ARVO 2016 Annual Meeting Abstracts
111 RPE
Sunday, May 01, 2016 8:30 AM–10:15 AM
Exhibit/Poster Hall Poster Session
Program #/Board # Range: 232–268/B0238–B0274
Organizing Section: Retinal Cell Biology
Program Number: 232 Poster Board Number: B0238
Presentation Time: 8:30 AM–10:15 AM
Comparative proteomic profiling of cultured RPE cells derived
from human embryonic stem cells vs. rhesus monkey
Bruce A. Pfeffer1, 2, Chengjian Tu3, Jun Li3, Jun Qu3, 4,
Steven J. Fliesler1, 2. 1Research Service, VAWNYHS, Buffalo, NY;
2
Ophthalmology, Biochemistry, and SUNY Eye Institute, University
at Buffalo, Buffalo, NY; 3Pharmaceutical Sciences, University at
Buffalo, Buffalo, NY; 4SUNY Eye Institute, University at Buffalo,
Buffalo, NY.
Purpose: Cultured retinal pigment epithelial (RPE) cells from nonhuman primate, and those derived from human embryonic stem cells
(hesc), each with their own strategic and translational merits and
deficiencies, are potential substitutes for RPE cell lines and cultures
established from human donor eyes. A fair consensus with regard
to human RPE signature gene and protein expression has emerged
from analysis of native and cultured RPE in published reports. We
generated proteomic profiles to compare cultured hesc-derived and
rhesus macaque RPE cells in terms of correlation with human RPE
signature genes and proteins.
Methods: Human (hesc-derived) and rhesus RPE cells (4th and
3rd passage, respectively) were maintained as stable, confluent
monolayers for 8 weeks. Both strains displayed a polarized,
pigmented epithelial phenotype in vitro. Triplicate sets of cell lysates
were subjected to on-pellet proteolytic digestion and separations by
nano-reverse-phase LC/ion-current-based tandem mass spectroscopy
[Tu et al., Mol Cell Proteomics 2013]. Identifications from the
UniProt human protein database were made on the basis of at
least two unique peptides for each protein. Proteins detected were
quantified by AUC using SIEVE, and ranked by relative abundance.
Results: Approximately 1100 – 1300 proteins per sample were
identified. Intensity vs. retention time plots showed consistent
alignment for replicates within each species (87.6% and 95.1%
for hesc- and rhesus RPE, respectively), but only a mean of 41.3%
between species. For the 110 most abundant proteins detected in
either hesc- or rhesus RPE, 70.0% vs. 77.3% of these overlapped
with the other subset, respectively. Of the 50 most abundant proteins
detected, 35 from either hesc-derived or rhesus cultured RPE
corresponded to an entry from at least one comprehensive, recently
published list of human RPE signature genes or proteins [Strunnikova
et al., Hum Mol Genet 2010; West et al., Mol Cell Proteomics
2003]. Cytoskeletal, retinoid-processing, ion and small molecule
transport, and antioxidant/pro-survival functional categories were
highly represented for both species. RPE65, an exemplary signature
protein for native RPE, was detected in both hesc- and rhesus RPE,
with comparative normalized abundance values of 0.041 and 0.350,
respectively.
Conclusions: Proteomic profiling may be used to authenticate
cultured hesc-RPE and non-human primate RPE.
Commercial Relationships: Bruce A. Pfeffer, None; Chengjian Tu,
None; Jun Li, None; Jun Qu, None; Steven J. Fliesler, None
Support: NIH EY007361 (SJF); U54HD071594 and HL103411
(JQ); Center of Protein Therapeutics Industrial Award (JQ); RPB
Unrestricted Grant (SJF); and VAWNYHS facilities and resources
(SJF, BAP)
Program Number: 233 Poster Board Number: B0239
Presentation Time: 8:30 AM–10:15 AM
In vivo quantification of macular RPE cell density in infant
monkeys using fundus photography
Lauren M. Renner1, Emily Johnson1, Kasie W. Paul1, Travis B. Smith2,
Trevor J. McGill2, 1, Martha Neuringer1, 2. 1Neuroscience, Oregon
Health & Science University, Beaverton, OR; 2Casey Eye Institute,
Oregon Health & Science University, Portland, OR.
Purpose: The density of retinal pigment epithelial (RPE) cells in
adult human and monkey eyes peaks in the fovea and decreases
with eccentricity, as demonstrated by histology and by AOSLO
autofluorescence, but has not been examined during development.
Surprisingly, we have been able to visualize macular RPE cells
in infant monkeys with standard fundus photography techniques,
possibly as a result of the lack of RPE and choroidal pigmentation,
and/or increased magnification due to small eye size. This study
utilized this phenomenon to quantify the development of the RPE cell
density profile longitudinally in infant monkeys in vivo.
Methods: RPE cells were visualized longitudinally in infant rhesus
macaques (n=11) at 2, 4, 8, 16 and 24 weeks of age by color and redfree fundus photography and fluorescein angiography using a Zeiss
FF450 fundus camera. For each animal and time point, the clearest
central fundus image of any imaging modality showing visible RPE
cells was selected. Images were analyzed with Mosaic Analytics
Software (courtesy of Joseph Carroll) with manual correction. A
custom MATLAB program was used to calculate RPE density in
the 0.5 mm diameter foveal center and in three concentric annuli at
0.25-0.5, 0.5-0.75, and 0.75-1.00 mm eccentricities, with additional
measurements at greater eccentricities when possible.
Results: At 2 weeks of age, RPE cells were visible in the macula
of all 11 infant monkeys. Density was highest in the central fovea
(2,400 cells/mm2) and decreased progressively with eccentricity. By
8 weeks of age, RPE density increased in all regions; the increase was
greatest in the central 0.5 mm, with density reaching 3,000 cells/mm2.
The area in which RPE cells were visible decreased as pigmentation
increased, and in some infants RPE cells were visible only in the
central 0.5 mm of the fovea. By 16 weeks, RPE cells were no longer
visible in most infants.
Conclusions: RPE cell density can be visualized and quantified in the
infant primate macula using standard ophthalmic imaging techniques.
Quantification of RPE cell density revealed the presence of a foveal
peak as early as 2 weeks and a progressive density increase similar to
the developmental pattern of foveal cone photoreceptor density.
Commercial Relationships: Lauren M. Renner; Emily Johnson,
Abbott Nutrition (F); Kasie W. Paul, Abbott Nutrition (F);
Travis B. Smith, None; Trevor J. McGill, Abbott Nutrition (F);
Martha Neuringer, Abbott Nutrition (F)
Support: Abbott Nutrition through the Center for Nutrition,
Learning, and Memory at the University of Illinois and NIH grant
P51OD011092
Program Number: 234 Poster Board Number: B0240
Presentation Time: 8:30 AM–10:15 AM
Impact of platelet-derived growth factor-C on junctional
adhesion molecule-C expression in human blood-retinal barrier
cells
Xu Hou. ophthalmology, Eye institute of Chinese PLA, Xi'an, China.
Purpose: To investgate the expression change of junctional adhesion
molecule-C (JAM-C) in human retinal pigment epithelial cells
(RPEs) and retinal capillary endothelial cells (RCECs) induced by
platelet-derived growth factor-C (PDGF-C).
Methods: Human RPE cells and RCECs were treated by VEGF-A
or PDGF-C, respectively. The expression quantity of JAM-C
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ARVO 2016 Annual Meeting Abstracts
was analyzed by real-time PCR, flow cytometry and western
blot, and the expression distribution of JAM-C was observed by
immunofluorescence staining.
Results: After the treatment with VEGF-A, the total JAM-C
expression in the two kinds of cells was not significantly changed.
Meanwhile, the expression level in cytoplasm was decreased, and
the expression level in membrane was increased. After the treatment
with PDGF-C, the total JAM-C expression in the two kinds of cells
was increased. The expression level in both cytoplasm and membrane
were increased.
Conclusions: VEGF-A may promote the transfer of JAM-C
expression from cytoplasm to cell membrane. PDGF-C may not only
promote the transfer of JAM-C expression, but also promote the total
JAM-C expression. Thus, PDGF-C should play a bigger role in the
regulation of JAM-C in the two kinds of blood-retinal barrier cells.
Commercial Relationships: xu hou, None
Program Number: 235 Poster Board Number: B0241
Presentation Time: 8:30 AM–10:15 AM
Assessment of Phagocytic Function of Human Retinal Pigment
Epithelium in Vitro
George Inana1, Chris Murat1, Weijun An1, Ian Harris2, Jing Cao2.
1
Bascom Palmer Eye Institute, Miami, FL; 2Janssen Research and
Development, Spring House, PA.
Purpose: The diurnally regulated phagocytosis of the outer tips of
photoreceptors that occur every morning is a key function of the
retinal pigment epithelium (RPE) in sustaining the health of the
light-sensing photoreceptors. Its importance is illustrated in the
well-known animal model of retinal degeneration, the Royal College
of Surgeons rat, in which a defect in the RPE phagocytic function
leads to retinal degeneration. In fact, the genetic defect (mutation
in the Mertk gene) causing this defect has also been found in some
humans with retinitis pigmentosa. This brings up the possibility that a
defect in the RPE phagocytic function may play a role in other retinal
degenerative conditions, including age-related macular degeneration
(AMD), especially since AMD is characterized by the accumulation
of abnormal intra- and extracellular debris (lipofuscin, drusen) which
have been shown to be composed of material from the photoreceptors
which ended up in the RPE via phagocytosis. With the goal of
studying the status of phagocytic function in normal and AMD
human RPE, we first set up an in vitro phagocytosis assay system for
the normal human RPE and carried out an initial assessment of the
phagocytic function.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Methods: Harvesting of human RPE, culture, isolation of rod outer
segments (ROS) and labeling with fluorescein, and phagocytosis
assay were mostly based on the methods used for rat RPE
phagocytosis assay that we have published (McLaren, Inana, Li IOVS
1993). RPE was obtained from 8 post-mortem human eyes. ROS was
prepared from human, rat, and pig retinas.
Results: Feeding of fluorescently labeled ROS to human RPE
cultured for at least 1 week demonstrated evidence of phagocytosis
of the ROS, clearly distinguishable microscopically as ingested
phagosomes, after ~8 hours of incubation as seen in our rat RPE
phagocytosis assays. The kinetics of phagocytosis by the human RPE
was also similar to that of the rat, with binding of the ROS first, then
ingestion after 7-8 hours. The human RPE preferred the human ROS
over the rat and pig for phagocytosis. A baseline of phagocytic level
was established for the normal human RPE, and a moderate decrease
in the level was observed with age (28 to 79yo).
Conclusions: Characteristics of ROS phagocytosis by the human
RPE were similar to that for the rat, and the initial findings will be
useful for the analysis of AMD eyes in the near future.
Commercial Relationships: George Inana, Janssen R&D
(R), Janssen R&D (F); Chris Murat, None; Weijun An, None;
Ian Harris, Janssen R&D; Jing Cao, Janssen R&D
Support: NIH Center Core Grant P30EY014801, Research to
Prevent Blindness Unrestricted Grant to the University of Miami
Program Number: 236 Poster Board Number: B0242
Presentation Time: 8:30 AM–10:15 AM
Semaphorin 4D inhibits photoreceptor outer segment
phagocytosis by retinal pigment epithelial cells
Ayelen Bulloj, Silvia C. Finnemann. Department of Biological
Sciences, Center for Cancer, Genetic Diseases, and Gene Regulation,
Fordham University, Bronx, NY.
Purpose: Daily clearance phagocytosis by the retinal pigment
epithelium (RPE) of photoreceptor outer segment fragments (POS)
is critical for photoreceptor function and longevity. In this process,
POS bind to ανβ5 integrin to stimulate signaling via Mertk and
AKT kinases, which ultimately target Rac1-dependent F-actin
reorganization that is required for POS engulfment. Semaphorins
belong to a diverse family of secreted or membrane associated
glycoproteins that are ligands for the plexin receptor family. The
receptor for semaphorin 4D (Sema4D) is plexinB1 (PlB1). PlB1
can reorganize the cytoskeleton by interacting with active Rac1 in
a ligand dependent manner and/or by promoting integrin-dependent
activation of AKT through RhoA/Rho kinase (ROCK). Here, we
set out to study if Sema4D and its receptor PlB1 contribute to the
molecular mechanism of RPE phagocytosis.
Methods: Bound and internalized POS were quantified by
feeding primary fetal human RPE cells with POS with or without
recombinant Sema4D followed by confocal microscopy (CM)
analysis. RhoA and Rac1 activity were measured by ELISA. Retinal
Sema4D protein levels before and after light onset were studied by
immunoblotting (IB) and by live tissue staining followed by CM
in wild-type (wt) and Mertk null (RCS) rat eyes. Phosphorylation
of PlB1 in rat eyes were analyzed by anti-phosphotyrosine
immunoprecipitation followed by IB. Phagosome quantification and
retina structure analysis were performed on sections of wt, Sema4D
and PlB1 null mouse retina.
Results: Added Sema4D abolished POS internalization by RPE
cells in culture without affecting binding. ROCK inhibition or Rac1
activation restored POS internalization blocked by Sema4D. PlB1
phosphorylation and Sema4D levels were reduced during the peak of
phagocytosis in wt rat eyes. This pattern was altered in RCS rat eyes,
in which POS internalization does not occur. In mice with Sema4D
or PlB1 gene deletion the RPE showed increased POS phagosome
content, but otherwise their retina lacked obvious structural defects.
