secretions of the RPE and contact with the RPE could supplement
or replace media as promoters of RPC differentiation, facilitating the
creation of a transplantable tissue.
Commercial Relationships: Peter Y. Zhao, None; Shaomin
Peng, None; Lilangi Ediriwickrema, None; Caihong Qiu, None;
Katherine J. Davis, None; Ron A. Adelman, None; Lawrence J.
Rizzolo, None
Support: NIH CTSA-TL1 TR000141 (PYZ), National Natural
Science Foundation of China 30772381 (SP), Newman’s Own
Foundation (RAA), Leir Foundation (RAA), Connecticut Stem Cell
Research Fund 10SBC02 (LJR)
Program Number: 3996 Poster Board Number: D0055
Presentation Time: 3:45 PM–5:30 PM
Silane-modified substratum improves cell attachment of human
embryonic stem cell-derived retinal pigment epithelial cells
Kati M. Juuti-Uusitalo1, 3, Anni Sorkio1, 3, Elli Käpylä2, 3, Shokoufeh
Teymouri2, 3, Kimmo Lahtonen4, Leena Vuori4, Mika Valden4,
Heli Skottman1, 3, Minna Kellomäki2, 3. 1Institute of Biomedical
Technology, Univeristy of Tampere, Tampere, Finland; 2Dept. of
Electronics and Communications Engineering, Tampere University
of Technology, Tampere, Finland; 3BioMediTech, Tampere, Finland;
4
Surface Science Laboratory, Tampere University of Technology,
Tampere, Finland.
Purpose: In in vivo assessments, such as live cell imaging, it would
be beneficial to grow and assess human embryonic stem cell-derived
retinal pigment epithelial (hESC-RPE) cells on thin, transparent and
rigid surfaces such as cover glasses. Thus, the objective of this study
was to assess how silanization with 3-aminopropyltriethoxysilane
(APTES) and 3-(trimethoxysilyl)propyl methacrylate (MAPTMS) or
Ormocomp® affects the surface properties of glass, protein binding
and finally the maturation of hESC-RPE cells.
Methods: Borosilicate glass coverslips were silanized either with
commercially available silanizing agents, APTES or MAPTMS, or
Ormocomp®. The properties of the surface treated coverslips were
assessed with contact angle measurements, X-ray photoelectron
spectroscopy (XPS) and atomic force microscopy (AFM). Lastly, the
cell adherence and proliferation were evaluated by culturing hESCRPE cells on collagen IV coated untreated or silane-treated surfaces
for 42 days.
Results: In the contact angle measurements, there was no difference
between the untreated, APTES or MAPTMS treated surfaces, but the
Ormocomp®-treatment induced a statistically significant increase
in the hydrophobicity. XPS results indicated that the Ormocomp®
treatment increased the amount of C-O and C=O bonds on the
glass surface. All the silane treatments increased surface roughness
detected in AFM, although only with Ormocomp® the change was
statistically significant. The protein binding test confirmed that the
Ormocomp® treated surfaces bound more collagen IV than APTES
or MAPTMS treated surfaces. Finally, all the silane treatments
increased the number of attached cells when compared to unsilanized
substrata. The highest cell numbers were detected on Ormocomp®
treated surfaces.
Conclusions: This study clearly demonstrated that Ormocomp®
treatment increased the surface hydrophobicity, surface roughness
and collagen IV binding compared to the commonly used commercial
silanizing agents APTES and MAPTMS. Furthermore, when
comparing to the unsilanized or APTES or MAPTMS treated
substrata, the Ormocomp® treatment had the most significant effect
in augmenting hESC-RPE cell attachment. We can conclude that the
Ormocomp® silanization promotes the attachment of hESC-RPE
cells on glass, thus enabling the use of microscopic live cell imaging
methods also for hESC-RPE cells.
Commercial Relationships: Kati M. Juuti-Uusitalo, None; Anni
Sorkio, None; Elli Käpylä, None; Shokoufeh Teymouri, None;
Kimmo Lahtonen, None; Leena Vuori, None; Mika Valden, None;
Heli Skottman, None; Minna Kellomäki, None
Program Number: 3997 Poster Board Number: D0056
Presentation Time: 3:45 PM–5:30 PM
Functional Analysis of Human Protein Induced Pluripotent Stem
Cell-derived Retinal Pigment Epithelium
Jie Gong, Mark A. Fields, Ernesto F. Moreira, Yiannis Koutalos,
Zsolt Ablonczy, Lucian V. Del Priore. Ophthalmology, MUSC Storm
Eye Institute, Charleston, SC.
Purpose: Retinal pigment epithelium (RPE) derived from human
induced pluripotent stem (iPS) cells (iPS-RPE) may provide a
promising source of cells for transplantation. Physiological tests
are required to establish the ability of iPS to function as RPE prior
to using these cells as a source of replacement RPE. Critical RPE
functions include phagocytosis of shed photoreceptor rod outer
segments (ROS) and the barrier function of the tight junction. Herein
we analyze the function of these ips-derived RPE as measured by
phagocytosis and TER (trans epithelial resistance).
Methods: Protein-induced iPS (piPS) from System Biosciences (SBI,
Mountain View, CA) were seeded directly onto Matrigel coated plates
in differentiation medium. From day 0-6, a combination of Noggin,
DKK1, IGF-1 and bFGF were added to the base medium. From day
6-14, Activin A, SU5402 and VIP were added. Subsequently, purified
piPS-derived RPE were digested and expanded in RPE medium.
Adult bovine rod outer segments were labeled with FITC, fed to
the PiPS-RPE cells and analyzed by fluorescent microscopy and
flow cytometry. These RPE monolayers were cultured on permeable
transwell membranes were measured TER by using an epithelial
voltohmmeter.
Results: Undifferentiated piPS cells expressed all pluripotent
embryonic cell markers SSEA4, TRA-1-60, OCT4, and TRA1-81, and no RPE markers were detected. After 30 days, cells
formed a hexagonal monolayer exhibiting an RPE phenotype and
pigmentation. Monolayer cell domes were observed, suggesting
apical-basal fluid transportation. Differentiated piPS-RPE cells
expressed mature RPE markers Bestrophin, MITF and RPE65, and
the tight junction marker ZO-1. Transepithelial resistance was tested
at an average of 154.8 + 0.23 Ω*cm2. Phagocytosis function test
showed that piPSC-RPE also ingested fluorescently-labeled isolated
bovine rod outer segments. Flow cytometry of piPSC-RPE revealed
90% of cells contained fluorescently labeled outer segments.
Conclusions: PiPS-RPE exhibited similar physiological properties
to those of adult RPE cells. Additional studies are required to analyze
RPE cell function further, including retinoids uptake and processing;
and analyzing cell survival and function in animal models of retinal
degeneration.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Jie Gong, None; Mark A. Fields,
None; Ernesto F. Moreira, None; Yiannis Koutalos, None; Zsolt
Ablonczy, None; Lucian V. Del Priore, None
Support: Foundation Fighting Blindness, Research to Prevent
Blindness
Program Number: 3998 Poster Board Number: D0057
Presentation Time: 3:45 PM–5:30 PM
EXAMINATION OF GAP JUNCTION INTERCELLULAR
COMMUNICATION IN HUMAN PLURIPOTENT STEM
CELL-DERIVED RETINAL PIGMENT EPITHELIUM
Clara Iglesias1, Akshayalakshmi Sridhar1, Sarah Ohlemacher1, Jason
S. Meyer1, 2. 1Biology, IUPUI, Indianapolis, IN; 2Stark Neuroscience
Research Institution, Indiana University, Indianapolis, IN.
Purpose: Retinal pigment epithelial (RPE) cells serve to support
retinal photoreceptors and are adversely affected in many blinding
disorders such as AMD. While many current strategies are focused
on the replacement of RPE cells using hPSCs, the demonstration
of the proper functionality of these stem cell-derived RPE remains
incomplete. Previous studies have identified gap junctions as
important for communication between RPE cells in vivo. However,
there are limited studies testing the functionality of these cells derived
from a hPSC source. Thus, efforts were undertaken to examine the
role of gap junction proteins in intercellular communication in hPSCderived RPE.
