Retinal Cell Biology (RC)

ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
141 Cell Differentiation and Cell Death in the Retina and RPE
Sunday, May 6, 2012, 3:15 PM - 5:00 PM
Hall B/C Poster Session
Program #/Board # Range: 1120-1150/A585-A615
Organizing Section: Retinal Cell Biology
Program Number: 1120 Poster Board Number: A585
Presentation Time: 3:15 PM - 5:00 PM
Canonical Wnt Signaling Modulates Chick Retina Regeneration
Jie Zhu, Julia Zavada, Amy Burke, Katia Del Rio-Tsonis. The Department of
Zoology, Miami University, Oxford, OH.
Purpose: The embryonic chick is able to regenerate its retina after retinectomy
when exposed to fibroblast growth factor (FGF). Regeneration occurs via the
activation of stem/progenitor cells present in the ciliary margin (CM) of the eye and
by transdifferentiation of Retina Pigmented Epithelium (RPE). Canonical Wnt
signaling plays a critical role in the development of peripheral eye structures which
house retina stem/progenitor cells and in RPE specification during eye
development. Thus, we wanted to study the role of canonical Wnt signaling during
retina regeneration from both cellular sources to better understand the molecular
basis of retina regeneration.
Methods: Immunohistochemistry was used to determine the expression of
molecules of the canonical Wnt pathway during retina regeneration. To examine
the role of this pathway during retina regeneration, RCAS retroviruses were used to
overexpress pathway activators or inhibitors. Eyes were collected at different stages
of regeneration and processed for histology and immunohistochemistry.
Results: Activation of canonical Wnt signaling assessed by nuclear b-catenin
immunostaining was detected in the CM as well as in the RPE in embryonic day 4
(E4) chick eyes. At E5 and E6 when the RPE is unable to transdifferentiate and the
CM has reduced regenerative ability, active b-catenin is absent in the RPE. After
retina removal at E4, b-catenin activation is reduced in the CM and increased in the
RPE cells to protect their phenotype. Intriguingly, Wnt signaling is completely
absent in FGF-induced regeneration from the CM and RPE transdifferentiation.
Over-expression of a negative lef1 construct (to inhibit canonical Wnt signaling) in
chick eyes after retinectomy was sufficient to induce regeneration from both
sources in the absence of FGF. Robust proliferation was observed in regenerating
retina and no cell death was detected by 7 days post-retinectomy. The regenerated
neuroepithelium differentiated into all major retina cell types with proper
Conclusions: Canonical Wnt signaling is downregulated in the CM after
retinectomy to allow those cells to undergo regeneration; on the contrary, the
canonical Wnt signaling is upregulated in RPE cells to protect their phenotype.
Inhibition of canonical Wnt signaling is sufficient to induce retina regeneration via
the activation of stem cells and by RPE transdifferentiation in the absence of FGF.
Commercial Relationships: Jie Zhu, None; Julia Zavada, None; Amy Burke,
None; Katia Del Rio-Tsonis, None
Support: NEI
Program Number: 1121 Poster Board Number: A586
Presentation Time: 3:15 PM - 5:00 PM
New Ex-Ovo Electroporation Technique Offers High Transfection Efficiency
for Primary Retinal Cell Cultures
M Natalia Vergara, Christian Gutierrez, M Valeria Canto-Soler. Ophthalmology,
Johns Hopkins School of Medicine, Baltimore, MD.
Purpose: In vitro culture systems not only complement in vivo studies to further
our understanding of retinal cell biology, but are also powerful tools for drug
development applications and the identification of factors that promote cell survival
and differentiation. For these and other purposes, the manipulation of gene
expression in primary retinal cell cultures is of critical importance, yet achieving
appropriate transfection efficiencies has remained challenging. The purpose of this
work was to develop an ex-ovo electroporation technique that would allow
transfection of chick retinal cells for primary cultures with higher efficiency than
other currently available protocols.
Methods: Embryonic chick eyes were enucleated, devoid of RPE and sclera, and
placed in an electroporation chamber filled with a plasmid solution. Square-wave
electroporation was applied and, after removing the lens and vitreous, retinas were
dissociated and cultured following standard techniques. Different parameters
including voltage and number of pulses, plasmid concentration and location in
relation to the retina, electrode type, and embryonic stage, were tested in order to
optimize the electroporation conditions. Plasmids encoding GFP or Pax6-IRESGFP under the control of a CAG promoter, were used. Transfection efficiency, cell
viability and cell differentiation were analyzed with a Cellomics ArrayScan at 1
and 4 days in culture. Pax6 expression was analyzed by immunohistochemistry and
Western blot.
Results: The best results were obtained by using custom-made electrodes devised
in our laboratory. The optimized protocol delivered transfection efficiencies in the
order of 20-30% (a significant improvement over the 5-8% transfection efficiency
obtained with other methods). Cell survival and cell-type ratios within the culture
were not significantly affected by the procedure, and both photoreceptors and nonphotoreceptor neurons were similarly transfected. A Pax6 encoding plasmid was
electroporated as an example of overexpression of a biologically significant gene,
resulting in Pax6 protein up-regulation and a concomitant decrease in
photoreceptor cell differentiation.
Conclusions: Our goal was to provide the eye research community with an
effective and efficient tool to manipulate gene expression in retinal cells in culture.
This protocol offers a significant improvement over other currently available
techniques, and we expect that it will prove useful to researchers working with this
Commercial Relationships: M Natalia Vergara, None; Christian Gutierrez,
None; M Valeria Canto-Soler, None
Support: NIH Grant EY04859, and Core Grant EY1765, RPB
Program Number: 1122 Poster Board Number: A587
Presentation Time: 3:15 PM - 5:00 PM
Dissecting the biological functions of Pumilio 2 in the developing visual system
Dani Sarkis1,2, Diane M. Cockburn2, Jason P. Charish2, Nardos G. Tassew1,2,
Philippe P. Monnier1,2. 1Genetics and Development, Toronto Western Research
Institute, Toronto, ON, Canada; 2Physiology, University of Toronto, Toronto, ON,
Purpose: The purpose of this study is to characterize the role of Pumilio2 (Pum2),
a founding member of the Puf family of mRNA binding proteins and translational
regulators, in the developing visual system. Particularly, the aim is to identify the
effects of Pum2 on eye development and axonal outgrowth.
Methods: The experimental model used in this study is the chicken visual system.
To examine the role of Pum2 in axonal outgrowth, retinal flat mount preparations
were electroporated with cDNA constructs. For the purpose of these experiments,
constructs over expressing, down regulating (Pum2 miRNA), and a dominant
negative form of Pum 2 (dnPum2) were cloned. For in vivo studies, the optic
vesicles of embryonic day 1.5 (E1.5) chicken embryos were electroporated with the
above constructs and eye size was measured at later stages of development.
Results: In situ hybridization confirmed the expression of Pum2 transcript in the
retina, including the retinal ganglion cell (RGC) layer, of chicken embryos.
Immunohistochemical staining revealed the expression of Pum2 in the optic fiber
layer of the retina. Also, when retinal explant preparations were stained for Pum2,
the protein was detected in the axons and growth cones of RGCs. When Pum2 is
electroporated into dissociated neurons, the length of neurites is approximately 84.7
± 2.7% (p < 0.0001) and 64.2 ± 8.2% (p = 0.001) shorter when compared to control
GFP and dnPum2 transfected neurons, respectively. Furthermore, a small eye
phenotype (micropthalmia) was observed at E9 following Pum2 overexpression in
the developing eye at E1.5.
Conclusions: Pum2 overexpression significantly decreased the length of neurites in
dissociated neurons and the eye size at E9. These preliminary data indicate a
significant involvement of Pum2 in the developing eye and axonal outgrowth.
More experiments are underway to investigate other functional aspects of Pum2 in
eye development.
Commercial Relationships: Dani Sarkis, None; Diane M. Cockburn,
None; Jason P. Charish, None; Nardos G. Tassew, None; Philippe P. Monnier,
Support: Vision Science Research Program Scholarship
Program Number: 1123 Poster Board Number: A588
Presentation Time: 3:15 PM - 5:00 PM
A Small Population of Nestin Positive Cells from Adult Human Neural Retina
Hui Lin, Lamei Deng, Ting Xie. Room A305, Medical Science Building, Tsinghua
University, Beijing, China.
Purpose: To establish human retinal cell lines with retinal stem cell potential from
primary culture of neural retina.
Methods: A total of 28 donor healthy human eyes were obtained within 24h post
mortem from the Tongren Eye Bank with full ethical approval, under the tenets of
the Declaration of Helsinki. The neural retina was gently seperated for culture.
Serum free medium with EGF and bFGF was used. When cells proliferated to form
confluent monolayer colonies, different morphological colonies were digested and
cultured respectively. Differentiation Media was maintained for 1 month for retinal
neurons induction. Standard protocols were used for IF staning of the cultured cells
at P2-P8 in serum-free medium and differentiation medium.
Results: Single cell suspensions from adult neuroretina were plated on gelatincoated plates. Only very few cells attached the plates within the first days, and even
less went proliferation after 4-6 days. Several confluent colonies with different
morphologies were formed after 2-3 weeks. For most colonies, cells could only
proliferated limited days and arrest the growth at P1. For some other type of
colonies, cells have short-term quick proliferation abilities and can be passaged for
2-3 times. For very few colonies (less than 0.5%) with the morphology (small size,
obvious nuclei and short spindle shape), cells can proliferate and maintained for
more than 8 passages. For this small polulation of cells, immunofluorescence
staining showed positive staining of Nestin and A2B5 in cell surface and
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
cytoplasma, and Sox2 and Islet1 in the nuclei. Pax6 staining was found both in the
cytoplasma and nuclei in these cells. More interesting, the putative Müller glia
marker, GS, which is expressed in the cytoplasma in Müller cells, was positive
staining in the nuclei of these retinal cells. For the other colonies derived cells, few
cells were positive staining with nestin and other stem cell marker. However, rare
cells with differentiation retinal neuron markers were identified in these cells in
differentiation media.
Conclusions: Although a small population of cells with retina stem cell markers
expression can be isolated from adult human neural retina and expanded ex vivo,
these cells are still hardly to be induced into retinal neurons.
Commercial Relationships: Hui Lin, None; Lamei Deng, None; Ting Xie, None
Support: NSFC81000403
Program Number: 1124 Poster Board Number: A589
Presentation Time: 3:15 PM - 5:00 PM
Human Retinal Pigment Epithelium Cell Secretions Promote Human Retinal
Progenitor Cell Differentiation into Photoreceptors
Matthew G. Trese1, Murilo B. Abud, Sr.2A, Caio V. Regatieri3, Michael J. Young2B.
Schepens Eye Rsch Inst/Boston Univ, Boston, MA; ARetina, BSchepens Eye
Research Inst, 2Harvard Medical School, Boston, MA; 3Ophthalmology, Schepens
Eye Res Inst - Harvard Medical, Boston, MA.
Purpose: This in vitro study developed a co-culture system that mimicked outer
retinal architecture and exposed human retinal progenitor cells (hRPCs) to the
apical secretions of a mature retinal pigment epithelium (RPE). In doing so we
were able to establish a relationship between RPE cell secretions and hRPC
Methods: In 20% oxygen conditions, ARPE19 cells were differentiated on the
underside of 12 well tissue culture inserts, until they attained a transepithelial
resistance (TER) value that was consistent with functional maturity. Mature
ARPE19 cell cultures were added to hRPC cultures for a period of seven days. At
the end of the co-culture period, hRPCs and ARPE19 cell cultures were analyzed
using immunocytochemistry (ICC) quantification and semi-quantitative reverse
transcriptase polymerase chain reaction (SQ-PCR). This study was repeated in both
20% and 3% oxygen tensions because environmental oxygen has a profound effect
on the proliferative capacity of RPCs.
Results: This study showed that a functionally mature RPE monolayer can be
derived in both 3% and 20% oxygen (TER values p< .05). Additionally, we
demonstrated the viability of a complex co-culture system as evidenced by cellular
survival and function throughout the co-culture period. Finally, this study showed
that the apical secretions of ARPE19 cells altered the gene and protein expression
of hRPCs resulting in the up regulation of the photoreceptor markers CRX,
Recoverin, Rhodopsin and Opsin Blue. Conversely, the gene and protein
expression of the immature markers PAX6, SOX2, KI-67, CMYC and nestin
decreased between control and co-culture groups. As expected cellular proliferation
markers increased in 3% oxygen when compared to 20% oxygen.
Conclusions: This study shows that mature RPE cell secretions promote the
differentiation of human RPCs into photoreceptors. Additionally this study
provides a spring board for future investigations into the specific trophic factors
that most efficiently drive photoreceptor specific differentiation.
Commercial Relationships: Matthew G. Trese, None; Murilo B. Abud, Sr.,
None; Caio V. Regatieri, None; Michael J. Young, None
Support: None
Program Number: 1125 Poster Board Number: A590
Presentation Time: 3:15 PM - 5:00 PM
Human Embryonic Stem Cell (hESC)-derived Retinal Pigment Epithelium
(RPE) as a Model to Screen Agents that Induce Autophagy
Tave van Zyl1A, Shawn M. Ferguson1B, Caihong Qiu1B, Lilangi S. Ediwickrema1A,
Lina Li1B, Ron A. Adelman1C, Lawrence J. Rizzolo1A. AOphthalmology/Surgery,
Cell Biology, COphthalmology, 1Yale University School of Medicine, New
Haven, CT.
Purpose: Pharmacological enhancement of autophagy in RPE has been proposed
as a strategy to reduce the burden of immunogenic debris associated with drusen
formation and Age-Related Macular Degeneration (AMD). In order to investigate
compounds altering autophagic flux in human RPE, we sought to establish a
method for monitoring autophagy in hESC-derived RPE cell lines, as well as assess
nuclear translocation of Microphthalmia Transcription Factor (MITF) as a novel
readout of autophagy.
Methods: RNA-sequencing identified hESC-derived RPE that most closely
resembled native RPE on a whole transcriptome level. The effects of starvation and
treatment with the mTOR inhibitors rapamycin and Torin were compared in hfRPE
and hESC-derived RPE, both in the presence and absence of the lysosomal
inhibitor, chloroquine. Degradation of long-lived proteins (standard autophagy
substrates) was quantified by pulse-chase labeling with 14[C]-valine, and overall
autophagosome formation was assessed by morphometric quantitation of
immunofluorescent LC3-positive punctate structures as well as immunoblotting of
LC3-I to LC3-II conversion. Additionally, changes in autophagy-related gene
transcription were measured via qPCR. Specific changes in nuclear abundance of
MITF were monitored by both immunofluorescence and subcellular fractionation.
Results: hESC-derived RPE was found to express 72% of autophagy-associated
mRNAs in similar levels as hfRPE. Induction of autophagy in response to either
nutrient starvation or pharmacologic inhibition of mTOR was observed in both
hESC-RPE and hfRPE as demonstrated by time- and dose-dependent increases in
degradation of long-lived proteins, LC3-positive punctate staining and LC3
conversion and turnover. Detection was enhanced by use of chloroquine to block
autophagosome turnover. In parallel with increases in standard autophagy markers,
we also observed a dramatic translocation of MITF to the nucleus.
Conclusions: We have established an effective system for monitoring autophagy in
hfRPE and hESC-derived RPE using a combination of classic (LC3-based) and
novel (MITF-based) approaches. This system will facilitate investigation of smallmolecule autophagy enhancers as ocular therapeutics to protect against and/or
repair age-related damage in the retina.
Commercial Relationships: Tave van Zyl, None; Shawn M. Ferguson,
None; Caihong Qiu, None; Lilangi S. Ediwickrema, None; Lina Li, None; Ron
A. Adelman, None; Lawrence J. Rizzolo, None
Support: Yale Endowed Research Fund,Connecticut Innovations, 10SBC02, Leir
Foundation, Newman’s Own Foundation
Program Number: 1126 Poster Board Number: A591
Presentation Time: 3:15 PM - 5:00 PM
Adult Human RPE Can Be Activated Into Multipotent Stem Cell That
Produces Mesenchymal Derivates
Enrique Salero1,2, Timothy A. Blenkinsop1, Barbara Corneo1, Ashley Harris1,3,
David Rabin1, Jeffrey Stern1, Sally Temple1. 1Neural Stem Cell Institute,
Rensselaer, NY; 2Ophthalmology, Bascom Palmer Eye Institute, Miami, FL;
University of Michigan, Ann Arbor, MI.
Purpose: RPE is a monolayer of cells underlying and supporting the neural retina.
It begins as a plastic tissue, capable, in some species, of generating lens and retina,
but differentiates early in development and remains normally nonproliferative
throughout life. Adult human RPE cells can be activated in vitro to a self-renewing
cell, the retinal pigment epithelial stem cell (RPESC) that loses RPE markers,
proliferates extensively and can re-differentiate into stable cobblestone RPE
monolayers. The purpose of this work is to explore whether this plasticity might
explain human pathologies in which mesenchymal fates are seen in the eye.
Methods: RPE tissue were obtained from postmortem donors and dissected and
gently dissociated in single cells. RPE cells were transferred into non adherent
plates and growth as spheres and then transferred into culture plates. After 2 weeks,
the cells became confluent. For clonal analysis single RPE cell was growth and
expanded. To determine their plasticity, both RPE culture types were examined for
their potential to form mesenchymal derivate by inductive treatments for neural,
osteogenic, chondrogenic, myogenic, adipogenic lineages. RPE cells were
dissociated and cultured as aggregates for 1 day and inoculated onto the CAMs
chick embryos and grown for 7 days. Masses were dissected, and cryostat sectioned
using mesenchymal markers for immunohistochemical analysis.
Results: RPE cells proliferate actively and self-renew over many passages, and
they express markers associated the stem cell state. We found that under
appropriate culture conditions RPE cells are highly proliferative and clonal studies
demonstrate that RPESCs are multipotent and in defined conditions can generate
both neural and mesenchymal progeny. This multipotency observed in human RPE
can be generalized across mammalian species showed the same capabilities.
Conclusions: RPE can be activated in vitro to a multipotent stem cell, the RPESC,
which can be expanded to produce stable RPE or readily differentiated into
mesenchymal lineages. This study establishes the RPESC as an accessible, human
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
CNS-derived multipotent stem cell can be expanded considerably even from
elderly donors and could be valuable to generate stable RPE cells suitable for
autologous or allogeneic replacement therapy and for producing in vitro models of
RPE disease.
Commercial Relationships: Enrique Salero, None; Timothy A. Blenkinsop,
None; Barbara Corneo, None; Ashley Harris, None; David Rabin,
None; Jeffrey Stern, None; Sally Temple, None
Support: New York State Department of Health, New York Stem Cell
(NYSTEM): C024399.
Program Number: 1127 Poster Board Number: A592
Presentation Time: 3:15 PM - 5:00 PM
Potential Of Undifferentiated Cells Isolated From Human Periodontal
Ligament To Generate Retinal Cells
Li Huang1, Yiqun GENG2, Jiajian LIANG2, Herman S CHEUNG3, Wai-ming
TSANG4, XiaoWu YAO5, Chi-Pui PANG1, Hin-Fai YAM1. 1Ophthalmology and
Visual Sciences, The Chinese University of Hong Kong, Hong Kong, China; 2Joint
Shantou International Eye Centre, Shantou, China; 3Department of Biomedical
Engineering, University of Miami, Miami, FL; 4Oral Maxillofacial Surgery and
Dentistry, North District Hospital, Hong Kong, China; 5Second Affiliated Hospital,
Shantou, China.
Purpose: We investigated the differentiation of the undifferentiated cells isolated
from neural crest-derived periodontal ligament (PDLUCs) into retinal neurons and
whether it can be an alternative source for retinal repair and regeneration.
Methods: PDL tissues were obtained from teeth collected from healthy donors,
aged 13 to 30, by manual scrapping and digestion with collagenase type I/III. The
dissociated cells were cultured in DMEM/F12 supplemented with 10% fetal bovine
serum and 2% penicillin/streptomycin for investigation of the expression profiles
by imunohistochemistry and RT-PCR. To imitate forebrain development, PDLUCs
were exposed to a retinal determination condition, i.e., antagonism of Wnt and
BMP signaling, with IGF-1 and bFGF. Their capacity to differentiate into retinal
cells was evaluated by expressions of retinal-specific markers using quantitative
PCR and confocal double immunofluorescence.
Results: Cultured PDLUCs at passages 2 to 5 expressed the mesenchymal and
neural crest markers CD90, CD44, p75/NGFR and Nestin, and the embryonic cell
antigen SSEA-4. They also expressed mRNA transcripts for Oct4, Sox2, Nanog
and c-Myc. Under retinal determination condition, PDLUCs yielded neurospheres
within 24 hours with an age-dependent decrease of neurosphere formation. These
neurospheres, when plated on matrigel-coated surface, formed neural rosettes and
proliferated. On day 20, Pax6 and rhodopsin were increased up to 50 folds. Retina
genes RX, DCX and Lhx2 were also significantly up-regulated. Similar
observations were made in a parallel experiment using human H9 embryonic stem
Conclusions: Neural crest-derived PDLUCs residing in human tooth exhibited
properties of cells at embryonic state and the potential to undergo retinal lineage
differentiation. They could provide an easily accessible and promising source for
retinal cell replacement.
Commercial Relationships: Li Huang, None; Yiqun Geng, None; Jiajian
Liang, None; Herman S Cheung, None; Wai-ming Tsang, None; XiaoWu Yao,
None; Chi-Pui Pang, None; Hin-Fai Yam, None
Support: Internal funding from DOVS, CUHK
Program Number: 1128 Poster Board Number: A593
Presentation Time: 3:15 PM - 5:00 PM
Pre Differentiated Human Retinal Progenitor Cells Integrate and Express
Mature Markers on the Host Retina
Caio V. Regatieri1,2, Petr Baranov1, Ana Carvalho1, ChiWan Ko1, Sarah Tao3,
Michael J. Young1. 1Ophthalmology, Schepens Eye Res Inst - Harvard Medical,
Boston, MA; 2Ophthalmology, Federal University of Sao Paulo, Sao Paulo, Brazil;
Advanced Development Center, CooperVision, Inc., Pleasanton, CA.
Purpose: Retinal degenerations cause permanent visual loss and affect millions of
people worldwide. Previous work has demonstrated the utility of using
biodegradable polymer scaffolds to induce differentiation and deliver retinal
progenitor cells for cell replacement therapy. In this study, we engineered a
biodegradable polycaprolactone (PCL) thin film scaffold with integrated submicron topography and analyzed its effect on the differentiation of human retinal
progenitor cells (hRPCs).
Methods: The PCL polymer was microfabricated with ridge-grooves or posts on
the surface. hRPCs were isolated from human retina of 14 to 18 weeks gestational
age and expanded in vitro, in low-tension oxygen (3%). At passage five the cells
were seeded in an 8-well slide (control group) and on three different types of PCL
scaffolds (smooth surface, ridge-groove and post). After one-week, real time
polymerase chain reaction (PCR) and immunocytochemistry (ICC) assays were
performed on hRPCs cultured on the biodegradable polymer and on the control
group, in order to evaluate the differentiation of hRPCs into photoreceptors.
Explant experiments and subretinal transplantation of pre differentiate hRPCs were
performed to analyze the cell migration and integration into the host retina of
rhodopsin -/- mice.
Results: Microfabricated topography in a PCL thin film enhanced the attachment
and organization of hRPCs compared to unstructured surfaces. hRPCs adherent to
PCL differentiated toward mature photoreceptor phenotypes as evidenced by
changes in mRNA and protein levels. Using real time quantitative PCR and ICC we
observed a statistically significant upregulation in the expression of rhodopsin,
CRX, recoverin and a statistically significant downregulation in SOX2 (a marker
for undifferentiated progenitor cells) and PAX6 compared to cells grown on
polystyrene. Additionally, transplanted pre differentiated hRPCs migrated into the
outer nuclear layer of the host retina and exhibited mature marker photoreceptor
Conclusions: This unique structured PCL thin-film scaffold provides a means to
organize and differentiate RPCs in a controlled manner. Moreover the pre
differentiate cells were able to integrate into a degenerated host retina.
Commercial Relationships: Caio V. Regatieri, None; Petr Baranov, None; Ana
Carvalho, None; ChiWan Ko, None; Sarah Tao, None; Michael J. Young, None
Support: Foundation Fighting Blindness, CAPES - Brazil, Pan American
Association of Ophthalmology
Program Number: 1129 Poster Board Number: A594
Presentation Time: 3:15 PM - 5:00 PM
MicroRNAs 204/211 Promotes Differentiation Of Human Retinal Pigment
Epithelial (RPE) Cells
Jeffrey Adijanto, John J. Castorino, Gerald B. Grunwald, Nancy J. Philp.
Anatomy, Pathology, & Cell Biology, Thomas Jefferson University, Philadelphia,
Purpose: Studies in various cell systems demonstrated the importance of
microRNAs in cell differentiation. In the RPE, miR-204 and -211 are the two most
highly expressed miRNAs and are important for epithelial function (Wang et al,
FASEB J, 2010). The goal of this study was to investigate the role of miR-204/211
in RPE differentiation.
Methods: Human fetal RPE (hfRPE) grown on transwells were used as a model
system. MicroRNA microarray was performed using human miRNA Microarray
(V2) from Agilent Technologies. hfRPE cells were transfected (Dharmafect 4) with
miR-204/211 mimics or inhibitors (AppliedBiosystems) once during seeding and
another three days later. Total RNA of samples were extracted using mirVana kit
and reverse transcribed using superscript III (Invitrogen). Changes in cell
morphology were evaluated using immunofluorescence confocal microscopy and
protein expression was analyzed by western blotting.
Results: In primary hfRPE cultures, seeding at low density induced RPE
dedifferentiation with a concomitant downregulation of miR-204, -211, and RPEspecific genes. Transfection of hfRPE cells seeded at low density with miR204/211 mimics prevented dedifferentiation and cells exhibited characteristic RPE
morphology as well as upregulation of genes and proteins that are critical for RPE
function (RPE65, CRALBP, MCT3). On the other hand, transfecting hfRPE cells
seeded at high density with miR-204/211 inhibitors prevented differentiation as
characterized by downregulation of RPE genes and loss of RPE morphology.
Microphthalmia-associated Transcription Factor (MITF) is a master regulator of
RPE differentiation and its expression was downregulated in dedifferentiated
hfRPE cells. Silencing MITF with siRNA resulted in decreased expression of miR204/211 and loss of epithelial morphology. Importantly, co-transfection of miR204/211 mimics with MITF siRNA rescued the RPE phenotype.
Conclusions: Our data demonstrate that miR-204/211 play a key regulatory role in
RPE differentiation and suggest that miRNA-based therapy could be a viable
approach for the treatment of ocular diseases that involve RPE dedifferentiation
such as PVR and AMD.
Commercial Relationships: Jeffrey Adijanto, None; John J. Castorino,
None; Gerald B. Grunwald, None; Nancy J. Philp, None
Support: EY012042
Program Number: 1130 Poster Board Number: A595
Presentation Time: 3:15 PM - 5:00 PM
Two-photon Microscopy and Fluorescence Lifetime Analysis of Lipid
Peroxidation Product in Photoreceptor Outer Segment and in Retinal Pigment
Epithelial Cell
Yoko Miura1A, Gereon Huettmann1A, Márta Szaszák1B, Regina OrzekowskySchroeder1A, Norbert Koop1A, Philipp Steven2, Ralf Brinkmann1A. AInstitute of
Biomedical Optics, BInstitute of Medical Microbiology and Hygiene, 1University of
Luebeck, Luebeck, Germany; 2Ophthalmology, University Hospital of Cologne,
Cologne, Germany.
Purpose: Lipid peroxidation products as 4-hydroxynonenal (4-HNE) are known to
form fluorescent protein adducts, and considered to be involved in the
lipofuscinogenesis in retinal pigment epithelial (RPE) cell. We have previously
shown that acute oxidative stress induces the appearance of lipofuscin-like bright
fluorescent inclusions in RPE cell. The purpose of this study is to elucidate the
pathogenesis of these inclusions in relation to the fluorescence properties of
associated lipid peroxidation products.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Methods: RPE-choroid tissue was isolated from porcine eye and preserved in the
culture medium. Lipid peroxidation was induced by exposing tissue to ferrous
sulfate and laser photocoagulation (Nd.YAG laser, λ=532 nm, 0.1 s, 300 µm) on
the RPE. RPE-autofluorescence (AF) was investigated with two-photon
microscopy combined with fluorescence lifetime measurement (FLIM). AF of
photoreceptor outer segments (POS) from porcine retina was also investigated
under normal and lipid peroxidation conditions to elucidate its association with the
AF in RPE cells. After examination of RPE-AF, the tissue was fixed and
immunolocalization of opsin, cellular retinalaldehyde binding protein (CRALBP)
and 4-HNE adducts were examined by multi-staining immunofluorescent method.
Results: Laser photocoagulation and exposure to ferrous ion induce the appearance
of bright AF granules inside and around RPE cells. The emission maximum of the
AF granules is at 450-500 nm with a two-photon excitation maximum at 710-750
nm. Their mean fluorescence lifetime (FL) is 0.655 ns. Addition of ferrous ion to
POS increases the intensity of their AF and shifts the emission maximum from 380450 nm to 450-500 nm and the mean FL from 1.3 ns to 0.65 ns. The extent of FL
reduction is correlated with the extent of intensity increase. Immunolocalization of
bright AF granules inside and around the RPE cells is coincident with the
localization of opsin and CRALBP, respectively, and also coincident with the
strong staining of 4-HNE.
Conclusions: These results suggest that heat- and ferrous ion-induced bright AF
granules inside and around RPE cells detected with two-photon microscopy are
phagocytised POS and retinoid-storing inclusions under lipid peroxidation,
respectively. Moreover, it is suggested that 4-HNE adducts may be one of the main
fluorophores of these AF granules.
Commercial Relationships: Yoko Miura, None; Gereon Huettmann,
None; Márta Szaszák, None; Regina Orzekowsky-Schroeder, None; Norbert
Koop, None; Philipp Steven, None; Ralf Brinkmann, None
Support: BMBF 01EZ0733
Program Number: 1131 Poster Board Number: A596
Presentation Time: 3:15 PM - 5:00 PM
An S-opsin Knockin Mouse (F81Y) Has Reduced Expression That Is Restored
By (exogenous) 11-cis Retinal
Lauren L. Daniele1A, Christine Insinna1A, Jason Davis2, DeLaine Larsen1A, Colleen
Kuemmel3, Jianhua Wang1A, Sergei S. Nikonov2A, Barry E. Knox3, Edward N.
Pugh, Jr.1B. ANeuroscience, BPhysiology, 1University of California, Davis, Davis,
CA; ANeuroscience, 2University of Pennsylvania, Philadelphia, PA; 3Neurosci &
Phys and Ophthal, SUNY Upstate Medical Univ, Syracuse, NY.
Purpose: To investigate the mechanisms of reduced S-opsin expression in
Opn1swF81Y/F81Y mice
Methods: We created a knockin mouse with a point mutation (F81Y) in S-opsin
that shifted the (λmax) from 360 to 425 nm, near that of human S-opsin, but
unexpectedly resulted in its reduced expression. The following methodology was
applied in parallel to WT and Opn1swF81Y/F81Y littermates to investigate the
mechanisms for the reduced S-opsin expression. Opsin expression was probed with
qRT-PCR and quantitative Western blot analysis. Electron microscopy was applied
to fixed retinal tissue to assess cone morphology. Cone function was assessed with
single cone recordings (sunction pipette recording). Immunohistochemistry
methods were employed to investigate S-opsin distribution throughout the cone
cell. Immuno-precipitation was performed in order to probe association of S-opsin
with ER associated degradation (ERAD) machinery. WT and Opn1swF81Y/F81Y
littermate pairs were injected with 11-cis retinal or vehicle on alternate days for a
10 day period.
Results: Our results indicate that S-opsin expression in the outer segments of
Opn1swF81Y/F81Y mice was reduced to 30-50% of the WT level, with no more than a
15% decrease in transcription. Despite a significant decrease of S-opsin expression,
Opn1swF81Y/F81Y mouse cones were morphologically normal and responded to light
with normal kinetics and the predicted shift in λmax. Mistrafficking of the mutant
opsin from ER/golgi to other cellular compartments does not account for its
reduced expression. Our immunohistochemistry experiments show a substantial
percentage (~25%) of total S-opsin signal outside of the outer segment
compartment, with comparable fractions in each compartment in both
Opn1swF81Y/F81Y and WT mice. However, F81Y S-opsin showed an increased
association with components of ERAD, ER degradation-enhancing alphamannosidase-like 1 (EDEM1) and valosin-containing protein (p97/VCP). The Sopsin in Opn1swF81Y/F81Y mice was restored to WT levels in littermate mouse pairs
receiving exogenous 11-cis retinal.
Conclusions: Despite reduced opsin expression, Opn1swF81Y/F81Y mouse cone
photoreceptors are healthy. However our results indicate that the mutant S-opsin is
less efficient at attaining a properly folded mature form in the ER, and a large
fraction of nascent S-opsin is degraded by ERAD. Our 11-cis retinal
supplementation results suggest that chromophore binding of nascent cone opsins is
critical for efficient biosynthesis and export from the ER.
Commercial Relationships: Lauren L. Daniele, None; Christine Insinna,
None; Jason Davis, None; DeLaine Larsen, None; Colleen Kuemmel,
None; Jianhua Wang, None; Sergei S. Nikonov, None; Barry E. Knox,
None; Edward N. Pugh, Jr., None
Support: EY-02660
Program Number: 1132 Poster Board Number: A597
Presentation Time: 3:15 PM - 5:00 PM
Rab38 Deficiency Leads To Loss Of Cathepsin D And a Defect In Rhodopsin
Degradation In Mouse Retinal Pigment Epithelium
Tanya Tolmachova, Miguel C. Seabra. Molecular Medicine, NHLI, Imperial
College London, London, United Kingdom.
Purpose: Rabs are small GTP-binding proteins that regulate intracellular vesicular
traffic. There are 60 known mammalian Rabs, some of which regulate general steps
and are expressed ubiquitously, while others perform a specialised function and are
expressed in a more restricted pattern. Rab38 was shown in the past to be expressed
in pigmented cells such as melanocytes and retinal pigment epithelium (RPE) and
be important for the delivery of tyrosinase to melanosomes. Deficiency in Rab38
leads to a significant reduction in a number of mature melanosomes in the RPE of
the Rab38 mutant mice (chocolate). The aim of this study was to investigate the
role of Rab38 in the phagocytosis of rod outer segments (ROS) in mouse RPE cells.
Methods: RPE cells were isolated from the eyes of Rab38 cht/cht, Rab38 cht/+ and
C57Bl6 mice and maintained in culture. Phagocytosis was studied in primary
mouse RPE cultures using FITC-labelled mouse ROS and data was collected by
FACS. Degradation of rhodopsin was followed using antibody specific to the Cterminus (1D4) in FITC-positive cells. Secretion of cytokines was measured in
primary RPE cultures by ELISA. The protein level of rhodopsin and cathepsin D
was analysed by FACS and Western blotting in RPE cells collected from the mouse
eyes 6 hours after the light onset.
Results: In primary Rab38cht/cht RPE cultures fed with FITC-labelled ROS we
detected a significant reduction in the rate of rhodopsin degradation in comparison
to Rab38 cht/+ and C57Bl6 RPE cultures. This result was confirmed by FACS in
non-cultured RPE cells where we detected a significant increase in rhodopsin
staining in Rab38cht/cht RPE in comparison to Rab38 cht/+ and C57Bl6 RPE.
Cathepsin D level was significantly reduced in Rab38cht/cht, in comparison to Rab38
and C57Bl6. Secretion of IL-6 (but not LIF) was specifically upregulated in
Rab38cht/cht in comparison to Rab38 cht/+ and C57Bl6 mice.
Conclusions: Rab38 is important for rhodopsin degradation. Cathepsin D is
severely reduced in the absence of Rab38 which suggests that Rab38 might be
involved in cathepsin D trafficking.
Commercial Relationships: Tanya Tolmachova, None; Miguel C. Seabra,
Support: WT093445MA
Program Number: 1133 Poster Board Number: A598
Presentation Time: 3:15 PM - 5:00 PM
Study Of The Mertk Mediated Phagocytosis Using Environmental Scanning
Electron Microscope (esem) And Atomic Force Microscope (afm)
Qingxian Lu1, Lixia Han2, Yong Tang2, Qian Liu2, Jiayi Xie3, Jiantao Feng3, Dong
Han3, Qingjun Lu2. 1Ophthalmology and Visual Sciences, Kentucky Lions Eye Ctr,
Univ of Louisville, Louisville, KY; 2Beijing Ophthalmology adn Visual Science
Key Laboratory, Capital Medical University Beijing School of Ophthalmology,
Beijing, China; 3National Center of Nanoscience and Technology, Chinese
Academy of Sciences, Beijing, China.
Purpose: MerTK-mediated phagocytosis is crucial for professional phagocytes,
e.g., macrophage, dendritic cell and retina pigment epithelium, to clear apoptotic
and spent cell debris. This study was planned to test how MerTK affected
phagosome uptaking; and whether the MerTK mutant phagocyte was able to uptake
the large targets, if yes, by what mechanism.
Methods: The macrophage and RPE were isolated from wild-type and MerTK
knockout mice and cultured in vitro for 24 hours before the assays. The time course
of the FITC-labeled apoptotic lymphocytes was compared between two groups.