Conclusions: Our findings identify a new function for the ligandreceptor pair Sema4D/PlB1 in the retina and add new players to the
photoreceptor outer segment renewal mechanism that is essential to
maintain health and functionality of the retina.
Commercial Relationships: Ayelen Bulloj, None;
Silvia C. Finnemann
Support: NIH grant R01-EY026215
Program Number: 237 Poster Board Number: B0243
Presentation Time: 8:30 AM–10:15 AM
Contribution of annexin A5 to diurnal phagocytosis by the retinal
pigment epithelium
CHEN YU1, Luis E. Munoz2, Sebastian Boeltz2, Silvia C. Finnemann1.
1
Department of Biological Sciences, Center for Cancer, Genetic
Diseases and Gene Regulation, Fordham University, Bronx, NY;
2
Department of Internal Medicine 3, Rheumatology and Immunology,
Friedrich-Alexander-Universität Erlangen-Nürnberg, Erlangen,
Germany.
Purpose: Diurnal phagocytosis of spent photoreceptor outer segment
fragments (POS) by the retinal pigment epithelium (RPE) is critical
for vision. POS recognition/binding by apical αvβ5 integrin receptors
of the RPE triggers signaling that synchronizes POS engulfment in
the eye. Annexin A5 is a Ca2+ dependent phospholipid binding protein
with a binding domain for αvβ5 integrin. However, the physiological
function of such interaction remains unknown. Here, we examined if
and how annexin A5 contributes to RPE phagocytosis.
Methods: Annexin A5 expression in rodent retina/RPE was
examined using Western blotting and immunofluorescence
microscopy. Retinal cell marker labeling was used to compare retinal
morphology of annexin A5 knock-out (KO) and wild-type mice.
POS phagosomes in the RPE at different times after light onset were
quantified to assess the phagocytic activity of annexin A5 KO RPE
in situ. POS phagocytosis assays of unpassaged primary RPE or of
mouse embryonic fibroblasts (MEFs) were performed to assess the
effect of silencing or overexpressing annexin A5 on binding and
internalization of isolated POS particles.
Results: We found that annexin A5 is expressed in the neural retina,
RPE and choroid in mouse and rat eyes. 9-month old Annexin A5
KO mice showed normal retinal morphology. However, in situ
phagosome quantification revealed that annexin A5 KO RPE lacks
the peak of phagosome load at light onset that is characteristic for
wild-type RPE. Silencing annexin A5 decreased and overexpressing
annexin A5 increased POS binding by wild-type primary RPE cells
in culture. Silencing annexin A5 did not directly affect internalization
or F-actin phagocytic cup formation by primary RPE. Re-expressing
annexin A5 was sufficient to rescue the impaired binding capacity
of annexin A5 KO MEFs and to increase POS binding by wild-type
MEFs above normal. In contrast, overexpressing annexin A5 in
integrin β5 KO MEFs had no effect on POS binding.
Conclusions: Our results demonstrate that annexin A5 plays an
important role in diurnal outer segment renewal. Moreover, our
cell culture assays indicate that annexin A5 specifically of the
RPE contributes to this process. Like αvβ5 integrin receptors,
manipulating annexin A5 alters POS binding but not POS
internalization. Our results further suggest that the function in
phagocytosis of annexin A5 is dependent on αvβ5 integrin.
Commercial Relationships: CHEN YU, None; Luis E. Munoz,
None; Sebastian Boeltz, None; Silvia C. Finnemann, None
Support: NIH R01-EY026215
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 238 Poster Board Number: B0244
Presentation Time: 8:30 AM–10:15 AM
Discovering Novel Genes and Pathways Involved in Maintenance
of Epithelial Phenotype in Retinal Pigment Epithelium Cells
Justin R. Chang1, Balendu Shekhar Jha1, Madhu Lal2, Ruchi Sharma1,
Marc Ferrer2, Kapil Bharti1. 1National Eye Institute, National
Institutes of Health, Bethesda, MD; 2National Center for Advancing
Translational Sciences, National Institutes of Health, Rockville, MD.
Purpose: Proliferative vitreoretinopathy (PVR) causes retinal
pigment epithelium (RPE) dedifferentiation where RPE loses its
epithelial characteristics. The goal of this study is to perform a series
of high throughput screening assays and bioinformatics analyses to
identify novel genes and pathways that are involved in maintenance
of RPE epithelial phenotype to facilitate vision recovery after PVR
surgery.
Methods: RPE cells derived from a reporter line of human induced
pluripotent stem cells (iPSCs) expressing RPE-specific GFP and
constitutive housekeeping RFP were optimized to be cultured in
384-well plates. A library of 875 miRNA mimics was tested, and
top 5% GFP up- and down-regulators (GFP, RFP signals measured
by MetaXpress) were identified. Multiple miRNA databases were
used to analyze these selected mimics for their target genes. Targets
that had >90% scores and common occurrences between multiple
databases per miRNA were chosen for subsequent siRNA screening.
siRNA screening results and Ingenuity Pathway Analysis (IPA)
software were used to compile a list of genes for further assays.
Results: The miRNA assay had a high reproducibility with
correlation coefficients of 0.87, 0.94, 0.91 in GFP, RFP, Hoechst
signals, respectively, between two identical screenings. The top 5%
of miRNA mimics (21 mimics) that decreased GFP by 50% compared
to control and the top 5% miRNA mimics (10 mimics) that increased
GFP by 250% compared to control were selected for target analysis.
miRNA databases and IPA predicted novel genes and pathways that
are involved in regulating the RPE epithelial phenotype. The results
were confirmed by a subsequent siRNA screening comprising of 122
siRNAs. Small molecule inhibitors and activators of these pathways
are currently being tested to modulate in vitro RPE phenotype.
Conclusions: HTS on iPSC-RPE identified novel genes and
pathways involved in RPE epithelial phenotype maintenance, and
potential therapeutic targets for RPE-associated conditions like PVR.
Additionally, these pathways can provide potential targets to enhance
the protocol of RPE differentiation from pluripotent stem cells.
Commercial Relationships: Justin R. Chang, None; Balendu
Shekhar Jha, None; Madhu Lal, None; Ruchi Sharma, None;
Marc Ferrer, None; Kapil Bharti, None
Program Number: 239 Poster Board Number: B0245
Presentation Time: 8:30 AM–10:15 AM
F-actin organization as potency indicator for high-quality,
functional human RPE cell culture
Claudia Müller, Silvia C. Finnemann. Department of Biological
Sciences, Center for Cancer, Genetic Diseases and Gene Regulation,
Fordham University, Bronx, New York, NY.
Purpose: Retinal pigment epithelial (RPE) cell replacement
therapies for treatment of age-related macular degeneration are under
development. Cells used for transplantation must establish critical
functional interactions with photoreceptors in the diseased retina.
Phagocytosis of shed photoreceptor outer segment fragments (POS)
is one important property of differentiated RPE. We investigated
cellular markers that may predict robust phagocytic capacity.
Methods: RPE cell lines derived from adult human RPE stem
cells (RPESC-RPE) passage 2 were used for experiments. Western
blot analysis, immunostaining and confocal microscopy were used
to study cell morphology and marker protein expression. Protein
secretion was assessed by ELISA. Binding and internalization of
purified POS were quantified to assess phagocytic capacity of RPE
cells in culture.
Results: Phagocytosis assays showed that RPE cell lines could
be categorized as either highly phagocytic (with 55-80% of cells
engulfing POS in our assay) or poorly phagocytic (with 5-40% of
cells engulfing POS). Regardless of phagocytic capacity, all RPE
lines tested expressed known proteins of the phagocytic pathway
and secreted phagocytic ligands (MFG-E8 and ProteinS). However,
in poorly phagocytic cells, POS surface binding and apical ezrin
localization were dramatically reduced indicating abnormal
microvilli. In highly phagocytic cells, the majority of cells showed
cobblestone epithelial morphology with an F-actin-rich apical brush
border, a circumferential apical adhesion belt and basal F-actin at
substrate attachment sites. In poorly phagocytic cells, less than 10%
of cells showed a circumferential F-actin belt, and most of these cells
contained less apical F-actin and abundant central and basal F-actin
stress fibers. Fewer than 1% of cells expressed the mesenchymal
marker smooth muscle actin (SMA). Inhibition of Rho-associated,
coiled-coil protein kinase (ROCK) was sufficient to improve
epithelial F-actin characteristics and to increase the fraction of cells
engulfing POS.
Conclusions: Our results suggest that F-actin cytoskeleton analysis
is sufficient to assess epithelial morphology of RPE cells in culture.
Moreover, alterations in F-actin directly correlate with and may
contribute to diminished phagocytic function of RPE in culture.
Commercial Relationships: Claudia Müller; Silvia C. Finnemann,
None
Support: Empire State Stem Cell Fund through New York State
Department of Health Contract #C028505
Program Number: 240 Poster Board Number: B0246
Presentation Time: 8:30 AM–10:15 AM
Molecular Mechanisms of Oxytocinergic Signaling and its
Inhibition of Kir7.1 in the RPE
Nathaniel York1, 2, Patrick Halbach1, Michelle Chiu1, Ian Bird1,
De-Ann M. Pillers1, 2, Bikash R. Pattnaik1, 2. 1Pediatrics, University
of Wisconsin - Madison, Madison, WI; 2McPherson Eye Research
Institute, Madison, WI.
Purpose: Oxytocin (OXT) is a neuropeptide that activates the
oxytocin receptor (OXTR), a rhodopsin family G-protein coupled
receptor. We have localized OXTR to the retinal pigment epithelium
(RPE) and OXT has been found in the adjacent cone photoreceptors.
We hypothesize that there is OXTR signaling in the retina and sought
to characterize this signaling in the RPE and explore the downstream
effects of OXT on cellular signaling, focusing on the regulation of
inwardly rectifying K+ channel Kir7.1.
Methods: Ca2+ response to OXT was measured in cultured human
fetal RPE cells (hfRPE) using Fura-2AM in the presence of 2-APB
and nifedipine, pharmacological inhibitors of Ca2+ signaling
pathways. HEK-293 cells where used to establish stable expression
of human OXTR and signaling was visualized using live cell imaging
following transient expression of PH-GFP and PKC-GFP, monitors
of GPCR metabolites PIP2 and DAG. Whole cell patch clamp
electrophysiology was performed on HEK-OXTR cells transfected
with GFP-fused Kir7.1 as well as freshly isolated mouse RPE cells to
monitor Kir7.1 current.
Results: OXT treatment of RPE cells in culture resulted in a transient
increase in cytoplasmic Ca2+ that was reduced by 95% in the presence
of the IP3R antagonist, 2-APB (P<0.001). Upon bathing the cells
in Ca2+ free extracellular solution or nifedipine, the Ca2+ response
to OXT was not altered. While the amplitude of responses was not
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ARVO 2016 Annual Meeting Abstracts
altered, time to recover from the rise in [Ca2+]i peak was faster, with
time constants (τ) of 0.53 (r2 = 0.983), 0.93 (r2 = 0.975), and 1.7
(r2 = 0.987) min for Ca2+-free, nifedipine, and Ringer’s solution,
respectively. We also demonstrate that OXTR activation blunted
Kir7.1 channel current, which has a physiologic role in RPE function.
In isolated mouse RPE, we observed an average 61.81 ± 4.77 %
decrease in K+ inward current amplitude and an average 11.4 ± 3.2
mV depolarization in resting membrane potential.
Conclusions: We propose that OXTR utilizes multiple capacitative
Ca2+ entry (CCE) mechanisms to sustain an increase in Ca2+ driven
by intracellular signaling molecules coupled to OXTR/G-protein in
the RPE. This OXT-OXTR signaling in the RPE cell also integrated
mobilization of intracellular Ca2+ and the parallel modulation of
the Kir7.1 channel. We suggest that novel OXT-OXTR signaling
pathways in the outer retina will be of fundamental importance for
eye development, health and visual function.
Commercial Relationships: Nathaniel York; Patrick Halbach,
None; Michelle Chiu, None; Ian Bird, None; De-Ann M. Pillers,
None; Bikash R. Pattnaik, None
Support: Meriter Grant MSN180934; UW Foundation
Program Number: 241 Poster Board Number: B0247
Presentation Time: 8:30 AM–10:15 AM
Choroid endothelial cells regulate RPE tight junctions through
modulation of Bruch′s membrane assembly
Ignacio Benedicto1, Guillermo Lehmann-Mantaras1,
Michael Ginsberg2, Daniel J. Nolan2, Olivier Elemento4,
Nazia M. Alam3, 4, Glen T. Prusky3, 4, Arvydas Maminishkis6,
Sheldon S. Miller6, Shahin Rafii2, 5, Enrique J. Rodriguez-Boulan1.
1
Ophthalmology, Weill Cornell Medical College, New York, NY;
2
Angiocrine Bioscience, New York, NY; 3Burke Medical Research
Institute, White Plains, NY; 4Physiology and Biophysics, Weill
Cornell Medical College, New York, NY; 5Genetic Medicine, Weill
Cornell Medical College, New York, NY; 6NEI, NIH, Bethesda, MD.
Purpose: A hallmark of terminal retinal differentiation is the
coordinated maturation of RPE, Bruch′s membrane (BM) and choroid
vasculature. Recent studies demonstrate that endothelial cells (ECs)
play key instructive roles in the differentiation and maintenance
of parenchymal cells. We tested the hypothesis that choroid ECs
regulate terminal differentiation of RPE and the outer bloodretina barrier by modulating BM assembly, which in turn triggers
remodeling of RPE tight junctions (TJs).