Methods: To initiate differentiation hPSCs were grown in a medium
consisting of DMEM/F12 (1:1) containing 20% knockout serum
replacement, L-glutamine, and MEM non-essential amino acids.
Within eight weeks of differentiation, RPE cells were readily
identified by their hexagonal cobblestone shape and accumulated
pigmentation. RPE was isolated by microdissection and expanded in
the presence of FGF-2, EGF and heparin. To confirm the identity of
these cells, the expression of genes and proteins characteristic to RPE
were analyzed using both RT-PCR and immunocytochemistry.
Results: hPSC-derived RPE cells expressed characteristic markers,
such as MITF, RPE65 and ZO-1, as assessed by RT-PCR and
immunocytochemistry. The expression of gap junction genes within
these hPSC-derived RPE cells was then examined by RT-PCR, in
which the most prevalent gap junction gene was found to be GJA1,
encoding for the protein Cx43. Subsequent immunocytochemistry
analysis confirmed the presence of Cx43 at points of contact between
neighboring RPE cells. Current studies are underway to test the
ability of hPSC-derived RPE to effectively communicate through gap
junction channels.
Conclusions: hPSC-derived RPE are demonstrated to possess a large
complement of native RPE-associated characteristics, including the
expression of gap junction proteins. The presence of these proteins
will likely be important for the maturation and functionality of these
RPE cells. The results of these studies serve to provide a greater
understanding of hPSC-derived RPE functionality, which may be
essential for the development of translational applications for these
cells.
Commercial Relationships: Clara Iglesias, None; Akshayalakshmi
Sridhar, None; Sarah Ohlemacher, None; Jason S. Meyer, None
Support: BrightFocus Foundation Grant# G2012027, Fight For Sight
Program Number: 3999 Poster Board Number: D0058
Presentation Time: 3:45 PM–5:30 PM
Comparative analysis of retinal layers after subretinal stem cell
implantation in Yucatan mini-pigs
Francisco R. Stefanini1, 2, Michael J. Koss1, 3, Paulo Falabella1,
2
, Marcel Pfister1, David R. Hinton3, Biju B. Thomas3, Padmaja
Thomas1, Dennis O. Clegg4, Mark S. Humayun3. 1Doheny Eye
Institute, Los Angeles, CA; 2Ophthalmology, Federal University
of Sao Paulo, Sao Paulo, Brazil; 3Ophthalmology, University of
Southern California, Los Angeles, CA; 4Univ of California-Santa
Barbara, Santa Barbara, CA.
Purpose: To compare the thickness of the retinal layers in
hematoxylin and eosin (H&E) stained histologic tissue sections after
surgery for implantation of human embryonic stem cell-derived
retinal pigment epithelium (hESC-RPE) monolayer in Yucatan minipigs.
Methods: Five Yucatan mini-pigs underwent vitrectomy with the
creation of a localized subretinal BSS bleb before the implantation
of hESC-RPE polarized cells cultured on parylene membranes into
the subretinal space. One month after surgery, Aperio Scanscope
landmark analysis from a standardized area of the retina was
performed. It included the measurement of ganglion cells (GCL),
nerve fiber (NFL), inner nuclear (INL), inner plexiform (IPL), outer
nuclear (ONL), outer plexiform layer (OPL), photoreceptors (PR),
and total retina thickness including the RPE (RT) in the anatomical
site of the bleb (BS), the site of implant (IS) and the non-bleb
retina (NBS). Three different areas per site were analyzed at 40x
magnification from 3 H&E sections.
Results: All animals reached the 1 month follow-up with thorough
in vivo and ex vivo documentation. The implant region had an
artifactual retinal detachment in all histologic sections, thus the PR
and RT was not measureable. Considering the distance between the
GCL/NFL to the ONL, the average overall thickness was as follows:
BS (166.5 ± 12.7 μm), NBS (138.9 ± 9.9 μm), IS (147.1 ± 15.1 μm).
IPL thickness was as follows: NBS (33.4 ± 6.8 μm) in BS (43.0 ±
7.3 μm) and IS (44.8 ± 7.6 μm). The ONL was as follows: IS (18.5 ±
5.2),BS (37.7 ± 11.1) and NBS (35.5 ± 3.6).
Conclusions: Overall there were minimal changes in the retina
in the different sites after surgical implantation. The procedure of
BSS infusion to create a subretinal pocket into which a hESC-RPE
polarized cells cultured on parylene membranes is implanted was
well tolerated. More studies are ongoing to further evaluation of this
approach.
Commercial Relationships: Francisco R. Stefanini, None; Michael
J. Koss, None; Paulo Falabella, None; Marcel Pfister, None; David
R. Hinton, None; Biju B. Thomas, None; Padmaja Thomas, None;
Dennis O. Clegg, None; Mark S. Humayun, Baush & Lomb (C)
Support: California Institute for Regenerative Medicine (CIRM):
DRI-01444; National Eye Institute (NEI): #EY03040
Program Number: 4000 Poster Board Number: D0059
Presentation Time: 3:45 PM–5:30 PM
Retrobulbar transplantation of mouse Adipose tissue derived
stem cells rescues NaIO2 induced Retinal Degeneration Mouse
Model
Sang-Joon Lee1, Jeong Hoon Heo1, Hyun Woong Kim2, YehChan Ahn3. 1Ophthalmology, Kosin University, Busan, Republic
of Korea; 2Ophthalmology, Inje University, Busan, Republic of
Korea; 3Biomedical Endgineering and center for marine integrated
Biomedical technology, Pukyong National University, Busan,
Republic of Korea.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: We investigate whether retrobulbar transplantation of
mouse adipose tissue derived stem cells (mADSC) have a rescue
effect on the retinal degeneration of mouse induced by NaIO3.
Methods: mADSCs which were harvested and characterized by
surface markers (CD 105, CD 73, and CD 90 (≥95 %), and lack
expression of CD 45, CD 34, CD 14 or CD 11b, CD 79) were stained
by BrdU before transplantation. Retinal degeneration was induced
by intraperitoneal injection of NaIO3 (50 mg/mL) on 7 C57BL6
mice. 3 days after the induction, mADSCs (2 x 104 cells/uL) were
transplanted on the retrobulbar space of the left eye (treated group)
and normal saline was also injected on the retrobulbar space of
the right eye (control group). 3 weeks after transplantation, 7 mice
were sacrificed and enucleated. The area of photoreceptor outer
segment(OS), outer nuclear layer(ONL), and inner nuclear layer(INL)
in 10 um length of the retina located within 2 disc diameter from the
disc were analyzed in the control group, treated group, and normal
control group. Immunohistochemistry of BrdU, GFAP, carbindin,
vimentain, CRALBP, SDF-1, and CXCR4 was done.
Results: The areas of OS, ONL, and INL in the normal C5BL6 mice
were 31.28 um2, 46.30 um2, and 29.69 um2. The areas of NaIO2
injected mice retina thicknesses which were OS; 2.19 um2, ONL;
5.69 um2, INL 20.66 um2 decreased significantly. In the mADSCs
treated group, the areas of OS (8.57 um2), ONL (13.93 um2), and
INL (24.97 um2) were significantly different comparing to the control
group. The BrdU stain was not observed in the intraocular tissue
including retina. The retina of the treated group were stained with
SDF-1 staining more than the control group.
Conclusions: The retrobulbar transplantation of mADSCs have
rescue effect for the NaIO3 damaged retina, which could imply
indirect effect of the transplanted stem cells on the degenerated
retina.
Commercial Relationships: Sang-Joon Lee, None; Jeong Hoon
Heo, None; Hyun Woong Kim, None; Yeh-Chan Ahn, None
Then, 5x5 mm neuroretinal explants were obtained from porcine
eyes, as previously described by our group (Fernandez-Bueno
et al. 2008), and explanted on the superior surface of the TWM.