The process of phagocytic cup formation was studied by environmental scanning
electron microscope (eSEM) using the 7-µm beads pre-coated with the whole
protein extract from apoptotic lymphocytes. The pulling strength generated by
cytoskeletal and dynamic proteins during engulfment and phagosome formation
was measured and analyzed by atomic force microscope (AFM).
Results: The cultured MerTK mutant macrophages were able to uptake apoptotic
lymphocytes, although at a significant low level compared to the wild-type
counterparts. However, they adapted a macropinocytotic mechanism. In the wildtype phagocytes, the phagocytic cup formation was initiated by denting the
membrane at the cell-cell contacting region and protruding the peripheral
membrane to sheathe the target particle, during which process the inner membrane
of the growing phagocytic cup was in close contact with the target surface. On the
other hand, the MerTK mutant phagocytes ate the targets by membrane ruffling,
and rolling the particles with membrane wave until engulfment was completed, a
typical manifestation of macropinocytosis as described by Swanson et al., (Mol.
Cell. Biol. 2008, 9:639). The AFM measurement of the particle-pulling strength
suggested that the MerTK-mediated phagocytosis generated a vertical pulling force,
which was necessary for up-taking the engulfed particles. Whereas the MerTK
mutant cells decreased this type of vertical pulling power, but instead, generated
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
additional horizontal pushing force, conceivably from the membrane ruffling when
the particle was endocytosed.
Conclusions: Our study showed that the MerTK mutant cells were still able to
uptake the targets although with inefficacy. The MerTK mutant cells preserved the
macropinocytosis mechanism for enclosing the large bound-particles. While the
wild-type phagocytes generated a vertical power for pulling the bound targets into
the cytoplasm, the mutants were defective, but replace with a dominant horizontal
pushing force to close-in the bound particles.
Commercial Relationships: Qingxian Lu, None; Lixia Han, None; Yong Tang,
None; Qian Liu, None; Jiayi Xie, None; Jiantao Feng, None; Dong Han,
None; Qingjun Lu, None
Support: NIH R01-EY018830; RPB special award
Program Number: 1134 Poster Board Number: A599
Presentation Time: 3:15 PM - 5:00 PM
In Vivo Analysis Reveals Novel Aspects of Retinal Disease Progression in Rsh1/Y
Mice but no Therapeutic Effect of Carbonic Anhydrase Inhibition
Ahmad Zhour1, Christian Grimm2, Marijana Samardzija2, Tobias Peters1, Torsten
Strasser1, Sylvia Bolz1, Bernhard H. F. Weber3, Eberhart Zrenner1, M. Dominik
Fischer1,4. 1Centre for Ophthalmology, University of Tuebingen, Tuebingen,
Germany; 2Lab for Retinal Cell Biol, Ophthalmology, University of Zurich,
Schlieren / Zurich, Switzerland; 3Institute of Human Genetics, University
Regensburg, Regensburg, Germany; 4Nuffield Laboratory of Ophthalmology,
University of Oxford, Oxford, United Kingdom.
Purpose: X-linked juvenile retinoschisis (XLRS) is the most common juvenile
maculopathy in men and is caused by mutations in the gene encoding retinoschisin
(RS1). Evidence in the literature on the therapeutic effect of carboanhydrase
inhibitors (CAIs) to treat schisis formation in the retina has remained equivocal.
Here, we evaluate the effect of the CAI dorzolamide on the structural and
functional disease progression in the mouse model for XLRS (Rs1h-/y).
Methods: Rs1h-/y mice were treated unilaterally with dorzolamide eye drops
(Trusopt® 20mg/ml) every 12 hours for 2 weeks starting on postnatal day 14
(n=27). Changes of retinal structure were monitored by confocal scanning laser
ophthalmoscopy and spectral domain optical coherence tomography at the age of 4,
6, 8, 12 weeks and 7 months. Changes in inner retinal thickness (IRT, as measure
of schisis formation) and outer retinal thickness (ORT, as measure of photoreceptor
degeneration) were quantified at all time-points. ERG was performed at the age of
8 and 12 weeks. At each time point two mice were sacrificed for histology.
Results: Schisis formation (peak at 3 months) preceded photoreceptor degeneration
and hyperfluorescence (peak at 7 months). Structural pathology was most severe in
the superior hemiretina. Quantitative analysis showed no significant differences
regarding the inner or outer retinal thickness of the treated vs. untreated eyes
(IRTOS-OD = -1.29±1.45µm; ORTOS-OD = 0.61±1.34µm; mean±95%CI). Retinal
function showed no statistical significant treatment effect in amplitudes (a-waveOSOD = 5.87±27.04µV; b-waveOS-OD = 0.82±27.81µV).
Conclusions: Time line analysis after treatment with CAIs failed to show shortterm, intermediate or long-term evidence of structural as well as functional
improvement in Rs1h-/y mice. Schisis formation in the inner retina peaked at the
age of 3 months and was followed by photoreceptor degeneration and
hyperfluorescence suggesting photoreceptor debris accumulation. Schisis formation
and hyperfluorescent spots were more severe in the superior hemiretina.
Commercial Relationships: Ahmad Zhour, None; Christian Grimm,
None; Marijana Samardzija, None; Tobias Peters, None; Torsten Strasser,
None; Sylvia Bolz, None; Bernhard H. F. Weber, None; Eberhart Zrenner,
None; M. Dominik Fischer, None
Support: None
Program Number: 1135 Poster Board Number: A600
Presentation Time: 3:15 PM - 5:00 PM
Structural and Functional Protection of the Retina by N-Acylethanolamines in
Peter Koulen1,2, Kent D. Chapman2, Simon Kaja1, Stephanie L. Burroughs1.
Department of Ophthalmology and Vision Research Center, University of
Missouri-Kansas City, Kansas City, MO; 2Center for Plant Lipid Research,
Department of Biological Sciences, University of North Texas, Denton, TX.
Purpose: Anandamide, N-Arachidonoylethanolamine, as the most extensively
studied N-Acylethanolamine (NAE), is an endogenous ligand for cannabinoid and
vanilloid receptors. In response to injury, NAEs become upregulated to exert their
protective function as lipid signaling molecules. With little knowledge available
about the function and physiological targets of many NAEs, such as Npalmitoylethanolamine (NAE 16:0), N-lauroylethanolamine (NAE 12:0) and Nlinoleoylethanolamine (NAE 18:2), we measured their protective properties in a
surgical model of glaucomatous retinopathy. We hypothesized that NAEs that do
not bind cannabinoid and vanilloid receptors regulate intracellular calcium
homeostasis thereby protecting retinal ganglion cells (RGCs), optic nerve head
astrocytes (ONHAs) and the optic nerve from dysfunction and cell death.
Methods: Intraocular pressure was raised unilaterally in one eye of Brown Norway
rats by episcleral vein injection of hypertonic saline. NAEs were delivered
intravitreally or systemically (intraperitoneally) daily and visual acuity and contrast
sensitivity were measured with visuo-spatial behavior testing of the optomotor
reflex. Loss of function and viability of RGCs and ONHAs were assessed using
TUNEL histochemistry, cell specific immunohistochemistry and live cell imaging
of intracellular calcium signaling. The extent of optic nerve damage was
determined with quantitative histology.
Results: NAEs 16:0, 12:0 and 18:2, NAEs that do not bind cannabinoid and
vanilloid receptors, significantly reduced the loss in synaptic connectivity, loss of
axons and cell numbers of RGCs after systemic or intravitreal delivery. At the same
time, NAE administration also significantly reduced the decline in visual function
as measured by visuo-spatial behavior testing. In both RGCs and ONHAs, NAEs
attenuated calcium toxicity after disease onset. Treated animals had visual
performance and histological phenotypes indistinguishable from age-matched and
contralateral controls while visual performance, RGC and axon counts continued to
decline with age in vehicle treated controls.
Conclusions: Treatment with NAEs resulted in a highly significant, dosedependent reduction of glaucomatous retinopathy-induced RGC death and of the
loss in visual performance indicating that NAEs that do not bind to cannabinoid
and vanilloid receptors and lack endocannabinoid activity have significant
neuroprotective potential. These results provide a rationale for the use of NAEs as
potential therapeutic agents in primary open angle glaucoma and potentially other
forms of glaucomatous retinopathy.
Commercial Relationships: Peter Koulen, None; Kent D. Chapman,
None; Simon Kaja, None; Stephanie L. Burroughs, None
Support: Supported in part by NIH grants EY014227, RR022570, RR027093, and
AG010485, AHAF grant G2010006 and the Felix and Carmen Sabates Missouri
Endowed Chair in Vision Research (P.K.).
Program Number: 1136 Poster Board Number: A601
Presentation Time: 3:15 PM - 5:00 PM
ERK-mediated Activation of Fas Apoptosis Inhibitory Molecule (Faim2)
Prevents Photoreceptor Apoptosis
Cagri G. Besirli, Qiong-Duon Zheng, David N. Zacks. Kellogg Eye Center,
University of Michigan, Ann Arbor, MI.
Purpose: To evaluate the role of Fas apoptosis inhibitory molecule 2 (Faim2), an
inhibitor of the Fas signaling pathway, and its regulation by stress kinase signaling
during Fas-mediated apoptosis of photoreceptors.
Methods: Photoreceptor (661W) cells were treated with a Fas-receptor activating
antibody and the levels of Faim2 were examined. Activation of the stress-kinase
pathways was assayed by immunoblotting of kinases and their downstream targets.
The levels of Faim2 were examined in the presence of stress-kinase inhibitors in
Fas-treated 661W cells. The effect of stress-kinase inhibition on Fas-mediated
photoreceptor apoptosis was evaluated by caspase 8 activation and measuring cell
viability. Faim2 expression was inhibited by siRNA knock-down in 661W cells and
caspase 8 activation was measured after Fas activating antibody treatment.
Results: Treatment of 661W cells with a Fas-activating antibody led to increased
levels of Faim2. Both ERK and JNK stress kinase pathways were activated in Fastreated 661W cells, but only the inhibition of the ERK pathway reduced the levels
of Faim2. Blocking the ERK pathway using pharmacological inhibitors increased
the susceptibility of 661W cells to Fas-induced caspase activation and apoptosis.
When the levels of Faim2 were reduced in 661W cells by siRNA knock-down, Fas
activating antibody treatment resulted in earlier and more robust caspase 8
Conclusions: Faim2 acts as a neuroprotectant during Fas-mediated apoptosis of
photoreceptors. The expression of Faim2 is regulated, at least in part, by the ERK
signaling pathway. Modulation of ERK signaling to increase Faim2 expression may
be a potential therapeutic option to prevent photoreceptor death.
Commercial Relationships: Cagri G. Besirli, None; Qiong-Duon Zheng,
None; David N. Zacks, None
Support: Michigan Eye Bank and Transplantation Center (DNZ); NEI R01020823 (DNZ); and Departmental Core Center for Vision Research NEI-EY07003. CGB is supported by The Heed Ophthalmic Foundation Fellowship.
Program Number: 1137 Poster Board Number: A602
Presentation Time: 3:15 PM - 5:00 PM
Ataxin-1 Poly-Q-induced Proteotoxic Stress And Apoptosis Are Attenuated By
Docosahexaenoic Acid-derived Neuroprotectin D1 In Human Retinal Pigment
Epithelial Cells
Jorgelina M. Calandria, Nicolas G. Bazan. Neuroscience Center, LSU Health
Sciences Center, New Orleans, LA.
Purpose: Poly-glutamine-dependent misfolding induces toxicity and consequently
contributes to degeneration in the central nervous system. In Spinocerebellar ataxia
1 (SCA1), Ataxin-1 mutation causes the accumulation and partial loss of function
of the protein leading to cytotoxicity. Ataxin-1 interacts with transcription factors
and spliceosome components. In addition to an involvement of the cerebellum in
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
SCA1, the retina also undergoes degeneration (J. Neurol. Neurosurg. Psychiatry
77:1293, 2006), thus providing the basis for applying this model to retinal studies.
We hypothesize that Neuroprotectin D1 (NPD1), a lipid mediator derived from
DHA, promotes the survival of the RPE cells undergoing proteotoxic stress by
increasing the turn-over of the poly-Q proteins.
Methods: Ataxin-1 containing 82 glutamines-tract was expressed in ARPE-19
cells. Apoptosis was assessed using activated caspase-3 immunostaining, TUNEL
and Hoechst assay. Akt phosphorylation status on NPD1 treated cells was
determined by western blot. Localization and accumulation of Ataxin-1 was
determined by immunocytochemistry.
Results: Ataxin-1 82Q expression in RPE cells induced apoptosis in a timedependent manner. This was evidenced by the increase in the percentage of
TUNEL and Hoechst positive cells when compared with the controls expressing
wild-type protein.Caspase-3 also showed the same pattern of activation. NPD1
(100 nM) decreased apoptosis induced by Ataxin-1 82Q. The expression of the
mutant Ataxin-1 also induced accumulation of the Serine-776 phosphorylated form
in the nucleus. ARPE-19 cells expressing Ataxin-1 82Q in the presence of NPD1
presented lower levels of phosphorylated Ataxin-1 signal in the nucleus. The
translocation into the nucleus and phosphorylation of Serine-473 of Akt, the main
kinase that phosphorylates Ataxin-1, was increased differentially when the 82Q
form was expressed even in the presence of NPD1, suggesting that NPD1 does not
prevent the phosphorylation of Ataxin-1 and may be acting on its
dephosphorylation instead.
Conclusions: These results demonstrate that: 1) the RPE cellular model depicts
similar features to the changes triggered by the mutation in Purkinje cells, laying
the groundwork for unveiling new pathways and therapeutic approaches in other
misfolded protein-induced pathologies in the retina and 2) NPD1 rescues the
deleterious effects of proteotoxic stress caused by extended polyQ tract Ataxin-1
expression in RPE cells due to the increase in turnover. These findings may lead to
new therapeutic applications for DHA and NPD1 for preventing and attenuating the
initiation of retinal degenerations.
Commercial Relationships: Jorgelina M. Calandria, None; Nicolas G. Bazan,
Support: NEI EY005121; Research to Prevent Blindness, Inc.
Program Number: 1138 Poster Board Number: A603
Presentation Time: 3:15 PM - 5:00 PM
Evidence of Type II Cell Death in P23H-Light Induced Retinal Degeneration
in a Xenopus laevis Model of Retinitis Pigmentosa
Tami P. Bogea, Beatrice T. Tam, Orson L. Moritz. Ophthalmology & Visual
Sciences, University of British Columbia, Vancouver, BC, Canada.
Purpose: Type II cell death, also described as cell death with autophagy, has been
recently implicated in the regulation of photoreceptor degeneration in several
mouse models . In the present study, we report the occurrence of type II cell death
in a light induced model of retinal degeneration where Xenopus laevis tadpoles
express a bovine form of the rhodopsin mutant P23H (bP23H).
Methods: Transgenic tadpoles expressing bP23H were transferred to a 12L: 12D
regimen to induce retinal degeneration. They were euthanized at various time
points. Transgenic tadpoles expressing an inducible form of caspase 9 (iCasp9)
were reared similarly, and retinal degeneration was induced by administration of
AP20187. Tadpole eyes were subsequently processed for high resolution electron
microscopy according to standard procedures.
Results: We observed defects in rod outer and inner segment membranes in
tadpoles expressing bP23H, which were aggravated by exposure to cyclic light.
Rod outer segments were shorter and exhibited vesiculations and altered disk
morphology, and were rapidly phagocytosed by the retinal pigment epithelium.
Phagocytosis was mostly complete by 36 hours. In rod inner segments, we
observed autophagic compartments within the rough endoplasmic reticulum. In
contrast, these defects were not observed in rod outer and inner segment
membranes of tadpoles expressing iCasp9, where the photoreceptors completely
degenerated within 24 hours.
Conclusions: Our results indicate the presence of ultrastructural defects in both the
outer and inner segment membranes of bP23H expressing photoreceptors. These
defects differed, however, from those observed in the drug induced rod apoptosis
model, indicating that they are associated with the mechanism of light-induced
retinal degeneration. We suggest that light-induced retinal degeneration occurs via
type II cell death (cell death with autophagy), which is consistent with the
instability of bP23H rhodopsin in outer segments and in the secretory pathway
upon light exposure.
Commercial Relationships: Tami P. Bogea, None; Beatrice T. Tam,
None; Orson L. Moritz, None
Support: Foundation Fighting Blindness - Canada, CIHR
Program Number: 1139 Poster Board Number: A604
Presentation Time: 3:15 PM - 5:00 PM
Lateral Stretch Of The In Vitro Retina - Implications For Cell Survival
Linnéa T. Taylor1A, Damian Moran1B, Karin Arnér1A, Eric Warrant1B, Fredrik
Ghosh1A. AOphthalmology, BBiology, 1Lund University, Lund, Sweden.
Purpose: To investigate tissue stretch in the lateral plane as a factor for retinal cell
survival in vitro.
Methods: Full-thickness retinal sheets (~12x12mm) were isolated from adult
porcine eyes and cultured for 10 days under four conditions: 1) standard procedure
with the retinal explant placed on a culture membrane; 2) addition of lateral stretch
using a magnet system (ring magnet under the culture membrane with four small
magnets on top of the retina fixing it to the membrane); 3) addition of the ring
magnet, but no stretch; and 4) free-floating in culture medium. The grafts were
analyzed morphologically using hematoxylin and eosin staining and
Results: Hematoxylin and eosin (H&E) staining showed that after 10 days group 1
(standard) had degenerated into a pyknotic cell mass with no discernible nuclear
layers. Inner (IS) and outer segments (OS) were absent. GFAP labeling showed a
strong up-regulation. Group 2 (stretch) displayed a laminated morphology with
well-populated nuclear layers and well preserved IS and OS in H&E staining.
GFAP labeling displayed weak labeling of the nerve fiber layer (NFL) and vertical
fibers terminating in the outer limiting membrane. Group 3 (magnet control)
displayed laminar architecture of the retina in H&E staining, however, the sections
appeared edematous with widespread cell death and vacuolization in all layers.
Some IS, and occasional OS were present. GFAP labeling showed low-level upregulation in the NFL, as well as weak labeling of vertical fibers. H&E of group 4
(free-floating) showed a complete loss of retinal architecture with only a few
remaining cells. GFAP labeling of scattered structures was present. Statistical
analysis of rows of rhodopsin and NeuN labeled cells showed that the number of
surviving cells in group 2 (stretch) was far greater than in any other group
(p<0.001; one-way ANOVA).
Conclusions: Biomechanical factors related to cell survival within the retina are
yet to be extensively investigated. In vivo, the retina is stretched against the retinal
pigment epithelium mechanically through the force of the intraocular pressure. In
this study, we have created an in vitro model to explore the biomechanical
stretching force, and we here show that it is an important factor for preservation of
the retinal laminar structure as well as cell survival.
Commercial Relationships: Linnéa T. Taylor, None; Damian Moran,
None; Karin Arnér, None; Eric Warrant, None; Fredrik Ghosh, None
Support: Faculty of Medicine, Lund Univeristy; The Swedish Research Council
no90247201; The Princess Margaretas Foundation for Blind Children; The
Wallenberg Foundation MMW 2011.0009
Program Number: 1140 Poster Board Number: A605
Presentation Time: 3:15 PM - 5:00 PM
Contribution of Calpain and Caspases to Cell Death in Monkey RPE Cells
Cultured Under Hypoxia or with A2E
Ryan D. Walkup1,2, Katherine B. Hammond1,2, Emi Nakajima1,2, Thomas R.
Shearer2, Mitsuyoshi Azuma1,2. 1Senju Laboratory of Ocular Sciences, Senju
Pharmaceutical Co. Ltd., Beaverton, OR; 2Integrative Biosciences, Oregon Health
& Science University, Portland, OR.
Purpose: Age-related macular degeneration (AMD) is the leading cause of vision
loss after age 65. Several causative mechanisms have been proposed. 1) Agerelated failure of the choroidal vasculature leads to loss of the retinal pigment
epithelium (RPE). 2) Accumulation of phagocytized but unreleased A2E causes
RPE dysfunction. A2E is an auto fluorescent conjugation product of two molecules
of all-trans-retinal and ethanolamine. 3) Zinc deficiency activates calpain and
caspase proteases, leading to cell death. Thus, the purpose of the present study was
to compare the activation of calpain and caspase pathways in monkey RPE cells
cultured separately with either A2E or under hypoxic conditions.
Methods: Monkey primary RPE cells were cultured with synthetic A2E or cultured
under hypoxic conditions in a Gaspak pouch. Cell viability was assessed by the
MTS assay. Immunoblotting was used to detect activation of calpain and caspase
molecules, and also to identify enzyme-specific, α-spectrin breakdown products
(SBDP). Calpain inhibitor, SNJ-1945, and pan-caspase inhibitor, z-VAD-fmk, were
used to confirm activation of the proteases.
Results: 1) A2E and hypoxia each decreased viability of RPE cells in a time
dependent manner. 2) Incubation with A2E alone induced activation of calpain,
mitochondria-dependent caspase-9, and executer caspase-3. Appearance of the
calpain-specific SBDP at 145 kDa and the caspase-3-specific SBDP at 120 kDa
were also observed. SNJ-1945 inhibited calpain activation and slightly inhibited
caspase activation. z-VAD-fmk inhibited caspase activation and slightly inhibited
calpain activation, suggesting inter-activation of one protease system with the
other. 3) Incubation under hypoxia alone induced activation of calpain, but not
caspases. Only the calpain-specific SBDP was formed. SNJ-1945 inhibited calpain
activation, but z-VAD-fmk did not.
Conclusions: A2E activated both calpain and caspase pathways in monkey RPE
cells, but hypoxia activated only the calpain pathway. Further investigation is
necessary to determine how A2E and hypoxia signal the activation of specific
calpain or caspase systems and how these systems interact. This may become
important in human AMD treatment because inhibitor drugs against calpain and/or
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
caspase may be used to prevent RPE dysfunction caused by the putative effectors
A2E and hypoxia.
Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are
employees of, Senju Pharmaceutical Co. Ltd.
Commercial Relationships: Ryan D. Walkup, Senju Pharmaceutical Co. Ltd.
(E); Katherine B. Hammond, Senju Pharmaceutical Co. Ltd. (E); Emi Nakajima,
Senju Pharmaceutical Co. Ltd. (E); Thomas R. Shearer, Senju Pharmaceutical Co.
Ltd. (C); Mitsuyoshi Azuma, Senju Pharmaceutical Co. Ltd. (E)
Support: None
Program Number: 1141 Poster Board Number: A606
Presentation Time: 3:15 PM - 5:00 PM
Cone Photoreceptor Death Occurring Secondary To Retinal Pigment
Epithelium Loss Assessed In Vivo In Transgenic Fluorescent Reporter Mice
Haidong Shan, Peter C. Issa, Sher A. Aslam, Alun R. Barnard, Robert E.
MacLaren. Nuffield Laboratory of Ophthalmology, University of Oxford, Oxford,
United Kingdom.
Purpose: To investigate the cone photoreceptor loss secondary to disruption of the
underlying retinal pigment epithelium (RPE) in vivo as a model of the potential
mechanism of sight loss in age-related macular degeneration (AMD).
Methods: Sodium iodate solution was injected intraperitoneally in mice (100
mg/kg) to induce patchy RPE cell death. A transgenetic strain of mouse (B6.CgTg(OPN1LW-EGFP), OPN1-EGFP) and C57BL/6 wild type mice were used in this
study. The mutant mice contain cones expressing enhanced green fluorescent
protein (EGFP) under control of the human long-wavelength-sensitive cone opsin
promoter. Phosphate buffered saline (PBS) was injected as the control in both
strains. Fundus imaging was performed at 1, 2, 4 and 7 weeks after treatment
(WK1, 2, 4 and 7, respectively) with a confocal scanning laser ophthalmoscope
(cSLO) using various imaging modes. These included lipofuscin-related blue (488
nm) and melanin-related near-infrared (790 nm) autofluorescence (AF), as well as
near-infrared reflectance (IR, 820 nm). At WK7, electroretinography (ERG) data
were recorded and all eyes were collected for histological processing.
Results: Compared to PBS controls, sodium iodate treated mice showed a
pronounced patch-like hyperfluorescence on near-infrared fundus AF images,
suggesting changes of the RPE melanin-compartment. There were faint
corresponding alterations on blue AF and IR images in C57BL/6 wild type mice.
The abnormal appearance was found at WK1 and remained stable until WK7. In
OPN1-EGFP mice, EGFP positive cones could individually delineated on blue AF
images. Detectable patchy hyperfluorescence was found at WK2. Localized loss of
EGFP-expressing cones correlated well with the patchy hyperfluorescent areas on
near-infrared AF recordings. Seven weeks after sodium iodate treatment, a
significant decrease of a-wave and b-wave amplitudes was recorded in both strains
compared to PBS controls. Retinal histology confirmed cell death in the outer
nuclear layer in regions overlying the disrupted RPE at WK7.
Conclusions: Systemic application of sodium iodate results in RPE-alterations that
can be visualized by cSLO imaging. Secondary photoreceptor loss as shown in the
EGFP reporter mouse can be tracked over time in vivo, providing a useful model
for potential treatments that might be developed to prevent sight loss in AMD.
Commercial Relationships: Haidong Shan, None; Peter C. Issa, None; Sher A.
Aslam, None; Alun R. Barnard, None; Robert E. MacLaren, None
Support: The Health Foundation, the NIHR Biomedical Research Centre, the
Wellcome Trust (WT 086868),China State Scholarship,the Winkler Oxford Award
of China Oxford Scholarship,Henry Lester Trust scholarship
Program Number: 1142 Poster Board Number: A607
Presentation Time: 3:15 PM - 5:00 PM
Genome-Wide Expression Profiling in the Retina of Vldlr Knockout Mouse
Svetlana V. Kiosseva1, Lily L. Wong1, Xiaohong Zhou2, James F. McGinnis1.
Ophthalmology, Dean A McGee Eye Inst, Univ of OK, Oklahoma City, OK;
Massachusetts Eye and Ear Infirmary Howe Lab, Harvard Med School, Boston,
Purpose: To identify candidate genes and pathways that are involved in the
pathophisiology of Age related Macular Degeneration (AMD) by microarray
analysis of the retinas of Very low density lipoprotein receptor (Vldlr-/-) knockout
mouse, an animal model for a form of AMD called Retinal Angiomatous
Proliferation (RAP). We focused on characterization of the early events of retinal
defects at postnatal day (P)14 when subretinal neovascularization begins and in the
newly matured (P28) retina.
Methods: Labeled and amplified cRNAs from wild type (WT) and Vldlr-/- mice
on a C57/BL6J background were hybridized to Illumina’s Sentrix Mouse-6
Expression BeadChip arrays containing~47,000 transcripts. Gene expression from
scanned images was quantified, analyzed and shown as fold change when
comparing Vldlr-/- to WT mouse retinas. Genes that changed by 1.8 fold and
pFDR<0.05 were subsequently analyzed. Ingenuity Pathway Analysis (IPA) was
used to search for biological processes, pathways and networks. The expression of
several genes with notable fold change values was confirmed by qRT-PCR and
differential protein expression by Western blots.
Results: Two hundred eleven genes (P14) and 229 genes (P28) were differentially
expressed in Vldlr-/- retina. Sixty six genes (P14) and 80 genes (P28) were upregulated, and 145 (P14) and 140 (P28) genes were down-regulated, respectively.
The top up-regulated genes were Serpina3n and Gdpd3 at P14, Serpina3n and Edn2
at P28, and down-regulated genes were Guca1b and Tomm22 at P14, Tomm22 and
Mns1 at P28. Fourteen up-regulated genes were common to two point times
including Gdpd3, Rgs5, Serpina3n, Crym, Tomm22, and 40 common genes were
down-regulated (Guca1b, Pde6c, Myo7, Picalm, Ccl21b). The differentially
expressed genes included those involved in developmental and genetic disorders,
ophthalmic, neurological and metabolic diseases. The top canonical pathways for
up-regulated genes were intrinsic prothrombin activation and cell cycle checkpoint
control (P14), acute phase response and oncostatin M signaling (P28), while the
phototransduction pathway was the top canonical pathway for the down-regulated
Conclusions: This study demonstrates novel genome-wide transcriptional profiling
data of retinal development in a mouse model of AMD and further provide new
insights into the molecular pathways and networks that can be targets for new
Commercial Relationships: Svetlana V. Kiosseva, None; Lily L. Wong,
None; Xiaohong Zhou, None; James F. McGinnis, None
Support: NIH: P30-EY12190, COBRE-P20 RR017703, R21EY018306,
R01EY018724. FFB C-NP-0707-0404-UOK08. NSF:CBET-0708172.
Unrestricted funds from PHF and RPB and an RPB SSI award to JFM.
Program Number: 1143 Poster Board Number: A608
Presentation Time: 3:15 PM - 5:00 PM
Nuclear p120-catenin Unlocks Mitotic Block of Contact-inhibited Human
Retinal Pigment Epithelial Cells without Disrupting Adherent Junction
Yingting Zhu1, Hung-Chi J. Chen2, Szu-Yu Chen1, Scheffer C. Tseng1. 1Research,
Ocular Surface Ctr and Tissue Tech, Miami, FL; 2Ophthalmology, Chang Gung
Memorial Hospital, Taoyuan, Taiwan.
Purpose: Contact-inhibition ubiquitously exists in non-transformed cells and
explains the poor regenerative capacity of in vivo human retinal pigment epithelial
cells (RPE) during aging, injury and diseases. RPE injury or degeneration may
unlock mitotic block mediated by contact inhibition but may also promote
epithelial-mesenchymal transition (EMT) contributing to retinal blindness.
Methods: ARPE-19 cells cultured to 7 days after post-confluence were treated with
100 nM siRNA to p120, α-catenin, β-catenin, N-cadherin and ZO-1 for 48 h with or
without microtubule disrupting or stabilizing agents, 5 µg/ml CT-04 or 10 µM
Taxol. Before termination, cells were further treated with 10 µM BrdU for 4 h.
Immunostaining was performed to monitor cytolocalization of p120, α-catenin, βcatenin, N-cadherin, ZO-1 and BrdU labeling. Western blotting was used to
measure their protein levels in the nuclear compartment.
Results: This study demonstrated that p120-catenin siRNA knockdown uniquely
unlocked mitotic block by p120-catenin nuclear translocation to relieve repression
by Kaiso in contact-inhibited ARPE-19 cells. This signaling is associated with
activation of RhoA-ROCK signaling, destabilization of microtubules, but not with
activation of Wnt/β-catenin signaling. Consequently, proliferating human RPE
maintained their phenotype with expression of junctional N-cadherin, ZO-1, and
membranous Na-K-ATPase. Further expansion of contact-inhibited ARPE-19 cells
with a normal phenotype and a higher density became feasible by prolonging p120catenin siRNA knockdown for 15 days followed by withdrawal for 10 days.
Conclusions: This new strategy of perturbing contact inhibition by selective
activation of p120-catenin/Kaiso signaling without disrupting adherent junction
may be deployed to engineer surgical grafts containing normal RPE to meet global
RPE shortage and for RPE transplantation.
Commercial Relationships: Yingting Zhu, None; Hung-Chi J. Chen,
None; Szu-Yu Chen, None; Scheffer C. Tseng, investor (I)
Support: NIH, NEI, R43 EY022502
Program Number: 1144 Poster Board Number: A609
Presentation Time: 3:15 PM - 5:00 PM
Insights into Caveolin-1 Function: Quantitative Proteomic Analysis of a Novel
Retina-Specific Knockout Mouse Model
Michael H. Elliott1, Mikhail Dozmorov2, Xiaoman Li1, Henna Iqbal1, Mark
McClellan1, Jonathan D. Wren2, Guangwen Cao3, Timothy C. Thompson4, Marius
Ueffing5,6, Stefanie M. Hauck6. 1Ophthalmology/Dean McGee Eye Institute, Univ
of OK Health Sci Ctr, Oklahoma City, OK; 2Arthritis & Clinical Immunology
Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK;
Department of Epidemiology, Second Military Medical University, Shanghai,
China; 4Department of Genitourinary Medical Oncology-Research, University of
Texas MD Anderson Cancer Center, Houston, TX; 5Institute for Ophthalmic
Research, University Eye Hospital, Tuebingen, Germany; 6Department of Protein
Science, Helmholtz Center Munich, Neuherberg, Germany.
Purpose: Caveolin-1 (Cav-1) is a lipid raft protein implicated in autoimmune
uveitis, diabetic retinopathy, and glaucoma. Given its broad tissue distribution,
Cav-1 functions in the retina have been difficult to elucidate. We developed a
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
mouse model in which Cav-1 was specifically deleted in the retina (ret-CavKO)
leaving other tissues Cav-1-competent. To determine retina-intrinsic Cav-1
functions, we carried out quantitative proteomic analyses of ret-CavKO.
Methods: Ret-CavKO mice were generated by crossing Cav-1 “floxed” mice (loxP
sites inserted flanking exon 2) with mice expressing Cre recombinase under control
of the Chx10 promoter. Controls were floxed littermates that did not express Cre.
Retinal membrane proteins were subjected to tryptic digestion and analyzed by
liquid chromatography mass spectrometry (LC-MSMS, OrbiTrap). Peptides were
aligned, statistically analyzed for differential abundance, and identified by database
search (Ensembl, species Mus musculus). Gene Set Enrichment Analysis (KEGG
pathways, gene ontologies, transcription factor binding sites) and Ingenuity
Pathway Analysis identified functions and canonical pathways affected by retinaspecific Cav-1 deletion. Candidate proteins were validated by Western blotting and
immunohistochemistry (IHC).
Results: Deletion efficiency assessed by Western blotting of retinal proteins
indicated >70% reduction in Cav-1 in agreement with quantitative proteomics.
Given that, by design, the retinal vasculature retained Cav-1 expression, we
estimate the deletion efficiency to approach 90% in cells specifically targeted by
Chx10-driven Cre. By IHC, Cav-1 ablation was evident in Müller glia which
abundantly express Cav-1. As designed, expression was retained in retinal
vasculature and RPE demonstrating specificity. Quantitative proteomics identified
1591 proteins of which 233 were differentially represented between ret-CavKO and
control (p ≤ 0.05). Both KEGG and Ingenuity analyses identified transmembrane
molecular transport as the major downregulated pathway in ret-CavKO. Several
amino acid transporters (glutamate, glycine, glutamine) and nearly all solute
carriers were downregulated. Oxidative metabolism and mitochondrial dysfunction
were also significantly represented with nearly all redox carrier complexes affected.
Conclusions: Our results suggest that Cav-1 plays a key role in Müller glial
transport activities and that loss of Cav-1 in these cells leads to metabolic
dysfunction in the retina.
Commercial Relationships: Michael H. Elliott, None; Mikhail Dozmorov,
None; Xiaoman Li, None; Henna Iqbal, None; Mark McClellan,
None; Jonathan D. Wren, None; Guangwen Cao, None; Timothy C. Thompson,
None; Marius Ueffing, None; Stefanie M. Hauck, None
Support: NIH R01 EY019494 (MHE), NIH RR017703, NIH EY12190, OCAST
(MHE), RPB (MHE), NIH R01 CA68814 (TCT), DOD DAMD17-98-1-8575
(TCT), Fed Ministry of Ed. and Res: SysTec-Verbund IMAGING FKZ 0315508A
Program Number: 1145 Poster Board Number: A610
Presentation Time: 3:15 PM - 5:00 PM
Inhibition of β-secretase Results in a Retinal Phenotype Involving Both the
Vasculature and the RPE
Xiaoping Qi1A, Jun Cai1A, Quin Ruan1A, Lutgarde Serneels2, Zhijuan Chen1A, Paul
Saftig3, Todd Golde1B, Maria B. Grant1C, Bart de Strooper2, Michael E. Boulton1A.
Anatomy and Cell Biology, BNeuroscience, CPharmacology and Therapeutics,
University of Florida, Gainesville, FL; 2Center for Human Genetics and Leuven
Institute for Neurodegenerative Diseases (LIND), KU Leuven, Leuven, Belgium;
Biochemical Institute, Christian-Albrecht’s University, Kiel, Germany.
Purpose: β-secretase 1 (BACE1) is an aspartyl protease that is strongly expressed
in the brain and implicated in Alzheimmer’s disease. BACE2 has also been
identified which shares around 68% homology with BACE1. Since BACE
inhibitors are in clinical trials for Alzheimer’s disease we wished to determine the
expression pattern of BACE1 and BACE2 in the retina and ascertain if BACE1
and/or BACE2 inhibition affects retinal structure and function.
Methods: The retina and choroid from BACE1-/- and BACE1-/- BACE 2-/knockout mice were assessed by routine pathology, immunohistochemistry for
vascular density and confocal microscopy for lipofuscin accumulation. BACE1 and
BACE2 expression in mouse and human retina/choroid were assessed by PCR and
immunohostochemistry. In vitro angiogenesis was assessed using bovine retinal
microvascular endothelial cell following exposure to VEGF, PEDF and
VEGF+PEDF in the presence or absence of a β-secretase inhibitor. Lipofuscin
accumulation was assessed by FACS analysis in human retinal pigment epithelial
cells maintained for up to 28 days in the presence or absence of either a β-secretase
inhibitor or BACE siRNA.