Methods: We co-cultured in Transwell chambers human fetal RPE
(hfRPE) and either human umbilical vein ECs or mouse choroid ECs
isolated by in vivo labeling of VE-cadherin and flow cytometry cell
sorting. We assessed the effect of ECs on hfRPE TJs by measuring
transepithelial electrical resistance (TER) and occludin subcellular
localization by cell surface biotinylation and quantitative microscopy.
We studied extracellular matrix (ECM) deposition by hfRPE by
scanning electron microscopy and immunofluorescence assays. Lysyl
oxidase activity, which catalyzes collagen and elastin crosslinking,
was inhibited in EC-conditioned media by β-aminopropionitrile
(BAPN) treatment. We carried out RNAseq analyses of choroid ECs
from developing (P5) and adult (P30) mouse retinas. We evaluated
newborn mice treated for 30 days with BAPN for visual function by
optomotor response and for BM maturation by transmission electron
microscopy.
Results: EC-conditioned media induced a specific increase in hfRPE
TER and the accumulation of occludin along hfRPE TJs. These
effects were significantly impaired by BAPN treatment and EC lysyl
oxidase-like 2 (LOXL2) knockdown. ECs enhanced the appearance
of bundled collagen I-positive fibers in the ECM deposited by hfRPE,
and increased the levels of active β1 integrin at the hfRPE basal
plasma membrane. RNAseq analyses showed that several ECMrelated genes were differentially expressed in developing versus
adult choroid ECs. In particular, LOXL2 was significantly more
expressed in P5 than in P30 choroid ECs. In vivo, BAPN treatment
during mouse terminal retinal differentiation induced defects in BM
structure and visual function.
Conclusions: Our results suggest that the regulation of BM
composition and structure by choroid ECs is key for the maturation
of RPE TJs and the establishment of the outer blood-retina barrier,
essential for proper vision.
Commercial Relationships: Ignacio Benedicto, None;
Guillermo Lehmann-Mantaras, None; Michael Ginsberg,
Angiocrine Bioscience, Angiocrine Bioscience (I); Daniel J. Nolan,
Angiocrine Bioscience (I), Angiocrine Bioscience; Olivier Elemento,
None; Nazia M. Alam, None; Glen T. Prusky, CerebralMechanics
(I); Arvydas Maminishkis, None; Sheldon S. Miller; Shahin Rafii,
Angiocrine Bioscience (I); Enrique J. Rodriguez-Boulan, None
Support: NIH/NEI R01 EY08538-24-29
Program Number: 242 Poster Board Number: B0248
Presentation Time: 8:30 AM–10:15 AM
Helping retina to breathe by increasing fetal hemoglobin
production in RPE: relevance to outer retinal preservation in
hypoxic conditions characteristic of aging and diabetes
Pamela M. Martin1, 2, Wanwisa Promsote1, Folami L. Powell1,
Alan Saul2, Vadivel Ganapathy3, Pamela M. Martin1, 2. 1Biochemistry
& Molecular Biology, Georgia Regents University, Augusta, GA;
2
Ophthalmology and Culver Vision Discovery Institute, Georgia
Regents University, Augusta, GA; 3Cell Biology & Biochemistry,
Texas Tech University Health Science Center, Lubbock, TX.
Purpose: In normal adults, 97% of total red cell hemoglobin (Hb)
is adult Hb (HbA). Elevated fetal Hb (HbF) levels post-infancy
generally indicate underlying metabolic stress and tissue hypoxia.
HbF binds oxygen with higher affinity than HbA; increased HbF
production is regarded as a “rescue” attempt by the body to increase
oxygen extraction and subsequent delivery to tissues under low
oxygen conditions. Hb production by RPE brings new perspectives
regarding the understanding of outer retinal oxygenation under
normal conditions. In light of this discovery, the known high
metabolic activity and consequent demand of photoreceptors for
oxygen, here we evaluated whether HbF modulation confers benefit
in retina under hypoxic conditions characteristic of aging and
diabetes. Additionally, we investigated the underlying molecular
mechanisms responsible.
Methods: Mice engineered to produce human Hb were used in this
study: abnormal Hb (HbSS)-producing mice to study the effects of
HbF augmentation on chronic, low-grade hypoxia that occurs in
aging and, normal Hb (HbAA)-producing mice ± STZ, to examine its
effects on diabetes-induced hypoxia. Animals were treated ± the HbFinducer monomethylfumarate (MMF; 15 mg/ml in drinking water).
Retinal health/function was monitored by ERG, OCT, and routine
histologic, RNA and protein analyses. MMF-induced alterations
in gene/protein expression were evaluated also in ARPE-19 cells
cultured ± MMF (100 μM) under normal, hyperglycemic (25 mM
glucose) or hypoxic (100 μM CoCl2 or 1% oxygen) conditions.
Results: Oral MMF-therapy increased γ-globin mRNA/HbF
production systemically and in retina, reduced oxidative stress/
inflammation, and preserved outer retinal morphology/function
positively in chronically hypoxic retinas (HbSS and HbAA+STZ).
MMF induced Nrf2 and downregulated Bcl11A, a repressor of
γ-globin transcription and regulator of cell metabolism and
senescence. siRNA studies demonstrated the effect on Bcl11A to be
Nrf2-dependent.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Conclusions: This study exposes novel potential links between factor
that govern globin gene switching, redox status, metabolism and
senescence and suggests that HbF-inducing therapies might afford
multiple levels of benefit in hypoxic retina.
Commercial Relationships: Pamela M. Martin;
Wanwisa Promsote, None; Folami L. Powell, None; Alan Saul;
Vadivel Ganapathy, Georgia Regents University (P);
Pamela M. Martin, Georgia Regents University (P)
Support: NIH Grant EY022704
Program Number: 243 Poster Board Number: B0249
Presentation Time: 8:30 AM–10:15 AM
Functional studies of sodium/proton exchanger 8 in adult mouse
retina
Chun-hong Xia, Mei Li, Audrey Kim, Ian Ferguson, Xiaohua Gong.
School of Optometry and Vision Science Program, University of
California, Berkeley, Berkeley, CA.
Purpose: To determine the essential role of sodium/proton
exchanger 8 (NHE8) in the retina of adult mice and to investigate the
mechanism of how NHE8 regulates pH homeostasis in the retinal
pigment epithelium (RPE).
Methods: Using the novel clustered regularly interspaced short
palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from
Neisseria meningitidis (NmCas9), we studied the role of NHE8
in the retina of adult mice. Recombinant adeno-associated viruses
(AAV) carrying NmCas9 and guide RNAs were used to knockdown
NHE8 in the RPE of 4-week-old adult wild-type mice by subretinal
injection; eye samples were collected 4 weeks post injection;
immunostaining was performed to determine the distribution of
NHE8 and other proteins in mouse retina; and NHE8 expression
was examined by real-time PCR. Cultured RPE cells infected with
AAV-NHE8-pHluorin-mcherry were used for pH measurements in
intracellular compartments by monitoring the fluorescent intensity
ratios between pH-sensitive ecliptic pHluorin and pH-insensitive
mcherry.
Results: Our previous studies revealed that NHE8 knockout and
NHE8-M120K point mutant mice developed retinal degeneration
associated with aberrant RPE with disrupted cell polarity; NHE8
proteins were predominantly colocalized with the Golgi complex and
intracellular vesicles in the RPE as well as in the inner segments of
rod photoreceptors. Current study showed a loss of photoreceptor
cells in eyes injected with AAV-Cas9 and AAV-sgRNA target NHE8,
while normal retinal morphology was observed in mice injected with
AAV-Cas9 and AAV-control sgRNA. Real-time PCR analysis showed
about 32% reduction of NHE8 transcripts in NHE8-sgRNA injected
retinas compared to control-sgRNA injected retinas. Fluorescent
intensity ratios revealed different pH values in some of intracellular
compartments including protein trafficking pathways between RPE
cells expressing wild-type NHE8 and mutant NHE8-M120K, infected
with AAV-NHE8-pHluorin-mcherry and AAV-NHE8-M120KpHluorin-mcherry.
Conclusions: Sustained function and regulation of NHE8 are
essential for maintaining the polarity and function of RPE cells in
the retina of adult mice. NHE8 regulates pH homeostasis in some
intracellular compartments of RPE. Impaired NHE8 such as mutant
NHE8-M120K would disrupt the protein trafficking or recycling to
affect the polarity, phagocytosis and other functions of RPE cells,
leading to photoreceptor cell death.
Commercial Relationships: Chun-hong Xia, None; Mei Li;
Audrey Kim, None; Ian Ferguson, None; Xiaohua Gong, None
Support: A grant from The East Bay Community Foundation.
Program Number: 244 Poster Board Number: B0250
Presentation Time: 8:30 AM–10:15 AM
Anticancer Drug Cisplatin Differentially Affects Retina Pigment
Epithelial Cells Depending on Mitochondrial Backgrounds
Tej Patel, Cristina M. Kenney, Catherine Y. Liu, Marilyn Chwa,
Shari R. Atilano, Jon L. Norman. Gavin Herbert Eye Institute, Irvine, CA.
Purpose: Mitochondrial (mt) DNA can be classified into haplogroups
based upon single nucleotide polymorphism patterns that define racial
populations. The anticancer drug, cisplatin, is known to cause toxicity
to the retina, including retinal pigmentary changes. Our hypothesis
is that retinal pigment epithelial (RPE) cells with the same nuclear
DNA but containing different mtDNA backgrounds are differentially
affected by cisplatin.
Methods: Cytoplasmic hybrids (cybrids), cell lines with identical
nuclear genomes but different mitochondrial genomes, are created by
fusion of platelets (with mtDNA but no nuclear DNA) collected from
various individuals to RPE cells devoid of mtDNA (H haplotype,
3 cell lines; J haplotype, 3 cell lines). H and J cybrids were treated
for 48 hours with 25 or 50 µM cisplatin and cell viabilities were
measured using trypan blue exclusion. H and J cybrids were also
treated with 40µM Cisplatin for 48 hours and the mitochondrial
membrane potential (ΔΨm) was measured to test for early apoptotic
changes. QPCR was used to analyze cellular gene expression
changes. Untreated cultures served as the controls. Statistical analysis
was done using GraphPad Prism 5.
Results: With 25 uM and 50 uM cisplatin treatment, J cybrids
show decreased viability to 65% (p=0.043) and 42% (p=0.002) of
untreated, respectively. H cybrids had cell viability that was less
effected by cisplatin: 87% (p=0.58) and 62% (p=0.051) of untreated
H-cybrids, respectively. Cisplatin-treated and untreated H-cybrids
had similar ΔΨm: 5.3% ± 7.8% (p=0.5). In contrast, treated J-cybrids
s showed a 24.0% ± 4.1% (p<0.0001) decrease in ΔΨm compared
to untreated. Comparison of gene expression in J-treated versus
H-treated cybrids showed increase in apoptotic gene BAX (p=0.03,
fold 1.4), but a decrease in genes such as SFRP1 (p=0.0001, fold
0.21), DHRS2 (p=0.041, fold 0.58) and EFEMP1 (p=0.009, fold
0.45).
Conclusions: Retinal Pigment Epithelial cells with identical
nuclear DNA but H versus J mtDNA haplogroups show significant
differences in cell viability, mitochondrial membrane potential and
cellular gene expression levels when treated with the cancer drug
cisplatin. We show that mitochondrial DNA changes alone can affect
cellular response. These findings may help identify individuals who
may be more susceptible to cisplatin retinal toxicity depending on
their mitochondrial background.
Commercial Relationships: Tej Patel, None; Cristina M. Kenney,
None; Catherine Y. Liu, None; Marilyn Chwa, None;
Shari R. Atilano, None; Jon L. Norman, None
Support: This work was supported by Discovery Eye Foundation,
Guenther Foundation, Beckman Initiative for Macular Research,
Polly and Michael Smith Foundation, Max Factor Family
Foundation, Iris and B. Gerald Cantor Foundation.
Program Number: 245 Poster Board Number: B0251
Presentation Time: 8:30 AM–10:15 AM
Dissecting Exosome Biogenesis in Adult Human Retinal Pigment
Epithelial Cells (ARPE19): Specific Interaction of αB-crystallin
with Rab GTPase27b
Rajendra K. Gangalum1, Ankur M. Bhat1, Sirus A. Kohan3,
Suraj P. Bhat1, 2. 1Jules Stein Eye Institute, UCLA, Los Angeles, CA;
2
Molecular Biology Institute and Brain Research Insitute, UCLA, Los
Angeles, CA; 3Brain Research Insitute, UCLA, Los Angeles, CA.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Purpose: We have previously demonstrated that the small heat
shock protein, αB-crystallin (αB) is secreted from ARPE19 cells
in culture via exosomes (ARVO 2009; J Biol Chem. 286:3261-9,
2011). Exosomes are 50 -200 nm proteolipid vesicles that carry
molecular information (proteins and RNA) from one cell to the
other and therefore represent potential vectors of intercellular
communication between cells in the RPE; they may also have a role
in the maintenance of this epithelial layer as a physiological and
physical barrier at the blood/retina interface. As currently understood
exosomes are produced from the endocytic pathway that maintains
membrane protein (receptor) homeostasis. Here we examine the
status of this pathway in ARPE19 cells that express αB-crystallin and
those that do not.