These three physically separated cellular layers were co-cultured in
Neurobasal A/DMEM (1:1) medium during 6 days. In parallel, cells
and neuroretinas were individually culture as controls. RPE and
AD-MSCs viability, proliferation and/or phenotype were determined.
Cryostat neuroretina sections were immunostained to evaluate retinal
degeneration process
Results: RPE and AD-MSCs adequately survived and proliferated
in the experimental condition. AD-MSCs showed significantly
decreased alpha-SMA (fibroblasts) and increased recoverin
(photoreceptors) immunoexpression, while CK8/18 (epithelial) and
nestin (stem cells) immunoexpressions were similar to respective
controls. Neuroretina immunoexpression of GFAP and CRALBP
(reactive gliosis) in the experimental condition was similar to
spontaneously degenerated controls at 6 days
Conclusions: The triple-layer co-culture model developed, not
previously described, could mimic an ex vivo subretinal space to
evaluate cell therapy potential in retinal degeneration. In this model,
AD-MSCs potentially expressed retinal cell proteins, however, were
unable to preserve retinal tissue from spontaneous degeneration. This
finding suggests that external biomolecules are necessary to reinforce
AD-MSCs neuroprotective effects on degenerated neuroretina
Commercial Relationships: Ivan Fernandez-Bueno, None; David
Rodriguez-Crespo, None; Salvatore Di Lauro, None; Amar K.
Singh, None; Maite Garcia-Gutierrez, None; Manuel GarrosaGarcía, None; Jose-Carlos Pastor, None
Support: National Plan of I+D+I 2008-2011 and ISCIIISubdireccion General de Evaluación y Fomento de la Investigación
(PS09/00938) (MICINN) co-financed by FEDER; Castilla y León
Regenerative Medicine and Cell Therapy Network Center; and JCYL
BIO/39/VA26/10 and VA386A12-2, Junta de Castilla y León; Spain.
Program Number: 4001 Poster Board Number: D0060
Presentation Time: 3:45 PM–5:30 PM
A Triple-layer Co-culture Model Of Neuroretina, RPE
And Adipose-MSCs To Evaluate Cell Therapy In Retinal
Degeneration
Ivan Fernandez-Bueno1, 2, David Rodriguez-Crespo1, Salvatore
Di Lauro1, 3, Amar K. Singh1, Maite Garcia-Gutierrez1, 4, Manuel
Garrosa-García5, Jose-Carlos Pastor1, 3. 1IOBA (Eye Institute),
University of Valladolid, Valladolid, Spain; 2Regenerative Medicine
and Cell Therapy Networking Center of “Castilla y Leon”, Valladolid,
Spain; 3Ophthalmology, Hospital Clinico Universitario, Valladolid,
Spain; 4CIBER de Bioingenieria, Biomateriales y Nanomedicina
(CIBER-BBN), Carlos III National Institute of Health, Valladolid,
Spain; 5Cellular Biology, Histology and Pharmacology, University of
Valladolid, Valladolid, Spain.
Purpose: To develop and standardize a co-culture model with three
layers, neuroretina, retinal pigment epithelium (RPE) and adiposederived mesenchymal stem cells (AD-MSCs), for evaluating ADMSCs potential in retinal degeneration
Methods: This study followed the tenets of the Declaration of
Helsinki and was approved by the IRC of the University of Valladolid
(Spain). RPE cells from porcine eyes and AD-MSCs from human
lipo-aspirates were isolated, maintained in culture and characterized
as previously described by our group (Srivastava et al., 2011; Singh
et al., 2013). RPE cells were seeded (30,000 cells/cm2) on the
bottom of Transwell culture plates. AD-MSCs were seeded (30,000
cells/cm2) on the inferior surface of Transwell membranes (TWM).
TWM with growing AD-MSCs on its inferior surface were inserted
into culture plates with growing RPE cells on its bottom surface.
Program Number: 4002 Poster Board Number: D0061
Presentation Time: 3:45 PM–5:30 PM
Vascular stem cell therapy of the diabetic retina with COMPAng1 and endothelial progenitor cells
Judd M. Cahoon1, Paul R. Olson1, Christina O2, Reinhold J. Medina2,
Alan W. Stitt2, Balamurali Ambati1. 1Ophthalmology and Visual
Sciences, University of Utah, Salt Lake City, UT; 2Centre for Vision
and Vascular Science, Queen, Belfast, United Kingdom.
Purpose: Retinal ischemia is a leading cause of blindness. Diabetic
retinopathy is the most common vasodegenerative retinopathy.
Restoring vascular homeostasis and replacing lost endothelial cells
(reparative angiogenesis) could reduce the neurovascular damage
that occurs in diabetic retinopathy. Outgrowth endothelial cells
(OECs) are a specific subtype of endothelial progenitor cell that
have the potential to reintegrate into damaged retinal vascular beds
and promote reparative angiogenesis. The purpose of this study was
to determine whether a novel angiopoietin-1 analog, COMP-Ang1,
could enhance OEC vasculogenic properties in vitro and increase
OEC integration into the diabetic retina.
Methods: OECs were harvested from the mononuclear layer
of donated cord blood and plated on collagen-coated plates
supplemented with EBM-2 MV media. OECs were exposed to
increasing doses of COMP-Ang1, or control (PBS), and assessed for
migration (scratch-migration assay), tube formation (3D Matrigel
assay), and intracellular signaling pathways (Western blot). Finally,
labeled OECs were administered via intravitreal injection into the
eyes of 7 month old diabetic mice treated two weeks previously with
an adenovirus expressing COMP-Ang1 (AAV2.COMP-Ang1) or
control (AAV2.GFP). Three days later retinas were harvested and
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
analyzed with confocal microscopy to assess OEC integration into
the retinal vasculature.
Results: COMP-Ang1 increased OEC migration speed and tube
formation in a dose-dependent manner compared to control treated
OECs (P < 0.001). Additionally, compared to control, COMP-Ang1
increased Akt phosphorylation, an important downstream effector of
the Tie2 receptor, in OECs (P < 0.001). Preliminary results suggest
that diabetic mice treated with COMP-Ang1 had increased OEC
integration into the retinal vasculature compared to control treated
mice.
Conclusions: COMP-Ang1 increases vasculogenic properties of
OECs and may be of use in therapeutic angiogenic strategies to
treat retinal ischemic diseases (e.g., diabetic retinopathy). Future
studies will determine the effect OECs and COMP-Ang1 have on the
cytokine millieu of the diabetic retina (e.g., VEGF, HIF-1α, TNF-α)
and whether OECs and COMP-Ang1 can play a functional reparative
role in diabetic retinopathy by decreasing leakage and promoting
neural function.
Commercial Relationships: Judd M. Cahoon, None; Paul R.
Olson, None; Christina O, None; Reinhold J. Medina, None; Alan
W. Stitt, None; Balamurali Ambati, None
Support: NIH5T32DK091317
Program Number: 4003 Poster Board Number: D0062
Presentation Time: 3:45 PM–5:30 PM
The relationships between endothelial progenitor cells,
inflammation, and diabetic retinopathy
Dawn A. Sim1, 2, Pearse A. Keane1, 2, Catherine A. Egan1, Adnan
Tufail1, 2, Marcus Fruttiger2. 1NIHR Biomedical Research Centre,
Moorfields Eye Hospital NHS Foundation Trust, London, United
Kingdom; 2Cell Biology, UCL Institute of Ophthalmology, London,
United Kingdom.
Purpose: To assess the relationship between endothelial progenitor
cells, inflammation, and retinopathy phenotype in type 2 diabetes.
Methods: 26 patients with type 2 diabetes and 30 healthy controls
were included. Patients were classified according to retinopathy
grade. Mononuclear cells were isolated from peripheral blood, and
endothelial progenitor cells (EPCs) identified by flow cytometry.
Monocyte profiles were also assessed.