Results: Assessment of the BACE 1-/- and combined BACE 1-/- BACE2-/knockout mice demonstrated a significantly reduced retinal and choroidal vascular
density, retinal thinning, apoptosis, thinning of Bruch’s membrane and a greater
than 2-fold increase in lipofuscin in the RPE. BACE2-/- mice exhibited a relatively
normal neural retina although occasional foci of neural retinal hyperplasia are
observed. However, these animals exhibited a highly disrupted choroid together
with swelling and lipofuscin accumulation in the overlying RPE but these changes
are less severe than in BACE1-/- animals. Both BACE1 and 2 were expressed
throughout the retina and choroid but BACE1 expression was highest in the neural
retina and BACE2 highest in the RPE/choroid. PEDF induced an increase in
BACE1 expression in endothelial cells but had no effect on the constitutive
expression of BACE1 in RPE cells. PEDF-inhibition of VEGF-induced
microvascular endothelial cell migration, proliferation and tubule formation were
abolished by BACE1 inhibition. Furthermore, BACE1 inhibition in cultured RPE
cells resulted in a greater than 2.5 fold increase in lipofuscin accumulation over a
28 day period which was associated with an increase in lysosomal pH and a
decrease in cathepsin D activity.
Conclusions: Both BACE1 and BACE2 play an important role in retinal
physiology and caution should be undertaken when developing BACE inhibitors
for Alzheimer’s disease as they may have off target effects in the retina.
Commercial Relationships: Xiaoping Qi, None; Jun Cai, None; Quin Ruan,
None; Lutgarde Serneels, None; Zhijuan Chen, None; Paul Saftig, None; Todd
Golde, None; Maria B. Grant, None; Bart de Strooper, None; Michael E.
Boulton, Eli Lilly; Amgen Inc (C)
Support: NIH grant EY018358
Program Number: 1146 Poster Board Number: A611
Presentation Time: 3:15 PM - 5:00 PM
Characterization of the Localization and Cleavage of Protocadherin-21 in Rod
and Cone Photoreceptors of Several Species
Lee Ling Yang, Orson L. Mortiz. Ophthal & Visual Sciences, University of British
Columbia, Vancouver, BC, Canada.
Purpose: Previous studies in mice suggest that protocadherin-21 (pcdh-21) is
involved in photoreceptor disk synthesis. Using X. laevis as a model organism, we
aim to investigate the role of pcdh-21 in regulating rod and cone photoreceptor disk
Methods: We generated antibodies targeting the N- and C-terminus of pcdh-21 and
examined its localization in X.laevis photoreceptors using confocal microscopy. We
compared this localization with that of other species, including X.tropicalis,
zebrafish, mouse and pig. Immunoblotting was used to study the proteolytic
processing of pcdh-21 in these species.
Results: In X. laevis retinas, immunofluorescence staining showed that pcdh-21
was differentially localized in rods and cones. In rods, pcdh-21 labeling was most
intense in the inner and outer segment (OS) plasma membrane, and in most cases,
also appeared as a thin band at the base of the rod OS. High levels of pcdh-21 were
observed in the disk lamellae of a small minority of rods. Pcdh-21 expression was
higher in cones than rods. Longitudinal sections showed that pcdh-21 was localized
to the cone inner segment plasma membrane. Cross sections double labeled with
anti-acetylated tubulin and pcdh-21 antibodies showed that pcdh-21 was localized
to the rims of cone disks, except at the side closest to the connecting cilium. In
zebrafsh retinas, pcdh-21 was localized to the rims of cone lamellae but it was not
detected in rods. In mouse and pig retinas, pcdh-21 was concentrated at the base of
the OS. Immunoblot showed that a significant proportion of pcdh-21 was cleaved
in mouse, but not X. laevis, photoreceptors.
Conclusions: Our results indicate that pcdh-21 is differentially localized in rods
and cones, and that localization is not conserved across species. In mammals with
rod-dominant retinas, pcdh-21 localization was most intense at the base of the rod
OS. In lower vertebrate retinas with higher cone to rod ratio, pcdh-21 expression is
significantly greater in cone outer segments, where it is localized to the disk rim
region, whereas it is localized to the plasma membrane and base of the OS in the
rods. Proteolytic processing may be unique to mammalian pcdh-21.
Commercial Relationships: Lee Ling Yang, None; Orson L. Mortiz, None
Support: NEI 5R01EY006891 and the Foundation Fighting Blindness (Canada)
Program Number: 1147 Poster Board Number: A612
Presentation Time: 3:15 PM - 5:00 PM
Exosomal Export In RPE Cells With Lysosome Inhibition
Zhen-Yang Zhao, Yan Chen, Jian Wang, Jiyang Cai. Ophthalmology, Vanderbilt
Eye Institute, Nashville, TN.
Purpose: Exosomes are 40-100nm sized vesicles that are released by fusion of
multivesicular bodies (MVBs) with the limiting cell plasma membrane. The MVBs
are involved in delivery of cargo proteins, including those aggregated and
misfolded ones, to lysosome for degradation and detoxification. We hypothesized
that retinal pigment epithelial (RPE) cells with lysosome inhibition will have
disrupted sorting machinery of MVBs and will release aberrant cargos via
exosomes. The purpose of this study is to determine whether lysosome inhibition in
cultured human fetal RPE cells changed protein and RNA profiles of exosomes.
Methods: Cultured fetal RPE cells were treated with non-lethal dose of
chloroquine (CQ) to inhibit lysosome. Exosomes were harvested by differential
ultracentrifugation and their protein contents were measured by Western blot
analyses. Exosomal microRNAs were isolated by an ultrafiltration method and
analyzed by Exiqon microarrays. The array data were processed in Partek
Genomics Suite.
Results: Treatment with 20 µM CQ, which did not affect cell viability but inhibited
lysosome degradation, led to increased release of lipidated microtubule-associated
protein 1 light chain 3 (LC3-II), a marker of autophagosome in isolated exosomes.
The amount of CD9 and Hsc70 was not affected by CQ, indicating lysosome
inhibition only increased the contents but not the overall rate of exocytosis from the
RPE. The majority of RNA species in RPE exosomes are short non-coding RNAs.
Exosomes from CQ-treated RPE cells had higher amount of a number of
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Conclusions: Lysosome inhibition altered the exosomal RNA and protein contents
released by the RPE. Such mechanisms have implications in the functional
interactions between RPE, Bruch’s membrane and choroidal endothelial cells.
Further characterization of RPE exosomes might shed light on the mechanisms of
extracellular deposit in drusen formation and RPE/choroid degeneration in agerelated macular degeneration.
Commercial Relationships: Zhen-Yang Zhao, None; Yan Chen, None; Jian
Wang, None; Jiyang Cai, None
Support: International Retinal Research Foundation, NIH grants EY019709,
EY07892, EY08126 and unrestricted department grant from Research to Prevent
Blindness, Inc.
Program Number: 1148 Poster Board Number: A613
Presentation Time: 3:15 PM - 5:00 PM
Peropsin Is Expressed In The Eyes Of The Basal Arthropod Limulus
B A Battelle1, D R. Dugger, Jr.2, K E. Kempler1. 1Whitney Laboratory, University
of Florida, St Augustine, FL; 2Department of Ophthalmology, University of
Florida, Gainesville, FL.
Purpose: To characterize a peropsin homologue expressed in the basal arthropod,
Limulus polyphemus. Peropsins are among the least understood members of the
opsin superfamily.
Methods: A full-length Limulus peropsin-like sequence (Lp-perops) was cloned
from a cDNA library of Limulus ventral eye using standard procedures. The Lpperops sequence was compared to other peropsins, retinochromes and RGRs in
phylogenetic analyses. The distribution of the transcript was determined by PCR
using gene specific primers and in situ hybridization using as probe RNA encoding
the full-length open reading frame. A specific monoclonal antibody directed against
the C-terminus of Lp-perops was applied to Western blots and to fixed frozen
sections of Limulus eyes to determine the tissue and cellular distribution of the
protein. Immunostained sections were analyzed by confocal microscopy.
Results: In phylogenetic analyses, the predicted amino acid sequence of Lp-perops
clusters more closely with deuterostome peropsins than it does with the
retinochromes of mollusks or the RGRs of vertebrates. The Lp-perops transcript
was detected in RNA extracted from all three types of Limulus eyes: lateral
compound eyes (LE), median ocelli (ME) and ventral rudimentary eyes (VE). In
situ hybridization of VE showed the transcript is located in specialized glia which
tightly surround photoreceptors. On Western blots, a single band of Lp-perops
immunoreactivity at approximately 38 kDa, close to the protein’s predicted size of
39 kDa, was detected in membrane extracts of LE and VE but so far not in
membranes from ME. Using immunocytochemistry, Lp-perops-like
immunoreactivity was detected in glia of VE and ME that tightly surround
photoreceptors and are closely associated with photosensitive membranes. In LE, it
was detected in pigment cells that surround photoreceptors.
Conclusions: A homologue of deuterostome peropsin is expressed in the eyes of
the basal chelicerate arthropod Limulus polyphemus. Thus, peropsins evolved
before the protostome-deuterstome split. The close association between Lp-peropsexpressing cells and photoreceptors suggests Lp-perops is involved in vision. The
function of this association remains a puzzle because the photopigment in
rhabdomeral photoreceptors is bistable.
Commercial Relationships: B A Battelle, None; D. R. Dugger, Jr., None; K. E.
Kempler, None
Support: Whitney Lab internal funds, NSF and NIH
Program Number: 1149 Poster Board Number: A614
Presentation Time: 3:15 PM - 5:00 PM
Epithelial-mesenchymal transition (EMT) Caused by EGTA with EGF+FGF-2
via Activation of Wnt Signaling
Ek Kia Tan1, Ying-Ting Zhu1, Hung-Chi Chen2, Szu-Yu Chen1, Scheffer C.G.
Tseng1. 1R&D, Ocular Surface Res & Edu Fndtn, Miami, FL; 2Department of
Ophthalmology, Chang Gung Memorial Hospital and Graduate Institute of Clinical
Medical Sciences, Chang Gung University, Taoyuan,, Taiwan.
Purpose: Proliferation and epithelial-mesenchymal transition (EMT) of the retinal
pigment epithelium (RPE) is a hallmark of proliferative vitreoretinopathy. We aim
at clarifying the role of EGF, FGF-2, and TGF-β1 in controlling how RPE
proliferates while undergoing EMT.
Methods: Post-confluent Day 7 ARPE-19 cells were treated for 24 h with 1 mM
EGTA, 10 ng/mL EGF, 20 ng/mL FGF-2, 10 ng/mL TGF-β1, or 10 µM XAV939,
individually or in combination. Before termination, cells were further treated with
10 µM BrdU for 4 h. Immunostaining was performed to monitor cytolocalization of
RPE65, p120, Kaiso, α-catenin, β-catenin, N-cadherin, ZO-1, S100A4, Smad,
ZEB1/2, and BrdU labeling. Western blotting was used to measure their protein
levels in the nuclear compartment.
Results: When contact inhibition of post-confluent ARPE-19 cells was disrupted
by EGTA, an increase of BrdU labeling was noted only in the presence of EGF
and/or FGF-2, and was accompanied by EMT as evidenced by the loss of a normal
RPE phenotype (altered cytolocalization of RPE65, N-cadherin, ZO-1, and Na,KATPase) and the gain of a mesenchymal phenotype (increased expression of
S100A4 and α-SMA). EMT with proliferation by EGTA plus EGF+FGF-2 was
accompanied by activation of canonical Wnt signaling (judged by the TCF/LEF
promoter activity and increased nuclear levels of β-catenin and LEF1 proteins),
which was abolished by concomitant addition of Wnt inhibitor XAV939, but not
associated with p120-Kaiso signaling. Contact inhibition disrupted by EGTA in the
presence of TGF-β1 also led to EMT but suppressed proliferation and Wnt
signaling. The Wnt signaling triggered by EGF+FGF-2 was sufficient and
synergized with TGF-β1 in activating the Smad/ZEB1/2 signaling responsible for
Conclusions: These findings establish differential role of EGF, FGF-2 and TGF-β
and Wnt signaling when cell junction of RPE is disrupted to undergo EMT with
Commercial Relationships: Ek Kia Tan, TissueTech (E); Ying-Ting Zhu,
TissueTech (E); Hung-Chi Chen, None; Szu-Yu Chen, TissueTech (E); Scheffer
C.G. Tseng, TissueTech (I, E, C, P)
Support: NIH Grant RO1 EY 06819
Program Number: 1150 Poster Board Number: A615
Presentation Time: 3:15 PM - 5:00 PM
Integrated Signalling Between CTGF And TGF-β2 Leads To Divergent
Activation Of Canonical And Non-canonical Pathways Controlling EMT In
ARPE-19 Cells: Implications For The Pathogenesis Of Proliferative
Darrell C. Andrews1A, John Browne1A, Helen O' Donovan1A, Noel Faherty1A,
Noelynn Oliver2, Deborah Wallace1B, Emily Hughes1B, Colm O' Brien1B, John
Crean1A. ASchool of Biomolecular and Biomedical Science, BSchool of Medicine
and Medical Science, 1University College Dublin, Dublin, Ireland; 2Fibrogen Inc,
San Francisco, CA.
Purpose: Proliferative vitreoretinopathy (PVR) is considered the result of
erroneous wound healing, characterised by disruption of the epiretinal membrane.
Here we describe the interaction between CTGF and TGF-β2, signaling and
transcriptional outcomes leading to transdifferentiation, suggesting a requirement
for co-operation between these factors in PVR.
Methods: Human retinal pigment epithelial cells (ARPE-19) were cultured and
stimulated with TGF-β 2, CTGF and both combined. Activation of canonical
(Smad 2, Smad 3 Smad 1/5/8) and non-canonical (erk, p38, Akt, cdc42) pathways
was assessed by immunoblot. Transcriptional activation was assessed by promoter
reporter (3TP-Luc) assay. EMT was determined by immunocytochemistry and
characterized by staining for vimentin, smooth muscle actin and ZO-1. Migratory
capacity was assessed by wound healing assay. Gene expression profiles were
determined using DNA microarrays.
Results: Direct binding of CTGF to TGF-β2 was demonstrated using a solid-phase
binding assay. CTGF had no effect on Smad signalling but antagonised TGF-β2
induced phosphorylation of Smad2 and Smad3. Both CTGF and TGF-β2
phosphorylated erk and p38; phosphorylation was significantly enhanced upon costimulation, suggesting cooperative effect and a shift from canonical to noncanonical signaling. 3TP-luciferase promoter reporter activity in response to TGF-β
was inhibited by CTGF. These responses were reversed by the anti CTGF
monoclonal antibody FG-3019. The consequences of this signaling were inhibition
of migratory capacity and increased cell differentiation. Microarray studies
identified a number of genes that were co-regulated by CTGF and TGF-β including
several implicated in epithelial to mesenchymal transition.
Conclusions: These findings suggest that a CTGF-enriched permissive cellular
environment regulates the initiation and promotion of TGF-β signaling networks,
identifying a hitherto undescribed “switch”. The consequences of this switch are
altered phenotypic behaviour, representing a significant advance in our
understanding of the patho-mechanisms of PVR. Therapeutic strategies targeting
CTGF in PVR have the potential for significant therapeutic gain.
Commercial Relationships: Darrell C. Andrews, None; John Browne,
None; Helen O' Donovan, None; Noel Faherty, None; Noelynn Oliver,
None; Deborah Wallace, None; Emily Hughes, None; Colm O' Brien,
None; John Crean, None
Support: None
213 Development of the Retina and RPE
Monday, May 7, 2012, 8:30 AM - 10:15 AM
Grand A Paper Session
Program #/Board # Range: 1314-1320
Organizing Section: Retinal Cell Biology
Program Number: 1314
Presentation Time: 8:30 AM - 8:45 AM
The Expression Of Irx7 In The Inner Nuclear Layer Of Zebrafish Retina Is
Essential For A Proper Retinal Development And Lamination
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Yuk Fai Leung1, Yuqing Zhang1, Yifan Yang1, Caleb Trujillo1, Wenxuan Zhong2.
Biological Sciences, Purdue University, West Lafayette, IN; 2Statistics, University
of Illinois at Urbana-Champaign, Champaign, IL.
Purpose: Our previous investigation on Smarca4, a retinal dystrophic mutant, has
identified a number of genes for retinal differentiation (Leung et al 2008). Among
them, irx7 is expressed in the prospective INL at 52hpf (Hensley et al 2011) when
retinal lamination is being established, suggesting a potential role in this process.
The purpose of this study was to define the role of irx7 in retinal development.
Methods: The expression dynamics of irx7 was investigated by in situ
hybridization. Irx7 in developing embryos was knocked-down by microinjection of
morpholinos (MOs). The resulting retinal phenotypes were characterized by
immunostaining and in situ hybridization with various markers.
Results: During retinal development, irx7’s expression was found to appear
exclusively in the inner nuclear layer (INL) as soon as the prospective INL cells
withdraw from the cell cycle and during retinal lamination. In Irx7-knockdown
retinas, the formation of a proper retinal lamination was disrupted and the
differentiation of INL cell types, including amacrine, horizontal, bipolar and Muller
cells, was compromised. Despite irx7’s exclusive expression in the INL,
photoreceptors differentiation was also compromised in Irx7-knockdown retinas.
Compared with other retinal cell types, ganglion cells differentiated relatively well
in these retinas, except for their dendritic projections into the inner plexiform layer
(IPL). In fact, the neuronal projections of amacrine and bipolar cells into the IPL
were also diminished. The expression of known TFs that can specify specific
retinal cell type was also altered in Irx7-knockdown retinas.
Conclusions: These results indicate that cells in the INL and photoreceptor layers
are preferentially affected. Retinal lamination issue in the Irx7-knockdown retinas
is likely caused by the attenuation of neurite outgrowth of the cells in these layers.
Thus, the irx7 gene network is a novel regulatory circuit for retinal development
and lamination.
Commercial Relationships: Yuk Fai Leung, None; Yuqing Zhang, None; Yifan
Yang, None; Caleb Trujillo, None; Wenxuan Zhong, None
Support: The Knights Templar Eye Foundation; Office of Science (BER), U.S.
Department of Energy; NSF grant DMS 1120256; Hope for Vision; Showalter
Research Trust
Program Number: 1315
Presentation Time: 8:45 AM - 9:00 AM
Olig2 Defines Subpopulations Of Retinal Progenitor Cells Biased Towards
Specific Cell Fates
Brian P. Hafler1,2, Constance Cepko2. 1Department of Ophthalmology and Visual
Science, Yale School of Medicine, New Haven, CT; 2Department of Genetics,
Harvard Medical School, Boston, MA.
Purpose: Previous lineage analyses have shown that retinal progenitor cells
(RPCs) are multipotent throughout development, and expression profiling studies
have shown a great deal of molecular heterogeneity among RPCs. To determine if
the molecular heterogeneity predicts competencies or biases to produce particular
types of progeny, clonal lineage analysis was used to investigate the progeny of a
subset of RPCs, those that express the basic helix-loop-helix (bHLH) transcription
factor, Olig2.
Methods: In order to address whether the molecular heterogeneity among RPCs
correlates with the extensive clonal heterogeneity observed in the previous studies
of retrovirally marked clones, we used a novel type of retroviral clonal analysis
based upon infection of a defined type of RPC. An Olig2 knock-in line, which
expresses Cre, as well as TVA, was used for fate mapping. Olig2-tva-ires-cre+/mice were crossed with Cre-responsive GFP reporter mice, RC::ePE, in which
enhanced GFP (eGFP) is under the control of the constitutively active CAG
promoter in the ROSA26 locus. The Cre fate mapping studies suggested that
Olig2+ RPCs produced a particular set of daughter cell types. However, since the
TVA gene was also in the Olig2 locus, there was an opportunity to examine the
descendents of Olig2-expressing RPCs exclusively. Retinas of E13.5, P0, and P3
Olig2-tva-cre+/- mice were infected in vivo with murine retroviruses expressing the
marker genes, human placental alkaline phosphatase (AP) (LIA virus) or GFP
(pQCXIX-GFP virus).
Results: In contrast to the large and complex set of clones generated by viral
marking of random embryonic RPCs, the embryonic Olig2+ RPCs underwent
terminal divisions, producing small clones with only two of the five cell types
being made by the pool of RPCs at that time. The embryonically produced cell
types made by Olig2 RPCs were cone photoreceptors and horizontal cell (HC)
interneurons. Moreover, the embryonic Olig2+ RPC did not make the later Olig2+
RPC. The later, postnatal Olig2+ RPCs also made terminal divisions, which were
biased towards production of rod photoreceptors and amacrine cell (AC)
Conclusions: These data indicate that the multipotent progenitor pool is made up
of distinctive types of RPCs, which have biases towards producing subsets of
retinal neurons in a terminal division, with the types of neurons produced varying
over time. This strategy is similar to that of the developing Drosophila
melanogaster ventral nerve cord, with the Olig2+ cells behaving as ganglion
mother cells.
Commercial Relationships: Brian P. Hafler, None; Constance Cepko, None
Support: HHMI
Program Number: 1316
Presentation Time: 9:00 AM - 9:15 AM
SOX2 Regulates Neural Retina Development Through Antagonism of
Canonical Wnt Signaling
Whitney E. Heavner1A, Danielle Matsushima1B, Larysa H. Pevny1A. AGenetics,
Neuroscience Center, 1University of North Carolina at Chapel Hill, Chapel Hill,
Purpose: Around 10% of human individuals with anophthalmia (no eye) or severe
microphthalmia (small eye) carry a mutation in the gene encoding the HMG-box
transcription factor SOX2. Our lab has previously shown that ablation of SOX2 in
mouse optic cup progenitor cells results in cell fate conversion from neurogenic
neural retina (NR) to non-neurogenic ciliary epithelium (CE) (Matsushima et al.
2011). These data suggest that SOX2 is a decisive factor of neural competence in
the developing retina. Recent work in the Pevny lab has focused on determining
how SOX2 maintains the competence of retinal progenitor cells to become neurons.
Retinal progenitor cells provide an especially useful model for determining SOX2’s
role in neurogenesis given that these cells retain the ability to become nonneurogenic CE if SOX2 is lost. It has been demonstrated that expression of
stabilized β-catenin, the major transcriptional mediator of canonical Wnt signaling,
in retinal progenitor cells induces expression of CE-specific genes (Liu et al. 2007).
The current study examines the hypothesis that SOX2 maintains neural competence
in the developing retina in part by antagonizing canonical Wnt signaling.
Methods: Genetic epistasis analysis was used to determine the relationship
between SOX2 and β-catenin in the developing optic cup. Cell cycle kinetics was
analyzed using flow cytometry, global gene expression analysis and double
labeling with thymidine analogs.
Results: Deletion of Sox2 in retinal progenitor cells resulted in expanded Wnt
signaling into the central optic cup/prospective neural retina. At embryonic day (E)
16.5, the Wnt target gene CyclinD1 was upregulated in central Sox2-mutant cells
but not in more peripheral Sox2-mutant cells. The increase in CyclinD1 was
associated with a decrease p27kip1. The pattern of CyclinD1 upregulation was
opposite to the graded decrease in cell cycle rate. Thus, more peripheral Sox2mutant cells exhibited longer cell cycle times and down-regulated CyclinD1
expression. By postnatal day 0, Sox2-mutant cells had exited the cell cycle.
Deletion of both Sox2 and β-catenin in retinal progenitor cells partially rescued the
Sox2-mutant phenotype. However, CyclinD1 remained upregulated in doublemutant cells at E16.5.
Conclusions: Results from these studies suggest that SOX2 maintains neural
retinal identity in part by antagonizing canonical Wnt signaling upstream of βcatenin. Moreover, SOX2 may regulate retinal progenitor cell proliferation through
control of G1 cell cycle components. SOX2’s role in canonical Wnt signaling can
be separated from its role in progenitor cell proliferation given that CyclinD1
remains upregulated in Sox2-mutant cells that lack β-catenin.
Commercial Relationships: Whitney E. Heavner, None; Danielle Matsushima,
None; Larysa H. Pevny, None
Support: NIH Grant R0110-3374
Program Number: 1317
Presentation Time: 9:15 AM - 9:30 AM
Sox11 is Required for Proper Optic Fissure Closure and Photoreceptor
Lakshmi S. Pillai, Wen Wen, Ann C. Morris. Department of Biology, University of
Kentucky, Lexington, KY.
The SRY-Box transcription factor Sox11 is expressed in the developing central and
peripheral nervous system, and regulates the development and differentiation of
several organ systems. Sox11-/- mice are microphthalmic and exhibit Peter's
anomaly and anterior segment dysgenesis, suggesting that Sox11 is required for
proper ocular morphogenesis. Here, we have investigated the role of Sox11 in
ocular development in the zebrafish, using an in vivo knockdown approach.
Translation blocking morpholinos (MO) were injected into 1-cell stage wild type
and transgenic zebrafish embryos. The embryos were collected at 24, 48 and 72
hours post fertilization and processed for whole-mount in situ hybridization,
histological and immunohistochemical examination. Digoxigenin labeled
riboprobes were used to identify expression patterns of sox11a, sox11b, pax6a, and
crx. Apoptosis was analyzed via TUNEL labeling, and cell proliferation and
cellular differentiation were analyzed via immunohistochemistry for PH3, BrdU,
and cell type specific markers. Morpholino-injected embryos were examined by
bright field and fluorescence microscopy; area measurements of the eye were taken
by outlining the entire eye and the lens using Nikon Elements software.
Whole-mount in situ hybridization showed that zebrafish sox11 paralogs, sox11a,
and sox11b, have overlapping yet distinct expression patterns in the eye during
early development. Furthermore, both transcripts are present in mitotic retinal
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
progenitor cells as well as post-mitotic precursor cells. Microscopic and
immunohistological examination showed that knockdown of both Sox11a and
Sox11b resulted in micropthalmia, delayed lens development, coloboma, and
specific reduction of photoreceptor cells. These phenotypes were rescued by coinjection of mRNA specific for sox11a and sox11b. Additionally, Sox11
knockdown resulted in a decrease in the expression levels of pax6a, crx, and Nr2e3
along with an increase in the number of mitoses. No significant difference in
apoptosis was observed between control and Sox11 morphant eyes.
Our results show that reduction in the levels of Sox11 causes abnormal ocular
morphogenesis as well as coloboma. This phenotype is accompanied by a specific
deficit in photoreceptor cells in the dorsal retina. Such results are consistent with
the hypothesis that Sox11 regulates early ocular morphogenesis as well as
photoreceptor differentiation in the developing retina, possibly by regulating
expression of genes necessary for photoreceptor cell fate specification. Studies are
ongoing to characterize the mechanism of optic fissure closure defect in Sox11
deficient eyes.
Commercial Relationships: Lakshmi S. Pillai, None; Wen Wen, None; Ann C.
Morris, None
Support: Knights Templar Eye Foundation, Pew Scholars Program in the
Biomedical Sciences
Program Number: 1318
Presentation Time: 9:30 AM - 9:45 AM
Long-Term Culture and Efficient Photoreceptor Differentiation of Swine
Embryonic Retinal Progenitor Cells
Yongqing liu1, Xiaoqin Lu1, Wei Wang1, Liang Zhou1,2, Li Huang1, Henry J.
Kaplan1, Douglas C. Dean1. 1Ophthalmology and Visual Sciences, University of
Louisville, Louisville, KY; 2Ophthalmology, The 2nd Xiangya Hospital of Central
South University, Changsha, Hunan Province, China.
Purpose: Retina degeneration is a major cause of blindness, and the mammalian
retina shows only a limited capacity for regeneration following injury or disease.
Therefore, studies are underway assessing the ability of transplanted stem cells to
restore retinal function. Stem cell therapies have utilized embryonic stem cells and
induced pluripotent stem cells which are pluripotent and must be driven toward a
lineage prior to transplantation, and adult stem cells which are progenitors already
committed to an organ-specific lineage. Encouraging results are being obtained
with adult stem cells, but such adult stem cells have not been isolated from the
mammalian eye. However, photoreceptor progenitors are evident in the embryonic
eye. But, attempts to maintain and expand these progenitors in culture under
conditions where they can readily differentiate into photoreceptors has been largely
unsuccessful, leading to a focus on embryonic stem cells and induced pluripotent
cells for retinal transplant experiments. The purpose of this study is to optimize
conditions for maintaining and expanding swine embryonic retinal progenitor cells
capable of efficiently differentiating into photoreceptors.
Methods: Primary retinal cultures were generated from swine at different
embryonic ages. Culture media and substrate were varied and effects on cell
proliferation and ability to differentiate into photoreceptor lineages were assessed
by immunostaining with lineage markers.
Results: Based on immunostaining of developing retina, we found Pax6+ retinal
progenitors were evident between embryonic days 50 and 85. Primary retinal
cultures were generated during this developmental range. We found that it was
essential that the cells be maintained with diluted matrigel in neural medium to
prevent differentiation. Differentiation into photoreceptor lineages was initiated by
forcing the cells to form neurospheres in suspension culture followed by a
monolayer culture on matirgel in differentiation medium. The cells could be
transplanted into a swine model of rod photoreceptor degeneration, where they
integrated into the outer nuclear layer and expressed rhodopsin.
Conclusions: We have devised culture conditions for maintaining embryonic swine
retinal progenitor cells, and a protocol for efficient differentiation of these cells into
rod photoreceptors. These cells have the potential to integrate into the retina in a
swine model of rod photoreceptor degeneration.
Commercial Relationships: Yongqing liu, None; Xiaoqin Lu, None; Wei
Wang, None; Liang Zhou, None; Li Huang, None; Henry J. Kaplan,
None; Douglas C. Dean, None
Support: 5 P20 RR017702-09
Program Number: 1319
Presentation Time: 9:45 AM - 10:00 AM
Exploration of the mechanism by which Spleen Tyrosine Kinase (SYK)
inhibitor R406 promotes rhodopsin expression
Delphine M. Bonnet Wersinger1, Sarah E. Collins2A, Jun Wan2A, Cynthia A.
Berlinicke2A, Donald J. Zack2A,2B. 1Neuroscience Institute of Montpellier, INSERM
U1051, Montpellier, France; AOphthalmology, BNeurosciences, 2Johns Hopkins
University, Baltimore, MD.
Purpose: Through a small molecule screen we identified the SYK inhibitor R406
as being able to stimulate rhodopsin reporter activity in cultured murine primary
retinal cells. Since our data suggested that R406’s effect on rod differentiation was
independent of its SYK inhibitory activity, we sought to determine the signaling
pathway(s) mediating its photoreceptor activity. Based on other known R406
targets, we concentrated on the Jak and Flt3 pathways.
Methods: Dissociated retinal cells were prepared from postnatal C57BL/6J,
RhoGFP knock-in and Flt3-/- mice. Cultures were treated with R406 (Rigel
Pharmaceuticals, San Francisco), JAK or FLT3 pharmacological inhibitors (EMD
Chemicals). Rod photoreceptor differentiation was quantified through rhodopsin
reporter assays with Rho-EGFP knock-in or secreted luciferase reporters. Total
RNA and proteins were prepared from R406 treated retinal cells and assayed by
QPCR and Western-Blot analysis, respectively. R406 induced transcriptome
changes obtained by exon arrays were confirmed by QPCR.
Results: Administration of increasing doses of FLT3 inhibitors did not stimulate
rhodopsin GFP or luciferase reporter expression. In addition, retinal cultures from
Flt3-/- mice did not show enhanced rhodopsin reporter expression in luciferase
assays. Interestingly, Jak mediated phosphorylation of STAT3 was dosedependently reduced by R406. In addition, R406 pre-treatment prevented CNTF
dose-dependent activation of STAT3. Moreover, the JAK inhibitor I competed with
R406 in terms of its ability to induce rhodopsin reporter expression. Muller glial
specific RNA transcripts were identified as 31% of the genes regulated by R406 in
the microarray study. Quantitative PCR verified significant decrease of several glial
genes, including Cryab, GFAP, Msn, Rgs5 and Vim. The major Notch target genes
Hes1 and Hes5 were significantly downregulated in postnatal retinal cells after
R406 treatment.
Conclusions: The effect of R406 on rhodopsin expression in primary retinal
cultures does not appear to be mediated by inhibition of FLT3 or SYK activity. Our
data suggests that Notch inhibition and modulation of Jak/STAT3 signaling may be
involved in R406’s activity, and that there may be a relationship with glial cell
differentiation. Further definition of these pathways may provide novel insights
into photoreceptor development.
Commercial Relationships: Delphine M. Bonnet Wersinger, None; Sarah E.
Collins, None; Jun Wan, None; Cynthia A. Berlinicke, None; Donald J. Zack,
Support: Wynn-Gund FFB Translational Grant, Guerrieri Family Foundation,
Robert and Clarice Smith gift, and EY core grant 5P30EY001765 (NEI)
Program Number: 1320
Presentation Time: 10:00 AM - 10:15 AM
Modelling Photoreceptor Development Using Embryonic Stem Cell-derived
3D Retinal Cultures
Emma L. West1, Anai Gonzalez Cordero1, Livia S. Carvalho1, Rachael A. Pearson1,
Jane C. Sowden2, Robin R. Ali1,3. 1Department of Genetics, UCL Institute of
Ophthalmology, London, United Kingdom; 2Developmental Biology Unit, UCL
Institute of Child Health, London, United Kingdom; 3NIHR Biomedical Research
Centre for Ophthalmology, London, United Kingdom.
Purpose: Photoreceptor degeneration is a leading cause of blindness in the western
world. The discovery of iPS cells and their potential use for disease modelling
means developmentally relevant methods for the differentiation of mature
photoreceptors are required. We sought to investigate photoreceptor differentiation
in ES cell-derived 3D retinal cultures to determine if this method models in vivo
photoreceptor development and ultimately produces structurally mature
Methods: Mouse ES cell lines (CCE and 129) were differentiated using a 3D
culture system similar to that described by Eiraku et al. 2011. The main difference
was that embryoid bodies were retained and the retinal neuroepithelia were not
cultured separately. This enabled photoreceptors to develop at the apical edge of
the neural epithelia and project into the luminal cavity of the embryoid body.
Photoreceptor differentiation was examined by RT-PCR and immunocytochemistry
over the 35 day culture period and the structural development of photoreceptors
was assessed by electron microscopy.
Results: Neural epithelia were present from day 9 in culture and displayed
polarised cell proliferation, similar to the retinal neuroblastic layer, at day 18.
Following 20 days of culture, Crx+ photoreceptor precursors were present and by
day 26 rhodopsin+ and recoverin+ cells maintained an ‘ONL-like’ layer at the apical
edge of the neural epithelia. Cralbp+ müller glial processes spanned this layer and
maintained ZO-1+ adherens junctions at the outer edge, reminiscent of the OLM.
Later stage photoreceptor markers, such as rod transducin, were also present from
day 28 of culture. Ultrastructural analysis demonstrated the presence of ‘inner-like’
segments from day 28 onwards, similar to the postnatal retina. However, ‘outerlike’ segments were not observed.
Conclusions: Photoreceptor development in 3D ES cell-derived cultures paralleled
in vivo development and ES cell-derived photoreceptors were ultrastructurally
comparable to postnatal photoreceptors. However, the organisation of these cells
was compromised following 35 days of culture; consequently it was unclear
whether these cells could progress to form ‘outer-like’ segments. Therefore further
modifications to increase the survival of these cells may be required to create an in
vitro model for photoreceptor degeneration.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: Emma L. West, None; Anai Gonzalez Cordero,
None; Livia S. Carvalho, None; Rachael A. Pearson, None; Jane C. Sowden,
None; Robin R. Ali, None
Support: MRC Grant (G03000341), Wellcome Trust (082217), The Royal Society,
NIHR BMRC for Ophthalmology
233 Immune and Inflammatory Mechanisms in the Retina and RPE
Monday, May 7, 2012, 8:30 AM - 10:15 AM
Hall B/C Poster Session
Program #/Board # Range: 1641-1673/D872-D904
Organizing Section: Retinal Cell Biology
Contributing Section(s): Immunology/Microbiology
Program Number: 1641 Poster Board Number: D872
Presentation Time: 8:30 AM - 10:15 AM
Effect of Excess Complement Activation on AMD-like Pathology in the
APOE4/cfh Knockout Transgenic Murine Model: Does Loss-of-function
Exacerbate Retinal Disease?
Joseph G. Christenbury1A, Jin-Dong Ding1A, Una Kelly1A, Marybeth Groelle1A,
Catherine Bowes Rickman1B. AOphthalmology, BOphthalmology and Cell Biology,
Duke University Medical Center, Durham, NC.
Purpose: There is strong evidence implicating local inflammation and complement
dysregulation in AMD. Complement factor H (CFH) polymorphisms are the
strongest genetic risk factor, but little is known about how risk is conferred. Since
CFH is a known complement alternative pathway inhibitor, we studied the in vivo
effects of excess complement activation on AMD-like pathology using APOE/cfh-/double transgenic mice. Here, we analyzed whether this complement system
manipulation worsened or ameliorated the APOE4 AMD-like phenotype.
Methods: The APOE4 murine model manifests an AMD-like phenotype in animals
aged over 65 weeks and fed a high fat cholesterol-enriched (HFC) diet.
Homozygous APOE4/APOE4 mice were crossed with cfh-/- transgenic mice to
establish the APOE4/cfh-/- double transgenic line. Experimental animals were aged
and fed a HFC diet. We used electroretinograms (ERGs), histology,
immunohistochemistry, Western blots, C3a ELISA and C3 hemolysis assays to
study the impact of complement cascade dysregulation.
Results: Preliminary results show that the ERGs of aged APOE4/cfh-/- mice on
normal diet and on HFC display impaired visual function compared to APOE4
controls on normal diet. Histological review reveals that aged APOE4/cfh-/- mice on
and off HFC diet display a similar number of basal deposits and degree of
vacuolization as compared to APOE4 controls. However, RPE was more damaged
in APOE4/cfh-/- mice than APOE4 controls, especially in mice fed a HFC diet.