Methods: We have employed Flow cytometry, Immunofluorescence
and Confocal Microscopy, Western blotting, Co-immunoprecipitation
and Double Immunogold labeling and Electron Microscopy to
elucidate the status of the endocytic pathway in αB expressing and
non-expressing stable ARPE19 αBshRNA transfected) cell lines
generated in our laboratory.
Results: In cells not expressing αB, the distribution of CD63
(LAMP3), an exosome marker, is markedly altered from the normal
dispersed pattern to a stacked perinuclear presence. However
sucrose density gradient analyses of cellular extracts indicate
continued exosome synthesis under conditions of impaired exosome
secretion. Immunoconfocal microscopy shows increased presence
of CD63(LAMP3) and LAMP1 indicating enhancement of the
endolysosomal compartment. This is further corroborated by
increased labelling of this compartment by Rab7, a Rab GTPase
known to be associated with the late endosome maturation. No
change is however, seen with Rab5. Interestingly, we find that αB
interacts specifically with Rab GTPase27b and not with Rab GTPase
27a in these cells suggesting that this small heat shock protein may
have a role in collaboration with other small GTPases in regulating
the secretion of exosomes from these cells.
Conclusions: These data point to a regulatory role for αB in exosome
biogenesis possibly via its involvement at a branch-point in the
endocytic pathway populated by Rab GTPases known to be involved
in multiple phases of secretion from eukaryotic cells.
Commercial Relationships: Rajendra K. Gangalum, None;
Ankur M. Bhat, None; Sirus A. Kohan, None; Suraj P. Bhat, None
Support: NIH Grant to SPB 1R01EY024929
Program Number: 246 Poster Board Number: B0252
Presentation Time: 8:30 AM–10:15 AM
PYK2 plays a role in matrix contraction by RPE-derived cells
Shigeo Tamiya, Kevin McDonald, Henry J. Kaplan. Ophthalmology
and Visual Sciences, University of Louisville, Louisville, KY.
Purpose: Contractile fibroblastic/myofibroblastic cells derived from
retinal pigment epithelial (RPE) cells have been implicated in the
fibrotic complications of proliferative vitreoretinopathy. We have
previously demonstrated that matrix contraction by RPE-derived cells
can be inhibited by dasatinib, an FDA-approved cancer medication
that inhibits multiple tyrosine kinases. The purpose of this project
was to identify molecular targets affected by dasatinib that contribute
to matrix contraction.
Methods: Primary cultured porcine RPE cells were used between
passages 3 to 5. Cells were cultured for 3 days in 25% vitreous fluid
supplemented DMEM in the presence or absence of tyrosine kinase
inhibitors for the final 24 hours. Western blot analyses were used to
determine the phosphorylation status of phospho-tyrosine proteins.
A type I collagen contraction assay was utilized to examine matrix
contraction by cells.
Results: Dasatinib reduced tyrosine phosphorylation of multiple
proteins. Interestingly, while FAK auto-phosphorylation on Tyr397
was unaffected, auto-phosphorylation on Tyr402 of PYK2, also
known as FAK2, was significantly reduced. In agreement with the
Western blot data, PF431396, a dual inhibitor of PYK2 and FAK,
significantly reduced matrix contraction while PF573228, an inhibitor
of FAK but not PYK2, was without effect.
Conclusions: The inhibitory effect of dasatinib on matrix contraction
by RPE-derived cells is, at least in part, due to the prevention of
PYK2 activation, which plays a role in the contractile process.
Commercial Relationships: Shigeo Tamiya, None;
Kevin McDonald, None; Henry J. Kaplan, None
Support: DoD/USAMRAA BAA W81XWH-15-1-0298; Research to
Prevent Blindness, New York, NY
Program Number: 247 Poster Board Number: B0253
Presentation Time: 8:30 AM–10:15 AM
MDM2 inhibition suppresses NF-κB-mediated inflammation in
retinal pigment epithelium cells
Biraj Mahato1, 2. 1Cell Biology and Immunology, University of North
Texas Health science Center, Fort Worth, TX; 2Laboratory of Ratinal
Rehabilitation, North Texas Eye Research Institute, Fort Worth, TX.
Purpose: Our group has shown that Nutlin-3, a MDM2 inhibitor
inhibits pathologic retinal endothelial cell proliferation through a
p53-dependent mechanism, but its p53-independent functions and
effect on retinal pigment epithelial (RPE) cell viability have not been
adequately elucidated. TNF-α induced RPE inflammation through
NF-κB is a known underlying mechanism of uveitis. Here, we
sought to investigate the non-canonical, p53-indendepent function
of Nutlin-3, and hypothesized that MDM2 inhibitors possess an
anti-inflammatory function benefiting patients suffering from retinal
inflammatory diseases.
Methods: For all experiments, ARPE-19 cells were cultured to >95%
confluence using standard methodology. In vitro cell viability was
assessed using a Hemacytometer and the MTT assay. To assess retinal
toxicity in vivo, 200 mM of Nultin-3 or vehicle (DMSO) (n=12
rat eyes per condition) was injected once weekly for four weeks.
Retinal toxicity was assessed using histopathology, ERG, SD-OCT,
and fundus photography. To assess the anti-inflammatory effect of
Nutlin-3, ARPE-19 cells were infected with lenti-virus carrying an
NF-κB luciferase reporter.
Results: Nutiln-3 did not demonstrate a statistical difference in cell
viability at doses lower than 45 mM (N=3, p>0.5). Rats did not show
evidence of retinal toxicity, but some rats did develop cataracts,
which may be related to the vehicle or injection technique. ARPE-19
cells were pretreated with Nutlin-3 10 μM (a dose that does not alter
RPE cell viability) for 30 minutes to 7 hours, and then stimulated
with TNF-α (50 ng/ml) for 24 hours. TNF-α alone induced NFκB luciferase reporter gene activity, and Nutlin-3 pre-treatment
significantly attenuated this TNF-α response (p<0.05).
Conclusions: Based on these experiments, these data suggest that
Nutlin-3 does not cause retinal toxicity at doses less than 45 mM in
vitro and 200 mM in vivo. Interestingly, Nutlin-3 appears to have
a novel p53-indepenent function in RPE cells by inhibiting TNF-α
mediated NF-κB RPE inflammation.
Commercial Relationships: Biraj Mahato, None
Support: R01 EY026201-01
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
to access the versions of record.
ARVO 2016 Annual Meeting Abstracts
Program Number: 248 Poster Board Number: B0254
Presentation Time: 8:30 AM–10:15 AM
Glutaredoxin 2 (Grx2) Protects Retinal Pigment Epithelial Cells
from Oxidative Damage by Regulating Autophagy
Xiaobin Liu1, 2, Christy Xavier1, 2, Hongli Wu1, 2. 1North Texas Eye
Research Institute, Fort Worth, TX; 2Pharmaceutical Sciences,
University of North Texas Health Science Center, Fort Worth, TX.
Purpose: Glutaredoxin 2 (Grx2) is an oxidoreductase present in the
mitochondria where it protects the organelle from oxidative damage
and maintains its redox homeostasis. The purpose of this study is to
evaluate the cytoprotective effects of Grx2 in human retinal pigment
epithelial (RPE) cells and characterize its potential function in
regulating autophagy.
Methods: Primary RPE cells were isolated from Grx2 knockout
(KO) mice and treated with or without 400 µM H2O2 for 4 h. Human
retinal pigment epithelial (ARPE-19) cells were transfected with
either a human Grx2 cDNA-containing plasmid (pCR3.1-hGrx2)
or an empty vector pCR3.1. Cells were treated with or without 200
µM H2O2 for 16 h. Grx2 protein expression was detected by western
blot analysis. 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. The level of protein glutathionylation (PSSG) and
autophagy pathway proteins were measured by immunoblotting.
Results: Primary RPE cells laking Grx2 were more sensitive to
oxidative damage. On the other hand, Grx2 overexpression protected
RPE cells from H2O2-induced cell viability loss. Assessment of
apoptosis indicated that cells transfected with Grx2 were more
resistant to H2O2 with fewer cells undergoing apoptosis as compared
to vector control cells. PSSG accumulation was also attenuated by
Grx2 overexpression with acute H2O2 challenge. Furthermore, the
protein level of Beclin-1, a key molecule to initiate autophagy, was
inhibited in Grx2 overexpressed cells with H2O2 treatment. LC3II, a
protein that finalizes autophagosome maturation, was also increased.
Conversely, primary Grx2 KO RPE cells showed higher levels of
Beclin-1 and LC3II under oxidative stress.
Conclusions: Grx2 rescues RPE cells from lethal oxidative damage,
possibly through alleviation of ROS-triggered autophagy and
prevention of PSSG accumulation.
Commercial Relationships: Xiaobin Liu; Christy Xavier, None;
Hongli Wu, Reata Pharmaceuticals (F)
Support: BrightFocus Foundation for Macular Degeneration (Grant
No. M2015180 to Hongli Wu)
Program Number: 249 Poster Board Number: B0255
Presentation Time: 8:30 AM–10:15 AM
Optimization of Culture Conditions for Human Retinal
Progenitor Cells
Danhong Zhu2, Christine Spee1, David R. Hinton3, 1. 1Ophthalmology,
University of Southern California, Los Angeles, CA; 2Pathology,
University of Southern California, Los Angeles, CA; 3Pathology,
University of Southern California, Los Angeles, CA.
Purpose: The implantation of retinal progenitor cells (RPCs) is
under investigation for the therapy of retinal degeneration diseases.
However, methods to increase cell growth, promote survival, and
inhibit differentiation during in vitro culture remain a major challenge
in the production of quality RPCs for implantation. This study aimed
at optimizing the culture conditions to obtain high yield of quality
RPCs.
Methods: 1). Human retinas were isolated from fetal eyes at 18-20
gestational weeks, sheared into small cell aggregates by forcing them
through a 25-gauge needle several times, and cultured in suspension
in RPC medium for 24 hours. 2). RPC were then cultured in different
conditions: co-culture with polarized human embryonic stem cellderived (HES)-RPE cells, surface-attached culture, and suspension
culture. 3). RPCs cultured in different conditions and at certain time
points were collected and analyzed for gene profiles by qPCR.
Results: Using newly isolated retinas as a standard, RPCs in
suspension culture expressed the highest level of photoreceptor
marker genes, such as arrestin and rhodopsin, while RPC co-cultured
with polarized HES-RPE cells expressed the highest level of Ki67
(proliferation marker gene) among the three groups after 1 month
of culture. RPC in attached culture only expressed higher levels of
neuronal and photoreceptor marker genes at 15 days of culture, but
decreased the expressions of those genes at 1 month of culture.
Conclusions: The co-culture of RPCs with polarized RPE promotes
RPC growth and inhibits RPC differentiation; while suspension
culture increases RPC photoreceptor differentiation. Future studies
will focus on how culture conditions (co-culture and suspension
culture) may be used sequentially, to achieve the most efficacious
RPG for successful retinal integration, differentiation and function.
Commercial Relationships: Danhong Zhu; Christine Spee, None;
David R. Hinton, None
Support: DR3-07438, California Institute for Regenerative Medicine
(CIRM)
Program Number: 250 Poster Board Number: B0256
Presentation Time: 8:30 AM–10:15 AM
Computational Analysis of iPSC-Derived RPE Shape and
Primary Cilium Location to Create Predictive Models of Cell
Function
Nathan A. Hotaling1, 2, Nicholas Schaub2, Jorge Ferrari1,
Balendu Jha1, Carl Simon Jr.2, Kapil Bharti1. 1Unit on Ocular
and Stem Cell Translational Research, National Eye Institute,
Washington, DC; 2Biosystems & Biomaterials Division, National
Institute of Standards and Technology, Gaithersburg, MD.
Purpose: The emergence of primary cilium (PC) on human retinal
pigment epithelium (RPE) cells regulates maturation of RPE as
demonstrated by polarized expression of surface markers, tight
junction formation, enhanced phagocytosis of photoreceptor outer
segments (POS), and improved transepithelial potential/resistance
(TEP/TER). We hypothesized that by analyzing the location of the
PC as well as morphometric data from induced pluripotent stem cell
(iPSC) derived RPE we could predict RPE maturation as defined by
phagocytosis of POS, TER and TEP.
Methods: Primary human iPSC derived RPE were cultured in
porous transwell membranes over 6 weeks in the presence of PC
inducers/suppressors (Aphidicolin, PGE2, and HPI-4). At the end
of 6 weeks cells were stained for PC markers ARL13B, and GT335,
tight junction marker ZO-1 and actin cytoskeleton stain phalloidin.
Phagocytosis of labeled POS was quantified by flow cytometry.
TER and TEP was recorded using a modified Ussing chamber. Cell
morphometric quantification and location of PC, cellular center of
mass (COM) and cell centroid were assessed via a novel ImageJ
plugin. Linear and logistic regressions correlating POS level, TER
and TEP with mean distance of cellular PC from cell centroid and
COM as well as various morphological features were performed.
Results: Enhanced induction of PC in RPE cells improved cell
compactness and the number of neighbors per cell to a level similar
to that of native human RPE. The level of cellular phagocytosis of
POS, TER, and TEP were significantly correlated to PC distance from
both RPE centroid and COM. Additionally, models including cell
area, number of neighbors and aspect ratio of cell major and minor
axis were found to be significantly predictive of RPE functionality.