Results: The proportion of CD34+ EPCs co-expressing VEGFR2
was significantly greater in diabetes (5.77%±2.15) compared to
healthy controls (2.14±2.00) (p=0.001). In diabetes, low levels
of circulating CD34+ EPCs were also associated with monocytes
co-expressing the monocyte activation marker CD163, and proangiogenic VEGFR2 (r=-0.53, p=0.04). On examination of monocyte
subtypes- (1) pro-inflammatory monocytes (CD14+ CD16++) had a
higher expression of CD163 (p=0.03) in patients with a body mass
index of >30; (2) classical monocytes (CD14+ CD16-) had a higher
expression of VEGFR2 and CD163 (p=0.03) in macular oedema; and
(3) Intermediate monocytes (CD14+ CD16+) had a higher expression
of VEGFR2 (p=0.05) in macular ischaemia.
Conclusions: Evaluation of cellular participants of vascular repair
and chronic inflammation may offer an explanation as to why
different phenotypes of diabetic retinopathy manifest, provide
new insights into mechanisms of visual loss, and provide potential
avenues for novel therapeutic approaches in this disease.
Commercial Relationships: Dawn A. Sim, Allergan (F); Pearse
A. Keane, Allergan (F); Catherine A. Egan, None; Adnan Tufail,
Allergan (F), Novartis (F); Marcus Fruttiger, Amaken (F),
AstraZeneca (F), Novartis (F)
Support: Fight for Sight UK Grant: 1984
Clinical Trial: Study 10842 - Endothelial Progenitor Cells in
Diabetic eye disease
Program Number: 4004 Poster Board Number: D0063
Presentation Time: 3:45 PM–5:30 PM
IL-10-modified endothelial progenitor cells suppress the
progression of non-proliferative diabetic retinopathy
Zhuhong Zhang1, 2, Ying Wang1, Feng Jiang1, Hua Yan1. 1Department
of ophtalmology, Tianjin Medical University General Hospital,
Tianjin, China; 2Moores Cancer Center, University of California San
Diego, San Diego, CA.
Purpose: Interleukin 10 (IL-10) has been shown to suppress the
chronic inflammatory diseases. Our previous study has shown that
reduced numbers and impaired function of circulating endothelial
progenitor cells (EPCs) contributed to the pathogenesis of DR. In this
study we investigated the roles of IL-10 in proliferation, apoptosis
and migration of EPCs, the function of IL-10-modified EPCs in
progression of non-proliferative diabetic retinopathy (NPDR) and the
roles of IL-10-modified EPCs in suppressing the NF-κB pathway and
inflammatory environment.
Methods: We firstly established EPC-GFP-IL-10 stable cell line,
and used the cell cycle assay, Annexin V/PI staining and Transwell
assay to determine the functions of IL-10 in EPCs. We used Western
blot to test the activation of STAT3 pathway in EPCs. Furthermore,
we performed intravitreal injection of EPC-GFP-IL-10 cells into the
streptozotocin-induced diabetic rats. We used in situ hybridization to
test the distribution of EPC-GFP-IL-10 cells and HE staining, Evans
blue assay, and Immunofluorescence to test the progression of NPDR.
We performed RT-PCR and Western blot to assess the makers of DR.
In addition, we performed Western blot, Immunohistochemistry, and
Elisa to assess the activation of NF-κB pathway.
Results: IL-10 increased the proliferation and migration of EPCs,
and decreased the apoptosis of EPCs. IL-10 also activated the STAT3
pathway in EPCs. Furthermore, EPC-GFP-IL-10 cells delayed the
damage of blood-retinal barrier and down-regulated the expression
of DR makers in retina tissues. In addition, EPC-GFP-IL-10 cells
decreased the expression of p65, p50 IL-6, IL-8 and TNF-α in retina
tissues.
Conclusions: IL-10-modified EPCs suppress the progression of
NPDR by inhibition of NF-κB pathway, and establish a rationale for
developing IL-10 as a potential therapeutic agent to treat NPDR.
Commercial Relationships: Zhuhong Zhang, None; Ying Wang,
None; Feng Jiang, None; Hua Yan, None
Clinical Trial: 1920271
Program Number: 4005 Poster Board Number: D0064
Presentation Time: 3:45 PM–5:30 PM
ADIPOSE STROMAL CELLS ATENUATE P38 MAPK IN
RETINAL ISCHEMIA-REPERFUSION INJURY
Alexandra Vayl1, Ahmed Gomaa1, Gangaraju Rajashekhar1,
2 1
. Ophthalmology, Indiana University School of Medicine,
Indianapolis, IN; 2Cellular & Integrative Physiology, Indiana
University School of Medicine, Indianapolis, IN.
Purpose: Retinal ischemia-reperfusion (I/R) injury in the eye cause
neurodegeneration and capillary degeneration associated with
inflammation and apoptosis. Previously we have shown that adipose
stromal cells (ASC) rescue from I/R injury within 7 days post
transplantation. In this study, we hypothesized that attenuation of p38
MAPK, a stress kinase, by ASC conditioned media (ASC-CM) may
play a role in the observed beneficial effect.
Methods: Human ASC were cultured to confluence in serum free
conditioned media for 72h and cell free supernatant was collected.
Basal essential media (BEM) prepared in similar fashion without
cells served as control. Unilateral retinal I/R were done in adult
Lewis rats by transiently elevating the intraocular pressure for 1h.
Uninjured eyes served as I/R controls. After day 7 of reperfusion
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
the animals were randomized to receive intravitreal ASC-CM
(2μL) or BME. After further 6-7 days, retinal function was assessed
by Electroretinogram (ERG), retinal structure and thickness was
assessed by optical coherence tomography (OCT) and the retinal
extracts were processed by Western blot. Human retinal endothelial
cells (HREC) exposed to TNF-α were incubated with varying dose of
ASC-CM or BEM for 72hr and processed for p38 MAPK by Western
blot.
Results: Retinal I/R resulted in a significant reduction in “b”
wave amplitude (as measured by electroretinogram) compared
to anesthetized live un-injured control rats, and this I/R induced
reduction was significantly improved by ASC-CM at day-6 post
injection (p<0.05). Subsequently, ASC-CM injection rescued the
retinal ganglion cells layer and whole retinal thickness assessed by
OCT. Finally, retinal whole extracts from injured eyes that received
ASC-CM demonstrated a significant reduction in phosphorylated p38
MAPK compared to the injured eyes that received BEM. Cultured
HREC exposed to TNF-α that received ASC-CM demonstrated a
decreased level of phosphorylated p38 MAPK compared to the cells
that received BEM.
Conclusions: Our findings suggest that ASC-CM not only rescue
retinal ganglion cells but also improved the function of retina.
Although more studies warranted, attenuation of p38 stress MAPK
pathway seems to play a major role in the observed beneficial effect.
Commercial Relationships: Alexandra Vayl, None; Ahmed
Gomaa, None; Gangaraju Rajashekhar, None
Support: National Eye Institute EY023427
Program Number: 4006 Poster Board Number: D0065
Presentation Time: 3:45 PM–5:30 PM
CD146+ Adipose stromal cells localize and improve the retinal
function in I/R injury
Ahmed Gomaa1, Alexandra Vayl1, Gangaraju Rajashekhar1, 2.
1
Opthalmology, Indiana University School of Medicine, Indianapolis,
IN; 2Cellular and integrative physiology, Indiana University School
of Medicine, Indianapolis, IN.
Purpose: We have demonstrated previously that intravitreal injection
of adipose stromal cells (ASC) in the eyes of diabetic rats improved
the retinal function. Well defined ASC population with highest
safety and efficacy are needed to plan for future human clinical
trials. Recently, CD146+ population of vascular pericytes have been
recognized as potential utility in particular therapeutic areas. In this
study, we aim to identify if CD146+ population of pericyte ASC
have better ability to improve the retinal function in I/R model of
retinopathy.
Methods: Unilateral retinal I/R were done in adult Lewis rats by
transiently elevating the intraocular pressure for 1h. Uninjured eyes
served as I/R controls. After day 7 of reperfusion the animals were
randomized to receive intravitreal CD146+ ASC, CD146- ASC,
(10,000 cells) or saline injections. After further 6-7 days, retinal
function was assessed by Electroretinogram (ERG), retinal structure
and thickness was assessed by optical coherence tomography
(OCT) and retinal whole mounts and confocal microscopy for ASC
localization to retinal vasculature.