Analysis of complement components in plasma confirmed that there was less C3
and more C3b in APOE4/cfh-/- animals compared to APOE4 and APOE4/sCrry
(which produces excess complement inhibition) controls.
Conclusions: These initial findings suggest that the effect of excess complement
activation, modeling CFH loss-of-function, on an AMD-like murine model
damages visual function. This supports the central importance of complement
factor H in the pathophysiology of AMD and use of this animal model to study
complement dysregulation in AMD.
Commercial Relationships: Joseph G. Christenbury, None; Jin-Dong Ding,
None; Una Kelly, None; Marybeth Groelle, None; Catherine Bowes Rickman,
Support: NIH Grant EY019038, P30 EY005722, Research to Prevent Blindness,
Inc., Ruth and Milton Steinbach Fund, Macular Vision Research Foundation
Program Number: 1642 Poster Board Number: D873
Presentation Time: 8:30 AM - 10:15 AM
Complement Play A Minor Role In Mouse Laser-induced Choroidal
Stephen H. Poor, Elizabeth Fassbender, Siyuan Shen, Yubin Qiu, Amber
Woolfenden, John Demirs, Karen Anderson, Bruce D. Jaffee. Ophthalmology,
Novartis, Cambridge, MA.
Purpose: To investigate the role of complement in laser-induced choroidal
neovascularization (CNV) in mice
Methods: Retina photocoagulation lesions in C57BL/6 mice were generated with
an Iridex Oculight® GLx 532 nm laser (3 lesions/eye, 6 lesions/mouse, 10
mice/cohort, yielding 60 data points per cohort). On day 7, mice were injected iv
with vascular label and euthanized. CNV lesions were photographed by fluorescent
microscopy and lesion area was measured with Axiovision software. Data was
masked during image acquisition, application of exclusion criteria, and analysis.
Wild-type and complement-deficient (or drug-treated) mice in each experiment
were age and sex matched and derived from a single vendor. Inter-group
differences were analyzed by one-way analysis of variance (ANOVA) with a
Neuman-Keuls post hoc analysis or an unpaired two tailed t test. Mice investigated
included knockouts (KO’s) of C3, factor D, factor B, C5, C5a receptor or C6 on a
C57/Bl6 Jackson background and CFH KO’s and C3 KO’s on a Taconic
background. In some experiments, wild type mice were treated with complement
modifying drugs including systemic administration of cobra venom factor, a serine
protease inhibitor (Nafamostat), a covalent inhibitor of factor D (Biocryst), and a
LMW C5aR antagonist (PMX53) as well as intravitreal administration of PMX53.
Results: The areas of CNV were significantly larger in C3, C5 and C6 KO mice
compared to C57/Bl6 mice on a Jackson background. The areas in Factor D & B
KO’s were not different from C57/Bl6 controls. C3 KO mice on a Taconic
background developed CNV lesions with areas similar in size as C57/Bl6 controls.
Factor H KO and heterozygote mice on a Taconic background developed
significantly smaller areas of CNV compared to C57/Bl6 controls. All drugs tested
did not significantly inhibit the area of CNV. We also observed that Taconic mice
develop an area of CNV ~ 3 fold larger than mice from the Jackson lab.
Conclusions: We note that the mice derived from different vendors have markedly
different responses to laser photocoagulation. The differences in CNV area
observed between KO mice and controls may be explained by variation in the
origin of mice. We reccommend that sibling controls are used for future
Our experimental results to date indicate that complement deficiency plays at most
a minor role in mouse laser-induced CNV. Our observations are in contrast to
published findings from other groups that found complement deficiency reduced
the size or completely inhibited the area of laser-induced CNV. We note that laserinduced CNV in mice has a different pathogenesis than wet AMD
Commercial Relationships: Stephen H. Poor, Novartis Institute of Biomedical
Research, Department of Ophthalmology (F); Elizabeth Fassbender, Novartis
Institute of Biomedical Research, Department of Ophthalmology (F); Siyuan Shen,
Novartis Institute of Biomedical Research, Department of Ophthalmology (F);
Yubin Qiu, Novartis Institute of Biomedical Research, Department of
Ophthalmology (F); Amber Woolfenden, Novartis Institute of Biomedical
Research, Department of Ophthalmology (F); John Demirs, Novartis Institute of
Biomedical Research, Department of Ophthalmology (F); Karen Anderson,
Novartis Institute of Biomedical Research, Department of Ophthalmology (F);
Bruce D. Jaffee, Novartis Institute of Biomedical Research, Department of
Ophthalmology (F)
Support: None
Program Number: 1643 Poster Board Number: D874
Presentation Time: 8:30 AM - 10:15 AM
Localized dysregulation of complement is involved in the pathogenesis of LateOnset Retinal Macular Degeneration (LORMD)
Shyamanga Borooah1A, Bal Dhillon1, James Ironside1, Siddharthan Chandran1A,
Alan F. Wright2. AMRC Centre for Regenerative Medicine, 1University of
Edinburgh, Edinburgh, United Kingdom; 2MRC Human Genetics Unit, Edinburgh,
United Kingdom.
Purpose: LORMD is a fully penetrant, autosomal dominant retinal degeneration
resulting from mutation in the protein C1QTNF5. Clinically, LORMD replicates
many of the key features of AMD. Disease pathogenesis is currently incompletely
understood. This study aims to identify whether disease mechanisms already
identified in AMD are relevant to LORMD.
Methods: A LORMD donor eye was fixed in 4% formalin and paraffin embedded.
4µm sections were cut. Antigen retrieval was performed using a decloaking
chamber. Slides were heated to 90 centigrade for 20 minutes before cooling to 80
centigrade. Immuno-staining was performed for C1QTNF5, CFH, C3d, C5b-9 and
CD68. Perl’s stain was used to detect iron. Similarly prepared and stained control
aged donor eyes were also stained.
Results: Staining revealed prominent C3d localization to the sub RPE deposit and
perivascularly in the choroid. Significant C5b-9 staining was noted to run linearly
along the deposit centre and CFH was localized to deposit surfaces. C1QTNF5 was
also found on deposit surfaces and on the RPE. Occasional enlarged CD68 positive
macrophages and iron deposits were noted.
Conclusions: Previous studies have revealed that immunohistochemical staining of
sub-retinal deposits in LORMD resembles drusen. In this study, the presence of
C3d and C5b-9 provides strong evidence for complement activation in disease
pathogenesis in LORMD. Localization of C1QTNF5 and CFH together on deposit
surfaces supports evidence for an interaction between mutant C1QTNF5 and CFH
resulting in local complement dysregulation. This study suggests that similar
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
pathways are involved in the etiologies of both AMD and LORMD.
Commercial Relationships: Shyamanga Borooah, None; Bal Dhillon,
None; James Ironside, None; Siddharthan Chandran, None; Alan F. Wright,
Support: Wellcome Trust ref: 098000/Z/11/Z
Program Number: 1644 Poster Board Number: D875
Presentation Time: 8:30 AM - 10:15 AM
Effects Of The Absence Of both Complement Factor H And Apolipoprotein E
On Retinal Morphology In Mice
Laura Garcia-Garcia1, Patricia Fernandez-Robredo1, Sergio Recalde1, Maite
Moreno-Orduña1, Vanessa Fernandez-Garcia1, Josemaria Caire1, Miguel A.
Marchena-Fernandez2, Pedro de la Villa2, Alfredo Garcia-Layana3,1.
Experimental Ophthalmology Lab, Universidad de Navarra, Pamplona, Spain;
Physiology, University of Alcala, Alcala de Henares, Spain; 3Ophthalmology,
Clinica Universidad de Navarra, Pamplona, Spain.
Purpose: Apolipoprotein E deficient mouse (apoE-/-), experimental model of
genetic hypercholesterolemia, shows ultrastructural retinal alterations. Complement
factor H (CFH) is involved in inflammatory process associated to retinal
degenerations. We aimed to investigate the effect of the absence of CFH and apoE
genes in mouse retinal pigment epithelium (RPE) and Bruch´s Membrane (BM).
Methods: Eight WT, 15 CFH-/-/apoE-/- (DKO) and 19 CFH+/-/apoE+/- (DH)
mice older than 12 months were used (all mice were in C57BL6/J background).
Light microscopy was performed to analyze the following morphological
parameters in the retinal sections (Toluidine Blue). Apoptotic-like nuclei from the
outer nuclear layer (ONL) were counted using AnalySIS software. The thickness of
the whole retina and the outer nuclear layer section were measured by ImageJ
image processing program. Samples were also processed for Transmission Electron
Microscopy (TEM) visualization. For comparisons after a significant ANOVA
among groups, post-hoc tests were applied (SPSS v.15.0).
Results: Apoptotic-like nuclei were significantly higher (p<0.001) among WT and
genetically modified groups (p<0.001) and between DKO and DH groups (p<0.05).
However, no differences were found for ONL and total retina among groups. In the
TEM visualization structural abnormalities were found in DKO mice compared to
WT. BM appeared swelled and with vacuoles were shown inside the membrane.
Moreover, basal laminar deposits were observed in genetically modified groups
whereas in the RPE electrodense and electrolucent vacuoles, phagosomes and
swelling of interdigitations could be found in genetically mice.
Conclusions: These results show that the absence of CFH and apoE is involved in
the development of RPE and BM morphological alterations and apoptosis of ONL
cells. This animal model could be useful as a model of retinal degeneration.
Commercial Relationships: Laura Garcia-Garcia, None; Patricia FernandezRobredo, None; Sergio Recalde, None; Maite Moreno-Orduña, None; Vanessa
Fernandez-Garcia, None; Josemaria Caire, None; Miguel A. MarchenaFernandez, None; Pedro de la Villa, None; Alfredo Garcia-Layana, None
Support: supported by RETICS RD07/0062
Program Number: 1645 Poster Board Number: D876
Presentation Time: 8:30 AM - 10:15 AM
Complement Factor H Deficiency Results in Decreased Neuroretinal
Expression of CD59 in Aged Mice
Carsten Faber1, Jennifer Williams2, Helene Juel1, John Greenwood2, Mogens Holst
Nissen1, Stephen E. Moss2. 1Eye Research Unit, ISIM, University of Copenhagen,
Copenhagen, Denmark; 2Cell Biology, UCL Institute of Ophthalmology, London,
United Kingdom.
Purpose: The complement system plays a key role in age-related macular
degeneration (AMD). Several complement genes are expressed in retinal pigment
epithelium (RPE) and complement split-products and regulators accumulate in
drusen. Further, a common variant of complement factor H (CFH) confers
increased risk of developing AMD. To examine the role of CFH in the ocular
complement regulatory system, we analysed the expression of complement genes in
neuroretinas, RPE/choroid and livers of young and old WT and Cfh-/- mice.
Methods: Neuroretinas, RPE/choroid and liver tissue were isolated from young
(~50 days) and old (~500 days) WT and Cfh-/- C57Bl/6 mice. Ocular RNA from
two eyes/animal, four animals/group, was analysed with whole-transcript
microarrays. Differential gene expression was validated with qRT-PCR. Liver
RNA was analysed with qRT-PCR.
Results: In RPE/choroid complement genes associated with activation of both the
classical and alternative pathway including C1q, C3 and Factor B were upregulated
with age. This coincided with increased expression of the negative regulators Cfh
and Cd59 in neuroretina adding to the marked neuroretinal expression of negative
regulators. Lack of age-dependent neuroretinal upregulation of Cd59 represented
the main difference between WT and Cfh-/- mice. Hepatic expression of Cd59 and
C1q increased with age independently of genotype. Hepatic expression of C3 and
Factor B was equal in all groups and expression of Cfh was equal in young and old
WT animals.
Conclusions: The age-related upregulation of substrates, activators and inhibitors
of the complement system in the retina is abnormal in mice deficient in CFH. This
could explain the visual function deficits and morphological changes in the Cfh-/mouse retina with age. Further, these results suggest that neuroretinal expression of
CD59 depends on normal functioning CFH, thus pointing towards deficient
neuroretinal complement regulation as an early event in some forms of AMD.
Commercial Relationships: Carsten Faber, None; Jennifer Williams,
None; Helene Juel, None; John Greenwood, None; Mogens Holst Nissen,
None; Stephen E. Moss, None
Support: None
Program Number: 1646 Poster Board Number: D877
Presentation Time: 8:30 AM - 10:15 AM
Complement Regulatory Protein Expression by Retinal Müller Cells
Vijay P. Sarthy1, Alex Park1, V. Joseph Dudley1, Richard J. Johnson2. 1OphthalFeinberg Med Sch, Northwestern University, Chicago, IL; 2Research Exploratory
Sciences, Baxter Healthcare Corporation, Round Lake, IL.
Purpose: The complement system is important for immunosurveillance, and recent
studies indicate that uncontrolled activation of the complement cascade contributes
to the development and progression of AMD. Previous studies have characterized
expression and modulation of complement regulatory proteins in retinal pigment
epithelial cells (RPE). The goal of the present study was to identify complementary
regulatory proteins expressed by Müller cells and to examine whether oxidative
stress affects their expression.
Methods: Experiments were carried out using the human Müller cell line, MIOM1. Oxidative stress was induced by exposure to 0.05mM hydrogen peroxide.
Complement regulatory protein expression was determined by qPCR and flow
Results: Through real-time PCR, we found high levels of CD46 mRNA and
moderate levels of CD59 mRNA, whereas CD55 mRNA was undetectable. Flowcytometry showed moderate expression of CD46 protein and high expression of
CD59, CD55 was not detectable. Furthermore, we found moderate levels of C5aR
mRNA and a low level of C3aR. At the protein level, both receptors were low in
abundance. In MIO-M1 cells treated with 0.5 mM Hydrogen peroxide, both flow
cytometry and RT-PCR showed very little or no changes in CD46, CD55 and
CD59 levels.
Conclusions: Our studies show that Müller cells express several complement
regulatory proteins but their expression levels are not modulated by oxidative
stress. These results suggest complement regulation might be different in Müller
cells and RPE.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: Vijay P. Sarthy, Baxter Healthcare Corporation (F);
Alex Park, Baxter Healthcare Corporation (F); V. Joseph Dudley, Baxter
Healthcare Corporation (F); Richard J. Johnson, Baxter Healthcare Corporation
Support: Baxter-Northwestern Alliance Grant
Program Number: 1647 Poster Board Number: D878
Presentation Time: 8:30 AM - 10:15 AM
Complement Activation and Changes in RPE and Retina after Subretinal
Treatment of Polyethylene Glycol (PEG) in Mice
Valeriy V. Lyzogubov, Purushottam Jha, Ruslana G. Tytarenko, Nalini S. Bora,
Puran S. Bora. Ophthalmology, Jones Eye Institute - UAMS, Little Rock, AR.
Purpose: Age-related macular degeneration (AMD) is a leading cause of
irreversible blindness worldwide. Recently we reported that polyethylene glycol
(PEG) can activate the alternative pathway of the complement system and induce
CNV in mice. The aim of this study was to characterize changes in mouse RPE and
retina after PEG treatment.
Methods: We injected male C57BL/6 mice subretinally with 0.5 mg of PEG. We
used 2 µL of solution for injections. Sterile PBS was used as vehicle and was
injected in control groups. Eyes were harvested at day 1, 3 and 5 after injection and
processed for histological analysis. Semi thin (1 µm) sections of PBS and PEG
treated mouse eyes were stained with hematoxylin. Paraffin sections (5 µm) were
stained with hematoxylin and eosin and used for morphometry. Sections were
stained for complement component C3, membrane attack complex (MAC),
proliferating cell nuclear antigen (PCNA) and cytokeratin 18 (CK18) by IHC. Light
and laser confocal microscopy was used for image capturing. Measurement and
quantification of morphometric parameters was performed using ImageJ program.
Results: PEG increased deposition of C3 and MAC on RPE cells and on all retinal
layers. PEG induced loss of cellular contacts between RPE cells, migration of RPE
cells in subretinal space at day 1 after PEG injection. Increased size of RPE cells
and expression of PCNA was detected at day 3 and 5 after PEG treatment.
Apoptotic bodies were observed in ONL at day 3 and 5 after PEG treatment. RPE
cells with dark cytoplasm and condensed chromatin were observed. By day 5 after
subretinal injection of PEG we found decreased nuclei density in the ONL of the
retina (-49%), decreased the length of the photoreceptor outer and inner segments (61%), increased size of the RPE cells (+133%), and reduced pigmentation of the
RPE cells (-33%) compared to PBS injected control group.
Conclusions: Activation the complement system by PEG leads to morphological
changes in RPE and retina consistent with dry AMD. PEG is a very useful tool to
induce proliferation and death of RPE cells and death of photoreceptors in mice.
This simple and fast model may be used to investigate dry AMD pathogenesis.
Commercial Relationships: Valeriy V. Lyzogubov, None; Purushottam Jha,
None; Ruslana G. Tytarenko, None; Nalini S. Bora, None; Puran S. Bora, None
Support: This work was supported by Pat & Willard Walker Eye Research Center,
Jones Eye Institute, Little Rock, AR.
Program Number: 1648 Poster Board Number: D879
Presentation Time: 8:30 AM - 10:15 AM
Human RPE Cell Injury Mediated by Oxidative Stress and Alternative
Complement Cascade
Ping Yang1, Brett A. Toimil1, Jacob E. Berchuck1, Peter Baciu2, Glenn J. Jaffe1.
Ophthalmology, Duke University Medical Center, Durham, NC; 2Biology,
Allergan, Inc, Irvine, CA.
Purpose: Complement activation has been increasingly implicated in the
pathogenesis of AMD. RPE cells are continually exposed to oxidative stress.
Herein, we investigate the effect of repetitive and single exposure to hydroquinone
(HQ) and hydrogen peroxide (H2O2) on alternative complement pathway (AP)mediated injury in human RPE (hRPE) cells.
Methods: Cultured hRPE cells from 3 adult donors (CFHYY402, CFHYH402, and
CFHHH402) were stimulated with various concentrations of HQ and H2O2 for 1 week
(four hits) or for 90 minutes (one hit) and then primed with a complement-fixing
antibody for 30 minutes followed by incubation with C1q-depleted human serum
for various times. Tetrazolium salt WST-1, lactate dehydrogenase release and cell
count assays were used to assess cell permeability, cell viability and cell number,
respectively. DNA fragmentation was determined using ELISA and TUNEL
assays; cells transfected with Bcl-xL splice-switching oligonucleotide (SSO) to
induce apoptosis were used as a positive control.
Results: AP attack significantly increased RPE cell permeability, and decreased
cell viability and cell number in a dose-dependent manner in 3 donor RPE cells
(P<0.05). Repetitive hits with low dose HQ significantly increased cell viability
(P<0.01), and AP attack significantly reduced this HQ-induced effect (P<0.001).
Repetitive and single hits with either HQ or H2O2 significantly enhanced AP-driven
cell death in 3 donor RPE cells as compared to cells treated with HQ, H2O2 and AP
alone (P<0.05). Nuclear shrinkage was observed by DAPI stain in cells treated with
HQ+AP. Cells treated with H2O2+AP appeared bloated. DNA fragmentation was
modestly but significantly increased in cells treated with HQ+AP as compared to
cells treated with HQ (P=0.02) and AP alone (P=0.02). TUNEL positive cells were
detected in SSO transfected cells, but not in cells treated with HQ+AP or
Conclusions: For both the repetitive hit and single hit model, oxidative stress
renders RPE cells more susceptible to AP-induced cell death. The repetitive hit
model which simulates chronic oxidant exposure will be useful in future
investigations to explore mechanisms of observed differences in HQ- and H2O2enhanced AP-driven RPE cell death.
Commercial Relationships: Ping Yang, None; Brett A. Toimil, None; Jacob E.
Berchuck, None; Peter Baciu, Allergan, Inc (E); Glenn J. Jaffe, None
Support: NIH P30 EY-005722 (Core grant),Research to Prevent Blindness, Inc.
Program Number: 1649 Poster Board Number: D880
Presentation Time: 8:30 AM - 10:15 AM
All-trans-Retinal Sensitizes Human RPE Cells to Alternative Complement
Pathway-Induced Cell Death
Jacob E. Berchuck1, Ping Yang1, Brett A. Toimil1, Peter Baciu2, Glenn J. Jaffe1.
Ophthalmology, Duke University Medical Center, Durham, NC; 2Biology,
Allergan, Inc, Irvine, CA.
Purpose: RPE cells are exposed to numerous oxidants, including all-trans-retinal
(ATR), a chromophore released in photoreceptors after photon absorption. ATR is
the precursor of A2E, a fluorophore believed to cause RPE cell death, and ATR
itself is reportedly toxic to RPE cells, yet its direct role in the pathogenesis of AMD
is unclear. It is thought that chronic oxidative stress injures RPE cells, impairing
their ability to regulate surface complement deposition leading to uncontrolled
alternative complement pathway (AP) activation. In this study, we used an in vitro
model to determine whether pre-treatment with ATR sensitizes primary human
RPE (hRPE) cells to AP-induced cell death. Further, we explored the signal
transduction pathways by which ATR and AP exert their cytotoxic effects.
Methods: hRPE cells were treated with a non-lethal dose of ATR for 90 minutes,
followed by 30-minute incubation with a complement-fixing antibody. Cells were
then treated with C1q-depleted human serum (C1qD) to activate AP. After 90
minutes, the effect of ATR and AP activation on hRPE cell viability was assessed
by tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release assays.
Following the same treatment conditions, changes in ERK and P38 phosphorylation
were examined by Western blot.
Results: Treatment with ATR alone caused a dose-dependent decrease in hRPE
cell viability, as determined by LDH release and WST-1 assays. AP activation
alone also caused hRPE cell death. In both LDH (p<0.01) and WST-1 (p<0.005)
assays, AP activation in hRPE cells pre-treated with a non-lethal dose of ATR
decreased viability more than the additive effect of both independent treatments.
Following the same treatment conditions, when measured 5 minutes after C1qD
addition, P38 phosphorylation increased, while ERK phosphorylation decreased.
Conclusions: We have shown that ATR causes dose-dependent cell death in hRPE
cells. More importantly, at a non-lethal dose, ATR sensitizes hRPE cells to the
cytotoxic effects of AP activation and when combined, the two synergistically
cause hRPE cell death. Although their role in ATR and AP-induced hRPE cell
death is not yet defined, P38 and ERK may be important mediators and represent
potential targets to inhibit the oxidative stress and complement-induced injury that
contributes to AMD.
Commercial Relationships: Jacob E. Berchuck, None; Ping Yang, None; Brett
A. Toimil, None; Peter Baciu, Allergan, Inc. (E); Glenn J. Jaffe, None
Support: Core Grant NIH P30 EY-005722, Howard Hughes Medical Institute
Medical Research Fellowship, Foundation Fighting Blindness, Research to Prevent
Blindness, Inc.
Program Number: 1650 Poster Board Number: D881
Presentation Time: 8:30 AM - 10:15 AM
Oxidized Low Density Lipoprotein-Induced Injury in RPE Cells Alters
Expression of the Transmembrane Complement Regulatory Factors CD46
and CD59 through Exosomal and Apoptotic Bleb Release
Katayoon B. Ebrahimi, Lei Wang, Mizuki Tagami, Marisol Cano, Sonny Dike,
James T. Handa. Ophthalmology, Johns Hopkins University, Baltimore, MD.
Purpose: Studies have identified the alternative complement pathway as an
important component of AMD. Our lab has shown that oxidized lipoproteins
accumulate in drusen in early AMD and promote complement activation and
apoptosis in RPE cells. This study was conducted to determine the mechanisms by
which the CD46 and CD59 decrease and how these changes apply to AMD.
Methods: ARPE-19 cells were cultured and exposed to 100 µg/ml oxLDL for 24 h.
Protein was extracted. The supernatant was lyophilized. Western blot analysis was
performed using mouse monoclonal anti-human CD59, CD46, CD63. Smears of
the supernatant were prepared and sequential double immunofluorescent staining
for CD46 and EO6 was performed. Immunohistochemistry was performed on agematched normal (n=9) and early-stage AMD (n=9) autopsy eyes. Sections were
deparaffinized, antigens were retrieved, and incubated with mouse anti-human
CD59 monoclonal antibody and isotype control.
Results: Previously we demonstrated that treatment of ARPE-19 cells decreases
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
transmembrane CD46 with increased CD46 protein in the supernatant. Oxidized
phosphorycholine is recognized by the E06 antibody and decorates apoptotic blebs.
To demonstrate that the CD46 release is associated with apoptotic blebs, using
sequential double immunofluorescence labeling for CD46 and EO6, we identified
CD46 positive membrane bound particles in the supernatant. The EO6 bound to
these particles along with apoptotic cells in a punctuate pattern. OxLDL treatment
also decreased cellular CD59, but increased CD59 in the supernatant. The
tetraspanin CD63, is highly abundant on exosomes, was also increased in the
supernatant after oxLDL treatment. This suggests that decreased CD59 may be
related in part, to the release of CD63 containing exosomes. Macular and peripheral
sections of unaffected normal individuals did not show detectable CD59
immunostaining. In peripheral sections of early AMD patients, minimal CD59
labeling was seen in the RPE. In contrast, maculas from patients with early AMD
showed strong CD59 labeling apically throughout the RPE with occasional
basolateral staining.When overlying drusen, the RPE displayed weaker CD59
Conclusions: In early AMD, the identification of CD59 at the RPE-photoreceptor
junction suggests heightened complement activity. Our data support a novel
hypothesis that reduced CD59 through exosomal release leads to complement
activation in the RPE. We also suggest that the decreased CD46 was released in
subapoptotic blebs after an oxLDL stimulus. Like CD59, this not only reduces
complement control of the RPE, but also releases CD46 into BrM and drusen.
Commercial Relationships: Katayoon B. Ebrahimi, None; Lei Wang,
None; Mizuki Tagami, None; Marisol Cano, None; Sonny Dike, None; James T.
Handa, None
Support: NEI EY14005(JTH), EY019904(JTH), Thome Foundation Grant (JTH),
Unrestricted grant from RPB. Dr. Handa is the Robert Bond Welch Professor.
Program Number: 1651 Poster Board Number: D882
Presentation Time: 8:30 AM - 10:15 AM
X-box Binding Protein 1 Regulates Endothelial Inflammation and Bloodretinal Barrier Homeostasis in Diabetic Retinopathy
Jingming Li, Joshua J. Wang, Yimin Zhong, Chen Chen, Sarah X. Zhang.
Department of Medicine, Endocrinology and Diabetes, Harold Hamm Diabetes
Center, University of Oklahoma, Health Sciences Center, Oklahoma City, OK.
Purpose: Endoplasmic reticulum (ER) stress has been widely implicated in chronic
inflammatory diseases, such as diabetes. X-box binding protein 1 (XBP1) is a
central coordinator of cellular responses during ER stress. Previously we reported
that preconditioning with ER stress mitigated retinal endothelial inflammation via
activation of XBP1. Further, we found that retinal expression of XBP1 was
markedly reduced in diabetic mice. The objective of this study is to investigat the
role of XBP1 in retinal inflammation and vascular leakage associated with diabetic
retinopathy (DR).
Methods: Endothelium-specific XBP1 deficient (XBP1EC-/-) mice were generated
by using the Cre-loxp system. Diabetes was induced by intraperitoneal injection of
streptozotocin (STZ). Expression of adherent molecules and tight junction proteins,
leukostasis and vascular permeability were evaluated in diabetic XBP1EC-/- and
wild type (XBP1EC+/+) mice. Mouse brain endothelial cells were isolated from
both XBP1EC-/- and XBP1EC+/+ mice for in vitro study. Expression of tight
junction proteins and adherent molecules was determined by Western-blot analysis
or immunocytochemistry.
Results: Retinal expression of intracellular adherent molecule-1 (ICAM-1) and
vascular adherent molecule-1 (VCAM-1) was significantly increased, in parallel
with enhanced leukocytes adhesion to retinal vasculature in diabetic XBP1EC+/+
mice. When compared with XBP1EC+/+ mice, diabetic XBP1EC-/- mice showed
much higher levels of adhesion molecules and greater increase in leukostasis in the
retina. In addition, retinal expression of zonula occludens-1 (ZO-1), a major tight
junction protein, was much lower in XBP1EC-/- mice, indicating impaired bloodretinal barrier. Furthermore, XBP1EC-/- mice showed more severe retinal vascular
leakage compared with XBP1EC+/+ mice. In addition, endothelial cells isolated
from XBP1EC-/- mice expressed much higher level of ICAM-1, but lower level of
ZO-1 after incubation with pro-inflammatory cytokine TNF-α. This suggests that
loss of XBP1 sensitizes endothelial cells to inflammation and tight junction
Conclusions: Our results indicate that XBP1 plays a vital role in regulating
endothelial barrier function. Impaired XBP1 signaling may in part contribute to
retinal inflammation and vascular leakage in diabetic retinopathy.
Commercial Relationships: Jingming Li, None; Joshua J. Wang, None; Yimin
Zhong, None; Chen Chen, None; Sarah X. Zhang, None
Support: NIH grant EY019949; ADA Research Award 7-11-BS-182; AHAF grant
M2010088; OCAST Research Grant HR10-060; Dr. William Talley Research
Program Number: 1652 Poster Board Number: D883
Presentation Time: 8:30 AM - 10:15 AM
Recruitment of Bone Marrow-derived Stem cells to the Retina Following
Thermal Injury to the Retinal Pigment Epithelium
David L. Kent1, Carl M. Sheridan2, Sergio Li Calzi3A, Sergio Caballero, Jr.3B,
Maria B. Grant3A. 1The Vision Clinic, Kilkenny, Ireland; 2Dept of Eye and Vision
Science, University of Liverpool, Liverpool, United Kingdom; APharmacology and
Therapeutics, BPharmacology/Therapeutics, 3University of Florida, Gainesville, FL.
Purpose: Hematopoietic stem cells (HSCs) have a natural ability to home to areas
of injury for regeneration. The integrity of retinal pigment epithelium (RPE) is
important for the maintenance of a healthy retina. This study investigated the
recruitment and distribution of HSCs in the retina in a laser-induced RPE injury
model with the specific aim of delivering a sub-lethal injury to the RPE and
avoiding rupture of Bruch’s membrane.
Methods: Chimeric mice were generated by injection of sca-1+, c-kit+ HSCs from
green fluorescent protein (gfp) transgenic mice into lethally irradiated wild-type
C57BL/6J recipients. The gfp chimeric mice were subjected to a low-intensity laser
injury (532-nm diode, 100 milliwatts for 0.1 second) targeted on the RPE layer and
then sacrificed at 1, 4, 12, 24 and 48 hours post laser. Their eyes were enucleated,
fixed in 4% paraformaldehyde and embedded in paraffin wax for
immunohistochemical analysis of gfp expression to determine if there was any
recruitment of HSCs to the retina following the sub-lethal RPE injury.
Results: Intense staining of gfp was seen in the retinal ganglion cell layer as early
as 1 hour after injury. gfp fluorescence was detected in parts of the inner nuclear
layer and outer plexiform layer throughout different time points. However, higher
intensity of gfp was found in the RPE and choroid of mice sacrificed at later time
points (24 and 48 hours) post laser injury.
Conclusions: This study provides evidence of the potential of bone marrowderived HSCs to rescue a compromised RPE layer in a time-dependent manner.
Immunohistochemical studies of the immune and inflammatory responses are
currently underway.
Commercial Relationships: David L. Kent, None; Carl M. Sheridan,
None; Sergio Li Calzi, None; Sergio Caballero, Jr., None; Maria B. Grant,
Support: None
Program Number: 1653 Poster Board Number: D884
Presentation Time: 8:30 AM - 10:15 AM
The Protective Effect of AICAR on the Neural Retina during Inflammation in
Mamoru Kamoshita, Kenya Yuki, Shunsuke Kubota, Manabu Hirasawa, Toshio
Narimatsu, Norihiro Nagai, Kazuo Tsubota, Yoko Ozawa. Ophthalmology, Keio
University School of Medicine, Shinjyuku, Japan.
Purpose: Recently, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an
activator of AMP-activated protein kinase, has been shown to have an antiinflammatory effect. The aim of this study is to investigate the effect of AICAR on
the neural retina during inflammation, using a mouse model of endotoxin-induced
uveitis (EIU).
Methods: The EIU model was induced by an intraperitoneal injection of
lipopolysaccharide (LPS). Each animal was given an intraperitoneal injection of
AICAR (250mg/kg) or a vehicle 3 hours before the LPS injection. Effects of
AICAR were evaluated at 24 hours after LPS injection. Visual function was
analyzed by electroretinogram (ERG). Protein level of rhodopsin in the retina was
measured by immunoblot analysis, and the morphological change was analyzed in
the retinal sections.
Results: Impairment of a-wave response recorded by ERG, which represents
photoreceptor dysfunction, in the EIU model mice was avoided by administration
of AICAR. Rhodopsin protein level and the length of photoreceptor outer segments
were reduced in the retina of EIU model mice, however, these changes were both
avoided by AICAR treatment.
Conclusions: The AICAR had a neuroprotective effect on the photoreceptor cells
during retinal inflammation.
Commercial Relationships: Mamoru Kamoshita, None; Kenya Yuki,
None; Shunsuke Kubota, None; Manabu Hirasawa, None; Toshio Narimatsu,
None; Norihiro Nagai, None; Kazuo Tsubota, None; Yoko Ozawa, None
Support: None
Program Number: 1654 Poster Board Number: D885
Presentation Time: 8:30 AM - 10:15 AM
TGF-Beta 2 is the Predominant TGF-Beta Isoform Secreted by RPE in
Polarized and Non-Polarized Cultures
Louis K. Hirsch, Parameswaran G. Sreekumar, Christine Spee, Ernesto Barron,
Laurie Dustin, Stephen J. Ryan, David R. Hinton. Doheny Vision Research Ctr,
USC, Keck School of Medicine, Los Angeles, CA.
Purpose: Retinal pigmented epithelium (RPE) secretes fibrogenic proteins, e.g.
transforming growth factor beta 1 & 2 (TGF-β1 & -β2). While both isoforms may
promote fibrogenesis, TGF-β2 has a stronger association with ocular pathology,
such as in proliferative vitreoretinopathy (PVR). Since RPE cell polarity may be
altered in PVR, we studied the effects of polarity on TGF-β isoform secretion.
Methods: (1) Human Fetal RPE culture: RPE from 4 donors were cultured under 3
conditions for comparison: subconfluent nonpolarized, confluent nonpolarized, and
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
highly polarized monolayers. (2) Enzyme-linked immunosorbent assay (ELISA):
TGF-β1 and TGF-β2 were measured in media from apical and basal compartments
of polarized RPE transwell filters and from the single compartment of nonpolarized
RPE petri dishes. (3) Transepithelial Resistance(TER): To confirm RPE
differentiation, donors had TERs of >185 Ω·cm^2. (4) Normalization: Data
normalized by total cell protein assays.
Results: Comparing secreted concentrations of total TGF-β1 to -β2, -β2 was
secreted in higher concentrations than -β1 in all 3 culture conditions, polarized
(p<0.01), nonpolarized confluent (p<0.01), and nonpolarized subconfluent
(p<0.01). Total TGF-β is the sum of both latent and endogenous active forms. RPE
also secretes significantly more endogenous active -β2 during in nonpolarized
confluent (p<0.001) and subconfluent cultures (p<0.001).
Conclusions: RPE secretes higher concentrations of TGF-β2 than -β1 in both
polarized and non-polarized cell cultures. These data are consistent with previous
reports that -β2 is the predominant ocular isoform. These data contribute to our
understanding of the cytokine environment created by the RPE. Further
investigation into these cytokines may lead to targeted molecular therapies for
fibrogenic ocular diseases like PVR.
Commercial Relationships: Louis K. Hirsch, None; Parameswaran G.
Sreekumar, None; Christine Spee, None; Ernesto Barron, None; Laurie Dustin,
None; Stephen J. Ryan, None; David R. Hinton, None
Support: NIH Core Grant EY03040, & Research to Prevent Blindness Grant
Program Number: 1655 Poster Board Number: D886
Presentation Time: 8:30 AM - 10:15 AM
Association of Angiopoietin-like Protein 2 with Inflammatory Signals in the
Human Retinal Pigment Epithelium
Manabu Hirasawa1,2, Motoyo Endo3, Seiji Miyake1, Toshio Narimatsu1, Shunsuke
Kubota1, Misa Suzuki1,4, Susumu Ishida1,5, Kazuo Tsubota1, Yuichi Oike3, Yoko
Ozawa1. 1Ophthalmology, Keio University School of Medicine, Tokyo, Japan;
Ophthalmology, Tokyo Dental College Suidobashi Hospital, Tokyo, Japan;
Molecular Genesis, Kumamoto University, Kumamoto, Japan; 4Ophthalmology,
Yokohama City University School of Medicine, Yokohama, Japan;
Ophthalmology, Hokkaido Graduate School of Medicine, Sapporo, Japan.
Purpose: Angiopoietin-like proteins (Angptls) possess a coiled-coil domain at the
N-terminus for oligomerization and a fibrinogen-like domain at the C-terminus
similarly to angiopoietin. They promote chronic tissue inflammation in adipose
tissue as well as carcinogenesis. However, little is known about the role of Angptl2
in the ocular tissue. The final goal is to explore the association of Angptl2 with the
inflammatory signals in the human retinal pigment epithelium (RPE).
Methods: Cyrosections of postmortem human ocular tissue were prepared and
analyzed the expression of Angptl2, immunohistochemically. The human retinal
pigment epithelium (RPE) cell line, ARPE-19, was cultured in the in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
(FBS). Cells were applied and stimulated by pro-inflammatory cytokines, such as
Tumor Necrosis Factor (TNF)-alpha, Transforming growth factor (TGF)-beta,
connective tissue growth factor (CTGF), and Vascular Endothelial Growth Factor
(VEGF). Cells were collected 24 hours after the stimulation, and mRNA and
protein levels of Angplt2 were analyzed using real-time PCR and ELISA.