Conclusions: Using linear and logistic regression we show that
iPS cell-derived RPE maturation, as assessed by phagocytosis of
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
POS, monolayer TER and TEP can be predicted with high statistical
certainty using four metrics: either PC distance to RPE centroid or
COM, and cell area, number of neighbors, and the ratio of the cell
major and minor axis. Due to the significance of the predictive ability
of the model we conclude that analysis of fluorescent imaging of
cells can adequately predict functional cell phenotype in lieu of, or in
conjunction with, physiological tests.
Commercial Relationships: Nathan A. Hotaling; Nicholas Schaub,
None; Jorge Ferrari, None; Balendu Jha, None; Carl Simon Jr.,
None; Kapil Bharti, None
Support: NIH Grant EY000542-02
Program Number: 251 Poster Board Number: B0257
Presentation Time: 8:30 AM–10:15 AM
MicroRNA-34a inhibits proliferation, migration and adhesion of
RPEs through direct targeting LGR4
Qiang Hou, Linglin Zhou, Jiajia Tang, Nan Ma, Ancong Xu, Lili Tu.
Ophthalmology, Wenzhou Medical University, Wenzhou, China.
Purpose: We previously showed that microRNA-34a was
significantly downregulted in subconfluent in vitro cultured RPE
cells. Introduction of microRNA-34a or siRNA for its known
targets c-Met inhibited proliferation and migration of RPE cells. As
microRNAs can target multiple genes at the same time. We tried to
sought new target genes of microRNA-34a and investigate its role in
the regulation of RPE cells.
Methods: TargetScan Human 5.0 software was used to predict
potential targets of microRNA-34a. This was further validated by
double luciferase reporter assay and Western blot analysis. The
expression of target gene was knockdowned with siRNA interference
in RPE cells. Subsequently, the proliferative potential of RPE cells
was evaluated with MTS and Ki67 staining after transfection of
siRNA or microRNA-34a. The migrative ability was investigated
with transwell and in vitro scratch assays. The adhesion ability was
analyzed with cell attachment assay. The expression of downstream
molecules was detected by Western blot.
Results: TargetScan results suggested LGR4 is a putative target
of microRNA-34a. LGR4, also known as GPR48, is a member
of G-protein coupled receptor. Its role in development and caner
metastasis have been well defined. However, there is no report about
its role in RPE cells. We validated the direct regulation of LGR4
by microRNA-34a by double luciferase and Western blot assays.
Introduction of siRAN sequence or microRNA-34a inhibited the
proliferation, migration and adhesion of RPE cells. Furthermore,
LGR4 can regulate similar downstream molecules as microRNA-34a,
such as E2F1, p-CDC2, CDK2, CDK4 and CDK6.
Conclusions: Our results indicate that LGR4 is direct target of
microRNA-34a. MicroRNA-34a can inhibit the proliferation,
migration and adhesion of RPE cells through regulation of LGR4. As
microRNAs have promising therapeutical roles in many diseases, and
G-protein coupled receptors are ideal targets of drug development,
our results provide new insights in the prevention of PVR and other
related retinal diseases.
Commercial Relationships: Qiang Hou, None; Linglin Zhou,
None; Jiajia Tang, None; Nan Ma, None; Ancong Xu, None;
Lili Tu, None
Support: National Natural Science Foundation of China Grant
81100671, Zhejiang Provincial Natural Science Foundation of China
Grant LY16H120009 and Y2110609.
Program Number: 252 Poster Board Number: B0258
Presentation Time: 8:30 AM–10:15 AM
Primary Cilia Regulates Human iPSC-RPE Maturation via
Regulation of WNT Signaling
Qin Wan1, Balendu Jha2, Helen May-Simera3, Ruchi Sharma2,
Juliet Hartford2, Vladimir Khristov1, Kiyoharu Miyagishima1,
Mostafa R. Lotfi1, Sheldon S. Miller1, Kapil Bharti2. 1SERPD, NEI/
NIH, Bethesda, MD; 2OSCTR, NEI/NIH, Bethesda, MD; 3Institute of
Zoology, Johannes-Gutenberg University, Mainz, Germany.
Purpose: Induced pluripotent stem cell (iPSC) derivatives often
do not fully-mature in vitro, limiting their use in cell therapy and
disease modeling. Retinal pigment epithelium (RPE), a ciliated
monolayer critical for maintaining the health and integrity of
adjacent photoreceptors, is an attractive candidate for stem cell
therapies to treat blinding eye diseases including Age-related macular
degeneration. The primary cilium serves as a signaling and sensory
hub and controls developmental cellular processes in many cell types,
but the role of primary cilium in human RPE development remains
largely unknown. The goal of this study is to explore the function and
underlying mechanism of primary cilium in RPE development and
maturation.
Methods: Human iPSC derived RPE (iPSC-RPE) were grown
on semi-permeable transwells to generate a confluent monolayer.
Primary cilia in iPSC-RPE were manipulated using cilium inducers or
blockers, or canonical WNT agonist or antagonist. Electrophysiology,
immunocytochemistry, electron microscopy, gene expression, and
phagocytosis assays were used to determine the polarization and
maturity of iPSC-RPE monolayer.
Results: The progressive development of primary cilia in human
iPSC-RPE coincides with the formation of RPE tight junctions and
epithelial cell morphology. Experimentally enhanced activation
of primary cilia led to extensive apical processes, improved
pigmentation, increased expression of adult-specific RPE genes,
increased phagocytic capability, and significantly enhanced electrical
responses that mimic native human RPE, demonstrating improved
RPE maturation and functionality. Electrophysiological recordings in
combination with immunostaining and phagocytosis using canonical
WNT activator or inhibitors revealed that primary cilia-induced
iPSC-RPE maturation was regulated through the suppression of
canonical WNT signaling.
Conclusions: Our results demonstrate a developmental role for
primary cilia in human iPSC-RPE maturation, which is mediated
through the suppression of canonical WNT pathway. This study
provides a mechanistic tool to mature RPE or other epithelial cell
types derived from human iPSCs, and also provides insight into
retinal degeneration caused by ciliopathies.
Commercial Relationships: Qin Wan, None; Balendu Jha, None;
Helen May-Simera; Ruchi Sharma, None; Juliet Hartford,
None; Vladimir Khristov, None; Kiyoharu Miyagishima, None;
Mostafa R. Lotfi, None; Sheldon S. Miller, None; Kapil Bharti,
None
Support: NEI Intramural Funds, NIH CRM, and NIH Common Fund
Program Number: 253 Poster Board Number: B0259
Presentation Time: 8:30 AM–10:15 AM
Ex-vivo assessment of metabolic alterations of retinal
pigment epithelial cells using Fluorescence Lifetime Imaging
Ophthalmoscopy (FLIO)
Natalie Blimke1, Joachim Pruessner1, Lars Alt1, Gereon Huttmann2,
Ralf Brinkmann1, 2, Yoko Miura2, 1. 1Medical Lasercenter Luebeck,
Luebeck, Germany; 2Institut for Biomedical Optics, Luebeck,
Germany.
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ARVO 2016 Annual Meeting Abstracts
Purpose: Fluorescence lifetime (FLT) decays of endogenous
fluorophores in the fundus is becoming of more interest as a noninvasive and non-labeling indicator of retinal metabolic status. The
aim of this study is to investigate changes of FLT of retinal pigment
epithelial (RPE) cells in static organ culture over time and its
relevance to cell metabolic status.
Methods: Porcine RPE-choroid-sclera explants were cultivated
in static culture medium. At 2, 24 and 48h after the initiation of
cultivation the FLT of the RPE was measured using a fluorescence
lifetime imaging ophthalmoscope (FLIO: Heidelberg Engineering)
with 80 MHz pulsed excitation at 473 nm and two emission spectral
channels (channel 1: 498-560 nm, channel 2: 560-700nm) coupled
with a time-correlated single photon counting system. A custommade artificial eye model mimicking optical characteristics of the
human eye served as a measuring chamber for the explants. RPE
cell viability was examined using calcein-AM staining, and cellular
glucose uptake was estimated by measuring glucose concentration of
the conditioned medium using GlucCell® glucose meter.
Results: Calcein-positive living RPE cells in the culture was found in
99.4% ± 0.27 at 48h, which was not significantly different from at 2h.
There was an increase in cellular glucose uptake over time, from 48.0
± 14.2 mg/mm2/h at 2h to 56.2 ± 17.2 mg/mm2/h at 24h, and 56.3
± 17.3 mg/mm2/h at 48h (p=0.035). In FLIO, the FLT decay curve
of the RPE was well fitted to a biexponential curve. The t2 lifetime
(longer component) in channel 2 was significantly longer at 24h and
48h than at 2h (2308 ± 76 ps at 2h, 2473 ± 90 ps at 24h (p=0.006),
and 2430 ± 103 ps at 48h (P = 0.017)). The ratio of the amplitude of
t1 and t2 (a1/a2) in channel 2 also showed a significant decrease over
time, 12.4 ± 3.2 at 2h, 11.0 ± 1.7 at 24h, and 9.8 ± 1.4 at 48h (p=0.04
vs 2h).
Conclusions: Results suggest that FLIO might be useful to
investigate metabolic alterations of the ex-vivo RPE. The change
of cell energy metabolisms which increases glucose uptake might
result in the increase of the length of long lifetime component and its
amplitude. Further investigations will provide insight into its clinical
use, e.g. in evaluating cell health and metabolic activity of the in-vivo
RPE, or of the RPE sheet for RPE cell transplantation therapy.
Commercial Relationships: Natalie Blimke; Joachim Pruessner,
None; Lars Alt, None; Gereon Huttmann, None; Ralf Brinkmann,
None; Yoko Miura, None
Support: Heidelberg Engineering GmbH
Program Number: 254 Poster Board Number: B0260
Presentation Time: 8:30 AM–10:15 AM
Feedback Regulation of IRE1 During Unfolded Protein Response
in RPE
Sarah Melissa P. Jacobo, Maximilian J. Gerhardt, Arogya Khadka,
Daniel Diaz-Aguilar, Magali Saint-Geniez. Department of
Ophthalmology, Harvard Medical School / SERI, Boston, MA.
Purpose: Inositol-requiring enzyme 1 (IRE1) regulates the most
ancient branch of the unfolded protein response (UPR), and is present
in the endoplasmic reticulum (ER) of all eukaryotes. IRE1 signaling
is mediated by its splicing of X-box binding protein 1 (sXBP1), a
transcription factor that is essential for expression of UPR genes
that are required for ER stress relief. We previously reported that ER
stress induced HtrA1 expression in retinal pigment epithelial (RPE)
cells. This up-regulation was protective and essential for surviving
proteotoxic cell death. We tested the hypothesis that IRE1 influences
HtrA1 expression during ER stress in the RPE.
Methods: Chronic protein misfolding was stimulated in cultured
ARPE-19 cells using tunicamycin. HtrA1 expression was assayed
by immunoblot and real time qRT-PCR. Parallel experiments were
performed in the presence of 4μ8C (100 nM), an active site inhibitor
of IRE1α’s endoribonuclease activity. To test the effect of HtrA1
suppression on IRE1α signaling, we stably expressed HtrA1-specific
shRNA or control GFP shRNA. The total RNA pool was used in a
UPR array (Qiagen) and gene expression was compared for IRE1α
-related targets in vehicle- or tunicamycin-treated cells. For randomly
selected hits, qRT-PCR results were validated by immunoblot. Data
are representative of n>3 experiments, and *p<0.05 was considered
statistically significant.
Results: We mimicked conditions of proteotoxicity in RPE by
preventing the glycosylation and ER-to-Golgi export of nascent
peptides and found that intracellular HtrA1 protein accumulated in
a tunicamycin dose (0.01–11 μM) and time (0-24h) – dependent
manner. HtrA1 protein levels correlated with enhanced mRNA
transcription. The IRE1α ribonuclease blocker 4μ8C potently
inhibited the accumulation of sXBP1 and HtrA1 mRNA during
chronic protein misfolding. We examined the effect of HtrA1 on
IRE1α signaling in the face of ER stress. We found that expression
of IRE1α protein, and consequently that of sXBP1, was suppressed
after HtrA1 knockdown. Reduction in IRE1α and sXBP1 altered the
mRNA and protein expression of members of the IRE1 signaling
cascade. These include proteins which are critical to folding,
glycosylation, and export of nascent peptides.
Conclusions: Our results unravel a positive feedback loop that
integrates HtrA1 into the IRE1α pathway, and which has direct
consequences on RPE’s toolbox for combating proteotoxic stress.
Commercial Relationships: Sarah Melissa P. Jacobo, None;
Maximilian J. Gerhardt, None; arogya Khadka, None;
Daniel Diaz-Aguilar, None; Magali Saint-Geniez, None
Support: BrightFocus Foundation Macular Degeneration Award
M2014025
Program Number: 255 Poster Board Number: B0261
Presentation Time: 8:30 AM–10:15 AM
Usher proteins are engaged in vesicular trafficking in human
retinal pigment epithelial and fibroblasts cells
Bhagwat V. Alapure, Marianne Hathaway, Jennifer J. Lentz.
Neuroscience Center of Excellence, Louisiana State University
Health Sciences Centre, New Orleans, LA.