Results: Retinal I/R resulted in a significant reduction in “b” wave
amplitude (as measured by ERG) compared to anesthetized live
un-injured control rats. Retinal I/R induced reduction in ‘b’ wave
was significantly improved by CD146+ ASC compared with CD146ASC at day-6 post injection (165.3 ± 24 v/s 211.5 ± 35 mvolts <
0.0343; N=6). In addition, at day 7 retinal I/R resulted in a significant
reduction in total retinal thickness which was rescued with CD146+
ASC in I/R rats as assessed by OCT. Finally, confocal microscopy
performed on retinal whole mounts from injured eyes that received
CD146+ ASC demonstrated a significant localization and homing to
the retinal vasculature in comparison to CD146- ASC cells.
Conclusions: Our findings suggest that CD146+ ASC improves the
function of retina and rescues I/R injury induced retinal degeneration.
Although more studies are warranted to understand the therapeutic
potential of CD146+ ASC, our data suggests that CD146+ ASC have
the highest ability to home and integrate into retinal vasculature,
perhaps as perivascular pericytes to repair damaged retinal
vasculature.
Commercial Relationships: Ahmed Gomaa, None; Alexandra
Vayl, None; Gangaraju Rajashekhar, None
Support: National Eye Institute EY023427
405 Retinal Development
Wednesday, May 07, 2014 8:30 AM–10:15 AM
S 310E-H Paper Session
Program #/Board # Range: 4032–4038
Organizing Section: Retinal Cell Biology
Program Number: 4032
Presentation Time: 8:30 AM–8:45 AM
Retinoic acid signaling regulates expression of the tandemly
duplicated LWS1 and LWS2 genes in zebrafish.
Deborah L. Stenkamp1, Craig B. Stevens1, Ruth A. Frey1, Shoji
Kawamura2. 1Biological Sciences, University of Idaho, Moscow, ID;
2
Integrated Biosciences, University of Tokyo, Tokyo, Japan.
Purpose: Differentiation of diverse photoreceptor phenotypes in the
vertebrate retina requires multiple signaling pathways that activate
cascades of gene expression. The signaling molecule retinoic acid
(RA) is known to regulate rod and cone cell fate, differentiation, and
survival. The purpose of the current study is to identify photoreceptor
genes controlled by RA signaling in the embryonic retina of the
zebrafish.
Methods: We treated embryos with RA at 48 hours post-fertilization
(hpf) and isolated total RNA from eyes for microarray analysis at
75 hpf in order to identify genes responding to RA over the period
of photoreceptor differentiation. Differentially expressed genes
were validated by quantitative RT-PCR (qRT-PCR) and in situ
hybridization. Wild-type zebrafish and those carrying an RA signaling
reporter transgene (RARE:YFP) were used.
Results: We identified 180 genes with significantly altered gene
expression. Of interest was the long wavelength sensitive opsin 1
(LWS1) gene, which was upregulated in eyes of RA-treated embryos.
LWS1 is the upstream member of the tandemly duplicated LWS gene
array, and is normally expressed in red-sensitive cones of ventral
retina, but not until larval stages. qRT-PCR verified significant
upregulation of LWS1, but not LWS2, in eyes of RA-treated embryos.
In situ hybridization using probes specific for the 3’ UTR of each
LWS gene revealed that nearly no control retinas expressed LWS1,
while those of embryos treated with (9-cis or all-trans) RA from 48
hpf to 5 dpf all expressed LWS1, predominantly in the ventral half
of the retina. Control embryos all expressed LWS2 throughout their
retinas, while RA-treated embryos showed a dramatic reduction in
the number of cones expressing LWS2. The expression domain of
LWS1 in the photoreceptor layer of RA-treated embryos matched the
expression domain of a YFP reporter gene driven by a series of RA
response elements.
Conclusions: RA signaling regulates numerous molecular targets in
the developing eye, including the tandemly-duplicated LWS1 and
LWS2 genes. It is possible that the level of RA signaling within a
developing red-sensitive cone acts as a molecular toggle to favor the
expression of LWS1 and/or suppress the expression of LWS2. This is
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
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ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
the first evidence that an extracellular signal may regulate expression
of opsins in a tandemly duplicated gene array.
Commercial Relationships: Deborah L. Stenkamp, None; Craig
B. Stevens, None; Ruth A. Frey, None; Shoji Kawamura, None
Support: NIH Grant R01 EY012146
Program Number: 4033
Presentation Time: 8:45 AM–9:00 AM
Regulation of Spatial Pattering of Rods and Cones in the Larval
Zebrafish Retina
James M. Fadool, Karen Alvarez-Delfin, Orleiquis Guerra, Mailin
Sotolongo-Lopez. Biological Science, Florida State University,
Tallahassee, FL.
Purpose: Humans are largely dependent upon cone vision. The
highest cone density is found in the fovea positioned at the center of
gaze, with comparatively few present in the rod-dominated retinal
periphery. Surprisingly, few mechanisms are known that regulate
the spatial patterning of rod and cone photoreceptors. The larval
zebrafish retina is anatomically and functionally cone-dominated with
conspicuously few rods in the central retina and greater numbers in
the ventral retina and periphery. Our strategy takes advantage of these
regional differences in the numbers of rods to identify fundamental
mechanisms regulate their spatial patterning.
Methods: Immunolabeling and expression of cell-specific reporter
genes were analyzed by confocal microscopy. Zebrafish embryos
and larvae harboring mutations of lor/tbx2b, lak/ath7, lep/pct2, ljr,
or morpholino knockdown of six7 or trb2 were used throughout the
study. Transcriptional activity was measured using luciferase reporter
assays of HEK293 cells co-transfected with transcription factor
constructs and opsin promoter-luciferase reporter plasmids.
Results: In the central retina of larval zebrafish, cones outnumber
rods 20:1. Our published data demonstrate that genetic mutation of
tbx2b/lor leads to an increased number and the uniform distribution
of rods due to a cell fate switch of SWS1 cones into rods. However,
the increase in rod number following knockdown of six7 is associated
with continued cell proliferation in the central retina. Furthermore,
our data show that the effects are additive. Larvae double mutant
for tbx2b/lor and six7/ljr demonstrate twice the number of rods as
either mutation alone. Surprisingly, no changes in rod patterning were
associated with the increased photoreceptor numbers observed in lak/
ath7 or lep/pct2 mutant larvae. In luciferase reporter assays, tbx2b
alone had no effect upon transcription from opsin promoter constructs
but represses the Crx/Nrl-induced transcriptional activation of the
rho-promoter, and Crx activation of the SWS1 promoter. No effects
were observed using six7.
Conclusions: Our data show that spatial patterning of photoreceptors
can be regulated independently by factors controlling cell fate
or cellular proliferation; that tbx2b and six7 act through distinct
molecular mechanisms. However the absence of patterning defects in
lak/ath7 or lep/pct2 mutant larvae suggests that additional factors are
also involved.
Commercial Relationships: James M. Fadool, None; Karen
Alvarez-Delfin, None; Orleiquis Guerra, None; Mailin SotolongoLopez, None
Support: NIH Grant EY17753
Program Number: 4034
Presentation Time: 9:00 AM–9:15 AM
Retina formation requires suppression of BMP and Activin
pathways in pluripotent cells
Kimberly Wong1, 2, Michael A. Trembley3, Andrea S. Viczian2.
1
Neuroscience and Physiology, SUNY Upstate Medical University,
Syracuse, NY; 2Opthalmology and Center for Vision Research, SUNY
Upstate Medical University and SUNY Eye Institute, Syracuse,
NY; 3Pharmacology and Physiology, Aab Cardiovascular Research
Institute, University of Rochester School of Medicine and Dentistry,
Rochester, NY.