Results: Immunostaining for Angptl2 was observed in the cytosol of human RPE,
and its expression in RPE was confirmed in vivo. In the ARPE19, changes in
mRNA expression were observed after stimulation of either TNF-alpha or TGFbeta. Interestingly, the levels were significantly dropped(P<0.01)in a dosedependent manner. The level of Angptl2 protein was also decreased after TNFalpha stimulation.
Conclusions: Angptl2 has been known as a pro-inflammatory signal in some
tissue, however, in ARPE19, its expression was suppressed by stimulation with a
pro-inflammatory cytokine, TNF-alpha. Multiple roles might be involved in
Angptl2 in a context dependent manner. Further investigation is required to reveal
the role of Angptl2 in inflammation.
Commercial Relationships: Manabu Hirasawa, None; Motoyo Endo,
None; Seiji Miyake, None; Toshio Narimatsu, None; Shunsuke Kubota,
None; Misa Suzuki, None; Susumu Ishida, None; Kazuo Tsubota, None; Yuichi
Oike, None; Yoko Ozawa, None
Support: Grant-in-Aid for Young Scientists(A)
Program Number: 1656 Poster Board Number: D887
Presentation Time: 8:30 AM - 10:15 AM
The Role Of CC Chemokines And Their Receptors In The Retinal
Degeneration In Rd Mice
Huiyang Zeng, Qian Liu, Qing-jun Lu, Ke-gao Liu, Ning-li Wang. Being Tongren
Eye Center, Beijing Tongren Hosp, Capital Med Univ, Beijing, China.
Purpose: Our previous studies showed that CC chemokines and CCR1,one of the
CC chemokine receptors, were involved in the retinal degeneration in the rd mice.
The aim of current study was to further investigate the expression of CC
chemokines and their other receptors in the rd retina and explore their role in the
photoreceptor degeneration.
Methods: Expression levels of CC chemokine receptors transcripts including
CCR2,CCR3 and CCR5 in the whole retina in the control and rd mice at postnatal
days (P) 8,10,12,14,16 and 18 were determined by RT-PCR assay. Location of CC
chemokines including MCP-1,MCP-3, MIP-1a,MIP-1βand RANTES and their
above receptors were studied by immunohistochemical analysis. Expression of
gp91phox and iNOS in the rd retina at each age group was studied by real-time PCR
analysis and immunostaining. Cellular location of CCR5, iNOS and gp91phox in the
retina was determined by double labeling.
Results: CCR3 and CCR5 mRNA was significantly up-regulated in the rd retina
from P12 to P16 while CCR2 mRNA was down-regulated when compared with the
controls. RANTES immunoreactivity was seen on the CD11b-postive retinal
microglial cells while other CC chemokines were expressed in the neurons of inner
retina. Immunoreactivity of CCR5 on the activated retinal microglial cells were
found to be increased in the rd mice. CCR2 and CCR3 were expressed on the
neurons in the inner retina of rd and control mice. Expression of gp91phox and iNOS
transcripts began to increase in the rd retinas at P10d and P8d respectively, reached
peak at P12d and maintained a high level until P16d. In the rd retina,
immunoreactivity of gp91phox was observed on the microglial cells while iNOS was
observed on the photoreceptor cells. Part of gp91phox and iNOS were co-localized
with CCR5 and CCR1 (expressed on the photoreceptor as shown in our published
study) respectively.
Conclusions: Activation of CC chemokines receptors, especially CCR5 and CCR1,
as well as activation of oxidative markers on the same cell type, were correlated
with or precedes the occurrence (P10) and peak (P16) of photoreceptor apoptosis,
suggesting that CC chemokines may play a role in the retinal degeneration in rd
mice either indirectly through activation of microglial cells killing mechanism(
activation of CCR5) or directly through activation of chemokine receptors (CCR1)
on photoreceptor cells.
Commercial Relationships: Huiyang Zeng, None; Qian Liu, None; Qing-jun
Lu, None; Ke-gao Liu, None; Ning-li Wang, None
Support: from National natural science foundation of China
Program Number: 1657 Poster Board Number: D888
Presentation Time: 8:30 AM - 10:15 AM
Down-Regulation Of TXNIP Prevents Retinal Neurodegeneration By
Mitigating Inflammation And Vascular Injury
Mona El-Azab, Barbara A. Mysona, Mohammed A. Abdelsaid, Suraporn
Matragoon, Azza B. El-Remessy. Clinical & Administrative Pharmacy, University
of Georgia, Augusta, GA.
Purpose: Retinal ganglion cell (RGC) death is characteristic for several blinding
diseases such as glaucoma, diabetic retinopathy and retinal vein occlusion. Our
recent studies using N-methyl-D-aspartate (NMDA) neurotoxicity model identified
thioredoxin interacting protein (TXNIP) as potential mediator of glial inflammation
and RGC death. The aim of the current work is to elucidate the multiple roles by
which TXNIP mediates RGC death. In particular, we will examine its impact on the
expression of inflammatory mediators in Müller cells and retina microvasculature
that can sustain RGC death. The therapeutic potential of verapamil as TXNIP
inhibitor was also investigated.
Methods: Neurotoxicity was induced by intravitreal injection of NMDA (40
nmoles/eye) into wild type mice, TXNIP deficient (TKO), or wild type treated with
verapamil (10mg/Kg,po). Retinal neurotoxicity was examined by TUNEL assay
and neuronal count. Expression of GFAP and TNF-α was assessed via
immunohistochemistry. Expression of TXNIP, ASK-1, PARP, TNF-α, and IL-1β
was assessed by Western-Blot and nitrotyrosine via Slot-Blot. For in vitro, rMC-1
cultures were stimulated with 1, 10, and 100µM NMDA. Vascular permeability
was assessed by extravasation of fluorescein and development of acellular capillary
was assessed by trypsin digest.
Results: NMDA injection induced retinal neurotoxicity as indicated by significant
increase in TUNEL-positive apoptotic RGC and ~50% reduction of neuronal cell
count in GC layer in wild type but not TKO. These effects were paralleled with
increases in nitrotyrosine, apoptotic markers including ASK-1 and PARP as well as
inflammatory markers including GFAP and TNF-α. In Müller cultures, NMDA
induced dose-dependent increases in expression of TXNIP, TNF-α and IL-1β.
NMDA caused vascular injury as indicated by early vascular permeability and
significant increases in acellular capillary formation after 8-days in wild type but
not in TKO mice or wild type treated with verapamil. In parallel, GCs were
preserved in TKO- and verapamil-treated groups but not in wild type.
Conclusions: Down-regulation of TXNIP expression using genetic and
pharmacological approaches prevented early glial activation, retinal inflammation,
and RGC death. Furthermore, preventing secondary retinal vascular injury
significantly delayed RGC loss. Therapies that modulate multiple targets can offer
better neuroprotective options for retinal neurodegenerative diseases.
Commercial Relationships: Mona El-Azab, None; Barbara A. Mysona,
None; Mohammed A. Abdelsaid, None; Suraporn Matragoon, None; Azza B.
El-Remessy, None
Support: JDRF Grant 4.2008-149, IDB Postdoc Scholarship
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 1658 Poster Board Number: D889
Presentation Time: 8:30 AM - 10:15 AM
Mechanisms of Bone-Marrow Stem Cell-Mediated Delayed Ischemic
Neuroprotection in Rat Retina<!-- field: -->
Steven Roth1A, John C. Dreixler1A, Jacqueline N. Poston1B, Lai Xue1B, Afzhal R.
Shaikh1A, Kelsey Y. Tupper1A, Irina Balyasnikova1C, Maciej Lesniak1C. AAnesthesia
and Critical Care, BPritzker School of Medicine, CNeurosurgery, 1Univ of Chicago,
Chicago, IL.
Purpose: Delayed treatment after ischemia is often unsatisfactory. However, we
recently demonstrated that “post-conditioning” using a 24 h-delayed, transient
ischemic stimulus was neuroprotective in a rat model of retinal ischemia. We
hypothesized that delayed injection of bone marrow stem cells (BMSCs) after
ischemia could mimick the post-conditioning neuroprotection, and in this study we
characterized the functional and histological outcomes, differentiation and
migration of the BMSCs, and molecular mechanisms of neuroprotection.
Methods: Retinal ischemia was produced in adult Wistar rats by increasing IOP for
55 min. BMSCs were harvested from rat femur and injected into the vitreous 24 h
after the end of ischemia. Recovery was assessed one week after ischemia using
electroretinography (ERG). Scotopic a- and b waves, oscillatory potentials, and
STR were measured. We examined 4 micron thick paraffin-embedded sections at 7
d after ischemia. Immunohistochemistry was performed on 7 micron thick
cryosections. Double-labeling was performed with anti-neurofilament as a neuronal
marker, thy-1 for RGCs, PKCa for bipolar cells, syntaxin for amacrine cells, and
anti-GFAP and anti-vimentin as glial markers; TUNEL indentifed apoptotic cells.
Western blotting quantitated apoptosis and autophagy markers.
Results: BMSCs significantly improved recovery of the b-wave, OP, and P2
amplitudes after ischemia (Figure), but not STR. RGC cell loss after ischemia was
unchanged, but cell density in the inner and outer nuclear layers was preserved.
There was no co-localization with neurofilament or any other neuronal marker, but
some cells double-labeled for GFAP and vimentin. Injection of BMSCs
significantly reduced post-ischemic TUNEL and caspase-3 and -6 and maintained
autophagy vs controls.
Conclusions: Functional protection after ischemia by BMSCs is suggested to be
occurring in inner retinal cells other than RGCs since both STR and RGC numbers
were not altered by BMSCs. Neuronal differentiation is not required for BMSC
mediated neuroprotection, but suppression of apoptosis/maintenance of autophagy
are mechanisms uncovered by this
Commercial Relationships: Steven Roth, None; John C. Dreixler,
None; Jacqueline N. Poston, None; Lai Xue, None; Afzhal R. Shaikh,
None; Kelsey Y. Tupper, None; Irina Balyasnikova, None; Maciej Lesniak,
Support: NIH Grants EY10343 (SR), EY10343-16S2 (SR), CA122930 (ML),
Illinois Society for the Prevention of Blindness and the Dean’s Research Advisory
Committee Center-Style Award (SR, ML).
Program Number: 1659 Poster Board Number: D890
Presentation Time: 8:30 AM - 10:15 AM
Change In The Distribution And Phenotype Of Subretinal Macrophages With
Aging In C57BL/6 Mice
Bogale Aredo, kaiyan Zhang, Rafael L. Ufret-Vincenty. Ophthalmology, UT
Southwestern Medical Center, Dallas, TX.
Purpose: The role of subretinal microglia in maintaining homeostasis and/or
inducing disease is not well understood. For instance, there is significant debate
surrounding the role of macrophages-microglia in age-related macular degeneration
(AMD). In order to better understand subretinal microglia, we decided to explore
the natural history of their distribution and phenotype in wild type, B6-pigmented
Methods: Naïve C57BL/6 (B6) mice of different ages were used for this
experiment. Eyes were dilated and retinal images were taken using a Micron III
rodent fundus camera (Phoenix Research Laboratories). Yellow spots in the central
fundus (5 disc radius from the center of optic disc) were counted. Eyes were
enucleated and fixed in 4% paraformaldehyde. Posterior segment flat mounts
(sclera-choroid-RPE complex) were prepared and were single, double, or triple
stained for ionized calcium binding adaptor molecule 1 (Iba-1), mouse Macrophage
Mannose Receptor (MMR) or mouse FcγIII/II Receptor (CD16/CD32). Microglia
cells (Iba-1+, MMR+, and/or CD16/CD32+) were counted on choroid-RPE flat
mount images for both the central region (within a 5 disc diameter radius from the
optic nerve), and for the total RPE.
Results: The yellow fundus spots seen on B6 mice change in distribution at
different ages. Iba-1+ microglial cells seen in immmunohistochemistry of
flatmounts show a similar distribution pattern. In young mice (1-8 m), the fundus
spots and the corresponding microglial cells on RPE flatmounts are frequently
located at the anterior (far peripheral) retina. As the mice age, the distribution of
cells is more posterior. In addition, an increasing proportion of the microglial cells
also stain for MMR as mice age. Moreover, the microglial cell population becomes
increasingly MMR+CD16/CD32- with advancing age.
Conclusions: As B6 mice age, although there is no significant change in the total
amount of microglial cells in the sub-retinal space, the location of these cells seems
to shift. Furthermore, progressively more cells become MMR+CD16/Cd32-. Further
studies are needed to understand what triggers the changes in subretinal microglial
cell distribution and phenotype, and how that may differ in mice under oxidative
stress or inflammation.
Commercial Relationships: Bogale Aredo, None; kaiyan Zhang, None; Rafael
L. Ufret-Vincenty, None
Support: (1) Unrestricted Research Grant from Research to Prevent Blindness. (2)
Ophthalmology Department Core Grant from NIH-EY020799. (3) Disease
Oriented Clinical Scholars Grant.
Program Number: 1660 Poster Board Number: D891
Presentation Time: 8:30 AM - 10:15 AM
Analysis of Toll-like Receptor 3 and Cellular Signaling Pathways in RPE Cells
Amit K. Patel, Abigail S. Hackam. Ophthalmology, University of Miami, Miami,
Purpose: A leading cause of visual impairment is age-related macular degeneration
(AMD). The overall goal of this project is to examine the activity of toll-like
receptor 3 (TLR3), a mediator of innate immunity, in a cellular model of AMD
injury. Although genetic polymorphisms in TLR3 are associated with AMD, the
precise role of TRL3 in AMD is unknown. Several reports show that TLR3
activation leads to retina pigment epithelium (RPE) cell death, but other studies
indicate that TLR3 has cytoprotective activity. Here, we tested the hypothesis that
aberrant TLR3 activation regulates Wnt and STAT3 cellular survival pathways,
leading to altered RPE cell viability.
Methods: Viability was measured in primary RPE cultures prepared from wildtype mice and the ARPE19 cell line, using Cell Titer Blue assays. TLR3 signaling
was activated by 2-100 µg/ml Poly I:C and TLR3 expression was downregulated
using siRNA. Cell cultures were exposed to oxidative stress using 0.4mM H2O2
and 0.8mM paraquat to simulate AMD-like injury. STAT3 and p65 expression was
identified using IHC. Wnt signaling was induced using the Wnt3a ligand and
quantified with luciferase reporter assays.
Results: Activation of TLR3 resulted in a modest dose-dependent decrease of RPE
viability by up to 20% (n=5, p<0.05). In contrast, TLR3 activation combined with
oxidative stress significantly increased RPE cell viability by 50% (n=5, p<0.05)
compared with oxidative stress conditions alone. This protective effect was
reversed using TLR3 siRNA (n=5). STAT3 signaling was increased by TLR3
activation by about 60% (n=2). Furthermore, although Wnt signaling significantly
increased ARPE19 cell viability when oxidative stress was induced by 0.4mM
H2O2 (n=4, p<0.05) and 0.4mM paraquat (n=9, p<0.05), TLR3 activation
suppressed Wnt signaling by about 50% (n=3, p<0.05).
Conclusions: We have demonstrated that activation of the innate immunity
receptor TLR3 in the presence of AMD-like injury increased the viability of RPE
cells. Furthermore, TLR3 activation regulates the Wnt and STAT3 cell survival
pathways. TLR3 signaling and related downstream pathways could be further
investigated as targets for developing novel therapeutic strategies for AMD.
Commercial Relationships: Amit K. Patel, None; Abigail S. Hackam, None
Support: Karl Kirchgessner Foundation, NIH Center Core Grant P30EY014801,
Research to Prevent Blindness Departmental Support, Research to Prevent
Blindness Special Scholar Award
Program Number: 1661 Poster Board Number: D892
Presentation Time: 8:30 AM - 10:15 AM
Amyloid Beta Enhances Migration Of Endothelial Progenitor Cells Via
Upregulation Of CX3CR1
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Jiying Wang1A, Kyoko Ohno-Matsui1A, Manabu Mochizuki1A, Ikuo Morita1B.
Ophthalmology & Visual Sci, BSection of Cellular Physiological Chemistry,
Tokyo Medical & Dental Univ, Tokyo, Japan.
Purpose: Amyloid β (Aβ) is accumulated within drusen and reportedly critical for
pathogenesis of age-related macular degeneration (AMD). We previously reported
endothelial progenitor cells (EPC) contributed to the development of CNV
(ARVO2011). In the present study we investigated the molecular mechanism of the
recruitment of EPC to subretinal space secondary to Aβ accumulation.
Methods: EPC that isolated from human cord blood or peripheral blood of wild
type and Cx3CR1-/- mice were stimulated by Aβ. CX3CR1 mRNA expression and
protein production were analyzed by real-time PCR and western blot. Migratory
activity was measured by Boyden chamber assay.
Results: Aβ treatment induced a significant increase of CX3CR1 expression and
protein production. In Boyden chamber assay, fractalkine caused a remarkable
migration of human EPC and EPC isolated from wild type mice, but failed to
induce migration of EPC from CX3CR1-/- mice. Aβ enhanced fractalkine-induced
human and wild type EPC migration, but did not affect EPC from CX3CR1-/mice.
Conclusions: These results suggest the possibility that Aβ causes the migration of
EPC to the area of drusen specially via up-regulation of CX3CR1. This
phenomenon might be an important process for the CNV development in AMD.
Commercial Relationships: Jiying Wang, None; Kyoko Ohno-Matsui,
None; Manabu Mochizuki, None; Ikuo Morita, None
Support: 22390322 and 21659399
Program Number: 1662 Poster Board Number: D893
Presentation Time: 8:30 AM - 10:15 AM
Stressed-Induced Neuroprotection through the LIF/GP130/STAT3 Pathway is
Mediated by TLR2 Signaling
Jiangang Wang1, Ratanmani Joshi2, John D. Ash1. 1Ophthalmology, Florida
University, Gainesville, FL; 2Ophthalmology, University of Oklahoma Health
Sciences Center, Oklahoma City, OK.
Purpose: We have previously shown that the GP130 receptor and STAT3 are part
of an inducible protective pathway that is essential for delaying inherited retinal
degeneration. This pathway is activated by upregulation of leukemia inhibitory
factor (LIF) in Muller cells in response to cellular stress caused by a genetic
mutation or oxidative injury in neurons. The purpose of this study is to investigate
whether toll like receptor 2 (TLR2) is part of the signaling cascade by which
stressed neurons signal Muller cells to induce LIF expression.
Methods: Adult albino mice were exposed to damaging light (4000 lux) for 5
hours following 2 days after TLR2 agonist Pam3CYS injection. GP130 was
specifically deleted from retina by CHX10-cre driven recombination of a floxed
gp130 allele. Electroretinography was measured to identify the retinal function and
retinal histology was used to quantify the photoreceptor loss. For gene expression
profile after injection, retinas were collected before and 2 days after injection. Total
RNA was extracted using Trizol (Invitrogen) and Real-time PCR was used to
confirm expression changes.
Results: Activation of TLR2 by its agonist Pam3CYS strongly rescued
photoreceptor function and cells from light induced cell death. Expression analyses
show that a broad spectrum of cytokines including IL-6 family cytokines were
remarkably upregulated by Pam3CYS. In the absence of TLR2, LIF expression was
not induced by light stress, and retinas were more sensitive to light damage. The
neuroprotective effects of Pam3CYS were dependent on gp130 activation.
Conclusions: The results suggest that activation of TLR2 on Muller glial cells is
necessary for oxidative stress induced expression of LIF. The study suggests a
model where stressed neurons release ligands of TLR2, which when activated in
Muller cells leads to increased LIF expression. Increased LIF then activates gp130
on neurons to induce protection.
Commercial Relationships: Jiangang Wang, None; Ratanmani Joshi,
None; John D. Ash, None
Support: NIH P20-RR017703; P30-EY012190; R01-EY16459; Foundation
Fighting Blindness; and Research to Prevent Blindness, Inc.
Program Number: 1663 Poster Board Number: D894
Presentation Time: 8:30 AM - 10:15 AM
Localization of Inflammatory Cytokines in Human Eyes with AMD
Sijia Cao1, Jiangyuan Gao1, Jing Z. Cui1, Kailun Jiang2, Eleanor To1, Dean
Zhang1, Valerie A. White3, Joanne A. Matsubara1. 1Ophthalmology and Visual
Sciences, University of British Columbia, Vancouver, BC, Canada; 2Faculty of
Medicine, University of Manitoba, Winnipeg, MB, Canada; 3Pathology, Vancouver
General Hospital, Vancouver, BC, Canada.
Purpose: Earlier in vitro studies identified several inflammatory modulators
upregulated after stimulation of RPE cells with either amyloid beta (Aβ) or
advanced glycation end products, two known components of drusen. Our earlier
findings suggest that drusen may induce inflammatory cytokine overexpression in
RPE, and thus promote AMD disease progression. The purpose of this study is to
examine the cellular localization of inflammatory cytokines in the outer retina of
the human AMD eye.
Methods: Six AMD (4 exudative and 2 atrophic) and 30 normal post-mortem eyes
were consented for research, embedded in paraffin and processed for
immunohistochemistry using antibodies against five proteins of interest from our
earlier studies: amyloid precursor protein (APP), interleukin 1beta (IL-1β),
CXCL11, interleukin 1 receptor antagonist (IL-1ra), and interleukin 10 (IL-10).
Control sections were run using a non-immune IgG at the same concentration as the
primary antibody.
Results: APP was strongly expressed in the cytoplasm of RPE cells in close
proximity to drusen sites in the AMD eye (e.g. distance of <10 RPE cell diameters).
IL-1β, a proinflammatory cytokine, was also expressed in cytoplasm of RPE cells
in close proximity to drusen, as well as in the drusen sites themselves in the AMD
eye. CXCL11 was expressed in drusen, choroid and Bruch’s membrane of the
exudative, but not the atrophic, AMD eye. IL-10, an anti-inflammatory cytokine,
was strongly expressed in drusen and choroid of the AMD eye. IL-1ra, another
inhibitor of inflammation, was expressed in RPE and choroid in AMD eyes. In
normal eyes, IL-1ra was strongly expressed in eyes with drusen (vs eyes without
drusen) and in the peripheral (vs macular) retina.
Conclusions: Our study demonstrated a unique pattern of immunoreactivity
associated with the human AMD eye that is consistent with our earlier in vitro
stimulation studies on RPE cells. The presence of APP in RPE cells in close
proximity to drusen, may suggest that Aβ, an APP cleavage product found in
drusen, might originate from RPE dysfunction. Interestingly, both proinflammatory
and inhibitors of inflammation appear to be highly expressed in RPE cells near
drusen sites. While often considered a consequence of the disease process, our data
suggest that drusen may promote AMD progression by proinflammatory
Commercial Relationships: Sijia Cao, None; Jiangyuan Gao, None; Jing Z.
Cui, None; Kailun Jiang, None; Eleanor To, None; Dean Zhang, None; Valerie
A. White, None; Joanne A. Matsubara, None
Support: CIHR Grant (MOP-97806)
Program Number: 1664 Poster Board Number: D895
Presentation Time: 8:30 AM - 10:15 AM
Characterisation of the Effects of Ageing on the Cfh-/- Mouse Retina
Jennifer A. Williams, E. Rebecca Longbottom, John Greenwood, Stephen E. Moss.
Cell Biology, Institute of Ophthalmology, UCL, London, United Kingdom.
Purpose: Dysregulation of the complement system in the eye is a feature of agerelated macular degeneration (AMD), the leading cause of visual impairment in the
UK. Several mutations in complement genes have been shown to increase an
individual’s susceptibility to AMD. The Y402H single nucleotide polymorphism in
complement factor H (CFH) is present in over 50% of AMD patients. CFH is a
secreted regulator of the alternative complement pathway and therefore key to
controlling the inflammatory response. Previous work in our group has shown that
CFH is required for normal visual function in 2 year mice. In this study we
characterise the retinas of Cfh-/- mice at 7-8 weeks and 1 year to gain insight into
the effects of Cfh deletion on retinal ageing.
Methods: 7-8 week and 1 year old Cfh-/- mouse retinas and age-matched wild-type
controls were processed for morphological and ultrastructural analysis. Fixed eyes
were sectioned or whole-mounted for immunohistochemistry. Electroretinograms
were performed on 1 year old Cfh-/- mice and age-matched wild-type controls.
Results: Morphological analysis revealed a reduction in photoreceptor density in
wild-type mice with age and a further reduction with age in Cfh-/- mice.
Immunohistochemical analysis showed that loss of CFH led to early signs of retinal
stress and re-distribution of complement regulators at 1 year. Analysis of visual
function by ERG revealed a significant increase in time to peak of the scotopic awave in 1 year old Cfh-/- mice.
Conclusions: The loss of CFH in mice does not have a significant impact on the
retina at 7-8 weeks of age. However, by 1 year the retina displays early signs of
stress and accelerated features of ageing. We show that the retina is able to
compensate for the deficiency of CFH by enhancing other complement regulators.
Overall, this study reveals the importance of CFH in maintaining retinal health and
visual function with age.
Commercial Relationships: Jennifer A. Williams, None; E. Rebecca
Longbottom, None; John Greenwood, None; Stephen E. Moss, None
Support: Medical Research Council and Wellcome Trust
Program Number: 1665 Poster Board Number: D896
Presentation Time: 8:30 AM - 10:15 AM
Novel Role For The Innate Immune Receptor Toll-like Receptor 4 (TLR4) In
The Regulation Of The Wnt Signaling Pathway And Photoreceptor Apoptosis
Abigail S. Hackam, Hyun Yi. Bascom Palmer Eye Institute, University of Miami,
Miami, FL.
Purpose: Innate immunity is proposed to play a role in the pathogenesis of agerelated macular degeneration (AMD), but its precise contribution is unknown.
Recent evidence has implicated innate immunity in reduced neuronal survival
elsewhere in the CNS during various disease conditions. Here, we examined the
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
role of the innate immunity receptor TLR4 in a cellular model of AMD.
Methods: Primary photoreceptor-Muller glia cocultures were prepared from
C57Bl/6 mice. Photoreceptor viability was measured by Cell Titre Blue and
caspase assays, in the presence of oxidative stress (0.4 mM H2O2) to simulate
AMD-like injury. Wnt signaling was measured by luciferase reporter assays and
Western blotting for phospho-LRP6.
Results: TLR4 activation by lipopolysaccharide (LPS) (0.05-50 ug/ml)
significantly reduced photoreceptor survival in primary Muller glia-photoreceptor
cocultures exposed to oxidative stress, whereas blocking endogenous TLR4
activation protected against oxidative-stress induced cell death. With respect to
mechanism, TLR4 suppressed Wnt signaling by 42% (p<0.05, n=5), decreased
phosphorylation and activation of the Wnt receptor LRP6 by 50% (p<0.05, n=5)
and blocked the protective effect of the Wnt3a ligand. Paradoxically, TLR4
activation prior to oxidative injury protected photoreceptors by five-fold (p<0.05,
n=4) in a phenomenon known as preconditioning. Furthermore, expression of TNFalpha and its receptors TNFR1 and TNFR2 decreased during preconditioning, and
preconditioning was mimicked by TNF-alpha antagonists, but was independent of
Wnt signaling.
Conclusions: TLR4 may regulate photoreceptor death at various stages of retinal
injury in AMD by regulating the Wnt and TNF-alpha pathways. These data indicate
a novel mechanism of neurotoxicity in the retina, and suggest that TLR4 signaling
could be further investigated as a target for developing novel therapeutic strategies
for AMD.
Commercial Relationships: Abigail S. Hackam, None; Hyun Yi, None
Support: NIH, KKF, RPB
Program Number: 1666 Poster Board Number: D897
Presentation Time: 8:30 AM - 10:15 AM
Signaling Molecules Regulating Microglia/macrophage Recruitment To CNV
Hu Huang, Rachel Parlier, Jikui Shen, Gerard A. Lutty, Vinores A. Vinores.
Ophthalmology, Johns Hopkins Wilmer Eye Inst, Baltimore, MD.
Purpose: Pathological conditions in neovascular age-related macular degeneration
(nvAMD) cause the migration of microglia/macrophage to choroidal
neovascularization (CNV), which in turn exacerbates the pathogenesis and
progression of nvAMD. The mechanisms regulating the recruitment process are not
completely understood. This study is focused on the cytokine and chemokine
signaling molecules that regulate microglia/macrophage recruitment to laserinduced CNV, an experimental model of nvAMD.
Methods: CNV was generated in mice by laser injury. The CNV lesions were
isolated by laser capture micodissection (LCM) and RNAs were isolated from the
LCM-isolated CNV and the surrounding tissues at 3, 7, and 14 days after lasering.
The RT2 Profiler PCR Arrays were used to examine the expression profile of
cytokine, chemokines and receptors. RT-PCR, real-time PCR and
immunofluorescence (IF) staining were used to determine gene and protein
expression. Neutralizing antibodies specific for various target molecules, such as
MF1 (VEGFR1) and DC101 (VEGFR2), were used to block their respective
signaling activity. A panel of antibodies to microglia/macrophage: Iba1, CD45, and
Mac-1 (CD11b) were used to confirm the identity of microglia/macrophage in the
retinal pigment epithelium (RPE)/choroidal flat mounts or retinal cross sections.
Quantification of digitized images was accomplished with ImageJ.
Results: With the LCM-isolated CNV, mRNA of VEGFR1 and its two ligands,
PlGF and VEGF-B, were detected in 3-, 7-, and 14-day CNV lesions. VEGFR2 was
not detected at 3-day, but was at 7- and 14-day CNV. IF staining detected protein
expression of placental growth factor (PlGF) and VEGF at the 3- and 7-day CNV.
Further gene expression profiling demonstrated that 9 out of 84 key chemokines,
cytokines and receptors represented in the array were expressed in the 3-day CNV;
they were Abcf1, Bcl6, CxCR5, Ccr1, Il1r2, Itgam, Mif, Spp1, Tgfb1. At 3-day
after laser, systemically-administrated MF1 but not DC101 significantly inhibited
recruitment of CD45 (+) and Mac-1(+) cells to CNV. At 14-day CNV,
microglia/macrophage expressed both VEGFR1 and VEGFR2 and both MF1 and
DC101 antibodies systemically-administrated markedly inhibited recruitment of
CD45 (+) and Mac-1(+) cells to CNV.
Conclusions: VEGFR1 and VEGFR2 can regulate CNV by controlling the
recruitment of microglia/macrophage independently and synergistically, depending
on the stages: VEGFR1 plays a dominant role at the 3-day CNV, whereas both
VEGF receptors play pivotal roles at 14-day CNV with the enhanced efficacy with
their combination. The microglia/macrophage populations recruited to CNV may
interact with the other cell types within CNV, such as RPE, affecting the
pathogenesis of CNV.
Commercial Relationships: Hu Huang, None; Rachel Parlier, None; Jikui
Shen, None; Gerard A. Lutty, None; Vinores A. Vinores, None
Support: EY017164; a stipend from Lilly/ImClone
Program Number: 1667 Poster Board Number: D898
Presentation Time: 8:30 AM - 10:15 AM
IL-17A and IL-17RC Expression in the Macula of the Human Eye with AgeRelated Macular Degeneration
Stanley Park1,2, De Fen Shen1, Lai Wei3, Yujuan Wang1, Charles Eberhart4, Robert
Nussenblatt3, Chi Chao Chan1. 1Immunopathology Section, Laboratory of
Immunology, National Eye Institute, National Institutes of Health, Bethesda, MD;
Howard Hughes Medical Institute, Chevy Chase, MD; 3Clinical Immunology
Section, Laboratory of Immunology, National Eye Institute, National Institutes of
Health, Bethesda, MD; 4Department of Pathology, Ophthalmology and Oncology,
Johns Hopkins University School of Medicine, Baltimore, MD.
Purpose: Age related macular degeneration (AMD) is the most common cause of
irreversible central blindness amongst the elderly. It is thought that various genetic
and environmental factors drive the dysregulation of macrophages, cytokines,
chemokines and complement factors. Though the ensuing inflammation and
oxidative stress have been well described, the precise pathogenesis remains
unknown. Th17 cytokines are implicated in several inflammatory diseases like
multiple sclerosis and uveitis. In AMD patients, complement factor C5b is capable
of inducing Th17 cytokines and both their levels are significantly elevated in the
peripheral blood. Preliminary data have shown that IL-17 is also locally elevated in
human AMD eyes and in those of a mouse model for AMD, Ccl2-/-/Cx3cr1-/- on
rd8 background. Moreover, there is an IL-17RC linked expression of CXCR4 in
peripheral CD14+ monocytes and there are elevated levels of CXCL12/SDF-1
found in the normal eye. This setup may predispose to a pathologic homing of these
monocytes to the eye and incite an inflammatory process. In addition, people with a
methylated IL-17RC promoter seem to be protected from developing AMD. Here
we further elucidate the role of IL-17 in the pathogenesis of AMD by investigating
IL-17A, IL-17F and IL-17RC expression in AMD and normal maculae.
Methods: The macular retina and choroid of 33 paraffin-embedded, archived AMD
eyes and 7 age-matched normal eyes were microdissected and their RNA was
isolated (Paradise reagent system) for cDNA synthesis (MX3000P QPCR system).
IL-17A, IL-17F and IL-17RC mRNAs were quantitated by real time PCR
monitored with SYBR green fluorescence.
Results: 40% of the 40 total microdissected samples (13 AMD and 3 normal eyes)
yielded valid data. Significantly elevated relative transcript levels of IL-17A
(AMD, 70.6±27.4; Normal, 4.59±3.4; p<0.034) and IL-17RC (AMD, 3260 ± 844;
Normal, 50.1 ± 28.9; p<0.002) were found in AMD maculae compared to normal
maculae. IL-17F mRNA was undetectable in all eyes. Of the 13 AMD samples, 3
of them had geographic atrophy while the remaining 10 had the exudative
neovascular form. There was no significant difference in the relative expression of
transcript levels of IL-17A and IL-17RC when geographic AMD was compared to
exudative AMD.
Conclusions: Significantly higher relative expression levels of IL-17RC and IL17A but not IL-17F are in AMD macula compared to the controls. These findings
support the aforementioned hypothesis of a monocyte driven inflammatory
pathogenesis. It also suggests that IL-17A may primarily induce the inflammatory
process through IL-17RC in the maculae.
Commercial Relationships: Stanley Park, None; De Fen Shen, None; Lai Wei,
None; Yujuan Wang, None; Charles Eberhart, None; Robert Nussenblatt,
None; Chi Chao Chan, None
Support: The NEI Intramural research program
Program Number: 1668 Poster Board Number: D899
Presentation Time: 8:30 AM - 10:15 AM
Inactivation Of Hif1a In Myeloid Cells Reduces Retinal And Choroidal
Clemens A. Lange1, Alessandro Fatin2, Laura Denti2, Alexander J. Smith1, Robin R.
Ali1, Christiana Ruhrberg2, James W. Bainbridge1. 1Genetics, Institute of
Ophthalmology, UCL, NIHR Biomedical Research Centre for Ophthalmology,
London, United Kingdom; 2Cell Biology, Institute of Ophthalmology, London,
United Kingdom.
Purpose: Bone marrow-derived myeloid cells have been implicated in the
development of retinal and choroidal neovascularisation. Hypoxia-inducible
transcription factors (HIF) are activated during inflammation and hypoxia and are
key drivers for the expression of the vascular endothelial growth factor (VEGF).
The aim of this study was to investigate the role of Hif1a and Vegf in myeloid cells
in the development of retinal and choroidal neovascularisation.
Methods: Conditional inactivation of Hif1a or Vegf in myeloid cells was achieved
by crossing Hif1a or Vegf conditional null (floxed) mice with transgenic mice
expressing cre-recombinase under the control of the myeloid-specific promoter
lysozyme M. Hif1a lysmCre and Vegfa lysmCre mutants and littermate controls
lacking lysmCre were studied in pathological retinal and choroidal angiogenesis in
the oxygen-induced retinopathy (OIR) and laser-induced choroidal
neovascularisation models.
Results: Myeloid cells accumulate in areas of retinal and choroidal
neovascularisation in the OIR and CNV mouse models. Immunofluorescence
labeling of blood vessels showed that inactivation of Hif1a or Vegfa in the myeloid
cell lineage reduces the area of neovascularisation in the OIR model by 51±18%
and 52±17%, respectively (p<0.0001). Fluorescein angiography 2 weeks after laser
injury demonstrated that myeloid-specific inactivation of Hif1a or Vegfa reduces
the area of choroidal neovascularisation by 59± 4% and 65±10%, respectively
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Conclusions: Both Hif1a activation and Vegf expression in myeloid cells
contribute substantially to the development of retinal and choroidal
neovascularisation. Targeting of Hif1a activation in myeloid cells might be an
effective strategy for therapeutic intervention.
Commercial Relationships: Clemens A. Lange, None; Alessandro Fatin,
None; Laura Denti, None; Alexander J. Smith, None; Robin R. Ali,
None; Christiana Ruhrberg, None; James W. Bainbridge, None
Support: None
Program Number: 1669 Poster Board Number: D900
Presentation Time: 8:30 AM - 10:15 AM
Increased Severity Of Choroidal Neovascularisation With Age Is Associated
With Enhanced CCL2-mediated Recruitment Of Neutrophils
Scott J. Robbie, Ulrich F. Luhmann, Barker E. Susie, Yanai Duran, Alexander J.