Purpose: Usher syndrome (Usher) is the most common genetic
condition that affects both hearing and vision. Sixteen genes are
associated with 3 clinical subtypes. Among the Usher genes, thirteen
are known and studies on their encoded proteins suggest that they
interact together and function in multiprotein complexes. Individual
Usher proteins, including myosin VIIa (USH1B), harmonin
(USH1C), cadherin 23 (USH1D), protocadherin 15 (USH1F), SANS
(USH1G), CIB2 (USH1J), usherin, (USH2A), VLGR1/GPR98
(USH2C), whirlin (USH2D), clarin-1 (USH3A), HARS (USH3B),
PZDZ7 (USH modifier) and CEP250 (atypical USH), are expressed
in hair cells and photoreceptors and are predicted to perform a wide
range of functions including intracellular trafficking, scaffolding,
cell adhesion and signaling. The exact function of these proteins and
mechanism by which they interact is unclear. The purpose of this
study is to determine the sub-cellular localization of Usher proteins
and the effect of blocking trafficking on their expression in human
retinal pigment epithelial (ARPE-19) and human dermal fibroblast
(hFB) cultured cells.
Methods: ARPE-19 and hFB cells were cultured with and without
trafficking inhibitors Brefeldin A (BFA) and Membrane Traffic
Inhibitor A5. The expression and sub-cellular localization of Usher
proteins were investigated using immunohistochemistry techniques.
Immunofluorescence intensity was quantified and analyzed using
ImageJ Software.
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ARVO 2016 Annual Meeting Abstracts
Results: All Usher proteins were found expressed in both ARPE-19
and hFB cells. All were found in the cytoplasm associated with the
endoplasmic reticulum. Myosin VIIa, harmonin and cadherin 23 were
also observed co-localized with the Golgi apparatus. Additionally,
myosin VIIa, harmonin, cadherin 23, protocadherin 15, SANS,
usherin and VLGR1 were found associated with F-actin showing a
punctate pattern; whereas SANS appeared to co-localize along the
long F-actin fibers. Lysosomal co-localization was not observed with
any of the Usher proteins. Reduced expression of all Usher proteins
was observed in the presence of BFA and A5 inhibitors.
Conclusions: These data suggest Usher proteins are involved in
intracellular trafficking between the ER and Golgi in ARPE-19 and
hFB cells.
Commercial Relationships: Bhagwat V. Alapure;
Marianne Hathaway, None; Jennifer J. Lentz
Support: The National Institutes of Health, Foundation Fighting
Blindness, Eye on Jacob Foundation, Usher in Sight and Sound, and
Usher 2020 Foundation.
Program Number: 256 Poster Board Number: B0262
Presentation Time: 8:30 AM–10:15 AM
Treatment of Haplogroup H and K ARPE-19 Cybrids with βAmyloid
Kunal Thaker, Marilyn Chwa, Shari R. Atilano, Tej Patel,
Cristina M. Kenney. Ophthalmology, Gavin Herbet Eye Institute UCI, Irvine, CA.
Purpose: Ashkenazi Jewish populations, mainly of the mitochondrial
(mt) haplogroup K, have been observed to have increased rates
of hypercholesterolemia, a risk factor for age-related macular
degeneration (AMD), compared to other Caucasian populations.
Apolipoprotein E (apoE) is a protein involved in lipid transport, and
processing the pathogenic peptide amyloid-β (am-β), known to have
a role in Alzheimer’s and AMD. Haplogroup H and K adult retinal
pigmental epithelial (ARPE) cytoplasmic hybrids (cybrids) were
treated with amyloid-β1-42 (am-β1-42) to determine if the differences in
APOE expression affected cellular response.
Methods: H (n=6) and K (n=8) haplogroup cybrids were created
using platelets from individuals. RNA and DNA were isolated from
cells cultured to the fifth passage. GeneChip Array Analysis was
performed to find the highest differential gene expression between
the two haplogroups and qPCR was performed on genes of interest.
Cells were plated on 96-well plates (5x104 cells per well) and treated
with 20µM am-β1-42 after 24 hours. An MTT assay was done to
observe cell viability 24 hours after treating. Each sample’s data
was normalized to its untreated average. Statistical significance was
determined through a student’s t-test.
Results: GeneChip Array Analysis found that the K cybrids had
increased gene expression in the atherosclerosis pathways compared
to H cybrids. QPCR showed that the K cybrids had a 9.3-fold
increase in the expression of APOE compared to the haplogroup
H cybrids (p<0.0001). APOC1, COL1A1, and MMP1 were also
measured but no significant difference was found between the H and
K cybrids. After treatment with am-β1-42 the cell viability in H cybrids
dropped 30% ± 2.3% (p<0.001), while the K cybrids decreased 16%
± 2.0% (p<0.0001) relative to their respective untreated controls. The
difference in viability between the H and K cybrids post treatment
was 14.7% (p<0.0001).
Conclusions: These findings demonstrate that cells with haplogroup
K mtDNA transcribe higher levels of APOE and are more resistant to
am-β1-42 toxicity compared to the cells with haplogroup H mtDNA. In
other systems, apoE enhances the elimination of am-β and provides
cyto-protection. Our results suggest that in human RPE cells, higher
levels of APOE expression may have protective functions against the
pathogenic effects of am-β. These findings indicate that mtDNA may
mediate levels of APOE expression and play a role in apoE-related
diseases.
Commercial Relationships: Kunal Thaker, None; Marilyn Chwa,
None; Shari R. Atilano, None; Tej Patel; Cristina M. Kenney,
None
Support: Discovery Eye Foundation, Guenther Foundation,
Beckman Initiative for Macular Research, Polly and Michael Smith
Foundation, Max Factor Family Foundation, Iris and B. Gerald
Cantor Foundation.
Program Number: 257 Poster Board Number: B0263
Presentation Time: 8:30 AM–10:15 AM
Validation of pluripotent stem cell derived RPE (scRPE) as
a pure and renewable source for disease modeling and assay
development
Ulrich F. Luhmann2, Sonja Schlicht1, Faye M. Drawnel2,
Silke Zimmermann1, Carolin Willburger2, Claire Hippert1,
Verena Küppers2, Jean-Baptiste Vallier1, Jean-Philippe Carralot1,
Marco Prunotto1, Mark Burcin1. 1Roche Pharma Research and Early
Development, Therapeutic Modalities, Roche Innovation Center
Basel, F. Hoffmann - La Roche Ltd., Basel, Switzerland; 2Pharma
Research and Early Development, Ophthalmology Discovery and
Biomarkers, Roche Innovation Center Basel, F. Hoffmann - La Roche
Ltd., Basel, Switzerland.
Purpose: Impaired function and loss of the retinal pigment
epithelium (RPE) impact on photoreceptor health and contribute
to vision loss in degenerative retinal diseases such as age-related
macular degeneration, one of the most common causes for blindness
in the developed world. To identify a reliable and renewable source
of human RPE cells for disease modelling and assay development
we implemented a differentiation protocol for pluripotent stem cell
derived RPE cells (scRPE) and characterized their biological activity
and function relative to ARPE19 cells and commercially available
primary human RPE under different culture and assay conditions.
Methods: To obtain RPE from pluripotent stem cells we developed a
stepwise differentiation protocol based on small molecules inhibiting
the Wnt and Nodal pathways during early cell development.
To be assay compatible, we developed cultivation conditions
to routinely expand, bank and mature scRPE. To validate their
functional properties and compare scRPE to primary human RPE
and ARPE-19 cells, we utilized gene expression profiling by PCR
and immunocytochemistry to identify gene products that represent
important functional properties of RPE. Further, we established assay
conditions to test scRPE for their oxygen consumption capacity, ROS
and cytokine production, cell polarization and barrier function.
Results: Under identical cultivation conditions scRPE show a similar
morphology to primary RPE. PCR analysis and immunostaining
show that scRPE and primary RPE express RPE specific marker
genes such as ZO-1 (tight junction), RPE65 (visual cycle),
CRALBP (visual cycle), Tyrosinase (melanin biosynthesis), CD68
(phagocytosis) and VEGFA (vascular endothelial cell proliferation
and survival). In comparison, ARPE-19 cells clearly show differences
in morphology and gene expression. By measuring oxygen
consumption, cell polarization, barrier function, and stressor-induced
ROS and cytokine production scRPE cells produce comparable
results to primary human RPE.
Conclusions: ScRPE obtained by our differentiation protocol provide
a reliable and renewable source of RPE cells that are functionally
comparable to primary human RPE in several disease-relevant assay
formats. This uniform source of scRPE cells will be useful for disease
modelling in vitro and the development of novel assays for drug
development.
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Commercial Relationships: Ulrich F. Luhmann, F. HoffmannLa Roche Ltd; Sonja Schlicht, F. Hoffmann-La Roche Ltd;
Faye M. Drawnel, F. Hoffmann-La Roche Ltd; Silke Zimmermann,
F. Hoffmann-La Roche Ltd; Carolin Willburger, F. HoffmannLa Roche Ltd; Claire Hippert, F. Hoffmann-La Roche Ltd;
Verena Küppers, F. Hoffmann-La Roche Ltd; Jean-Baptiste Vallier,
F. Hoffmann-La Roche Ltd; Jean-Philippe Carralot, F. HoffmannLa Roche Ltd; Marco Prunotto, F. Hoffmann-La Roche Ltd;
Mark Burcin, F. Hoffmann-La Roche Ltd
Program Number: 258 Poster Board Number: B0264
Presentation Time: 8:30 AM–10:15 AM
A Porcine Model of Retinal Pigment Epithelium (RPE)
Injury to Test the Efficacy of Human Induced Pluripotent Stem
Cell– derived RPE (hiPSC-RPE) Transplants
Juan Amaral1, 2, Maria M. Campos1, 3, Arvydas Maminishkis1, 4,
Vladimir Khristov1, 4, Raymond Zhou1, 4, Steve T. Charles5, 1,
Boris Stanzel6, 1, Balendu Jha1, 2, Irina Bunea1, 2, Sheldon S. Miller1, 4,
Kapil Bharti1, 2. 1National Eye Institute, Bethesda, MD; 2Ocular and
Stem Cell Translational Research, Bethesda, MD; 3NEI Histology
Core, Bethesda, MD; 4Section on Epithelial and Retinal Phisiology
and Disease, Bethesda, MD; 5Charles retina Institute, Memphis, TN;
6
Ophthalmology, University of Bonn, Bonn, Germany.
Purpose: RPE replacement therapy has potential for the treatment of
retinal degenerative diseases. Autologous hiPSC-RPE transplantation
avoids immune rejection and the complications of long-term
immunosuppression. We have developed a porcine model of localized
retinal degeneration and a surgical procedure to test the ability of
hiPSC-RPE cell transplants to protect RPE injury induced retinal
degeneration.
Methods: A micropulse laser with a 3% duty cycle and power
between 600-750 mW was used to create a 5.4 mm2confluent grid
lesion in 3 months old pigs. Histologic evaluation (n=15) was done
on days 0, 2, and weeks 1, 2, 3 and 6 after laser. In selected cases,
(n=14) 2 days after laser a standard 25G 4 port pars plana vitrectomy
was done and a localized retinal detachment (RD) was created with
a 38G cannula. Using a custom made injection tool, hiPSC-RPE
cells on a PLGA biodegradable scaffold were introduced into the
subretinal space. The retina was attached using fluid air exchange
(FAX) without retinopexy.
Results: Laser treatment leads to an immediate RPE detachment with
edema. Bruch’s membrane and choriocapillaries are not damaged.
By day 2 there is extensive RPE and photoreceptor damage but the
photoreceptor nuclear layer (ONL) is still present. Over the next 3
weeks the ONL shrinks in the areas not transplanted by the hiPSCRPE scaffold with slow recovery after 5 - 6 weeks. During surgery
a localized RD is induced in the laser treated area. The injector is
introduced into the subretinal space through a retinotomy and the
RPE scaffold is released. FAX is used to attach the RD and to close
the enlarged sclerotomy via surface tension. Major complications
include hemorrhage, proliferative vitreous retinopathy, and immune
reaction against human cells. The overall success rate of the
procedure is more than 50%.
Conclusions: We have developed a porcine laser model for RPE
ablation with selective outer retina damage. Our model and surgical
techniques are suitable for in- vivo functional evaluation of hiPSCRPE transplants and their ability to rescue laser-damaged outer retina.
Commercial Relationships: Juan Amaral, None;
Maria M. Campos, None; Arvydas Maminishkis, None;
Vladimir Khristov, None; Raymond Zhou, None;
Steve T. Charles, None; Boris Stanzel, None; Balendu Jha, None;
Irina Bunea, None; Sheldon S. Miller, None; Kapil Bharti, None
Program Number: 259 Poster Board Number: B0265
Presentation Time: 8:30 AM–10:15 AM
Development of a high content imaging photoreceptor outer
segment phagocytosis assay in inducible pluripotent stem cell
(iPSc)-derived RPE cells
Megan Jabour, Heather MacLeod, Frada Berenshteyn,
Arnaud Lacoste, Kathryn McAllister. Novartis Institutes for
BioMedical Research, Cambridge, MA.
Purpose: Daily phagocytosis of shed photoreceptor outer segments
(OS) by the retinal pigmented epithelium (RPE) is essential to
maintaining the health of the retina, and dysfunction of this process
has been implicated in retinal degeneration. Prior studies have
focused on OS phagocytosis by transformed RPE cell lines or freshly
isolated RPE cells. The aim of these studies is to use high content
imaging to develop an OS phagocytosis assay with iPSc derived RPE
cells.