Purpose: Retina formation requires the correct spatiotemporal
expression of key regulatory proteins. Signaling through the bone
morphogenetic protein (BMP) pathway represses the formation of
neural and retinal fates. It has been shown that Noggin acts as a
morphogen to specify neural cell types at low concentrations, and
retinal cell types at higher concentrations. The aim of our study is
to determine if the higher concentration of Noggin affects signaling
pathways other than BMP.
Methods: We treated pluripotent Xenopus laevis tissue (animal
caps) with chemical inhibitors and function-altering components of
the BMP and Activin/Nodal signaling pathway. Animal caps were
removed from the embryos at the blastula stage and cultured until
neural plate stage. Their effect on retina formation was determined
using the Animal Cap Transplant (ACT) assay, in which the animal
caps were transplanted into the eye field of sibling embryos.
Signaling activity was determined by Western blot and semiquantitative PCR (RT-PCR) to measure downstream protein and gene
target expression.
Results: Overexpressing Noggin in animal caps resulted in a
concentration-dependent suppression of both Smad1 and Smad2
phosphorylation, which act downstream of BMP and Activin/Nodal
receptors, respectively. This caused a decrease in downstream
transcriptional ability, reflected by the reduced expression of
mesodermal marker, Xbra, and endothelial marker, Xk81. However,
we also observed that Cerberus was less effective at blocking
Smad1/5/8 phosphorylation, yet it can specify retina as efficiently
as Noggin in ACT assays. Cerberus has been shown to block the
Activin/Nodal pathway, as well as the BMP pathway, suggesting
that there is a specific balance between the two pathways that is
required to direct a retinal fate. The use of dominant negative BMP
and activin receptors revealed that retinal specification was increased
when both pathways were inhibited simultaneously. Similar results
were observed when the chemical inhibitors Dorsomorphin and
SB431542 were used to inhibit Smad1 and Smad2/3 phosphorylation,
respectively.
Conclusions: Thus, the dual inhibition of BMP and activin pathways
promotes retinal specification in Xenopus tissue. Future studies will
translate these findings to a mammalian culture assay, in order to
efficiently produce a large percentage of retinal cells in vitro.
Commercial Relationships: Kimberly Wong, None; Michael A.
Trembley, None; Andrea S. Viczian, None
Support: NEI R01 Grant EY019517, Research to Prevent Blindness
(RPB), Lions District 20Y1
Program Number: 4035
Presentation Time: 9:15 AM–9:30 AM
HDAC inhibition protects degenerating cones in the cpfl1 mouse
Dragana Trifunovic, Blanca Arango-Gonzalez, Klaudiaj Masarini,
Norman Rieger, Michelle Dierstein, Marius Ueffing, Francois
Paquet-Durand. Institute for Ophthalmic Research, University of
Tuebingen, Tuebingen, Germany.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Purpose: Understanding the mechanisms of cell death during
inherited retinal degeneration may allow the definition of novel
targets for neuroprotection. The cpfl1 mouse is a model for cone
cell death characterized by fast and progressive cone degeneration.
Additionally, the Pde6c mutation governing cpfl1 cone degeneration
leads to an impaired cone migration during retinal developement
by unknown mechanisms. The cpfl1 cone degeneration follows
a non-apoptotic cell death mechanism and is characterized by
cGMP accumulation and increased activities of PKG and calpains
(Trifunovic et al., J Comp Neurol., 518(17):3604-17, 2010). In
the present study, we asked whether the epigenetic modifications
contributing to primary rod degeneration in Pde6b-mutant rd1 mice
(Sancho-Pelluz et al., Cell Death Dis., 1:e24, 2010), are also involved
in primary cone degeneration.
Methods: We assessed the correlation of increased HDAC activity
and cone degeneration in cpfl1 retina using an HDAC in situ activity
assay. The neuroprotective properties of the HDAC inhibitor,
Trichostatin A (TSA), were assessed using a cpfl1 ex vivo retinal
explant system, followed by immunohistological detection of
characteristic cone markers.
Results: Similar to corresponding observations in the rd1 model,
cpfl1 cone photoreceptor cell death is associated with increased
HDAC activity. TSA inhibition of the HDAC activity in cpfl1
retinal explant cultures resulted in a significant improvement in
cone survival. At the same time, TSA treatment did not negatively
affect wild-type cones. Notably, HDAC inhibition also significantly
improved developmental cone migration compared to non-treated
retinas.
Conclusions: Our finding that primary cone photoreceptor
degeneration is associated with increased HDAC activity suggests
the existence of cell death mechanisms common to both rod and
cone degeneration. This raises the possibility that equivalent
neuroprotective strategies may be used to prevent both types of
photoreceptor degeneration. Indeed, HDAC inhibition emerges
as a novel neuroprotective approach for the treatment of primary
cone degeneration, and provides an exciting new possibility for
a preservation of useful vision in patients suffering from cone
dystrophies.
Commercial Relationships: Dragana Trifunovic, None; Blanca
Arango-Gonzalez, None; Klaudiaj Masarini, None; Norman
Rieger, None; Michelle Dierstein, None; Marius Ueffing, None;
Francois Paquet-Durand, None
Support: DRUGSFORD is a Collaborative Project funded by the
European Commission under the Seventh Framework Programme
(HEALTH-F2-2012-304963), ALCON
Program Number: 4036
Presentation Time: 9:30 AM–9:45 AM
Inhibitor of Apoptosis Stimulating Protein of p53 (iASPP) is
required for retinal ganglion cell survival after axonal injury
Ariel Wilson1, Vince Chiodo2, Sanford L. Boye2, Nicholas Brecha3,
William W. Hauswirth2, Adriana Di Polo1. 1Neuroscience, University
of Montreal, Montreal, QC, Canada; 2Ophthalmology, University of
Florida, Gainesville, FL; 3Neurobiology and Medicine, University of
California Los Angeles, Los Angeles, CA.
Purpose: p53 apoptotic activity is tightly regulated by the apoptosisstimulating proteins of p53 (ASPP) family members: ASPP1, ASPP2
and iASPP. We previously showed that pro-apoptotic members
ASPP1 and ASPP2 contribute to the p53-dependent death of retinal
ganglion cells (RGC), however the role of the p53 inhibitor iASPP
in the central nervous system is unknown. Here, we addressed the
role of iASPP on RGC survival in a model of acute optic nerve injury
(axotomy) using loss-of-function and gain-of-function experiments
in vivo.
Methods: iASPP knockdown was carried out by intravitreal injection
of small interference RNA (si-iASPP). Overexpression of iASPP
in RGCs was achieved by intraocular delivery of a tyrosine mutant
serotype 2 adeno-associated virus (AAV.iASPP). Phosphoserine
immunoprecipitation was performed on retinal lysates of intact,
axotomized, and iASPP overexpressing retinas. iASPP, Fas/
CD95, PUMA, Noxa and Bax protein levels were examined by
retinal immunohistochemistry and western blot analysis. RGC
immunolabeling was performed with an RBPMS antibody. RGC
densities were assessed by quantification of Brn3a-positive cells on
retinal whole mounts followed by statistical analysis using one-way
analysis of variance (ANOVA) followed by Bonferroni’s multiple
comparison post-test.
Results: Our data demonstrate that iASPP is expressed by intact and
injured RGCs, and that iASPP phosphoserine levels, which increase
iASPP affinity towards p53, are significantly reduced following
axotomy. We show that iASPP downregulation by siRNA exacerbates
RGC death, whereas selective AAV-mediated overexpression of
iASPP promotes robust RGC survival. iASPP overexpression results
in an increase of iASPP phosphoserine levels following axotomy
compared to retinas treated with a control virus. Analysis of p53
downstream targets demonstrate that increasing iASPP levels in
RGCs leads to downregulation of pro-apoptotic PUMA and Fas/
CD95.
Conclusions: Our study demonstrates a novel role for iASPP in the
death of RGCs, and provides further evidence of the importance of
ASPP family in CNS neuronal survival after axonal injury.
Commercial Relationships: Ariel Wilson, None; Vince Chiodo,
None; Sanford L. Boye, None; Nicholas Brecha, None; William W.