Smith, Robin R. Ali, James W. Bainbridge. Genetics, UCL Institute of
Ophthalmology, London, United Kingdom.
Purpose: Laser-induced choroidal neovascularisation (CNV) is more severe in
older mice. The mechanism for this is unknown. The purpose of this work was to
determine whether upregulation of the chemokine CCL2 in the choroid with age
results in the enhanced recruitment of pro-angiogenic inflammatory cells that
mediate CNV severity.
Methods: Using 6-colour flow cytometry, we established the proportions of
resident innate immune cells in the retinae and choroids of wild-type (WT) and
CCL2-/- mice and the changes in these populations with increasing age. The effects
of these aging changes on CNV lesion size in WT and CCL2-/- mice were then
determined using the laser model. We conducted serial fluorescein angiography to
determine differences in the rate of development of CNV between young and old
WT animals. We then induced CNV in <4 month, 12 month and 24 month old WT
and CCL2-/- mice and analysed innate immune cells in retinal and choroidal tissue
by flow cytometry at 3 days post-laser, normalising to age-matched non-injured
Results: We observed increases over time in CD11b+ Gr-1Low/Mid F4/80High
CD11cMid cell populations (likely activated macrophages) in the uninjured retinae
and choroids of both WT and CCL2-/- mice from an early age. Increases in the
choroid appear to precede those in the retina. CNV lesion size increased with age in
both WT and CCL2-/- animals but was consistently reduced in the latter. The effect
of aging on CNV severity appeared attenuated in CCL2-/- animals. Whilst CNV
lesion size stabilises at 3 days post-induction in young animals, lesion growth
continues up to 7d in old WT mice. Analysis of tissue obtained at 3d post-CNV
reveals more neutrophils in the choroids of old WT animals and this effect is
attenuated in CCL2-/- animals. Induction of CNV results in the emigration of
macrophages from the retinae and choroids of both genotypes.
Conclusions: We found no evidence that increased CNV lesion size is associated
with the proportions of macrophages in uninjured retina and choroid. However, the
enhanced recruitment of neutrophils, partly mediated by CCL2, may account for
the increase in CNV lesion size observed with age.
Commercial Relationships: Scott J. Robbie, None; Ulrich F. Luhmann,
None; Barker E. Susie, None; Yanai Duran, None; Alexander J. Smith,
None; Robin R. Ali, None; James W. Bainbridge, None
Support: NIHR
Program Number: 1670 Poster Board Number: D901
Presentation Time: 8:30 AM - 10:15 AM
Novel Ccr3 Antagonists Are Effective Mono- And Combination Inhibitors Of
Cnv Growth And Vascular Permeability
Norihiro Nagai1, Kanako Izumi-Nagai1, Scott J. Robbie2A, James W. Bainbridge2,
Peter S. Adamson3, Yin-shan Ng1, David T. Shima2B. 1ORBIT, University College
London, London, United Kingdom; AGenetics, BOcular Biology and Therapeutics,
UCL Institute of Ophthalmology, London, United Kingdom; 3Ophthalmology
R&D, GSK, London, United Kingdom.
Purpose: We explored the functional role of CCR3 in laser-induced and
spontaneous choroidal neovascularisation.
Methods: A novel model of spontaneous CNV and laser-induced CNV models
were used for assessing the novel CCR3 antagonists. CCR3 ligands, receptors, and
leukocytes were analysed by immunostaining. Pharmacological blockade of mouse
CCR3 was performed by using the CCR3 antagonist GW766994, and in the
primate laser-induced CNV model, a second antagonist, GW782415 was used.
Systemic and topical routes of administration were explored. CNV number, area
and permeability were evaluated by fluorescein angiography and immunostaining
in the mouse, and by fluorescein angiography in the primate.
Results: In rodent studies, CNV was associated with increased expression of
CCR3, and both the number and size of spontaneous CNV were significantly
reduced in a dose-dependent manner by a systemic CCR3 antagonist. Similarly,
oral dosing with CCR3 antagonist dose-dependently reduced laser-induced CNV
development. Furthermore, topical administration of the CCR3 antagonist was also
effective in reducing spontaneous CNV area and number, with efficacy comparable
to that of the systemic CCR3 antagonist. Combination treatment with systemic anti-
VEGFR2 antibody and CCR3 antagonist resulted in an additive effect in reducing
CNV number, area, and permeability. In the primate, oral dosing of the CCR3
antagonist GW782415 resulted in a significant suppression of grade IV CNV
lesions 16, 24 and 30 days following laser photocoagulation.
Conclusions: We have demonstrated a direct functional role of CCR3 in the
development and hyper-permeability of CNV, and also the additive therapeutic
effect of a CCR3 antagonist in combination with VEGFR2 blockade. Our data
suggest that CCR3 signalling may be an attractive therapeutic target for
neovascular AMD and is distinct from the VEGF signalling pathway.
Commercial Relationships: Norihiro Nagai, None; Kanako Izumi-Nagai,
None; Scott J. Robbie, None; James W. Bainbridge, None; Peter S. Adamson,
GSK (E); Yin-shan Ng, GSK (F); David T. Shima, GSK (F)
Support: None
Program Number: 1671 Poster Board Number: D902
Presentation Time: 8:30 AM - 10:15 AM
Secretion Of Vascular Growth Factors/inflammatory Cytokines By Rpe Is
Inhibited By Ranibizumab
Murugeswari Ponnalagu1, Alan W. Stitt2, Kim Ramasamy3, Muthukkaruppan
Veerappan1. 1Immunology and Cell Biology, Aravind Medical Research
Foundation, Madurai, India; 2Centre for Vision & Vascular Science, Queens
University Belfast, Belfast, United Kingdom; 3Vitreous and Retina Service,
Aravind Eye Hospital, Madurai, India.
Purpose: To understand the effects of VEGF and IL-6 neutralization on the
secretion of cytokines and angiogenic growth factors by ARPE-19 cells in vitro
Methods: ARPE-19 cells were exposed to different concentrations of anti-VEGF
antibody (ranibizumab; RZB) (0.125 mg/ml and 0.25 mg/ml) or anti-IL6 antibody
(0.05 µg/ml, 0.1 µg/ml and 0.2 µg/ml) for 24 hrs. The conditioned medium was
analyszd for 12 cytokines (IL-1α,IL1-β,IL-2, IL-4,IL-6,IL-8,IL-10,IFN-γ,TNFα,VEGF,MCP-1 and EGF) by Cytokine Biochip Array. mRNA expression was also
studied using SYBR green real-time PCR analysis.
Results: Among the cytokines measured, significant levels of VEGF
(687.2±9.3pg/ml), IL-6 (25.2±0.3pg/ml) IL-2 (5.1±1.2pg/ml), IFN-γ
(22.4±4.7pg/ml), MCP-1(>840pg/ml) and IL-8(253.6±5.6pg/ml) were observed in
24 hrs culture supernatant. A significant reduction of VEGF (P<0.001) and also IL6 and IL-2 (P<0.001) was observed on treatment with RZB. After 24hrs treatment,
cells were cultured without RZB for 48 hrs and still VEGF was not detected in the
conditioned medium. Moreover, neutralizing antibody to IL-6 reduced the secreted
levels of VEGF (P<0.001) in the cell supernatant. However, subsequent culturing
without anti-IL6 antibody did not affect the secretion of VEGF by ARPE 19 cells.
These antibodies did not influence the secretion of other cytokines (IFN-γ and
MCP-1). No remarkable changes in the mRNA expression of VEGF and IL-6 were
found when treated with antibodies.
Conclusions: Anti-VEGF antibody reduced the secretion of IL-6 in addition to
VEGF. Neutralizing IL-6 antibody also reduced VEGF expression by ARPE-19
cells. Further studies are required to understand the molecular mechanism between
cytokines and angiogenic growth factor regulation in RPE.
Commercial Relationships: Murugeswari Ponnalagu, None; Alan W. Stitt,
None; Kim Ramasamy, None; Muthukkaruppan Veerappan, None
Support: Aravind Medical Research Foundation
Program Number: 1672 Poster Board Number: D903
Presentation Time: 8:30 AM - 10:15 AM
Deficiency of CXCR3 Prevents Inflammation and Neuronal Damage in Retinal
Ischemic Injury
Wenbo Zhang1A, Hua Liu2A, Zhimin Xu2A, Harumasa Yokota2A, Jun Wang1A,
Subhadra P. Narayanan2A, Modesto A. Rojas2A, Massoud Motamedi1A,1B, Robert W.
Caldwell2B, Ruth B. Caldwell2A,3. AOphthalmology and Visual Sciences, BCenter for
Biomedical Engineering, 1University of Texas Medical Branch, Galveston, TX;
Vascular Biology Center, BPharmacology and Toxicology, 2Georgia Health
Sciences University, Augusta, GA; 3VA Medical Center, Augusta, GA.
Purpose: Retinal neuronal death is a common pathological feature of many vision
threatening diseases, such as diabetic retinopathy, retinal vascular occlusion and
glaucoma. Recent studies indicate that inflammation could influence crucial steps
of tissue injury. The goal of this study is to determine the role of the chemokine
receptor CXCR3, a critical mediator of leukocyte recruitment and activation, in the
retinal neuronal injury.
Methods: Retinal neuronal injury was studied with a mouse model of retinal
ischemia-reperfusion (IR). IR injury was generated in C57/BL6 wild type (WT)
and CXCR3-deficient mice by increasing the intraocular pressure to 110 mm Hg
for 40 minutes followed by reperfusion. Bone marrow transplants were performed
by administration of bone marrow cells of EGFP mice to lethally irradiated WT
Results: CXCL10 is a ligand for CXCR3. Its mRNA level was markedly elevated
following IR with a peak increase (175.5±23.1 fold) at 6 hours after reperfusion.
Immunoreactivity for CXCL10 protein was also increased by IR and was evident in
retinal neurons including ganglion cells. Immunolocalization studies using IbaI, a
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
microglia/macrophage marker, revealed a prominent increase in these cells within
the inner retina at 24 hours after reperfusion. Bone marrow transplant experiments
revealed that IbaI positive cells included monocytes from circulation and retinal
local microglia. CD11b, a marker for monocyte/microglia activation, and levels of
the inflammatory molecules interleukin-1 beta and E-selectin, were also
significantly increased following IR. The above changes were associated with
increases in peroxynitrite formation, retinal cell apoptosis and loss of neurons
within the ganglion cell layer. These retinal alternations were significantly
attenuated in CXCR3-deficient mice.
Conclusions: These results indicate that CXCL10/CXCR3 pathway is upregulated
and is critically involved in IR-induced inflammatory reactions, oxidative stress
and neuronal damage.
Commercial Relationships: Wenbo Zhang, None; Hua Liu, None; Zhimin Xu,
None; Harumasa Yokota, None; Jun Wang, None; Subhadra P. Narayanan,
None; Modesto A. Rojas, None; Massoud Motamedi, None; Robert W.
Caldwell, None; Ruth B. Caldwell, None
Support: AHA11SDG4960005 and JDRF 10-2009-575 (W.Z.); NIH Grants
EY11766 (R.B.C. and R.W.C.), EY04618 (R.B.C.), HL70215 (R.W.C.), VA Merit
Award (R.B.C.)
Program Number: 1673 Poster Board Number: D904
Presentation Time: 8:30 AM - 10:15 AM
Effects Of Lipofuscin Photoreactivity On RPE Lysosomal Membrane Stability
And Activation Of The NALP3 Inflammasome
Carolina Brandstetter1A, Jürgen Kopitz2, Eicke Latz1B, Frank G. Holz1A, Tim U.
Krohne1A. AOphthalmology, BInstitute of Innate Immunity, 1University of Bonn,
Bonn, Germany; 2Institute of Pathology, University of Heidelberg, Heidelberg,
Purpose: Lipofuscin accumulation in the retinal pigment epithelium (RPE)
secondary to impaired lysosomal function represents a hallmark of age-related
macular degeneration (AMD). Both lipofuscin photoreactivity and local
inflammation have been implicated in the progressive RPE dysfunction and
degeneration in AMD. This study employs an in vitro model of lipid peroxidationmediated lipofuscinogenesis to investigate the effects of lipofuscin photoreactivity
on RPE lysosomal membrane stability and activation of the NALP3 inflammasome.
Methods: Isolated porcine photoreceptor outer segments (POS) were modified
with the lipid peroxidation products 4-hydroxynonenal (HNE) or malondialdehyde
(MDA). Confluent monolayers of the human RPE-derived cell line ARPE-19 were
incubated with modified POS for 7 days and subsequently exposed to blue light
(wavelength 455-460 nm; irradiance 0.8 mW/cm2). Cytotoxicity was assessed by
LDH release and DNA fragmentation assays. Lysosomal membrane
permeabilization was analyzed by acridine orange staining and activity of
lysosomal marker enzyme N-acetyl-glucosaminidase (NAG) in isolated cytosolic
fractions. Following priming of cells with IL-1α, NALP3 inflammasome activation
was investigated by release of IL-1β.
Results: Phagocytosis of HNE- or MDA-modified POS induced pronounced
cellular lipofuscinogenesis. Subsequent blue light irradiation for 9 hours resulted in
a reduction of cell viability to 17%. In contrast, control cells incubated with
unmodified POS were unaffected by light treatment (viability 97%). Lipofuscinassociated cytotoxicity was mediated by apoptosis. Lysosomal membrane
permeabilization occurred after blue light irradiation in a dose-dependent manner
and was associated with activation of the NALP3 inflammasome as evident from
expression and release of mature IL-1β. Inhibition of caspase activity using ZVAD-FMK suppressed IL-1β release.
Conclusions: Lipofuscinogenesis induced by lipid peroxidation-related protein
modifications renders RPE cells susceptible to light-induced lysosomal
destabilization and apoptotic cell death. Lipofuscin photoreactivity-mediated
lysosomal membrane permeabilization is associated with activation of the NALP3
inflammasome and release of IL-1β. Via this mechanism, lipofuscin accumulation
in the RPE may contribute to local inflammation in AMD.
Commercial Relationships: Carolina Brandstetter, None; Jürgen Kopitz,
None; Eicke Latz, None; Frank G. Holz, None; Tim U. Krohne, None
Support: Dr. Eberhard and Hilde Rüdiger Foundation, University of Bonn
BONFOR Program
249 Diabetic Changes in Retinal Vascular Cell Biology - Minisymposium
Monday, May 7, 2012, 1:45 PM - 3:30 PM
Floridian BCD Symposium
Program #/Board # Range: 1704-1708
Organizing Section: Retinal Cell Biology
Program Number: 1704
Presentation Time: 1:50 PM - 2:10 PM
Retinal Ischemia in Diabetic Retinopathy
Elia J. Duh. Ophthalmology, Johns Hopkins/Wilmer Eye Institute, Baltimore, MD.
Commercial Relationships: Elia J. Duh, None
Support: None
Program Number: 1705
Presentation Time: 2:10 PM - 2:30 PM
Targeting the Arginase Pathway to Limit Vascular Dysfunction/Injury in
Diabetic Retinopathy
Ruth B. Caldwell. Vascular Biology Center, Georgia Health Sciences University,
Augusta, GA.
Commercial Relationships: Ruth B. Caldwell, None
Support: VA Merit Review, NIH EY11766, NIH EY04618
Program Number: 1706
Presentation Time: 2:30 PM - 2:50 PM
Tight Junctions In Vascular Permeability And Growth Control
David A. Antonetti. Ophthalmology and Visual Sciences, University of Michigan,
Ann Arbor, MI.
Abstract.Abstract1:Well-developed tight junctions contribute to the formation of
the blood-retinal barrier and control of vascular permeability. Diabetic retinopathy
induces alterations in both retinal vascular permeability and vascular angiogenesis
through production of vascular endothelial growth factor (VEGF). We
hypothesized that post-translational modifications to the tight junction protein
occludin contributes to control of vascular permeability in response to VEGF.
Through the use of mass spectrometry analysis, we have identified novel occludin
phosphorylation sites and have developed phosphospecific antibodies. Mutational
analysis of these sites demonstrates the role of occludin phosphorylation in barrier
regulation and response to VEGF and identifies Ser490 as a downstream target of
protein kinase C beta. In addition, occludin phosphorylation contributes to growth
control with increased Ser490 phosphorylation promoting both endothelial
permeability as well as cell proliferation and tube formation. Phosphorylated
occludin demonstrates novel centrosomal localization that may contribute to
endothelial cell proliferation. These data provide new understanding regarding the
role of occludin in both regulation of tight junction strand breaks and barrier
properties as well as a novel centrosomal localization of occludin contributing to
mitotic entry and growth control. Targeting occludin phosphorylation may provide
novel therapeutic approaches to control both vascular permeability and
Commercial Relationships: David A. Antonetti, Akebia Therapeutics (C), Use
of aPKC to treat vascular permeability (P)
Support: NIH Grant EY012021; EY019392; Juvenile Diabetes Research
Foundation; Research to Prevent Blindness
Program Number: 1707
Presentation Time: 2:50 PM - 3:10 PM
Impact of DM on Intracellular Signaling Events that Govern Angiogenesis
Andrius Kazlauskas. Ophthalmology, Schepens Eye Res Inst/Mass Eye & Ear/
Harvard Med Schl, Boston, MA.
Abstract.Abstract1:The objectives were to investigate how diabetes mellitus
(DM) influences responsiveness of retinal neovessels to lysophosphatidic acid
(LPA), and to elucidate the underlying mechanism. To this end we used an ex vivo
assay in which neovessels sprouted from retinal explants (isolated from either
control or DM mice) when cultured between 2 layers of collagen and in the
presence of VEGF-A. While DM had no effect on the formation of neovessels, it
prevented LPA-induced regression. High glucose (HG)-treatment of retinal
explants mimicked the DM phenotype. Similarly, primary retinal endothelial cells
(RECs) that were subjected to HG-treatment organized into tubes that were
resistant to LPA. HG caused LPA resistance within RECs by elevating ROS, which
activated SFKs that stimulated the Erk pathway, which antagonized LPA-mediated
signaling events that were required for regression. This ROS/Src/Erk mechanism
appeared to be the same route by which DM-induced LPA resistance of retinal
neovessels. We conclude that DM/HG reprograms signaling pathways in RECs to
induce a state of LPA resistance.
Commercial Relationships: Andrius Kazlauskas, None
Support: JDRF 1-2008-905
Program Number: 1708
Presentation Time: 3:10 PM - 3:30 PM
Developmental Ocular Angiogenesis
Martin Friedlander. Cell Biology MB28, Scripps Research Institute, La Jolla, CA.
Commercial Relationships: Martin Friedlander, None
Support: None
266 Retinal Glial Cells
Monday, May 7, 2012, 1:45 PM - 3:30 PM
Hall B/C Poster Session
Program #/Board # Range: 1997-2027/D841-D871
Organizing Section: Retinal Cell Biology
Contributing Section(s): Visual Neurophysiology
Program Number: 1997 Poster Board Number: D841
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Presentation Time: 1:45 PM - 3:30 PM
The Expression of Acid-Sensitive Current by Solitary Salamander and
Monkey Müller Cells
Peter R. MacLeish, Xiaoming Chen, Tiandong Leng, Mian Xie, Zhigang Xiong.
Dept of Neurobiology, Morehouse School of Medicine, Atlanta, GA.
Purpose: Müller cells perform multiple functions in maintaining the health of the
retina and might be targets of chronic disorders given their intimate contact with all
classes of retinal neurons and blood vessels. Here, we investigated whether or not
Müller cells were able to respond to low pH since this condition accompanies a
variety of retinal disorders. In other systems, neurons and glial cells express AcidSensing Ion Channels (ASICs) whose activation by low tissue pH seems to
contribute to the pathophysiology of certain disorders.
Methods: Müller cells were isolated from the salamander (A. tigrinum) or rhesus
monkey (Macaca mulatta) retina following treatment with 7u/ml of papain and
trituration. Retinal cells were plated on coverslips coated with the antibody, Sal-1,
for salamander or 9B5 for monkey. Membrane current was measured under voltage
clamp conditions using the whole cell configuration. Electrodes contained CsF or
KCl at pH 7.3. Extracellular pH was changed by fast perfusion of buffered
solutions at pH 7.4, pH 6.0, or pH 5.0.
Results: For 40 salamander Müller cells, about 40% responded to pH changes from
7.4 to 6.0 (or 5.0) by a clear increase in inward current. All 5 monkey cells
responded with inward current. The amplitude of the induced current and the
overall time course were variable but resembled those of currents induced by
activation of acid-sensing ion channels (ASICs) in other systems. The range of
amplitudes of induced current was between 10 and several hundred pA. In 2
salamander cells where a complete current-voltage relationship was constructed, a
reversal potential approaching +60 mV was observed, suggesting the activation of a
Na+-selective channel. Interestingly, the currents in salamander Müller cells appear
to be resistant to amiloride, a commonly used non-specific blocker for ASICs. In
contrast, ganglion cells in the same dish had acid-sensitive inward currents that
were blocked by amiloride. The currents in monkey Müller cells were sensitive to
amiloride and Psalmotoxin 1 (PcTx1), a specific inhibitor of ASIC-1.
Conclusions: Müller cells from salamander and monkey express acid-sensitive
currents that were observed when the extracellular pH was changed from 7.0 to 6.0
or to 5.0. Work in other laboratories has reported the presence of acid-sensitive
currents on retinal ganglion cells so our finding indicates that other retinal cells
express acid-sensitive currents. Our results indicate that monkey Müller cells
express acid-sensing channels with properties of ASICs seen in other systems while
salamander Müller cells express channels with distinct pharmacological properties.
The molecular basis for this difference and the functional significance are of
interest for future studies.
Commercial Relationships: Peter R. MacLeish, None; Xiaoming Chen,
None; Tiandong Leng, None; Mian Xie, None; Zhigang Xiong, None
Support: U54-NS060659, R01-NS066027, R01-NS47506, 2G12RR003034,KL2
RR025009, S21MD000101, RR000165
Program Number: 1998 Poster Board Number: D842
Presentation Time: 1:45 PM - 3:30 PM
Muller Cells In The Salamander Retina Express Functional Ca2+-permeable
AMPA Receptors
Manuel Esguerra, Robert F. Miller. Neuroscience, University of Minnesota,
Minneapolis, MN.
Purpose: We have previously demonstrated that D-serine is a necessary co-agonist
for activation of NMDA receptors during synaptic transmission in the vertebrate
retina. However, the source of D-serine and the mechanisms for D-serine release in
the retina are still unknown. Several models suggest that functional AMPA-type
glutamate receptors are necessary for D-serine release, and our laboratory has
demonstrated that glial cells may be a prominent source of D-serine release in the
retina. Reconciling these two ideas for D-serine release requires that glial cells
express functional AMPA receptors. We tested this hypothesis by challenging
isolated Muller glia with AMPA receptor agonists and antagonists while measuring
changes in cytosolic free Ca2+ and transmembrane currents.
Methods: Muller glia and neurons were acutely isolated from larval tiger
salamander retinas by enzymatic digestion, loaded with fluorescent Ca2+-sensitive
dyes, and plated in a perfusion chamber on the stage of a fluorescence-equipped
inverted microscope. Intact Muller cells were selected for simultaneous whole-cell
patch recording and imaging of intracellular Ca2+. Agonists and antagonists of
AMPA receptors were applied by bath perfusion.
Results: Application of AMPA alone (up to 100µM) did not evoke either Ca2+
responses or conductance changes in isolated Muller cells. However, AMPA
application along with 50µM cyclothiazide, which prevents AMPA receptor
desensitization, evoked large Ca2+ increases (3-11x resting Ca2+) and large inward
currents from rest (Erev~ +3 mV). In 85% of cells, a selective antagonist of Ca2+permeable AMPA receptors, N-acetyl spermine, reduced Ca2+ mobilization and
evoked current amplitudes by 40%.
Conclusions: Our results indicate that isolated Muller glia express functional
AMPA glutamate receptors, which appear to be in a desensitized state at rest.
Sensitivity of the observed responses to N-acetyl spermine suggests that a large
proportion of glial AMPA receptors are a Ca2+-permeable variant. These results are
consistent with our earlier capillary electrophoretic analyses, which indicate that
cyclothiazide is necessary to evoke AMPA-receptor mediated increases in
extracellular D-serine levels in the mouse retina. Our results support the hypothesis
that glial cells may be a major source of D-serine released during light-evoked
activity in the vertebrate retina.
Commercial Relationships: Manuel Esguerra, None; Robert F. Miller, None
Support: NIH Grant EY03014
Program Number: 1999 Poster Board Number: D843
Presentation Time: 1:45 PM - 3:30 PM
Association of Interphotoreceptor Retinoid-Binding Protein (IRBP) with
Pericellular Outer Segment and Müller cell Domains
Mary Alice Garlipp1,2, Federico Gonzalez-Fernandez1,3. 1Research Service,
Veterans Affairs WNYHS, Veterans Administration, Buffalo, NY; 2Ophthalmology
& Pathology and Anatomic Sciences, SUNY Eye Institute /University at Buffalo,
Buffalo, NY; 3Ophthalmology & Pathology and Anatomic Sciences, SUNY Eye
Institute / University at Buffalo, Buffalo, NY.
Purpose: How IRBP accurately targets the delivery of 11-cis and all-trans retinol,
and 11-cis retinaldehyde between the RPE, rods, cones, and Muller cells in the
rod/cone visual cycles remains largely a mystery. Here we address whether IRBP
can associate with specific anatomic compartments along the cells bordering the
interphotoreceptor matrix.
Methods: Adult Xenopus lavis were selected for these experiments because of
their large photoreceptors, and that their retina can be detached in light and
maintained in organ culture. Retinas were detached directly under 4%
paraformaldehyde (“unwashed”), or Ringer’s saline followed by 3 saline washes
and fixation (“washed”). The distribution of IRBP was characterized by indirect
confocal immunofluorescence in wholemount and cross-section using rabbit antiXenopus IRBP serum, and double labeling with anti-opsin (mAb COS-1), and
fluorescently labeled lectins (PNA and WGA). The distribution of IRBP in the rod
and cone associated matrix was also studied by immunogold electron microscopy
in undetached retinas. Washed retinas were also incubated with purified Xenopus
IRBP-647, ovalbumin-Alexa 647, and Alexa-647 dye alone. Full-length Xenopus
IRBP was expressed in a soluble form as a thioredoxin fusion protein. The
thioredoxin was removed and the Xenopus IRBP purified by a combination of ion
exchange, and Ni2+ affinity chromatography.
Results: In unwashed retinas, IRBP was distributed throughout the IPM. Washing
removed the diffuse protein leaving behind a residual IRBP associated with specific
anatomic domains. This wash resistant IRBP typically rimmed the outer segments
of rods and in particular to that of cones. The above may correspond to a diffuse
and peri-cellular IRBP noted by immunogold EM. Recombinant Xenopus IRBP647 preferentially targeted the pericellular domain surrounding cone outer
segments, and to a lesser extent the rod outer segments. Binding of Xenopus IRBP647 was also observed in the region of the Muller cell villi. Immunogold showed
that IRBP is normally sequestered in this region. The above interaction was
abolished by incubating with a 50-fold excess of unlabeled Xenopus IRBP. A
similar binding was observed with bovine IRBP. No binding was detected with
ovalbumin Alexa-647, or Alexa-647 dye alone.
Conclusions: Our data indicates that IRBP is not simply loosely present in retina as
previously thought. Rather, it interacts with specific domains associated with the
pericellular region of the outer segments particularly that of the cones, and the
Müller cell villi. These findings may provide a mechanism through which IRBP
targets specific retinoids to the correct cells during the visual cycle.
Commercial Relationships: Mary Alice Garlipp, None; Federico GonzalezFernandez, None
Support: Veterans Affairs R&D grant I01BX007080, NIH/NEI grant EY09412,
and an Unrestricted Grant to the SUNY Ross Eye Institute from Research to
Prevent Blindness, Inc., New York, NY.
Program Number: 2000 Poster Board Number: D844
Presentation Time: 1:45 PM - 3:30 PM
Brain-Derived Neurotrophic Factor-Secreting Müller Cells Increase Survival
and Neurite Outgrowth of Rat Retinal Ganglion Cells in Vitro
Yuko Shinohara, Masayoshi Nakatani, Nobuharu Asai, Kan Ohtsuki.
Bioengineering Institute, R&D Division, NIDEK Co., Ltd., Gamagori, Japan.
Purpose: We examined the efficacy of brain-derived neurotrophic factor (BDNF)secreting Müller cells in supporting the survival and neurite outgrowth of rat retinal
ganglion cells (RGCs).
Methods: A Müller cell line (BD-TM4) stably expressing BDNF was established
by transfecting a BDNF gene into an immortalized rat Müller cell line (TR-MUL).
RGCs were isolated from 6-day-old rats and provided for co-culture on a porous
membrane insert, avoiding direct contact with BD-TM4 or TR-MUL. After culture
for 2 days in Neurobasal medium under conditions of neurotrophic factor
deficiency, the cells were exposed to 200 µM glutamate for 24 h or left unexposed.
RGC survival was evaluated by counting calcein AM-stained RGCs. Retinal
explants from adult rats 7 days after optic nerve crush were cultured in BD-TM4-
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
or TR-MUL-conditioned medium (CM) for 6 days. As a positive control,
recombinant human BDNF (rhBDNF) was added to the explant cultures. Numbers
of outgrowing neurites and surviving RGCs were counted by using
immunohistochemical techniques.
Results: The amount of BDNF released from BD-TM4 cells was approximately
1.0 ng/1×105 cells/day. Under conditions of neurotrophic factor deficiency without
glutamate exposure, BD-TM4 cells enhanced RGC survival (to 300% of that in the
control culture [i.e. culture of RGC cells alone]) significantly more than did TRMUL cells (to 150% of that in the control culture). Similarly, under glutamate
toxicity, more RGCs survived in the presence of BD-TM4 cells than in the
presence of TR-MUL cells. Methionine sulfoximine, a glutamine synthetase
inhibitor, significantly inhibited the protective effect of BD-TM4 (P < 0.05) or TRMUL cells (P < 0.01). TR-MUL-CM had little effect on neurite outgrowth in
retinal explants, whereas BD-TM4-CM significantly increased (by 320%) the
number of neurite outgrowths compared with those in unconditioned Neurobasal
medium. Similarly, rhBDNF-positive control medium significantly increased (by
380%) the number of neurite outgrowths compared with those in unconditioned
medium. After optic nerve crushing, more surviving RGCs were observed in retinal
explants cultured in BD-TM4-CM than in unconditioned medium.
Conclusions: Factors released from the BD-TM4 cells not only enhanced RGC
survival but also stimulated neurite outgrowth, suggesting that BDNF secretion
enhances the rescue effect of RGCs by the original cell line.
Commercial Relationships: Yuko Shinohara, NIDEK Co., Ltd. (E); Masayoshi
Nakatani, NIDEK Co., Ltd. (E); Nobuharu Asai, NIDEK Co., Ltd. (E); Kan
Ohtsuki, NIDEK Co., Ltd. (E)
Support: None
Program Number: 2001 Poster Board Number: D845
Presentation Time: 1:45 PM - 3:30 PM
Comparative Study Of The Cellular Triad - Müller Cells, Astrocytes, Vessels Between Normal And Dystrophin 71 Deficient Mice Using An AAV Variant
Targeting Müller Cells
Ophelie Vacca1, Deniz Dalkara2,3, Stephane Fouquet1, Romain Benard1, Sijia Cao1,
Michel Paques1, Jose-Alain Sahel1,4, Ramin Tadayoni1,5, Alvaro Rendon1. 1Institut
de la Vision/INSERM/UPMC Univ Paris 06/CNRS/CHNO des Quinze-Vingts,
Paris, France; 2Univ of California, Berkeley, CA; 3HWNI and CChem, Berkeley,
CA; 4Institute of Ophthalmology, University College of London, UK/ Fondation
Ophtalmologique Adolphe de Rothschild, Paris, France / Académie des SciencesInstitut de France, Paris, France; 5Ophthalmology Dept, Hôpital Lariboisière, APHP, Univ Paris Diderot, Paris, France.
Purpose: Dystrophin Dp71 is a membrane cytoskeletal protein expressed in Müller
cells, astrocytes and pericytes. We have shown that in comparison with wild-type
mice (WT), the absence of Dp71 induces a blood retinal barrier (BRB) breakdown
(Sene, 2009). A highly permeable BRB has been considered as a characteristic of
diabetic retinopathy. At this respect, Dp71-null mice could be a good model to
approach the study of this pathology. It has been shown that the AAV variant
ShH10-GFP, closely related to AAV serotype 6, is capable of efficient, selective
Müller cells infection through intravitreal injection (IVT) in rat retina (Klimczak,
2009). Here, we tested the efficiency of ShH10-GFP in mice retina and studied
Müller cells morphology and their relations with astrocytes and vessels in WT and
Dp71-null mice.
Methods: We performed IVT of ShH10-GFP at 4.10^11 vg/µl of 7-weeks-old
C57BL/6J WT and Dp71-null mice. Every week until one month after injection, we
have made fundus imaging and scanning laser ophtalmoscopy to follow ShH10GFP expression. Mice were sacrificed one month after injection. Retinae were
prepared for flatmounts or cryosections, and labeled with GFAP antibody and
Lectin marker. Cellular morphology was observed by confocal imaging and
analysed by Fiji software.
Results: As in rat, ShH10-GFP transduced Müller cells in WT and Dp71-null mice.
On fundus imaging, we have observed that ShH10-GFP is strongly expressed one
week after injection and its expression level is constant to at least one month after
injection. On cryosections, we have seen that transduced cells are specifically
Müller cells. On flatmount retinae, transduced Müller cells appear to be more
evenly transduced in Dp71-null mice than in WT. However, in absence of Dp71, (i)
interactions between Müller cells and vessels seem to be less tight and Müller cells
appear to be more evenly transduced in whole retina and (ii) astrocytes are
dramatically reduced in number and have more elongated shapes.
Conclusions: Here we demonstrate that (i) in mice, ShH10-GFP targets
specifically Müller cells and that (ii) in Dp71-null mice, Müller cells are deficient
and astrocytes are less well organized. This study also suggests the potential
therapeutic role of ShH10 genetic tool. Indeed, ShH10 could be used to restore
Dp71 expression in order to treat pathologies related to BRB breakdown.
Commercial Relationships: Ophelie Vacca, None; Deniz Dalkara,
None; Stephane Fouquet, None; Romain Benard, None; Sijia Cao,
None; Michel Paques, None; Jose-Alain Sahel, None; Ramin Tadayoni,
None; Alvaro Rendon, None
Support: None
Program Number: 2002 Poster Board Number: D846
Presentation Time: 1:45 PM - 3:30 PM
Effect of High Glucose on Connexin 43 (Cx43) Expression, Localization and
Intercellular Communication in Retinal Muller Cells and Retinal Pericytes
Tetsuya Muto, Casey Stottrup, Sayon Roy. Ophthalmology, Boston University
School of Medicine, Boston, MA.
Purpose: To study if high glucose alters gap junction protein Cx43 expression, its
distribution pattern, and intercellular communication in cultures of retinal Muller
cells, and in cocultures of retinal Muller cells and pericytes.
Methods: Retinal Muller cells (rMC-1) alone, and cocultures of rMC-1 and bovine
retinal pericytes (BRPs) were grown in normal (N; 5 mM glucose) or high (30 mM)
glucose medium for 7 days. Western blot analysis was performed to determine
expression of Cx43 protein, and scrape-load dye transfer (SLDT) technique was
performed to assess gap junction intercellular communication (GJIC) activity.
Additionally, Cx43 immunostaining was performed to determine Cx43 localization
and distribution pattern.
Results: Cx43 expression was significantly reduced in both rMC-1 cultures and
rMC-1 and BRP cocultures grown in high glucose medium compared to those
grown in normal medium (64.4±6.9% of control, p<0.01; 72±16.6% of control,
p<0.05,respectively). HG significantly reduced the number of Cx43 plaques in
rMC-1 cultures, and rMC-1 and BRP cocultures (66.4±6.7% of control, p<0.01;
70.7±11.4% of control, p<0.05, respectively). GJIC was also significantly reduced
in rMC-1 cultures and in rMC-1 and BRP cocultures compared to those grown in N
medium (55.5±6.2% of control, p<0.01; 61.2±6.0% of control,p<0.01,
Conclusions: Results indicate that Cx43 expression, localization, and GJIC activity
are reduced by high glucose in cultures of rMC-1, and in cocultures of rMC-1 and
BRPs. High glucose-induced loss of intercellular communication in retinal Muller
cells and between Muller cells and pericytes may negatively impact vascular cell
viability and vascular homeostasis, and contribute to retinal dysfunction associated
with the pathogenesis of diabetic retinopathy.
Commercial Relationships: Tetsuya Muto, None; Casey Stottrup, None; Sayon
Roy, None
Support: NIH, NEI EY018218
Program Number: 2003 Poster Board Number: D847
Presentation Time: 1:45 PM - 3:30 PM
Inflammatory Role of ProNGF/p75NTR in Müller cells of the Diabetic Retina
Barbara A. Mysona, Mohammed A. Abdelsaid, Su Matragoon, Bindu Pillai, Azza
El-Remessy. UGA Clinical Pharmacy program, University of Georgia, Augusta,
Purpose: Diabetic retinopathy, the leading cause of blindness in U.S. working age
adults, is characterized by neurodegeneration, inflammation, and vascular
dysfunction. Our recent studies using diabetic animals show that neurodegeneration
and vascular leakage are associated with an imbalance of nerve growth factor
(NGF) leading to accumulation of its precursor (proNGF) and its receptor, p75NTR.