Methods: iPSc derived RPE cells and the transformed RPE cell
line, ARPE-19, were cultured in 384-well plates for 3-5 weeks
prior to phagocytic challenge. Isolated bovine OS were labeled
with FITC isomer or Qtracker 800 nanocrystals. OS were incubated
with RPE cells for 2-24 hours at 37°C, washed, and fixed with
4% paraformaldehyde. Cells were then permeabilized with ice
cold methanol and stained for rhodopsin or ZO-1, to evaluate tight
junction formation. Images were acquired on the Molecular Devices
ImageXpress MicroXL, and OS counts quantified using MetaXpress
Transfluor HT analysis module.
Results: Both iPSc derived RPE and ARPE-19 cells form tight
junctions when cultured in 384 well format, but only iPSc derived
RPE develop the cobblestone morphology typical of RPE cells.
Visualization of RPE phagocytosis was achieved through direct
labeling of OS with either Qtracker nanocrystals or FITC isomer.
Complete colocalization with rhodopsin staining was observed
with FITC labeled OS. Further evaluation of serial diluted OS
demonstrate that ARPE-19 and iPSc derived RPE cells phagocytose
OS in a concentration-dependent manner with maximal phagocytosis
occurring at approximately 4 hours.
Conclusions: We have developed a 384-well high content
imaging assay allowing for visualization and quantification of OS
phagocytosis by iPSc derived RPE cells. This in turn may enable high
throughput evaluation and deconvolution of pathways involved in the
phagocytic process.
Commercial Relationships: Megan Jabour, Novartis;
Heather MacLeod, Novartis; Frada Berenshteyn, Novartis;
Arnaud Lacoste, Novartis; Kathryn McAllister, Novartis
Program Number: 260 Poster Board Number: B0266
Presentation Time: 8:30 AM–10:15 AM
Autophagy machinery is functional in human pluripotent stem
cell-derived retinal pigment epithelial cells
Kati M. Juuti-Uusitalo1, Niko Kivinen2, Johanna Viiri2,
Juha Hyttinen2, 7, Arto Koistinen3, Hannu M. Uusitalo4, 5,
Debasish Sinha6, Heli Skottman1, Kai Kaarniranta2. 1University
of Tampere, BioMediTech, Tampere, Finland; 2Department of
Ophthalmology, Institute of Clinical Medicine, University of Eastern
Finland, Kuopio, Finland; 3SIB Labs, University of Eastern Finland,
Kuopio, Finland; 4Department of Ophthalmology, University of
Tampere, Tampere, Finland; 5SILK,TAUH Eye Center, Tampere
University Hospital, Tampere, Finland; 6The Wilmer Eye Institute,
The Johns Hopkins University School of Medicine, Baltimore, MD;
7
Department of Ophthalmology, Kuopio University Hospital, Kuopio,
Finland.
Purpose: The impairment in autophagic and proteasomal cleansing
has been documented in retinal pigment epithelial (RPE) cells and
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ARVO 2016 Annual Meeting Abstracts
age-related macular degeneration (AMD) pathology, but not studied
with human embryonic stem cell (hESC)-RPE cells.
Methods: Here the role of autophagy and proteolytic machinery was
assessed in the regulation of melanocytic pigmentation with a tandem
fluorescently tagged LC3 (GFP-mCherry-LC3) transfected or nontransfected mature hESC-RPE cells which were treated either with
MG-132, AICA ribonucleotide, 5-aminoimidazole-4-carboxamide-1β-D-ribofuranoside (AICAR), bafilomycin A1 or their combinations.
The effects were analysed by western blotting (WB), confocal and
electron microscopy (EM) and calcein fluorescence.
Results: Inhibition of proteasomal function with MG-132, or
autophagy with bafilomycin A1 increased the accumulation of
premelanosomes seen in EM. The upregulation of autophagy
markers SQSTM/p62 and MAP1LC3A/LC3-II, and the perinuclear
localization of them was observed in WB and confocal microscopy,
respectively. The AMP-dependent protein kinase activator AICAR
treatment decreased the proteasome inhibitor -induced accumulation
of melanosomes observed in EM, and also eradicated the SQSTM/
p62 and LC3-II detected in WB. The autophagy flux was shown to be
induced during the MG-132 proteasome inhibition, and was further
enhanced by simultaneous AICAR treatment, observed in confocal
microscopy with the GFP-mCherry-LC3. Bafilomycin A1 increased
amount of autophagosomes, premelanosomes and LC3-II observed in
EM, calcein fluorescence or WB.
Conclusions: Our result suggests that proteasomal inhibition
increases the expression of the autophagy flux markers SQSTM1/p62
and LC3-II and lead to elevated accumulation of autophagosomes
and melanosomal granules in the hESC-RPE. Furthermore, the
AMPK activator AICAR promoted cleansing of up-regulated melanin
granules, SQSTM/p62 and LC3-II, indicating autophagic cleansing.
These results reveal that autophagy machinery is functional in hESCRPE cells and regulates cellular pigmentation with proteasomes.
Commercial Relationships: Kati M. Juuti-Uusitalo, None;
Niko Kivinen, None; Johanna Viiri, None; Juha Hyttinen, None;
Arto Koistinen, None; Hannu M. Uusitalo, None; Debasish Sinha,
None; Heli Skottman, None; Kai Kaarniranta, None
Program Number: 261 Poster Board Number: B0267
Presentation Time: 8:30 AM–10:15 AM
Ano2 contributes to the outwardly rectifying Ca2+-dependent Clconductance in the retinal pigment epithelium
Olaf Strauss, Nadine Reichhart, Susanne Keckeis. Experimental
Ophthalmology, Charite University Medicine Berlin, Berlin,
Germany.
Purpose: The retinal pigment epithelium (RPE) expresses a variety
of Cl channels which contribute to transepithelial transport of Cland water. The Ca2+-dependent Cl channel in the RPE can adopt
the transepithelial transport to the functional needs by rises of
intracellular free Ca2+ as second messenger. Patch-clamp data from
different groups have described the Ca2+-dependent Cl channel in
the RPE as an outwardly rectifying channel which shows properties
of anoctamin-2 (Ano2), a member of a Ca2+-dependent ion channel
family. The purpose of the study is to characterize the molecular
identity of the Ca2+-dependent Cl channel in the RPE.
Methods: Analysis of the expression pattern of Ano2 in the mouse
retina and human ARPE-19 cell line; patch-clamp analysis of ATPinduced Cl- conductance in ARPE-19 cells; siRNA approach against
Ano2.
Results: In sagittal sections of the mouse retina, we found Ano2
in the basolateral membrane of the RPE. ARPE-19 cells showed
Ano2 expression at the mRNA and the protein level. Stimulation
of ARPE-19 cells with 500µM ATP, which leads to a transient
increase in intracellular free Ca2+, transiently stimulated an outwardly
rectifying Cl- conductance which showed reversal potentials close the
Cl- equilibrium potential and was blocked by DIDS and niflumic acid.
siRNA knock-down of Ano2 in ARPE-19 cells reduced the Ano2
protein and Ca2+-dependent Cl- conductance.
Conclusions: Ano2 contributes to the basolateral Ca2+-dependent
Cl – conductance in the RPE. Its biophysical properties correspond
with that of previous publications. Thus Ano2 is a major Ca2+dependent Cl channel in the RPE.
Commercial Relationships: Olaf Strauss, None;
Nadine Reichhart, None; Susanne Keckeis, None
Support: DFG grant STR480/14-1
Program Number: 262 Poster Board Number: B0268
Presentation Time: 8:30 AM–10:15 AM
Evaluation of the proliferative capacity of canine retinal pigment
epithelial cells harvested from different regions of the fundus
Freya M. Mowat1, Jonathan Hash1, Philip Mzyk2, Jill Harned2,
Steven Nagar2, Mary C. McGahan2. 1Clinical Sciences, North
Carolina State University, Raleigh, NC; 2Molecular Biomedical
Sciences, North Carolina State University, Raleigh, NC.
Purpose: Regional susceptibility to disease of the foveomacular
region is challenging to model in rodents due to the absence of a
region of enhanced cone photoreceptor density in these species.
The canine retina has a well-defined cone photoreceptor enriched
region known as the area centralis. We tested the hypothesis that
the canine central retinal pigment epithelium (RPE) is less capable
of proliferation in response to stress in culture using an established
tissue culture model.
Methods: We harvested RPE cells from young adult healthy mixedbreed dogs (n = 6 eyes) following humane euthanasia. Cells were
harvested and separate low-density cultures were made from the RPE
of the area centralis, nasal visual streak, superior peripheral retina
and inferior peripheral retina. As cultures approached confluency,
cells were fixed and immunocytochemistry for RPE65 and the cell
proliferation marker Ki67 was performed. We quantified the number
of Ki67 positive nuclei compared with all nuclei (DAPI nuclear
counterstain). A repeated measures ANOVA with Bonferroni post-test
was used to compare values from the area centralis with other regions
of the eye.
Results: As expected, the RPE cells of the area centralis contained no
observable melanosomes. Cells from all regions expressed RPE65,
supporting an RPE cell-type. There was no significant difference in
the density of the cells, at the time of initial culture (p = 0.27), or
at the time of assay (p = 0.2). The area centralis region (6.1 ± 5.3%
SEM) contained a significantly smaller proportion of proliferating
cells than the superior peripheral retina (38.5 ± 9.1%, p <0.05).
Conclusions: Mature retinal pigment epithelium cells of the canine
area centralis region have reduced capacity to proliferate. Our results
are consistent with the hypothesis that the retinal pigment epithelium
of the cone-rich canine area centralis remains in a more quiescent
state. Regional cultures of dog retinal pigment epithelium could be
used to further study the underlying causes of regional susceptibility
to foveomacular disease involving the RPE.
Commercial Relationships: Freya M. Mowat, None;
Jonathan Hash; Philip Mzyk, None; Jill Harned, None;
Steven Nagar, None; Mary C. McGahan, None
These abstracts are licensed under a Creative Commons Attribution-NonCommercial-No Derivatives 4.0 International License. Go to http://iovs.arvojournals.org/
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ARVO 2016 Annual Meeting Abstracts
Program Number: 263 Poster Board Number: B0269
Presentation Time: 8:30 AM–10:15 AM
Increased expression and apical distribution of betaA3/A1Crystallin in polarized RPE cells
Hiroto Terasaki1, 2, Parameswaran G. Sreekumar1, Shozo Sonoda2,
Christine Spee3, Taiji Sakamoto2, David R. Hinton3, 4, Ram Kannan1.
1
Arnold and Mabel Beckman Macular Research Center, Doheny Eye
Institute, Los Angeles, CA; 2Ophthalmology, Kagoshima University
Graduate School of Medical and Dental Sciences, Kagoshima, Japan;
3
Ophthalmology, Keck School of Medicine of the University of
Southern California, Los Angeles, CA; 4Pathology, Keck School of
Medicine of the University of Southern California, Los Angeles, CA.
Purpose: While recent studies have shown that alpha-crystallins
play an important role in retinal function, not much is known about
expression and function of beta-crystallins in the retinal pigment
epithelium (RPE). The purpose of this study was to investigate the
expression of betaA3/A1-crystallin in human RPE cells and the effect
of polarity and oxidative stress on its expression.
Methods: Polarized human fetal RPE cells with a mean TER of 385
± 13.1 Ω·cm2 were cultured following an established protocol in our
laboratory (Sonoda et al. Nature protocols 2009). Initial microarray
analysis of polarized and non-polarized H-RPE cells (Lonza,
Walkersville, MD) was carried out using a commercial array service
(Agilent Expression Array; Takara Bio, Yokkaichi, Japan). Expression
of betaA3/A1-crystallin in polarized and non-polarized human
RPE cells was assessed by western blot and real-time PCR. Apical/
basolateral localization of beta A3/A1 crystallin was determined by
immunostaining. To study the effect of oxidative stress on expression
of betaA3/A1 crystallin, polarized RPE cell were exposed to 500uM
of H2O2 for 24 h and expression was determined by western blot and
real-time PCR analysis.
Results: Microarray analysis revealed that, among crystallins,
betaA3/A1 crystallin was highly expressed in polarized RPE cells
(about 6.5 times higher compared to non-polarized RPE cells). This
finding was confirmed by western blot, confocal microscopy and realtime PCR which showed higher expression of betaA3/A1 crystallin
in polarized RPE cells while non-polarized RPE cells had negligible
expression. Oxidative stress caused a decrease in gene and protein
expression of betaA3/A1 crystallin in polarized RPE cells.
Conclusions: BetaA3/A1 crystallin was highly expressed in
polarized RPE cells and its expression decreased with oxidative
stress. The increased expression in the apical domain suggests a
possible role for betaA3/A1 crystallin to protect the neural retina.
Commercial Relationships: Hiroto Terasaki, None;
Parameswaran G. Sreekumar, None; Shozo Sonoda, None;
Christine Spee, None; Taiji Sakamoto, None; David R. Hinton,
None; Ram Kannan, None
Support: EY01545 and a grant from the Arnold and Mabel Beckman
Foundation
Program Number: 264 Poster Board Number: B0270
Presentation Time: 8:30 AM–10:15 AM
Modulation of Epithelial Mesenchymal Transition (EMT)
Through the Notch Signaling Pathway in Human Retinal
Pigment Epithelial Cells
Jinggang Yin1, Junming Yue2, Gunnan Zhao2, Wenying Huo2,
Weihong Huo1, Edward Chaum1. 1Ophthamology, University of
Tennessee Health Science Center, Memphis, TN; 2Pathology,
University of Tennessee Health Science Center, Memphis, TN.