Hauswirth, None; Adriana Di Polo, None
Support: Canadian Institutes of Health Research (CIHR)
Program Number: 4037
Presentation Time: 9:45 AM–10:00 AM
Neuroprotective effect of KUS121, a VCP modulator, on a rat
model of ischemia and reperfusion-induced retinal degeneration
Masayuki Hata1, Hanako O. Ikeda1, Munemitsu Yoshikawa1, Tomoko
Hasegawa1, Noriko Nakano1, Yuki Muraoka1, Akira Kakizuka2,
Nagahisa Yoshimura1. 1Ophthalmology, Kyoto University, Kyoto,
Japan; 2Graduate school of biostudies, Kyoto University, Kyoto,
Japan.
Purpose: Valosin-containing protein (VCP) is a ubiquitously
expressed ATPase that is reported to be involved in several
physiological activities as well as neurodegeneration. Newly
synthesized compounds (Kyoto University Substances, KUSs) that
inhibit VCP ATPase activity have been shown to protect cells under
stress conditions. This study aimed to confirm whether KUS121, one
of KUSs has a neuroprotective effect on a rat model of ischemia and
reperfusion (I/R)-induced retinal degeneration.
Methods: KUS121 was orally administered to adult Thy1-GFP rats
(Magill CK et al. Arch Facial Plast Surg 2010) before I/R injury.
Retinal I/R injury in the rats was induced by elevating the intraocular
pressure for 60 minutes with subsequent reperfusion. Spectraldomain optical coherence tomography (SD-OCT) examinations
(Multiline OCT, Heidelberg Engineering) were performed to evaluate
the inner retinal thickness (IR: RNFL + GCL + IPL+INL) around the
optic nerve head 1, 4, and 7 days after I/R injury.
Results: The IR thickness of untreated I/R rats (n = 9) changed from
126.6 ± 14.7 mm at baseline to 133.9 ± 9.6 mm at day 1, 96.3 ± 14.0
mm at day 4, and 84.3 ± 15.3 mm at day 7 after I/R injury. In contrast,
the IR thickness of I/R rats treated with KUS121 (n = 9) changed
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
from 125.6 ± 9.7 mm at baseline to 126.0 ± 4.8 mm at day 1, 104.7 ±
21.9 mm at day 4, and 93.0 ± 29.8 mm at day 7 after I/R injury. The
mean IR thickness of the rats treated with KUS121 was greater than
that of the control rats 7 days after I/R (P = 0.008).
Conclusions: KUS121 has a neuroprotective effect on an I/R injury
rat model, suggesting the potential usefulness of the compound for
the treatment of human ischemic ocular diseases.
Commercial Relationships: Masayuki Hata, Kyoto University (P);
Hanako O. Ikeda, Kyoto University (P); Munemitsu Yoshikawa,
None; Tomoko Hasegawa, None; Noriko Nakano, None; Yuki
Muraoka, None; Akira Kakizuka, Kyoto University (P); Nagahisa
Yoshimura, Kyoto University (P)
Support: Astellas Foundation for Research on Metabolic Disorders,
the Japan Foundation for Applied Enzymology, the Uehara Memorial
Foundation, Mochida Memorial Foundation for Medical and
Pharmaceutical Research, YOKOYAMA Foundation for Clinical
Pharmacology (YRY1308), Japan Intractable Diseases Research
Foundation, Japan Research Foundation for Clinical Pharmacology
(IOH), and a Grant-in-Aid for Young Scientists (24791850)
Program Number: 4038
Presentation Time: 10:00 AM–10:15 AM
MANF in Retinal Therapies:Improving Regenerative Therapies
by Promoting Tissue Repair
JOANA NEVES, Deepak A. Lamba, Heinrich Jasper. Buck Institute
for Research on Aging, Novato, CA.
Purpose: Stem cell based therapies, have been shown to hold real
promise in the treatment of degenerative diseases of the retina.
However, the efficiency of such strategies is still considerably low.
Tissue repair mechanisms are conserved at the organism level and
enhance the regenerative process. We hypothesized that promoting
tissue repair may also enhance the efficiency of cell engraftment in
the retina. Key components of the retinal repair network have been
identified in Drosophila involving interactions between the damaged
retina and hemocytes. We have used the Drosophila to identify
hemocyte derived factors that can promote tissue repair in the retina
and have tested the conservation of their function in the mammalian
retina. Our work focused on Mesencephalic Astrocyte-derived
Neurotrophic Factor (MANF).
Methods: We have used UV induced retinal damage in flies and light
induced retinal damage in mouse as model systems to test the effects
of MANF. MANF was overexpressed in flies using the UAS/Gal4
system. In mice, recombinant protein was delivered by intravitreal
injection.
Results: We have identified MANF as a hemocyte derived protein
in Drosophila that can promote tissue repair in the fly retina, using
RNAseq. We show that MANF is expressed in hemocytes of
Drosophila larvae, it is secreted to the hemolymph and induced in
response to stress in a Pvf-1/PvR dependent manner. Hemocyte
specific MANF expression is sufficient to reduce tissue loss after
UV and genetically-induced photoreceptor apoptosis. Moreover,
stress induced MANF results in changes in the hemocyte population
correlating with increased lamellocyte differentiation. We have tested
the conservation of the pathway in mammalian retinal repair. As in
flies, MANF is induced in microglia/macrophages invading the retina
following light damage and this correlates with reduced tissue loss.
Importantly, intravitreal delivery of MANF recombinant protein is
sufficient to limit cell death following light damage and promotes
alterations in macrophages/microglia.
Conclusions: MANF is a conserved neuroprotective factor in the
retina. MANF acts as an immune-modifying factor to limit cell loss
following acute damage. . This work will serve as a proof of concept
to the use of tissue repair promoting factors as co-adjutants in stem
cell regenerative therapies.
Commercial Relationships: JOANA NEVES, None; Deepak A.
Lamba, None; Heinrich Jasper, None
Support: Glenn Foundation Postdoctoral fellowship to JN, NIH
Grant EY018177-04
421 Photoreceptor Degeneration
Wednesday, May 07, 2014 8:30 AM–10:15 AM
Exhibit/Poster Hall SA Poster Session
Program #/Board # Range: 4362–4390/C0145–C0173
Organizing Section: Retinal Cell Biology
Contributing Section(s): Retina
Program Number: 4362 Poster Board Number: C0145
Presentation Time: 8:30 AM–10:15 AM
Caspase-9-Mediates Photoreceptor Apoptosis After Blunt Ocular
Trauma
Richard J. Blanch1, 2, Zubair Ahmed1, Nsikan Akpan3, David R.
Snead5, Martin Berry1, Carol M. Troy3, Robert A H Scott2, 4, Ann
Logan1. 1Neurotrauma and Neurodegeneration, University of
Birmingham, Birmingham, United Kingdom; 2Academic Department
of Military Surgery and Trauma, Royal Centre for Defence Medicine,
Birmingham, United Kingdom; 3Department of Pathology & Cell
Biology, Columbia University, Birmingham, NY; 4Birmingham and
Midland Eye Centre, Sandwell and West Birmingham Hospitals NHS
Trust, Birmingham, United Kingdom; 5Histopathology, University
Hospitals Coventry and Warwickshire NHS Trust, Coventry, United
Kingdom.
Purpose: Commotio retinae (CR) involves photoreceptor outer
segment disruption after blunt ocular trauma and occurs in 0.4% of
civilian and 19% of military eye injuries. Photoreceptor degeneration
permanently reduces vision in 26% of patients with macula
involvement. In animal models of CR, photoreceptors die by both
necrosis and apoptosis.
Apoptotic mechanisms in CR have not been described. We assessed
the role of caspase-9 as a mediator of photoreceptor apoptosis in a rat
model of CR.