Here, we examine the role of proNGF/p75NTR in mediating retinal inflammation
and vascular dysfunction in the diabetic retina.
Methods: STZ-diabetes was induced in wild type and p75NTR knockout (KO) mice.
After 4 weeks, vascular permeability was assayed by extravasation of BSAconjugated fluorescein. Müller cells were cultured for 2 days in high glucose (25
mM), normal glucose (5 mM), or osmolar controls and stimulated with cleavageresistant proNGF in the presence or absence of compound E (an inhibitor of p75NTR
intracellular domain cleavage) and epicatechin (a nitration inhibitor that reduces
p75NTR expression). Expression of p75NTR, NGF, proNGF, and inflammatory
mediators were determined in retinal and cell lysates by Western-Blot. Animals
used for this research were handled in accord with ARVO Statement for the Use of
Animals in Ophthalmic and Vision Research.
Results: Deletion of p75NTR prevented diabetes-induced vascular permeability
detected in wild-type mice. In diabetic wild-type mice NGF protein levels
decreased by 50% while NFkB and TNF-α levels increased by 1.8 fold and 6 fold,
respectively (N=5-7, P < 0.01). These changes were not observed in diabetic
p75NTR KO mice. Interestingly, basal levels of NGF, NFkB, and TNF-α were
altered in p75NTR KO mice (N=5-7, P < 0.05). Müller cell studies showed that
addition of exogenous, cleavage-resistant proNGF enhanced high glucose induced
elevation of p75NTR. Elevation of NFkB and TNF-α observed in high glucose,
cleavage-resistant proNGF treated cells was reduced by co-treatment with
compound E and epicatechin (N = 4-6, P < 0.05).
Conclusions: Deletion of p75NTR or inhibition of its cleavage reduced the diabetes
induced elevation of inflammatory mediators that cause increased vascular
permeability. Inhibition of the proNGF/p75NTR inflammatory axis is a potential
therapeutic target in the diabetic retina.
Commercial Relationships: Barbara A. Mysona, None; Mohammed A.
Abdelsaid, None; Su Matragoon, None; Bindu Pillai, None; Azza El-Remessy,
Support: JDRF 2008-149
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2004 Poster Board Number: D848
Presentation Time: 1:45 PM - 3:30 PM
Müller Cell Is A Major Cellular Source Of Survival Signals For
Photoreceptors In Diabetes
Shuhua Fu1,2, Shuqian Dong1,3, Yun-Zheng Le1,4. 1Medicine and Cell Biology and
Harold Hamm Oklahoma Diabetes Center, University of Oklahoma Health
Sciences Center, Oklahoma City, OK; 2Ophthalmology, The Second Affiliated
Hospital of Nanchang University, Nanchang, China; 3Ophthalmology, Xiangya
Hospital, Central South University, Changsha city, China; 4Dean A. McGee Eye
Institute Oklahoma City, Oklahoma City, OK.
Purpose: Diabetic retinopathy (DR) is traditionally considered as a microvascular
complication in diabetic retinas . However, emerging evidences suggest that the
loss of retinal neuron function and the death of retinal neurons are involved in DR.
To determine whether Müller cell is a major cellular source of survival signals for
photoreceptors in DR, we generated a mouse model of Müller cell-specific
knockout for vascular endothelial growth factor receptor-2 (VEGFR2) . We then
determined the effect of loss of VEGF signaling in Müller cells on their own
survival and the survival of photoreceptors.
Methods: Diabetes was induced with streptozotocin. Retinal function was
measured with electroretinography (ERG). Retinal morphology was assessed with
hematoxylin & eosin (H&E) stained sections. The density of Müller cells and cone
photoreceptors was evaluated with immunohistochemistry. Gene expression was
analyzed with immunoblotting.
Results: While loss of VEGF signaling in Müller cells did not cause any apparent
alteration in the retina under normal conditions, the conditional VEGFR2 knockout
mice exhibited a significant loss of Müller cells, cones and rods, as well as both
scotopic and photopic ERG amplitudes under diabetic conditions. Mechanistic
studies regarding the diabetes-induced loss of photoreceptors are in progress.
Conclusions: Our results suggest that VEGF signaling is required for Müller cell
survival and Müller cell is a major cellular source of survival signals for
photoreceptors in DR.
Commercial Relationships: Shuhua Fu, None; Shuqian Dong, None; YunZheng Le, None
Support: NIH grants R01EY020900 and P20RR024215, ADA grant 1-10-BS-94,
OCAST contract HR09-058
Program Number: 2005 Poster Board Number: D849
Presentation Time: 1:45 PM - 3:30 PM
Neuroprotection and Inhibition of Blood-Retinal Barrier Breakdown In a
Transgenic Model of Conditional Müller Cell Ablation
Weiyong Shen1, Marcus Fruttiger2, Ling Zhu1, Sook H. Chung1, Nigel L. Barnett3,
Joshua Kirk1, Nathan J. Coorey1, Murray Killingsworth1, Mario R. Capecchi4,
Mark C. Gillies1. 1Clin Ophthal & Eye Health, University of Sydney, Sydney,
Australia; 2UCL Institute of Ophthalmology, London, United Kingdom; 3Centre for
Clinical Research, University of Queensland, Brisbane, Australia; 4Department of
Human Genetics, University of Utah, Salt Lake City, UT.
Purpose: Since Müller glia interact closely with both retinal neurons and blood
vessels, Müller cell dysfunction may disrupt both systems. We have developed a
novel transgenic model of conditional Müller cell ablation. This study aimed to test
strategies to prevent the photoreceptor death and blood-retinal barrier breakdown
that occur in this transgenic model.
Methods: We generated a DNA construct containing a Müller cell-specific
promoter driving a tamoxifen-inducible form of Cre recombinase (CreER). The
cell-specific promoter contained a fragment of regulatory region of the
retinaldehyde binding protein 1 gene (Rlbp1). Rlbp1-CreER transgenic mice were
crossed with Rosa-DTA176 mice for inducible, Muller cell-specific intracellular
expression of an attenuated form of diphtheria fragment A. Dynamic changes in
Müller glia, photoreceptors and retinal vasculature were investigated after
switching on the DTA176 toxic gene expression with tamoxifen. Cilliary
neurotrophic factor (CNTF) and VEGFB20.4.1.1, a VEGF antibody that binds both
human and murine VEGF-A, were injected intravitreally to examine their effects on
photoreceptor death and BRB breakdown at different stages after conditional
Müller cell ablation.
Results: Müller cell ablation led to rapid photoreceptor apoptosis, blood-retinal
barrier breakdown and, later, deep retinal neovascularization. Intravitreal injection
of CNTF inhibited photoreceptor apoptosis but had no effect on the vasculopathy.
Blocking VEGF action with intravitreal injection of VEGFB20.4.1.1 dramatically
attenuated vascular leak but did not rescue photoreceptors.
Conclusions: Our results demonstrate that Müller glia dysfunction can be the
upstream cause of both neuronal and vascular pathology, which can occur
independently in the retina. Our findings also show that Müller glia dysfunction
may have a so far unappreciated role and be a mechanistic link between neuronal
and vascular pathologies in retinal diseases.
Commercial Relationships: Weiyong Shen, None; Marcus Fruttiger,
None; Ling Zhu, None; Sook H. Chung, None; Nigel L. Barnett, None; Joshua
Kirk, None; Nathan J. Coorey, None; Murray Killingsworth, None; Mario R.
Capecchi, None; Mark C. Gillies, None
Support: Lowy Medical Research Institute, Ophthalmic Research Institute of
Australia, Rebecca L. Cooper Medical Research Foundation and University of
Sydney Bridging Grant
Program Number: 2006 Poster Board Number: D850
Presentation Time: 1:45 PM - 3:30 PM
Increased Retinal Müller Cell Immunolabeling For Glial Fibrillary Acidic
Protein In Rats Following Either Impairment Of Sympathetic Control Of
Choroidal Blood Flow By Removal Of The Superior Cervical Ganglion Or By
Normal Aging
Malinda E. Fitzgerald1A,2, Corey Haughey1B,2, Nobel Del Mar1B, Anton J. Reiner1A.
Anatomy, Neurobiolology and Ophthalmology, BAnatomy & Neurobiology,
UTHSC, Memphis, TN; 2Biology, Christian Brothers University, Memphis, TN.
Purpose: The Superior Cervical Ganglion (SCG) is the source of the
vasoconstrictory sympathetic innervation of choroidal vessels. Impaired
sympathetic control of choroid results in a failure of baroregulatory
vasoconstriction of choroidal vessels, and thus excessive choroidal blood flow
(ChBF) during high blood pressure. We have shown previously that disruption of
adaptive parasympathetic control of choroid during low systemic blood pressure
impairs retinal function as assessed by ERG, and increases glial fibrillary acidic
protein (GFAP) in retinal Müller cells (ARVO e abstract 4081, 2011). In this
current study, we characterized retinal pathology using GFAP
immunohistochemistry following removal of the SCG. We also investigated GFAP
immunolabelling in retina in normal aging.
Methods: Retinal morphology was examined in 26 rats. Thirteen rats served as
young controls and either received no removal of the SCG or an electrolytic lesion
in a brain region unrelated to sympathetic or parasympathetic ChBF control. Eleven
rats received bilateral SCG-ectomies and were allowed to recover for 4-8 weeks.
Four rats greater than a year old were also studied. Paraformaldehyde-perfusion
fixed retinas were sectioned on a cryostat (20µm), slide-mounted, and
immunolabeled for GFAP using peroxidase-antiperoxidase methods with
diaminobenzidine. GFAP immunolabeling was blindly analyzed as to group
category using a scoring system that reflected the extent of GFAP labeling in the
Müller cells and the abundance of labeled Müller cells.
Results: The retinas from SCG-ectomy rats showed a significant increase in GFAP
immunolabeling within the retina in comparison with retinas from control rats, with
the SCG-ectomy levels being more than 2x those in controls. A similar GFAP
increase was previously seen following disruption of parasympathetic ChBF
control (ARVO e abstract 4081, 2011). The old rats had GFAP levels 3.5x those in
Conclusions: Müller cells of the retina respond to ChBF deficiency caused by
disrupted sympathetic choroidal regulation, as they also do to disruption of the
parasympathetic control. These findings indicate that both the sympathetic and
parasympathetic regulation of ChBF play an important role in maintaining retinal
health. The basis of the great retinal GFAP increase with aging is uncertain, but
age-related changes in sympathetic and parasympathetic regulation of ChBF may
be contributors.
Commercial Relationships: Malinda E. Fitzgerald, None; Corey Haughey,
None; Nobel Del Mar, None; Anton J. Reiner, None
Support: NIH-EY-05298 (AR), the Department of Ophthalmology at UTHSC
Research to Prevent Blindness (MECF), NIH/NEI-5P30EY013080 (D, Johnson)
Program Number: 2007 Poster Board Number: D851
Presentation Time: 1:45 PM - 3:30 PM
Phenotype of the Müller Cells of the Crb1rd8 Mutant Mouse in vitro and in
Concepcion Lillo, Saúl Herranz-Martín, Antonio E. Paniagua, Almudena Velasco,
Juan M. Lara, José Aijón. Cell Biology and Pathology, INCyL, University of
Salamanca, Salamanca, Spain.
Purpose: Müller cells are the radial glial of the retina, and at the level of the retinal
outer limiting membrane (OLM), they establish adherens junctions with
photoreceptor cells. CRB1 is a transmembrane protein present in the Müller cells,
and localized in the subapical region (SAR) of the OLM. Mutations in the gene that
codifies for the CRB1 human protein lead to retinal degenerations, such as Leber
Congenial Amaurosis or Retinitis pigmentosa. The Crb1rd8 mouse has a mutation
identified as a deletion of a single base in the Crb1 gene, encoding a truncated
protein. There is a clear phenotype in the Crb1rd8 mouse retina, showing gaps in
the OLM, photoreceptor-rosettes and progressive photoreceptor death.
Nevertheless, little is known about the characteristics of the Müller cells in the
mutant retina. To characterize the modifications that the CRB1 mutant Müller cells
may experiment during the development of the retina, our purpose is to analyze
their behavior in vitro as well as in vivo comparing wild type and mutant cells.
Methods: Müller cells from mutant and wild type mouse retinas were isolated at
P7, cultured, and their viability was examined at different time points. We
performed western blot and immunofluorescence analysis with well-known
markers for this cell type in the cells in vitro and in the retinal tissue. Finally, we
have tested the possibility of transfecting Müller cells with Chariot©, a commercial
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
agent that introduce proteins in their native configuration in cells in vitro and in
Results: Müller cells primary cell cultures are viable at seven days in culture. We
found differences in the viability of the mutant cells compared with the wild type
cells at the different time points measured. Besides, there are also variations in the
amount of expression of several Müller cells markers, such as GFAP, glutamine
synthetase and SOX2 comparing wild type and mutant Müller cells in vivo and in
vitro. We have detected these differences from early development of the retina,
being more evident in the primary cultures. We have used the commercial
transfection agent Chariot© to introduce b-galactosidase in Müller cells in vitro and
in vivo from both phenotypes.
Conclusions: We have characterized the phenotype of the CRB1 mutant Müller
cells in vivo and in vitro from the development of the retina onwards. These cells
are viable in vitro and, as well as in vivo, they show a pattern of expression of
several Müller cells markers which is different to that of the wild type ones.
Finally, we have verified that Müller cells obtained from both phenotypes, mutant
and control, are susceptible of being transfected with the commercial agent
Chariot© with a high efficiency.
Commercial Relationships: Concepcion Lillo, None; Saúl Herranz-Martín,
None; Antonio E. Paniagua, None; Almudena Velasco, None; Juan M. Lara,
None; José Aijón, None
Support: Ministerio de Ciencia e Innovación (BFU2008-04490/BFI y BFU200911179), Junta de Castilla y León (Grupo de Excelencia, GR-183).
Program Number: 2008 Poster Board Number: D852
Presentation Time: 1:45 PM - 3:30 PM
Global Genomic Analysis of Retinae from Selective Müller Cell Knockout
Sook H. Chung1, Weiyong Shen1, Jean Yang2, Penghao Wang2, Nick Shackel3,
Mark C. Gillies1. 1Clinical Ophthal & Eye Health, Save Sight Institute, Sydney,
Australia; 2Mathematics and Statistics, the University of Sydney, Sydney,
Australia; 3Centenary Institute, Sydney, Australia.
Purpose: It has been suggested that müller cell dysfunction may contribute to
neuronal damage in some retinal diseases such as diabetic retinopathy and macular
telangeictasia type 2. The purpose of the study is to identify genes that are
differentially expressed (DE) between retinae of transgenic mice, in which patchy
müller cell ablation has been conditionally induced, and the wild type mice.
Methods: Transgenic mice, in which a müller cell specific promoter (Rlbp1) has
been linked to a gene encoding DTA176 toxin with a Cre-ER system, and wild type
mice received 4 consecutive tamoxifen injections to activate transcription of the
gene encoding the toxin. All mice were sacrificed at 3 different time points (1
week, 1 month and 3 months) after administration of tamoxifen and retinas were
collected. After RNA extraction (Qiagen cat#74104), 1st and 2nd strand cDNA
were synthesized, purified and biotin labelled for hybridization (Affy mouse gene
1.0 ST arrays). The scanned data were analysed and validated with qRT-PCR.
Results: At the 3 different time points, there were 395 (309 upregulated [U] and 86
downregulated [D]), 126 (58U, 68D), 123 (46U, 77D) DE genes respectively. Most
of genes were differentially expressed at 1 week. These either remianed upregulated or subsided in the later stages. Up-regulated DE genes were related to
proteolysis, apoptosis, prostaglandin biosynthetic process and MAPK activity,
whereas down-regulated genes were associated with glucose metabolism,
regulation of cell adhesion, cytolysis and actin cytoskeleton organization. qRTPCR result was consistant with microarray data.
Conclusions: Many DE genes found in this study were related to putative
consequences of müller cell dysfunction such as vascular leak, apoptosis and
glucose metabolism. Further research comparing tissue obtained from patches of
müller cell ablation using laser capture microdissection with normal retina will
shed further light on the contribution of müller cell dysfunction to retinal disease.
Commercial Relationships: Sook H. Chung, None; Weiyong Shen, None; Jean
Yang, None; Penghao Wang, None; Nick Shackel, None; Mark C. Gillies, None
Support: NHMRC
Program Number: 2009 Poster Board Number: D853
Presentation Time: 1:45 PM - 3:30 PM
Optic Nerve Head Astrocytes Increase Expression Of Tenascin C In Response
To Injury
Juan Qu, Tatjana Jakobs. Howe Laboratory of Ophthalmology, Massachusetts Eye
& Ear Infirmary, Boston, MA.
Purpose: Tennascin c (Tnc) is one of the astrocyte-secreted extracellular matrix
proteins of the central nervous system. It is involved in the remodeling of the
central nervous system, both during development and in adult. We previously
reported that mouse optic nerve head astrocytes go through a two-stage
morphological remodeling after optic nerve crush. It changes the architecture of the
optic nerve head. In this present study, we tested the hypothesis that Tnc is secreted
by optic nerve head astrocytes and may contribute to the tissue remodeling in
response to crush injury and in experimental glaucoma.
Methods: Optic nerve crush was conducted in 2-3 months old C57Bl/6 mice and
GFAP-Tomato mice. The optic nerve in one eye was crushed and the contralateral
eye served as control. Eyes of 10-12 months old DBA/2J mice that had developed
moderate or severe glaucoma were also studied. Eyes of age-matched DBA/2J mice
that had no glaucoma served as control. Q-PCR using RNA extracted from the
whole optic nerve head and single-cell RT-PCR using acutely dissociated optic
nerve head astrocytes were performed to determine the mRNA level of Tnc.
Immunohistochemistry was performed on transverse and longitudinal sections of
optic nerve head to localize Tnc protein.
Results: The mRNA level of Tnc in the whole optic nerve head of the crushed eye
significantly increased 24 hours after injury. The up regulation was sustained for at
least one week. It returned to resting levels by three weeks. Single-cell RT-PCR of
optic nerve head astrocytes confirmed that the source of the up regulation was in
astrocytes. In the glaucoma model, the mRNA level of Tnc also significantly
increased, especially the eyes with moderate glaucoma. Immunohistochemistry for
Tnc showed clear enhancement of Tnc protein in the optic nerve head both after
optic nerve crush and in glaucoma.
Conclusions: Optic nerve head astrocytes increase their expression of Tnc in
response to crush injury and in glaucoma. This indicates that astrocytes actively
respond to injury and disease. In addition, Tnc can interact with extracellular matrix
protein membrane receptors such as integrin, which regulates cell adhesion,
migration, and proliferation. Therefore Tnc may play an important role in the optic
nerve head tissue remodeling in response to injury.
Commercial Relationships: Juan Qu, None; Tatjana Jakobs, None
Support: NIH grant EY019703
Program Number: 2010 Poster Board Number: D854
Presentation Time: 1:45 PM - 3:30 PM
Modulation Of Gliosis Using shGFAP Or/and shvimentin To Determine Its
Role In Photoreceptor Transplantation Efficiency
Claire Hippert1, Amanda C. Barber1, Anastasios Georgiadis1, James W.
Bainbridge1, Alexander J. Smith1, Jane C. Sowden2, Robin R. Ali1, Rachael
Pearson1. 1Division of Molecular Therapy, UCL Institute of Ophthalmology,
London, United Kingdom; 2Developmental Biology Unit, UCL Institute of Child
Health, London, United Kingdom.
Purpose: Cell transplantation is a potential therapeutic strategy for the irreversible
loss of photoreceptors occurring in retinal degeneration. In the degenerating retina,
Müller glia undergo gliosis, a process that includes hypertrophy and increased
production of the intermediate filaments GFAP and vimentin. Glial scarring is
likely to have a major impact on regenerative strategies, including cell
transplantation, by acting as a physical barrier and/or as a reservoir of inhibitory
molecules. Here, we studied the role of gliosis in impeding photoreceptor
integration success in the rhodopsin knockout (rho-/-) model of retinal
degeneration. To examine the impact of GFAP and Vimentin upregulation on cell
integration efficacy, we downregulated GFAP and/or Vimentin in the degenerating
Methods: Gliosis was examined by western blot and immunostaining.
Photoreceptor transplantation was assessed by transplanting FACS-sorted rod
precursors into rho-/- recipients at early (4 week), mid (6 week) or late (10 week)
degenerative stages. GFAP and vimentin expression was manipulated using
AAV2/9-shGFAP, AAV2/9-shVimentin (Vim) or AAV2/9-lshGFAPshVim
vectors. Their efficacy was studied by qRT-PCR and immunostaining at 1 week
post subretinal injection.
Results: Total GFAP expression was higher in the rho-/- at 4 wks compared to
wildtype, and increased significantly between 4 and 10 weeks. The number of
GFAP+ve processes extending into the outer nuclear layer (ONL) also increased
together with the appearance of lateral glial scars at the outer edge of the ONL.
Transplanted photoreceptor integration was significantly lower in the rho-/- mouse
than in age-matched wildtype controls and declined markedly with degeneration.
Expression of GFAP and vimentin was specifically knocked down by subretinal
injection of AAV2/9-shGFAP or AAV2/9-shVim, which led to significant decrease
in GFAP (~80%) and Vimentin (~50%) respectively, compared to non-injected
Conclusions: There is an inverse correlation between increasing gliosis and
transplanted photoreceptor integration in the rho-/- mouse. We have demonstrated
the efficacy of our AAV2/9-shGFAP and shVim expression vectors in the retina.
We are using these vectors to determine whether or not GFAP and vimentin, the
hallmarks of gliosis, impede transplanted photoreceptor integration.
Commercial Relationships: Claire Hippert, None; Amanda C. Barber,
None; Anastasios Georgiadis, None; James W. Bainbridge, None; Alexander J.
Smith, None; Jane C. Sowden, None; Robin R. Ali, None; Rachael Pearson,
Support: BRPS (GR566), Wellcome Trust (082217) and Royal Society
(RG080398) Moorfields Eye Hospital and UCL Institute of Ophthalmology
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2011 Poster Board Number: D855
Presentation Time: 1:45 PM - 3:30 PM
Reduced GFAP Expression in Optic Nerve Head Astrocytes Linked To
Pannexin-mediated Release Of ATP
Jonathan M. Beckel1A, Jingsheng Xia1A, Jason C. Lim1A, Edward J. Macarak1A,
Claire H. Mitchell1A,1B. AAnatomy and Cell Biology, BPhysiology, 1University of
Pennsylvania, Philadelphia, PA.
Purpose: Astrocytes in the optic nerve head have been identified as key mediators
of mechano-sensitive change in glaucoma, and levels of activation marker GFAP
can rise in glaucoma. We have previously shown that astrocytes display mechanosensitive ATP release through a non-vesicular mechanism. This study further
examined the mechanisms of mechano-sensitive ATP release from rat optic nerve
head astrocytes (RONHA) and investigated the role purinergic signaling plays in
astrocyte activation.
Methods: RONHA from neonatal rats (PD 3-7) were cultured on 96 well plates,
with ATP concentrations determined using a luciferin-luciferase assay and a plate
reader. Stretched RONHA were seeded on a silicon substrate and subjected to a 5%
equilateral strain at 0.3 Hz for 4 hours using a specially designed pneumatic piston.
RNA was extracted 20 hours later and qPCR was performed using standard
procedures. Regulatory volume decrease (RVD) measurements were determined
from cells filled with calcein-AM and imaged on a fluorescent microscope.
Results: The application of P2 receptor analog ATPγS (100 µM) reduced the
expression of GFAP mRNA in RONHA when levels were measured 16 hrs after
stimulation. Application of mechanical strain to astrocytes by either stretching or
swelling the cells, led to a rapid release of ATP. The pannexin inhibitors
probenecid (1mM) and carbenoxolone (10µM) reduced this mechano-sensitive
release of ATP, suggesting release involved pannexin hemichannels. In contrast,
neither 18α-glycyrrhetinic acid (50µM), GdCl3 (50µM) nor the TRPV4 antagonist
HC067047 (1-10µM), inhibited the swelling-induced release of ATP. While cells
normally restored their volume soon after swelling, this regulatory volume decrease
was prevented by probenecid (1mM) and the ATPase apyrase (1U/ml), implying
that auto-stimulation of P2 receptors by released ATP is involved in maintaining
homeostasis after mechanical strain.
Conclusions: Mechanical strain triggers ATP released from RONHA through
pannexin hemichannels. Released ATP may act in an autocrine/paracrine manner
on astrocyte purinergic receptors to maintain homeostasis and lower GFAP levels.
Commercial Relationships: Jonathan M. Beckel, None; Jingsheng Xia,
None; Jason C. Lim, None; Edward J. Macarak, None; Claire H. Mitchell,
Support: NIH EY015537, EY013434
Program Number: 2012 Poster Board Number: D856
Presentation Time: 1:45 PM - 3:30 PM
Regulation of Chronic Oxidative Stress Responses in Adult Retinal Astrocytes
Jeremy M. Sivak, Adrian Nahirny, Xiaoxin Guo. Vision Sciences &
Ophthalmology, Toronto Western Hospital/University of Toronto, Toronto, ON,
Purpose: Astrocytes are the most prominent glial cell type found in the nerve fiber
layer (NFL) and optic nerve head (ONH). During ‘normal’ tissue function they
perform an essential role maintaining retinal ganglion cell (RGC) homeostasis.
Following retinal insult, they rapidly activate, undergoing a variety of cellular and
biochemical changes, including; secretion of cytokines and growth factors, and
producing antioxidants around the sensitive RGCs and NFL axons. The molecular
mechanisms regulating this response are still unclear, but oxidative stress has been
established as an important factor contributing to progression of a variety of
diseases affecting the inner retina, including glaucoma and diabetic retinopathy. In
order to investigate the influence of oxidative stress on NFL astrocytes directly, we
have developed a new model exposing primary adult rat NFL astrocytes to chronic
production of reactive oxygen species (ROS).
Methods: Primary NFL astrocytes from three to four week old Wistar rats were
isolated and cultured using a protocol adapted from methods developed for
neonatal brains. The resulting cells were characterized through morphology and
immunofluorescence staining by a panel of marker proteins. Astrocyte cultures
were then subjected to ROS exposure by acute and chronic methods, using H2O2,
or the redox cycling chemical paraquot, respectively. ROS generation, cell death,
and expression and concentration of markers for glial activation, and oxidative
stress were subsequently investigated.
Results: This culture system produced relatively pure populations of NFL
astrocytes with little or no contamination from other retinal cell types. Increasing
levels of ROS and cell death showed a clear correlation, with chronic exposure
producing more severe effects over time. Molecular and biochemical analyses
revealed concomitant changes in glial reactivity, and a panel of oxidative and
metabolic stress response factors, including increases in GFAP and HSP70, and a
surprising decrease in glutamate synthetase (GS).
Conclusions: This in vitro system demonstrates the damaging effect chronic ROS
exposure can have on this important mechanism regulating RGC homeostasis. We
propose this new model for further study of the factors regulating chronic oxidative
stress responses in adult NFL astrocytes.
Commercial Relationships: Jeremy M. Sivak, None; Adrian Nahirny,
None; Xiaoxin Guo, None
Support: CNIB, TGH & TWH Foundation
Program Number: 2013 Poster Board Number: D857
Presentation Time: 1:45 PM - 3:30 PM
Bmp Pathway And Retinal Astrogliosis
Subramanian Dharmarajan1, Teri Belecky- Adams1, Nader Sheibani2. 1Dept of
Biology, IUPUI, Indianapolis, IN; 2Dept of Ophthalmology and Visual Sciences,
University of Wisconsin, Madison, WI.
Purpose: All injury and disease states of the central nervous system, including the
retina, are accompanied by reactive gliosis and the formation of regenerationinhibiting glial scar. The primary components of the glial scar are astrocytes
marked by increases in proliferation, migration, and hypertrophy as well as an
increase in inhibitory chondroitan sulfate proteoglycans (CSPGs). Reactivity in
astrocytes is thought to be the result of secreted signals, such as bone
morphogenetic proteins (BMPs) and epidermal growth factor that are released at
the injury site to interact with quiescent astrocytes. While BMPs are known to be
upregulated following injury in the CNS, little information is available concerning
the role of BMPs in reactive gliosis of optic cup astrocytes. Our purpose in
performing these studies was to clarify the role of BMP7 in retinal astrocytes
reactive gliosis.
Methods: Mouse retinal astrocyte cells were incubated with 0.15 mM sodium
peroxynitrite (a strong oxidizing agent typically released by injured neurons) for 32
h or 40, 80 or 100 ng/ml of BMP7 for either 24 h or 36 h. Cells were lysed and total
protein or total RNA was isolated. Total protein was analyzed via western blot for
change in GFAP expression. While total RNA was reverse transcribed into cDNA
and further analyzed via real time PCR for change in levels of over 30 markers.
Results: A profile of reactivity was initially established by comparing mouse
retinal astrocytes treated with vehicle or sodium peroxynitrite. Treatment with
peroxynitrite for 32 h led to statistically significant increases in levels of mRNA in
7 of 30 markers evaluated by qPCR, including GFAP, Pax2, and a subset of
inhibitory chondroitan sulfate proteoglycans (CSPGs). In comparison to control
vehicle-treated retinal astrocytes, BMP7 treated samples showed similar increases
in the levels of mRNA encoding for GFAP, Pax2, and CSPGs. A comparison
between the profiles of peroxynitrite- and BMP7-treated cells showed largely (but
not completely) overlapping profiles of expression.
Conclusions: Our conclusions are as follows: 1) astrocyte reactivity can be
initiated by BMP7 treatment in vitro, and 2) reactivity may be the result of the
activation of more than one intracellular signaling pathway, each of which appears
to have an overlapping but distinct profile changes.
Commercial Relationships: Subramanian Dharmarajan, None; Teri BeleckyAdams, None; Nader Sheibani, None
Support: NIH 1R01EY019525-01
Program Number: 2014 Poster Board Number: D858
Presentation Time: 1:45 PM - 3:30 PM
Protein Kinase CK2 Inhibition Affects Actin-Myosin Cytoskeleton In
Cultured Human Astrocytes And Vascular Endothelial Cells
Andrei A. Kramerov1A, Alexander V. Ljubimov1B,2. AOphthalmology Research,
Ophthalmology Research Lab, 1Cedars-Sinai Medical Center, Los Angeles, CA;
David Geffen School of Medicine at UCLA, Los Angeles, CA.
Purpose: Protein kinase CK2 participates in the regulation of cellular morphology
and migration, and could be an important angiogenesis mediator. CK2 localized in
the retinal tissue mostly in astrocytes and vascular endothelial cells (EC), and CK2
inhibitors reduced retinal neovascularization and stem cell recruitment in the mouse
model of retinopathy. Recently, we have co-localized CK2 and F-actin in
contractile stress fibers in microvascular EC. The purpose was to examine a
possible role of CK2 in the regulation of actin-myosin-based contractility in
astrocytes and EC.
Methods: Cultured human astrocytes and vascular EC were treated by specific
inhibitors of CK2 (TBB, TBCA) alone, or in combination with inhibitors of Rhokinase (hydroxyfasudil) or myosin light chain (MLC)-kinase (ML7). After 1-18 hr
of treatment, cultured cells were fixed in 4% formaldehyde, permeabilized in 0.1%
Triton X100 and incubated with mouse anti-CK2 antibody followed by secondary
antibodies conjugated with fluorescein, or phalloidin conjugated with rhodamine.
For Western blotting of the cultured cell extracts, rabbit antibodies to phosphoSer19-MLC were used.
Results: We found that CK2 inhibitors caused disassembly of actin-myosin stress
fibers and cell shape changes, including cytoplasmic retraction and generation of
processes. Stress fiber formation is mainly regulated by phosphorylation of MLC
that leads to an increase in myosin II activity, which cross-links actin filaments and
generates contractile force. Interestingly, we discovered that low doses of inhibitors
of Rho-kinase and MLC-kinase that both phosphorylate MLC, potentiated the
effect on cell shape of suboptimal CK2 inhibition. Such striking morphological
effect of the combined action of the protein kinase inhibitors was accompanied by
decreased level of phospho-MLC, thus implicating CK2 in the regulation of actin-
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
myosin cytoskeleton.
Conclusions: These results suggest an important role of CK2 in control of
contractility and cell motility, which may account for suppressing effect of CK2
inhibition on retinal neovascularization in the mouse retinopathy model.
Commercial Relationships: Andrei A. Kramerov, None; Alexander V.
Ljubimov, None
Support: NIH Grant R01 EY13431, OneSight Research Foundation, Eye Defects
Research Foundation
active CNV. Thus, RAC exosomes may represent a novel treatment for CNV in
Commercial Relationships: Amir R. Hajrasouliha, None; Guomin Jiang,
None; Huayi Lü, None; Wei Wang, None; Henry J. Kaplan, None; Hui Shao,
Support: Supported in part by NIH grants NEI EY12974 (HS), Research to
Prevent Blindness (RPB) Lew R. Wasserman Merit Award (HS), and the
Department's RPB Unrestricted Grant (HK).
Program Number: 2015 Poster Board Number: D859
Presentation Time: 1:45 PM - 3:30 PM
Astroglial Changes Induced By The Absence Of Apolipoprotein E (ApoE) In
Mice Retina
Alberto Trivino1, Blanca Rojas1, Rosa De Hoz1, Roberto Gallego-Pinazo2, Vicente
Andrés3, Maria D. Pinazo-Duran4. 1Inst Invest Oftalmol Ramon Castroviej, Univ
Complutense de Madrid, Madrid, Spain; 2Ophthalmology, University and
Polytechnic Hospital La Fe, Valencia, Spain; 3Centro Nacional de Investigacio
Cardiovascular CNIC, Madrid, Spain; 4Ophthal Research Unit, University Hospital
Dr Peset, Valencia, Spain.
Purpose: To study astroglial changes in the retina of apoE knock-out (apoE-/-)
adult mice, and their relation with the vascular endothelial growth factor (VEGF)
Methods: apoE-/- C57BL/6 mice (n=12) and wild-type counterparts (WT) (n=12)
12 months old were used. Retinal whole mounts were immunostained with
antibodies against GFAP. The VEGF levels were determined by enzyme-linked
immunosorbent assay (ELISA) in the retinal homogenates.
Results: In comparison with naïve, astrocytes in apoE-/- mice showed several
changes: i) they were significantly reduced in number when the analysis was made
both by the whole retina (p<0.001) and by zones of the retina (p<0.003 for the
peripapillary zone and p<0.000 for the intermediate and peripheral zone; ii) their
GFAP immunoreactivity (IR) decreased; iii) they had fewer processes and most cell
bodies and processes were thinner; iv) the astroglial plexus was unrecognizable in
most areas of the retina. GFAP-IR of Müller cells in apoE-/- eyes was increased in
comparison with naïve. VEGF expression was significantly lower (p<0,001) in the
apoE-/- retina than in the WT.
Conclusions: Seeking to provide precise molecular facts that would help in
managing retino-vascular damage and visual impairment, we found that apoE
seems to be essential for retinal astrocyte survival and homeostasis and as a
consequence for neuroprotecion.
Commercial Relationships: Alberto Trivino, None; Blanca Rojas, None; Rosa
De Hoz, None; Roberto Gallego-Pinazo, None; Vicente Andrés, None; Maria D.
Pinazo-Duran, None
Support: None
Program Number: 2017 Poster Board Number: D861
Presentation Time: 1:45 PM - 3:30 PM
The Intracellular Traffic Of Mt1-mmp Is Regulated By α2-macroglobulin/lrp1
System In müLler Glial Cell.
Pablo F. Barcelona, Javier R. Jaldin, Gustavo A. Chiabrando, Maria C. Sanchez.
Bioquimica Clinica, CIBICI-Dpto de Bioq Clinica FCQ UNC, Cordoba, Argentina.
Purpose: Müller cells (MC) are known to undergo functional and morphological
changes related with the matrix metalloproteinases (MMPs) production during
retinal proliferative disorders. Previously, we demonstrated that MC express the
alpha2-Macroglobulin (α2M) receptor, LDL receptor-related protein 1 (LRP1), and
that under α2M treatment, LRP1 regulates the MMP-2 activity and cell migration
on different matrix-protein-coated surfaces, which suggest the participation of
MT1-MMP. Herein, we evaluated whether LRP1 regulates the MT1-MMP function
in MC stimulated by α2M. In addition, we investigated the subcellular distribution
of MT1-MMP and LRP1 and evaluated the cell migration induced by α2M on
different matrix-protein-coated surfaces.
Methods: Transient transfection of the MIO-M1 cells with MT1-MMP-GFP vector
was performed using Lipofectamine 2000. These cells cultured in Dulbecco’s
modified Eagle’s medium (DMEM) were stimulated with α2M (60 nM) for
different times. The cellular distribution of MT1-MMP and LRP1 was examined by
confocal microscopy using GFP-conjugated MT1-MMP, specific antibodies against
LRP1 and intracellular compartments of endocytosis. The molecular association of
MT1-MMP/LRP1 was analyzed by immunoprecipitation (IP). The protein silencing
of LRP1 was performed using siRNA LRP1 with siPORT Neo FX Transfection
Results: MIO-M1 cells, under α2M treatment, showed that LRP1 and MT1-MMP
were mainly co-localized in endosomal compartments characterized as EEA1 early
endosomes. By IP assay we showed a molecular association between MT1-MMP
and LRP1, which was increased by α2M stimulation. Finally, the LRP1 silencing
abrogated the MMP2 activation and cell migration capacity, promoted by α2M, in
this cells.