Purpose: Epithelial mesenchymal transition (EMT) of the RPE plays
a central role in the development of proliferative vitreoretinopathy
(PVR), a blinding clinical complication of retinal detachment and
trauma. The Notch signaling pathway has been shown to regulate the
expression of EMT proteins; however, its role in PVR is unknown.
We examined the impact of Notch signaling in the RPE using a
genome editing CRISPR/cas9 approach to knock out Notch 2/3
expression in ARPE19 cells and evaluated its impact on EMT protein
expression and cell phenotype.
Methods: Two gRNAs were cloned into lentiviral CRISPR/Cas9
vectors and used to target the Notch1-4 receptors. Lentiviruses were
packaged in HEK293FT cells and ARPE19 cells were transduced
with lentiviral Notch KO constructs. Transfected cells were isolated
using puromycin and KO status confirmed by sequencing. EMT
protein markers were evaluated in the Notch KO cell lines by
Western blotting, and the cell phenotype was examined including
proliferation, migration, and susceptibility to apoptosis.
Results: Disruption of Notch2 in ARPE19 cells leads to the
upregulation of N-cadherin, β-catenin, Snai1, α-SMA and
downregulation of the E-cadherin and ZO-1 compared to the
CRISPR/Cas9 empty vector infected controls. Knockout of Notch2
promotes ARPE19 cell migration.
Conclusions: Disruption of the Notch2 gene indirectly promotes
EMT-associated gene expression in ARPE19 cells. Notch pathway
signaling may play an important role in contributing to the PVR
phenotype expressed by transformed RPE cells following retinal
detachment.
Commercial Relationships: Jinggang Yin; Junming Yue, None;
Gunnan zhao, None; Wenying Huo, None; Weihong Huo, None;
Edward Chaum, None
Support: Research to Prevent Blindness, Plough Foundation,
Hamilton Eye Institute NEI Core Grant for Vision Research
(P30EY013080), US Army Medical Research and Materiel
Command (W81XWH-15-1-0023)
Program Number: 265 Poster Board Number: B0271
Presentation Time: 8:30 AM–10:15 AM
A cell-based model to study wound repair in retinal pigmented
epithelial cells
YingHsuan Shih1, 2, Monte J. Radeke1, Pete J. Coffey1. 1Neuroscience
Research Institute, University of California-Santa Barbara, Santa
Barbara, CA; 2Molecular, Cellular, and Developmental Biology,
University of California-Santa Barbara, Santa Barbara, CA.
Purpose: In rodents, RPE can repair laser-induced wounds by
migration and proliferation. Whether human RPE employs a similar
mechanism is unclear. To gain further understanding on wound
healing of differentiated human RPE, we developed a cell-based
system to study RPE behavior, morphology, and gene expression in
response to chronic wounds.
Methods: Human fetal RPE were cultured on Electric Cell-substrate
Impedance Sensing (ECIS) 96-well plates for 30-60 days to
differentiate. Each well contains 2 electrodes, which can introduce
elevated electrical pulses to kill overlying RPE and create 0.25mm2
wounds. To create chronic wounds, electrical pulses were applied to
kill RPE once a day. TGFβ1 was applied to study the roles of TGFβ
signaling pathway on RPE repair. Wound healing was monitored by
real-time impedance recording. Cell proliferation was detected using
EdU labeling and cell size was measured based on ZO-1 staining.
Gene expression was analyzed by RNA-Seq.
Results: Immediately after wounding, cell death was detected as an
abrupt drop in impedance. After 3 hours, bystander cells began to
repopulate the electrodes. After 15 hours, the wounds were enclosed
by cells that originated from the peripheral. Both EdU+ and EdUcells were observed in/outside of the enclosed wounds indicating
that monolayer repair resulted from cell proliferation and migration.
Cell size and density remained stable following a single wound.
Conversely, after 20 treatments, RPE became larger and less dense.
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ARVO 2016 Annual Meeting Abstracts
After repeated treatments, gene expression of the whole population
remained constant despite the substantial changes in cell behavior,
morphology and cell numbers. These results suggest that monolayer
repair resulted from localized signaling. Moreover, activation of
the TGFβ signaling pathway repressed cell proliferation, prolonged
repair, and led to larger and less dense RPE.
Conclusions: Here we used a cell-based system to study the
repair mechanism of differentiated human RPE in response to
chronic wounding. We showed that human RPE employs both cell
proliferation and migration to repair disruption of the monolayer.
However, the capability to repair is reduced by chronic cell loss and
activation of the TGFβ signaling pathway. This cell-based system not
only allows us to dissect the detailed mechanisms that regulate RPE
repair under different conditions, but may also provide a platform to
understand the lack of repair in diseased RPE.
Commercial Relationships: YingHsuan Shih, None;
Monte J. Radeke, None; Pete J. Coffey
Support: LA1-02086; Garland Initiative for Vision Research Grant
Program Number: 266 Poster Board Number: B0272
Presentation Time: 8:30 AM–10:15 AM
Fluorescence Lifetime Imaging Ophthalmoscopy of in-vitro
retinal pigment epithelial cells under different glucose conditions
Joachim Pruessner1, Natalie Blimke1, Lars Alt1, Gereon Huttmann2,
Ralf Brinkmann1, 2, Yoko Miura2, 1. 1Medical Laser Center Lübeck,
Lübeck, Germany; 2Institute of Biomedical Optics, University of
Lübeck, Lübeck, Germany.
Purpose: Fluorescence lifetime imaging ophthalmoscopy (FLIO)
is a new method for measuring fluorescence lifetimes (FLT) of
endogenous fundus autofluorescence (AF). The metabolically active
retinal pigment epithelium (RPE) may largely contribute to the
fundus AF signal, and thus it is of great importance to elucidate its
FLT characteristics under different pathological conditions. The aim
of this study is to utilize the FLIO system to investigate in-vitro RPE
cells cultivated under different glucose conditions.
Methods: Second passage confluent porcine RPE cells cultured
on 12-mm Transwell® membrane inserts were used for the study.
The cells were cultured under glucose concentrations of 1000 (low
glucose: LG), 4500 (normal glucose: NG) or 9000 (high glucose:
HG) mg/L for 6 days. FLT was measured on day 3 and day 6, using a
prototype system of FLIO (Heidelberg Engineering), which is based
on a laser scanning ophthalmoscope with excitation at 473 nm and
two emission spectral channels (ch. 1: 498-560 nm, ch. 2: 560-700
nm). Measurements were performed by placing the membrane insert
into a custom-made artificial eye model. RPE cell viability was
evaluated using calcein-AM cell viability indicator.
Results: There was no significant change in RPE cell viability among
different glucose conditions. A biexponential fit was performed for
FLT decay analysis, thus each FLT consists of the short (t1) and long
(t2) components. Under LG and HG conditions, elongation of t1 and
t2 was typically seen (e.g. in ch. 1 on day 3, t1 was 355 ps ± 6.6, 366
ps ± 12, and 368 ps ± 16, and t2 was 2101 ps ± 64, 2123 ps ± 129
and 2179 ps ± 100, under NG, LG and HG conditions, respectively).
The apparent difference between LG and HG conditions was shown
in tm (mean FLT) of both channels on both days, where tm under
LG is significantly longer than under other conditions. Regarding
component contribution ratio (a1/a2), LG and HG conditions led
to the significantly higher a1/a2 of ch. 2 on day 6 (2.43±0.03 and
2.57±0.22, respectively), compared to the NG condition (2.15±0.18).
Conclusions: Our original setup is useful for measuring FLT of
cultivated RPE cells while maintaining cell viability. The results
suggest that glucose concentration-induced alterations of RPE
metabolic status can result in change of the length and contribution
of autofluorescence lifetime components, which can be detected by
using FLIO.
Commercial Relationships: Joachim Pruessner; Natalie Blimke,
None; Lars Alt, None; Gereon Huttmann, None; Ralf Brinkmann,
None; Yoko Miura, None
Support: Financial support by Heidelberg Engineering, Germany
Program Number: 267 Poster Board Number: B0273
Presentation Time: 8:30 AM–10:15 AM
Phagocytosis of photoreceptor outer segments regulates
mTORC1 activity in the retinal pigment epithelium
Bo Yu, Pei Xu, Zhen-Yang Zhao, Yan Chen. Ophthalmology and
Visual Science, University of Texas Medical Branch, Galveston, TX.
Purpose: Mechanistic target of rapamycin complex 1 (mTORC1) is
a central regulator of metabolism and integrates environmental cues
to adjust cellular metabolic activities in a timely manner. Retinal
pigment epithelial (RPE) cells remove daily shed photoreceptor outer
segments (POS) by phagocytosis. Degradation of ingested POS and
recycling its components generate demanding metabolic loads to
the RPE. In this study, we investigated whether POS can stimulate
mTORC1 activity in the RPE and explored the underlying molecular
mechanisms.
Methods: RPE mTORC1 activity was monitored during morning
burst of OS disc shedding, the time when synchronized phagocytosis
occurs. RPE cells were harvested from C57BL6/J mice started 30
minutes before light on to 3 hours afterwards. mTORC1 activity was
analyzed by monitoring the phosphorylation status of downstream
substrate proteins S6 and 4EBP-1. Subcellular localization of
mTORC1 components was measured by immunostaining of RPE
flat mounts. For mechanistic studies, cultured human RPE cells
were treated with purified porcine POS. To investigate the roles of
lysosomes in POS-induced mTORC1 activation, cells were either
treated with lysosome inhibitor chloroquine, or transfected with small
interference RNA targeting p18, V-ATPase6V0C, V-ATPase6V1A,
or SLC38A9. The activity and distribution of mTORC1 were then
measured by western blot or immunostaining.
Results: RPE mTORC1 was transiently activated during morning
burst of phagocytosis. Treating RPE cells with purified POS also led
to activation of mTORC1 in culture. Co-localization of rhodopsin and
LAMP1 with mTOR, p18, or RagA was detected by immunostaining,
indicating their distribution on phagolysosomes. In contrast to
the response to nutrient signals, disrupting proton gradients of the
lysosome or downregulating the V-ATPase transporter protein did not
affect POS-induced mTORC1 activation.
Conclusions: POS are physiological stimuli of mTORC1 in the RPE.
The daily fluctuation in mTORC1 activity responding to POS can be
critical in maintaining normal functions of the RPE and retina.
Commercial Relationships: Bo Yu; Pei Xu, None; ZhenYang Zhao, None; Yan Chen, None
Support: NIH grant EY 019706 and the International Retinal
Research Foundation
Program Number: 268 Poster Board Number: B0274
Presentation Time: 8:30 AM–10:15 AM
hiPSC-derived RPE cells: Characterization of blood-retinal
barrier properties and drug permeability
Jenni J. Hakkarainen1, Julien Maruotti2, Aila Seppänen1,
Brigitte Onteniente2, Giedrius Kalesnykas1, Mika Reinisalo1. 1R&D
department, Experimentica Ltd., Kuopio, Finland; 2Phenocell, Evry,
France.
Purpose: The retinal pigment epithelium (RPE) has a central role in
maintenance of the outer blood-retinal barrier (oBRB). The oBRB
efficiently limits drug access to the retina from systemic circulation
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ARVO 2016 Annual Meeting Abstracts
(i.e. choroidal blood). The purpose of this study was to investigate the
permeability, tight junction proteins and oBRB properties of a novel
in vitro RPE cell model, derived from human induced pluripotent
stem cells (hiPSC).
Methods: hiPSC-derived RPE cells (PCi-RPE1426, Phenocell,
France) were cultured on permeable Transwell® cell culture inserts
and the apparent permeability coefficient (Papp) values for standard
molecules were determined. Expression of the tight junction proteins,
occludin and ZO-1, was characterized in the PCi-RPE1426 model
by immunostaining. In addition, tert-butyl hydroperoxide (tBHP)induced oxidative stress in PCi-RPE1426 cells was assessed by using
a resazurin cell viability assay.
Results: The experimentally determined Papp value for the low
permeability standard molecule 6-carboxyfluorescein was 1.0×10-6
cm/s, representative of a tight paracellular barrier between PCiRPE1426 cells. The dynamic range was >14 in PCi-RPE1426
indicating high discrimination between high and low Papp values.
Furthermore, PCi-RPE1426 cells showed low susceptibility to tBHPinduced oxidative stress (IC50 = 2.2 mM).
Conclusions: We provide evidence that PCi-RPE1426 cells form
differentiated cell monolayers with intercellular tight junctions
characteristic of the oBRB. This correlated with a decreased Papp for
the low standard molecule, representing a 3-7 fold tighter barrier
compared to commonly used ARPE-19 cells. In addition, PCiRPE1426 cells were able to tolerate high levels of tBHP-induced
oxidative stress suggesting a potent endogenous antioxidant defense
system.
Commercial Relationships: Jenni J. Hakkarainen, Experimentica
Ltd.; Julien Maruotti, Phenocell (I), Phenocell; Aila Seppänen,
Experimentica Ltd.; Brigitte Onteniente, Phenocell, Phenocell (I);
Giedrius Kalesnykas, Experimentica Ltd. (S), Experimentica Ltd.
(I), Experimentica Ltd.; Mika Reinisalo, Experimentica Ltd.
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