Methods: Bilateral CR was induced in rats using ballistic ocular
trauma. Caspase-9 activity was assessed by immunohistochemistry,
western blots and bVAD-fmk active caspase capture. Caspase-9
was inhibited using unilateral intravitreal injection of the highly
specific X-linked inhibitor of apoptosis-baculoviral IAP repeat 3
domain (XBIR3) linked to cell transduction peptide Penetratin 1
(Pen1) after ballistic injury compared with control eyes treated with
Pen1 injection alone and retinal function assessed by ERG a-wave
amplitude and photoreceptor survival by outer nuclear layer (ONL)
thickness. n=6-8 rats/analysis
Results: Cleaved caspase 9 immunolocalised to photoreceptor inner
segments and retinal levels of cleaved caspase-9 increased 5 hours
after injury (Fig 1A-B), whereas levels of catalytically active full
length caspase-9 increased up to 48 hours (Fig 1C-D). Caspase-9
inhibition by intravitreal injection of Pen1-XBIR3 preserved a-wave
amplitude and ONL thickness 14 days after ballistic injury compared
to control eyes (Fig 2).
Conclusions: Caspase 9 initiates photoreceptor apoptosis after CR
and its inhibition increases photoreceptor survival and function,
highlighting a new therapeutic angle in the treatment of ocular
trauma.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Commercial Relationships: Richard J. Blanch, None; Zubair
Ahmed, None; Nsikan Akpan, None; David R. Snead, None;
Martin Berry, None; Carol M. Troy, None; Robert A H Scott,
None; Ann Logan, None
Support: Ministry of Defence, UK; Drummond Foundation, UK; Sir
Ian Fraser Foundation, Blind Veterans UK
Program Number: 4363 Poster Board Number: C0146
Presentation Time: 8:30 AM–10:15 AM
A study of PDZD7 in the mouse retina
Jun Yang, Junhuang Zou, Tihua Zheng. Moran Eye Center, University
of Utah School of Medicine, Salt Lake City, UT.
Purpose: PDZD7 is a newly identified modifier and contributor gene
of Usher syndrome (USH). In the inner ear, PDZD7 colocalizes with
GPR98, an USH2C protein, at ankle links in cochlear and vestibular
hair cells. Therefore, PDZD7 is proposed to be a novel component
of the USH2 complex, which is composed of the three known USH2
causative proteins. In this study, we investigated PDZD7 expression
and its role in the organization of the USH2 complex in the retina.
©2014, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permission
to reproduce any abstract, contact the ARVO Office at pubs@arvo.org.
ARVO 2014 Annual Meeting Abstracts by Scientific Section/Group - Retinal Cell Biology
Methods: The expression of Pdzd7 was examined at the mRNA and
protein levels using RT-PCR, western blotting, and immunostaining
assays. A Pdzd7 knockout mouse was generated by gene trapping
and characterized phenotypically by immunostaining and
electroretinogram.
Results: Five Pdzd7 splice variants were identified from 19
independent RT-PCR clones in adult mouse retinas. All of them
are predicted as N-terminal but not full-length Pdzd7 isoforms.
At the protein level, full-length Pdzd7 was found in the mouse
retina during postnatal development. However, no Pdzd7 protein
expression could be detected in adulthood by either western blotting
or immunostaining. Pdzd7 knockout mice showed close to normal
distribution of USH2A, GPR98 and WHRN at the periciliary
membrane complex in photoreceptors. The knockout mice exhibited
normal ERG responses at one month of age.
Conclusions: Despite the existence of multiple splice variants,
PDZD7 expression at the protein level is very low in the retina.
PDZD7 is not as important as WHRN in organizing the USH2
complex in photoreceptors.
Commercial Relationships: Jun Yang, None; Junhuang Zou,
None; Tihua Zheng, None
Support: NIH grant EY020853, E.Matilda Ziegler Foundation for
the Blind, Inc, Research to Prevent Blindness
Program Number: 4364 Poster Board Number: C0147
Presentation Time: 8:30 AM–10:15 AM
Suppression of microglial activation is neuroprotective in a
mouse model of human retinitis pigmentosa
Bin Lin, Bo Peng. Anatomy, University of Hong Kong, Hong Kong,
Hong Kong.
Purpose: Retinitis Pigmentosa (RP) is a photoreceptor-degenerative
disease caused by various mutations that result in rod photoreceptor
cell death followed by gradual death of cone photoreceptors. The
molecular mechanisms that lead to the secondary cone death are
not yet fully understood. Neuroinflammation contributes to the
progression of many chronic neurodegenerative disorders. However,
the nature of microglia involvement in RP has not been documented.
In this study, we explored the role of microglia as a contributor to
photoreceptor degeneration in the rd10 mouse model of RP.
Methods: we backcrossed Cx3cr1 knockout mice, in which
the Cx3cr1 gene was replaced with a cDNA encoding green
fluorescent protein (GFP), into the rd10 background creating a new
line of mice. To validate the importance of inflammation in RP,
minocycline, an inhibitor of microglial activation, was injected into
the peritoneum of rd10 mice. We investigated the effect of microglia
activation on photoreceptor survival, using a combination of
immunocytochemistry, RT-PCR, western blot analysis, and a series of
simple visual tests.
Results: We found that microglia activation and migration into the
subretinal space preceded photoreceptor apoptosis in rd10 retinas.
Suppression of microglia activation by minocycline resulted in
down-regulation of pro-inflammatory cytokines and chemokines
and pro-apoptotic molecules, leading to significant structural and
functional rescue in rd10 retinas. We also identified that minocycline
exerted its neuroprotective effects through both anti-apoptotic and
anti-inflammatory mechanisms.
Conclusions: Our data demonstrated that activated microglia
contributed to the severity of RP disease and played an important role
in regulating the survival of photoreceptors in RP retinas. Therefore,
modulating microglia activation could be a potential treatment
strategy aimed at improving photoreceptor survival in human RP.
Commercial Relationships: Bin Lin, None; Bo Peng, None
Support: This work was supported by The University of Hong Kong
Seed Funding Program for Basic Research and General Research
Fund from the Hong Kong Research Grants Council (772810).
Program Number: 4365 Poster Board Number: C0148
Presentation Time: 8:30 AM–10:15 AM
Optimization of ImageJ for automated image analysis to assess
for photoreceptor cell death in retinal tissue sections
Boris Busov, Cagri G. Besirli. University of Michigan, Ann Arbor,
MI.
Purpose: To develop an automated, accurate way of analyzing
different parameters of retinal sections to determine the effects of
retinal detachment.
Methods: Experimental retinal detachments were created in adult
C57/B16 mice. Eyes were obtained 1 and 2 months post-retinal
detachment and processed for histologic analysis. Retinal sections
were stained and photographed. Sections were examined using
ImageJ, a Java-based image-processing program available by the
National Institutes of Health (NIH). Automated cell counting was
performed in the outer nuclear layer (ONL) of retinal sections
where photoreceptor nuclei are located. Mean size of photoreceptor
nuclei in attached and detached samples were compared. Parameters
in ImageJ were adjusted to improve the accuracy of automated
counting. Photoreceptor nuclei area was found. The number of nuclei
in the ONL was determined with ImageJ and compared to manual
counting. Two other measurements were also assessed: the ratio of
ONL area to total retinal section area and the ratio of ONL thickness
to total retinal thickness.
Results: Detached retinal sections had significantly lower
photoreceptor nuclei counts than attached retinal sections and
automated cell counting produced accurate results. Average cell
area was calculated and plotted. The cell area was used to obtain
a more accurate value for photoreceptor nuclei count—this was
accomplished by first determining the average cell area and a
standard deviation which were used to construct a confidence interval
to eliminate any outliers. Furthermore, the ratio of ONL area to total
retinal area showed a reduction in retinal area for the detached retinal
sections and the ratio of ONL to total retinal thickness showed that
detached retina had significantly reduced ONL thickness.
Conclusions: Automated analysis using an image analysis program
available publicly from the NIH is accurate and may be used in place
of manual evaluation to reduce the time needed for data collection
considerably.
Commercial Relationships: Boris Busov, None; Cagri G. Besirli,
None
Program Number: 4366 Poster Board Number: C0149
Presentation Time: 8:30 AM–10:15 AM
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