Conclusions: These data demonstrated that MT1-MMP is associated with LRP1 at
intracellular level in MIO-M1 cells stimulated with α2M, which suggest that this
receptor is regulating the intracellular traffic of MT1-MMP. In addition, the MT1MMP/LRP1 interaction induced by α2M, regulates the MMP-2 activation and
cellular migration of MC.
Commercial Relationships: Pablo F. Barcelona, None; Javier R. Jaldin,
None; Gustavo A. Chiabrando, None; Maria C. Sanchez, None
Support: SeCyT, FONCyT
Program Number: 2016 Poster Board Number: D860
Presentation Time: 1:45 PM - 3:30 PM
Exosomes From Retinal Astroglial Cells (RACs) Suppressed New Vessel
Formation In a Laser-induced CNV Model
Amir R. Hajrasouliha, Guomin Jiang, Huayi Lü, Wei Wang, Henry J. Kaplan, Hui
Shao. Ophthalmalogy & Visual Sciences, Kentucky Lions Eye Center, University
of Louisville, Louisville, KY.
Purpose: Choriodal neovascularization (CNV) is a devastating complication of
age-related macular degeneration (AMD), a leading cause of blindness in the
developed world. In this study, we tested whether the exosomes released from
normal tissue cells is an alternative and better therapy for CNV.
Methods: CNV in C57BL/B6 (B6) mice was induced by 4 laser spots (50um
diameter at 250mV, 0.5s energy) around the optic disc. Exosomes released from
cultured murine retinal astroglial cells (RAC), RPE, fibroblasts or dendritic cells
(DC) were isolated. Mice with CNV were injected periocularly (in sub-Tenon’s
space) with PBS or derived exosomes daily starting on day 0. On day 7 mice were
perfused with PBS containing 50 mg/ml fluorescein-labeled dextran (FITCdextran). The incidence of active CNV was observed in vivo by fluorescence
microscopy. The incidence and area of new vessel formation in the choroid was
determined in choroid flat mounts using fluorescent microscopy. Macrophage
infiltration into CNV lesions was evaluated by immunostaining with F4/80 Ab.
Results: Exosomes were detected in the retina 15 min after peri-ocular injection.
Active CNV was completely inhibited after treatment with RAC exosomes
compared to 42% incidence in PBS-treated mice. On choroid flat mounts incidence
of CNV was observed in 75% of control laser spots versus 40% in RAC-exosome
treated group. In addition, RAC exosome-treated mice had 83% less area of new
vessels in the choroid than those in PBS-treated mice. Macrophage infiltration was
also significantly reduced in RAC exosome-treated group compared to control (27
± 4 vs. 60 ± 13 macrophage per laser spot, respectively, P<0.05). Exosomes derived
from RACs had the strongest treatment efficacy compared to RPE, fibroblast and
DC derived exosomes.
Conclusions: RAC exosomes contain anti-angiogenic molecules that can inhibit
Program Number: 2018 Poster Board Number: D862
Presentation Time: 1:45 PM - 3:30 PM
N-methyl-d-aspartate (NMDA)-induced Excitotoxicity After Gliotoxin
Treatment In Mice
Akira Takamiya1, Youngseok Song 1, Akitoshi Yoshida2. 1Ophthalmology,
Asahikawa Medical Unversity, Asahiawa, Japan; 2Ophthalmology, Asahikawa
Medical University, Asahikawa, Japan.
Purpose: It is well known that gliotoxin-alpha amino-adipic acid (AAA), which is
the glutamate analogue, selectively enters astroglial cells and causes gliotoxicity for
the Muller glial cells and astrocytes in the retina. To determine whether the
treatment of AAA leads to suppress retinal neuronal cell death by inhibition of the
astroglial activity, adult wild type (WT) mice were examined in an experimental
model of excitotoxic neural damage.
Methods: Retinal neural damage was induced in adult WT mice by intravitreal
injection of N-methyl-D-aspartate (NMDA) (50 nmol) 2 days after gliotoxin or
PBS treatment. Retinal cell death was determined by TDT-mediated-dUTP nick
end labeling (TUNEL) at day 1 after NMDA treatment. At day 7 after the
treatment, survival retinal ganglion cells were immunostained with a primary
antibody against a ganglion cell specific marker, beta-III tubulin and the number of
cells was counted.
Results: At day 1 after NMDA treatment (at day 3 after AAA or PBS treatment),
more TUNEL+ cells were counted from retinal sections of PBS pretreated mice
than that of AAA treated mice. There were few TUNEL+ cells in AAA or PBS
treatment alone. At day 7 after NMDA treatment, more ganglion cells survived in
the retinas of AAA treated mice than that in PBS treated mice.
Conclusions: Our results suggest that AAA treatment may induce to protect the
retinal neuronal cells from excitotoxic neural injury by inhibition of the activity of
Muller glial cells and astroglial cells.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: Akira Takamiya, None; Youngseok Song ,
None; Akitoshi Yoshida, None
Support: Japan Society for the Promotion of Science KAKENHI 21791661 (grantin Aid for young Scientists A)
Program Number: 2019 Poster Board Number: D863
Presentation Time: 1:45 PM - 3:30 PM
Berberine Acts As A Novel Autophagy Blocker To Protect Human müLler
Cell From 4-hne Induced Cell Death
SHIHE YANG1A, Mingyuan Wu1A, Dongxu Fu1A,2, Junping Chen1A, Jing zhang1A,
Kenneth Wilson1A, Michael Elliot1B, Mei Du1A, Timothy Lyons1A. AHarold Hamm
Diabetes Center and Section of Endocrinology and Diabetes, Department of
Medicine, BDean McGee Eye Institute, 1University of Oklahoma Health Sciences
Center, Oklahoma City, OK; 2Department of Immunology, Harbin Medical
University, Harbin, Heilongjiang Province, China.
Purpose: 4-Hydroxynonenal (4-HNE, a component of oxidized lipoproteins) may
mediate retinal injury and promote diabetic retinopathy (DR). Berberine (BBR) has
favorable effects on glucose and lipid metabolism in animal and human clinical
studies, but its effects on retinal cells exposed to modified lipoproteins (as occurs in
DR) are unknown. Using 4-HNE to model effects of oxidized lipids and
lipoproteins on retinal cells in DR, we investigated the potential protective effects
of BBR.
Methods: Confluent human retinal Müller cells were exposed to 4-HNE (5, 10, 20,
or 40µM) for various periods (1h, 3h, 6h, 12h, or 24h) with/without BBR
pretreatment (1, 5, 10, or 20µM for 1h). Cell viability was
detected by CCK-8 assay. To investigate mechanisms of the potential protective
effects of BBR, cells were pretreated with BBR (5µM, 1h) prior to 4-HNE
treatment. Indices of autophagy and apoptosis were measured by western blot and
immunocytochemistry (ICC).
Results: 4-HNE induced dose- and time-dependent cell death (CCK-8 assay, n=3,
p<0.001), which was partially attenuated by BBR pretreatment. Western blot and
ICC demonstrated that 4-HNE induced markers of autophagy (LC3B) and
apoptosis (PARP, BAX/BCL2 ratio, and thereafter, increased TUNEL positive
cells). Pretreatment with BBR attenuated the over-expression of LC3B induced by
4-HNE. Such attenuation was also observed in cells pretreated with 3Methyladenine (3-MA, an inhibitor of autophagy, as positive control) (5mM, 2hr).
However, pretreatment with BBR did not inhibit apoptotic markers induced by 4HNE.
Conclusions: 4-HNE induced human retinal Müller cell death through both
autophagy and apoptosis pathways. BBR attenuated autophagy, but had no effect
on apoptosis. BBR may act as a novel inhibitor of autophagy, and has potential to
prevent Müller cell death and inhibit DR.
Commercial Relationships: SHIHE Yang, None; Mingyuan Wu,
None; Dongxu Fu, None; Junping Chen, None; Jing zhang, None; Kenneth
Wilson, None; Michael Elliot, None; Mei Du, None; Timothy Lyons, None
Support: NIH Grant 3P20RR024215-03S109; OCASTHR08-67
Program Number: 2020 Poster Board Number: D864
Presentation Time: 1:45 PM - 3:30 PM
Quantification Of Retinal Astrocytes Using A Novel Method For Objective
Cell Counting In Retinal Flatmounts
Felicitas Bucher, Sebastian Maier, Marc Leinweber, Andreas Stahl, Gottfried
Martin, Hansjürgen T. Agostini. University Eye hospital Freiburg, Freiburg,
Purpose: Quantification of cells and changes in cell number in retinal flatmounts is
an important method in basic and translational research. Unblinded and subjective
image analysis, however, may yield biased counting results. Using the example of
astrocytic density in the murine retina we demonstrate a novel method for valid and
objective cell quantification avoiding user-dependent selection bias.
Methods: Mice expressing histone-bound GFP under the Pdgfra promoter are used
to identify astrocytic nuclei in the retina. Retinal vessels in flatmounts are stained
with an antibody raised against collagen IV. GFP signal is photographed in the
green channel while collagen IV is photographed in the red channel. Images are
evaluated by an automated ImageJ macro (Astro Count Macro) which allows for
blinded selection of regions of interest (ROIs) and a documented counting process.
ROIs are chosen solely on the basis of the collagenIV-stained vasculature in the
murine flatmount without the GFP signal of retinal astrocytes being visible to the
user. The ROIs from the vascular image are then automatically projected onto the
astrocyte image on which the counting process is performed. Finally, the red and
green channels are merged and the counted cells labeled. The numeric results can
easily be exported to spreadsheet calculation programs.
Results: The nuclear GFP signal expressed under the Pdgfra promoter allows
quantitative analysis of retinal astrocytes. Using the Astro Count Macro, we show
that astrocytes are not equally distributed during the physiological development of
the murine retina. The density of astrocytes in the periphery at P7 exceeds their
density in the central retina. The blinded selection of ROIs with the Astro Count
Macro yields user-independent (R²= 0,96) and time-consistent results (R²= 0,98).
Manual and automated counting processes yield highly concordant results
Conclusions: The Astro Count Macro provides an objective and documented cell
counting method which is able to detect differences in retinal cell density. The
automated counting requires reasonably good image quality but is not limited to
astrocytes. The method might therefore be similarly useful for objective analysis of
other labeled retinal cells or structures like ganglion cells, apoptotic cells, and
Commercial Relationships: Felicitas Bucher, None; Sebastian Maier,
None; Marc Leinweber, None; Andreas Stahl, None; Gottfried Martin,
None; Hansjürgen T. Agostini, None
Support: None
Program Number: 2021 Poster Board Number: D865
Presentation Time: 1:45 PM - 3:30 PM
Astrocyte Migration And Vascular Development In The Retina are Regulated
By Laminin-Mediated Signaling Mechanisms
Gopalan Gnanaguru1A,2, Germán A. Pinzón-Duarte1A, Johnny Chew1B, Galina
Bachay1A,2, William J. Brunken3,2. AOphthalmology and Cell Biology,
Ophthalmology, 1SUNY Downstate Med Ctr, Brooklyn, NY; 2SUNY Eye
Institute, Brooklyn, NY; 3Ophthalmology and Cell Biology, SUNY Downstate
Medical Center, Brooklyn, NY.
Purpose: Retinal vascular development is dependent on astrocyte function. Our
aim is to study the mechanism by which basement membrane (BM) molecules
regulate astrocyte migration and subsequent retinal vascular growth.
Methods: Expression of laminin β2 and γ3 chains, integrins and integrin-linked
kinase (ILK) were studied by immunohistrochemistry (IHC). Astrocyte migration
and inhibition assays were studied using ex vivo culture methods. Vascular growth
was analyzed by IHC.
Results: Laminins containing β2 and γ3 chains are important for inner limiting
membrane formation and are differentially expressed in vascular BM. Analysis of
Lamc3 nulls showed slower astrocyte migration, reduced vascular growth
progression and excessive vascular branching. Deletion of Lamb2 and Lamb2:c3
genes severely affected astrocyte migration and distribution, as well as subsequent
vascular growth. Furthermore, in vitro study revealed that laminins act as
haptotactic factors in an isoform specific manner and exogenous addition of
laminins rescued astrocyte migration and spatial patterning in Lamb2:c3 null retina.
Integrin β1 has shown to be critical for cell migration. We found that in the Lamb2
and Lamb2:c3 nulls integrin β1 expression was specifically affected in astrocytes.
To test if laminin-integrin β1 interactions regulate astrocyte migration, we analyzed
the effect of integrin β1 antibody on ex-vivo WT retinal explants cultured on EHSLaminin coated coverslips. Anti-integrin β1 treatment affected both astrocyte
migration and cytoskeletal organization. This result demonstrates that laminins
regulate astrocyte migration via β1 integrins. Because integrin β1-BM interactions
activate integrin-linked kinase (ILK), which further activates cdc42 and Rac-1
promoting cytoskeletal reorganization and migration. We are currently
investigating if these BM mediated intracellular signaling events are affected in
laminin nulls. Our preliminary results demonstrate that WT astrocytes express ILK.
In contrast, ILK protein was absent from Lamb2 null astrocytes demonstrating a
defect in coupling of BM-integrin β1 mediated signaling during migration.
Conclusions: These data demonstrate that laminins containing β2 and γ3 chains
regulate astrocyte migration through an integrin β1 receptor-mediated mechanism.
Deletion of these laminin chains affects localization of integrin β1 receptors and
recruitment of ILK during astrocyte migration, which is critical for subsequent
Commercial Relationships: Gopalan Gnanaguru, None; Germán A. PinzónDuarte, None; Johnny Chew, None; Galina Bachay, None; William J. Brunken,
Support: EY12676, U54 HD071594
Program Number: 2022 Poster Board Number: D866
Presentation Time: 1:45 PM - 3:30 PM
Immunological Activation of Astrocytes by Deiminated Myelin Basic Protein
Mabel E. Algeciras1A, Di Ding1B, Sanjoy K. Bhattacharya1A, Horacio M. Serra2.
Ophthalmology, BBiochemistry, 1Univ of Miami/Bascom Palmer, Miami, FL;
Bioquimica Clinica, CIBICI, Fac ultad de Cs Quimicas UNC, Cordoba, Argentina.
Purpose: To determine whether astrocytes from lamina cribrosa region are
biochemically activated by peptide products of deiminated myelin basic protein
(MBP), resulting in expression of immune molecules and thus imparting the
capacity to present antigens to activate T cells.
Methods: We obtained deiminated and non-deiminated synthetic myelin basic
protein peptides, and subjected isolated lamina cribrosa astrocytes obtained from
C57BL/6J mice (about 3000 per plate for 24 hours) to biochemical stimulation with
either deiminated or non-deiminated peptide (control). Astrocyte activation was
measured by overexpression of morphological and other established activity
markers such as ALDH1-L1, S-100β and Aquaporin 4.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Immunological activation was assessed in terms of expression of the co-stimulatory
molecules B7-1 (CD80) and B7-2 (CD86), since these molecules are required for
activation of naïve T-cells. MHC class I and II expression was examined upon
priming of T-cells with non-activated and activated astrocytes. Animals were used
adhering to ARVO statement for utilization of animals in vision research.
Results: Expression of co-stimulatory and MHC class II molecules was
upregulated as detected by immunohistochemistry, ELISA and Western blot
analyses in activated astrocytes but not in control (non-activated) astrocytes derived
from C57BL/6J mice.
Conclusions: Biochemical stimulation of astrocytes by deiminated MBP peptides
increases their immunologic activation and antigen presentation capacity to T cells.
Commercial Relationships: Mabel E. Algeciras, None; Di Ding, None; Sanjoy
K. Bhattacharya, None; Horacio M. Serra, None
Support: RPB Career award (SKB), an unrestricted grant from RPB to University
of Miami, NIH grant P30EY014801.
Program Number: 2023 Poster Board Number: D867
Presentation Time: 1:45 PM - 3:30 PM
Intravitreal Bevacizumab Activates Müller Cells And Astrocytes And Reduces
Carbonic Anhydrase In Retinas Of Monkeys: A Possible Explanation For The
Reduction Of Retinal Edema
Sylvie Julien, Ulrich Schraermeyer. Experimental Vitreoretinal Surgery, Centre for
Ophthalmology, Tuebingen, Germany.
Purpose: To examine the effect of intravitreal bevacizumab on primate eyes with
particular focus on choroidal and retinal vessels.
Methods: Five cynomolgus monkeys received an intravitreal injection of 1.25 mg
bevacizumab with or without tritium labeling. The eyes were enucleated on days 1,
4, 7 and 14 and prepared for light (left eyes) and electron microscopy (right eyes).
Control eyes from 6 monkeys remained untreated. Bevacizumab, GFAP, Vimentin,
Iba1, CD32 and carbonic anhydrase were localized by immunocytochemistry.
Bevacizumab was additionally localized by autoradiography. Thrombocytes and
fibrin were stained by histochemistry. The diameter of the choriocapillaris was
measured by morphometry.
Results: Bevacizumab has a very strong immunoreactivity and totally or partially
filled the sectioned lumen of many blood vessels. Autoradiography showed
localization of bevacizumab at the inner sides of retinal vessel walls. At the
electron microscopical level, thrombocytes and granulocytes within the retinal
vessels exhibited typical signs of activation such as pseudopodia, vacuoles and
degranulation. Electron-dense granules resembling serotonin granules released
from thrombocytes were present in the bloodstream and endothelium of most
vessels. As detected by electron microscopy, felt-like structures (several µm thick)
were attached to the inner sides of retinal veins and interacted with blood cells.
Microthrombi were frequently observed, one retinal central vein was thrombotic.
The diameter of the choriocapillaris was significantly reduced in all treated eyes
compared to the control. Müller cells, astrocytes and microglia were activated,
FcgammaRIIa receptors were upregulated on the cell membranes of photoreceptors
and apical RPE, the ILM was detached and carbonic anhydrase IX was downregulated after bevacizumab injection.
Conclusions: Bevacizumab has several unexpected effects on the monkey retina. It
interferes with retinal and choroidal blood flow. Carbonic anhydrase activity is
known to be involved in retinal fluid absorption. Therefore, reduction of carbonic
anhydrase activity and activation of Müller cells may cause removal of retinal
edema in human retinas. Bevacizumab is known to interact with FcgammaRIIa
receptors on thrombocytes that are lacking in rodents. Therefore monkeys but not
rodents are relevant models for preclinical studies with intravitreal full length
antibodies or VEGF traps.
Commercial Relationships: Sylvie Julien, None; Ulrich Schraermeyer, None
Support: None
Program Number: 2024 Poster Board Number: D868
Presentation Time: 1:45 PM - 3:30 PM
Glia Alterations In An Experimental Autoimmune Glaucoma Model
Rozina Noristani1, Sandra Kuehn1, Mathias Kuehn1, Jennifer E. Schiwek2, Franz H.
Grus2, Burkhard Dick1, Stephanie C. Joachim1. 1Experimental Eye Research
Institute, Ruhr University, Bochum, Germany; 2Experimental Ophthalmology,
University Medicine, Mainz, Germany.
Purpose: Under pathological conditions glia cell invasion is associated with
neuronal decline. Over the past decades evidences obtained from experimental
studies supported the role of auto-antibodies in glaucoma which cause retinal
ganglion cell (RGC) loss and glia activation. The aim was to examine if glia
activity changes in retina after immunization with S100 calcium-binding protein
and optic nerve homogenate (ONA).
Methods: Rats were immunized with S100 and ONA in Freund’s Adjuvant and
Pertussis Toxin (n=6). Both groups were compared with NaCl as a control group
(n=6). After 28 days astrocytes and RGC density was quantified in retinal crosssections using glia fibrillary acidic protein (GFAP, Millipore) and brain specific
homeobox (Brn-3a, Santa Cruz) in double staining to investigate gliosis and loss of
RGC at once. Photos were taken in two central and two peripheral areas of four
sections per eye. GFAP intensity was scored ranging from 0 (no signal) to 3 (severe
signal) and a group comparison was performed using student t-test. Additionally,
immunostaining with anti-Iba1 and anti-ED1 was performed to gain further
knowledge about the role of activated microglia in retina.
Results: A significant lower number of RGCs was detectable in ONA and S100
group after 28 days (S100: p=0.005; ONA: p=0.0005). Findings of glia cells in
ONA immunized rats revealed a massive invasion of glia compared to control
group. A significant difference regarding GFAP staining intensity could be
observed between the ONA (mean score 2.3±0.70) and the control group (1.8±0.67,
p=0.000001). The mean GFAP score in the S100 group was not significantly higher
than in control group and stayed within the normal range (p=0.7).
Conclusions: Therefore, we conclude that the immunization with certain ocular
antigens such as ONA causes gliosis which is engaged in eliciting RGC death in
this model. Other antigens like S100 lead to RGC loss without significant gliosis.
The severity of gliosis seems to be highly antigen-dependent. These findings
indicate that the immunization with ONA causes a lower RGC density which is
linked to proliferation and invasion of glia in this model.
Commercial Relationships: Rozina Noristani, None; Sandra Kuehn,
None; Mathias Kuehn, None; Jennifer E. Schiwek, None; Franz H. Grus,
None; Burkhard Dick, None; Stephanie C. Joachim, None
Support: German Research Foundation (DFG JO-886/1-1), FoRUM Program
Program Number: 2025 Poster Board Number: D869
Presentation Time: 1:45 PM - 3:30 PM
IOP Induces Changes In The GFAP-Labelled Retinal Area But Not In
Astrocyte Number In Mice Retina Contralateral To Experimental Glaucoma
Jose-Manuel Ramirez1, Rosa De Hoz1, Juan J. Salazar1, Ana I. Ramirez1, Blanca
Rojas1, Beatriz I. Gallego1, Manuel Salinas-Navarro2, Arturo Ortin-Martinez2,
Maria P. Villegas-Perez2, Alberto Trivino1. 1Instit Invest Oftalmol Ramon
Castroviejo, Universidad Complutense de Madrid, Madrid, Spain; 2Facultad de
Med-Ophthal, University of Murcia, Espinardo, Murcia, Spain.
Purpose: To analyse the effects of laser-induced ocular hypertension (OHT) in the
astrocytes of contralateral eyes two weeks after lasering.
Methods: Adult Swiss mice were divided into two groups: naïve (n=6) and lasered
(n=6). Retinal whole-mounts were immunostained with antibodies against GFAP.
The GFAP-labelled retinal area (GFAP-RA) and the number of astrocytes were
Results: In comparison with naïve: i) astrocytes were more robust in contralateral
eyes and had a less intense GFAP immunoreaction (GFAP-IR) with fewer
secondary processes in OHT-eyes; ii) GFAP-RA increased in contralateral eyes
(p<0.032) and decreased in OHT-eyes (p<0.001); iii) there were no differences in
astrocyte number in contralateral or OHT-eyes. However, 57.02% (5523 of 9686)
of the astrocytes in OHT-eyes had weaker GFAP-IR and fewer secondary processes
than did naïve eyes.
Conclusions: Two weeks of laser-induced OHT caused changesin the GFAPlabelled retinal area but not in astrocyte number in both contralateral and OHTeyes. On the basis of the astroglial changes detected in the present work, the use of
the contralateral eye as an internal control in experimental model unilateral OHT
should be reconsidered.
Commercial Relationships: Jose-Manuel Ramirez, None; Rosa De Hoz,
None; Juan J. Salazar, None; Ana I. Ramirez, None; Blanca Rojas,
None; Beatriz I. Gallego, None; Manuel Salinas-Navarro, None; Arturo OrtinMartinez, None; Maria P. Villegas-Perez, None; Alberto Trivino, None
Support: Grant ISCIII RD07/0062/0000 and RD07/0062/0001; FMM Grant
4131173; Santander-UCM (GR58/08; GR35/10-A); Grant Regional Government of
Murcia Fundación Séneca 04446/GERM/07
Program Number: 2026 Poster Board Number: D870
Presentation Time: 1:45 PM - 3:30 PM
Microglial Population In Contralateral Mice Retina To Experimental
Glaucoma: A Qualitative And Quantitative Study
Blanca Rojas1, Juan J. Salazar1, Ana I. Ramirez1, Rosa De Hoz1, Beatriz I.
Gallego1, Manuel Salinas-Navarro2, Luis Alarcon-Martinez3, Manuel Vidal-Sanz3,
Alberto Trivino1, Jose-Manuel Ramirez1. 1Inves Oftalmol Ramon Castroviejo, Univ
Complutense de Madrid, Madrid, Spain; 2Facultad de Med-Ophthal, University of
Murcia, Espinardo, Murcia, Spain; 3University of Murcia, Murcia, Spain.
Purpose: To analyse the qualitative and quantitative changes of laser-induced
ocular hypertension (OHT) in microglial population of eyes with OHT (OHT-eyes)
and contralateral eyes two weeks after lasering.
Methods: Adult albino Swiss mice were divided in two groups: naive; (agematched control; n=6) and lasered (n= 6). Retinal whole-mounts were
immunostained with anti-Iba1, and the morphology, density, and distribution of
Iba1+ microglial cells were analysed.
Results: In the three study groups, Iba1+ microglias were observed throughout the
retina, distributed in the subretinal space (SS), outer plexiform layer (OPL), inner
plexiform layer (IPL), ganglion-cell layer (GCL) and nerve-fibre layer (NFL). In
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
naïve eyes, Iba1+ microglias in the OPL and IPL exhibited a branched morphology
emanating from small cell bodies. By contrast, Iba1+ microglias in the SS had
rounded bodies with fewer and shorter processes; in the NFL and in the GCL,
Iba1+ microglias were less ramified and were distributed parallel to axons of the
retinal ganglion cells. Iba1+ cells in contralateral and OHT-eyes exhibited
morphological features that differed from naïve:i) Iba1+ microglial bodies were
more robust; ii) Iba1+ microglial processes were thicker and more branched in
contralateral eyes and thicker and retracted in OHT-eyes. The number of Iba1+
microglias in OHT-eyes and contralateral untreated eyes increased in comparison to
naïve (p< 0.001 and p< 0.041 respectively).
Conclusions: Two weeks of laser induced-OHT triggered microglial retinal
changes in the contralateral untreated and OHT-eyes. The microglial behaviour in
contralateral untreated eyes, lead us to suggest that the use of contralateral eyes as
internal controls in experimental unilateral OHT, should be reconsidered.
Commercial Relationships: Blanca Rojas, None; Juan J. Salazar, None; Ana I.
Ramirez, None; Rosa De Hoz, None; Beatriz I. Gallego, None; Manuel SalinasNavarro, None; Luis Alarcon-Martinez, None; Manuel Vidal-Sanz,
None; Alberto Trivino, None; Jose-Manuel Ramirez, None
Support: Grant ISCIII RD07/0062/0000 and RD07/0062/0001; FMM Grant
4131173; Santander-UCM (GR58/08; GR35/10-A); Grant Regional Government of
Murcia Fundación Séneca 04446/GERM/07
Program Number: 2027 Poster Board Number: D871
Presentation Time: 1:45 PM - 3:30 PM
IOP Induces MHC-II Overexpression In Microglia And Macroglia In
Contralateral Mice Retina To Experimental Glaucoma
Beatriz I. Gallego1, Ana I. Ramirez1, Rosa De Hoz1, Juan J. Salazar1, Blanca
Rojas1, Alberto Trivino1, F Javier Valiente-Soriano2, Manuel Salinas-Navarro2,
Marcelino Aviles-Trigueros2, Jose-Manuel Ramirez1. 1Inves Oftalmol Ramon
Castroviejo, Univ Complutense de Madrid, Madrid, Spain; 2Ophthalmology,
Universidad de Murcia, Murcia, Spain.
Purpose: To analyze the effects of laser-induced ocular hypertension (OHT) in
MHC-II expression in the macro- and microglia of eyes with OHT (OHT-eyes) and
contralateral eyes two weeks after lasering.
Methods: Adult Swiss mice were divided into two groups: naïve (n=6) and lasered
(n=6). Retinal wholemounts were immunostained with antibodies against GFAP,
Iba-1 and MHC-II.
Results: In the naïve retinas, weak constitutive MHC-II expression was scarcely
found in some Iba-1+ microglial cells and rarely in GFAP+ astrocytes. Only a
small dendritiform subpopulation of Iba-1+ cells, located in the juxtapapillary area
and in the marginal region of the retina, had a strong MHC-II immunoreaction. In
comparison to naïve results, MHC-II in the contralateral eye was expressed in
Müller cells and was overexpressed in Iba-1+ cells- and astrocytes. Thus, in
contralateral macroglia, MHC-II was preferentially expressed by astrocytes. In
OHT-eyes, Iba-1+ cells showed MHC-II immunoreactivity similar to contralateral
and no MHC-II astrocytes were observed; however, MHC-II expression was
upregulated in several groups of Müller cells throughout the retina and was
preferentially located in the end-foot of the cells.
Conclusions: Two weeks of laser-induced OHT caused macro- and microglial
retinal changes in MHC-II expression both in contralateral and in OHT-eyes. Our
results suggest that the gliotic behaviour in contralateral untreated eyes could be
related to the immune response. On the basis of the glial changes observed, the use
of the contralateral eye as a control in experimental unilateral OHT should be
Commercial Relationships: Beatriz I. Gallego, None; Ana I. Ramirez,
None; Rosa De Hoz, None; Juan J. Salazar, None; Blanca Rojas, None; Alberto
Trivino, None; F Javier Valiente-Soriano, None; Manuel Salinas-Navarro,
None; Marcelino Aviles-Trigueros, None; Jose-Manuel Ramirez, None
Support: Grant ISCIII RD07/0062/0000 and RD07/0062/0001; FMM Grant
4131173; Santander-UCM (GR58/08; GR35/10-A); Grant Regional Government of
Murcia Fundación Séneca 04446/GERM/07
291 Fetal and Neonatal Retinal Angiogenesis
Monday, May 7, 2012, 3:45 PM - 5:30 PM
Hall B/C Poster Session
Program #/Board # Range: 2527-2558/D905-D936
Organizing Section: Retinal Cell Biology
Program Number: 2527 Poster Board Number: D905
Presentation Time: 3:45 PM - 5:30 PM
Prolactin In Milk Promotes The Regression Of Hyaloid Vasculature By
Increasing Intraocular Vasoinhibins
Zulma Duenas1, Gonzalo Martinez de la Escalera2, Carmen Clapp3. 1Physiological
Sciences, Universidad Nacional de Colombia, Bogota D.C., Colombia; 2Instituto de
Neurobiologia, Universidad Nacional Autonoma de Mexico, Queretaro, Mexico;
Instituto de Neurobiologia, Universidad Nacional Autonoma de Mexico,
Juriquilla, Querétaro., Mexico.
Purpose: The hormone prolactin (PRL) is present in milk from which it reaches the
circulation of the offspring. Ocular tissues incorporate systemic PRL and
proteolytically process it to vasoinhibins, a family of peptides with antiangiogenic
properties that promote the apoptosis-mediated regression of the ocular hyaloid
vessels. Here, we investigate whether a deficiency of PRL in milk reduces the
levels of intraocular vasoinhibins and the apoptosis of the hyaloid vasculature in
neonatal rats.
Methods: On postpartum days 4 to 18, lactating rats were injected with
bromocriptine, an inhibitor of pituitary PRL secretion, with bromocriptine and
ovine PRL (delivered via an osmotic minipump), or with vehicle. Serum PRL
levels in lactating mothers and pups were measured by the Nb2 cell-bioassay.
Apoptosis and vasoinhibin levels were measured by ELISA and Western blot,
respectively, in pups’ eye extracts.
Results: Bromocriptine reduced serum PRL by 75% in mothers and pups and was
associated with a significant decrease of offspring body weight at postnatal days
(PND) 16 and 18. Treating the bromocriptine-injected mothers with PRL increased
by two-fold the circulating levels of the hormone in both mothers and pups, and it
reversed the loss of offspring body weight. Moreover, bromocriptine lowered the
levels of retinal vasoinhibins in the nursing young. Consistent with the expected
times for the apoptosis of the ocular hyaloid vasculature, eye apoptosis increased
three-fold between PND 8 and 12, and declined on PND 18. Bromocriptine
significantly reduced ocular apoptosis on PND 8 and 12, and this reduction was
prevented by exogenous PRL.
Conclusions: Lowering PRL in the mother reduces circulating PRL, retinal
vasoinhibins, and ocular apoptosis in the nursing young. These findings are
consistent with PRL in milk entering the circulation of the nursing young and
contributing to the physiological involution of the hyaloid vasculature via its
intraocular conversion to vasoinhibins. These findings could help explain the
observation that breastfeeding protects against retinopathy of prematurity, perhaps
via milk PRL promoting vasoinhibin-mediated regression of the neovessels.
Commercial Relationships: Zulma Duenas, None; Gonzalo Martinez de la
Escalera, None; Carmen Clapp, None
Support: CONACYT 161594 (CC) and PLISSER /UNAL (ZD)
Program Number: 2528 Poster Board Number: D906
Presentation Time: 3:45 PM - 5:30 PM
Mixed Arterial-Venous PO2 Levels Critical to Ocular Markers of
Angiogenesis and Oxidative Stress in Neonatal Rats
William J. Brunken1A,2, Charles L. Cai1B, Dharmendra Kumar1B, Gloria B.
Valencia1B, Jacob V. Aranda1C,2, Kay D. Beharry3,2A. AOphthalmology and Cell
Biology, BPediatrics/Neonatology, CPediatrics/Neonatology and Ophthalmology,
SUNY Downstate Medical Center, Brooklyn, NY; APediatrics/Neonatology and
Ophthalmology, 2SUNY Eye Institute, Brooklyn, NY; 3Pediatrics/Neonatology,
University of California, Irvine, Irvine, CA.
Purpose: Aberrant angiogenesis and oxidative stress are hallmarks of oxygeninduced retinopathy (OIR). Incremental hyperoxia and the range of PO2 that affects
ocular angiogenesis and oxidative stress are unknown. We tested the hypothesis
that there is a critical threshold of inspired O2 that influences ocular biomarkers of
angiogenesis and oxidative stress in the newborn rat.
Methods: Within 2-6 hours of birth, 10 groups of rat pups (n=16 pups/group) were
exposed to stepwise increases in inspired O2 (10%, 21%, 30%, 40%, 50%, 60%,
70%, 80%, 90% or 100%) for 2 hours. Immediately following exposure, the pups
were euthanatized and mixed arterial-venous blood gases were determined. Levels
of VEGF, sVEGFR-1 (an endogenous VEGF trap), IGF-I, and 8-iso-PGF2α (a
reliable marker for oxidative stress) were determined using ELISA.
Results: Birth weights were comparable among the groups. Mixed venous-arterial
PO2 (mmHg) increased with increasing hyperoxia from 35.6±5.0 at 10% O2
(range: 31.5-39.8) to 108.5±25.0 at 100% O2 (range: 82.2-134.8). Mixed venousarterial PO2 at 21% O2 was 42.4±7.3 (range: 36.8-48.1). At 10% O2, VEGF levels
(111.5±22.0 pg/mg protein) were significantly higher than that at 21% (41.7±7.2,
p<0.01), 30% (43.3±5.8, p<0.05), 80% (45.2±17.0, p<0.05), 90% (49.8±11.0,
p<0.05), and 100% (46.8±8.3, p<0.05). A non-significant decrease in sVEGFR-1
was noted at 70% to 100% (92.4±11.7 to 94.6±14.0 pg/mg protein) vs. 10% to 60%
(range: 136±37.5 to 239.8±48.1). IGF-I levels were significantly higher at 70%
(385.5±64.2 ng/mg protein) than 80% (169.3±11.1, p<0.05), 90% (174.1±17.3,
p<0.05) and 100% (144.0±27.2, p<0.01). Similar increases in 8-iso-PGF2α
occurred at 70% (671.7±110.5 pg/mg protein, p<0.05), 80% (708.4±106.4, p<0.05),
90% (689.4±92.8, p<0.05), and 100% (672.9±±79.2, p<0.05) vs. 21%
(290.7±40.5). Linear regression analysis revealed a significant correlation between
PO2 and 8-isoPGF2α (r=0.26; p<0.001). Data are mean±SEM.
Conclusions: Data suggests that a critical threshold of inspired O2 exerts adverse
effects on ocular biomarkers for angiogenesis and oxidative stress. Exposure of the
immature eyes to >70% O2 for at least 2 hours may lead to pathogenic
Commercial Relationships: William J. Brunken, None; Charles L. Cai,
None; Dharmendra Kumar, None; Gloria B. Valencia, None; Jacob V. Aranda,
None; Kay D. Beharry, None
Support: U54 HD071594
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2529 Poster Board Number: D907
Presentation Time: 3:45 PM - 5:30 PM
Effect of Breast Milk Nutrition on a Rat Model of Oxygen-induced
Michiko Matsubara1A, Yuta Saito1A, Takako Nakanishi-Ueda1B, Junkichi
Kabayama1A, Toshihiko Ueda1A, Yoshihiro Wada1A, Tadashi Hisamitsu1B, Ryohei
Koide1A. AOphthalmology, BPhysiology, 1Showa Univ Sch of Medicine, Tokyo,
Purpose: To investigate the effects of abundant breast milk intake on body weight
gain, retinopathy, concentration of vascular endothelial growth factor (VEGF) in
retina and concentration of insulin-like growth factor-1 (IGF-1) in serum in a rat
model of oxygen-induced retinopathy (OIR).
Methods: Neonatal Sprague-Dawley rats were divided two groups; one was a
group consisted of seven rat pups (7-rats group) to let them have more amount of
milk intake. And the other was consisted of fourteen rat pups (14-rats group) as a
control. Each group was put in a cage with a mother rat. The rats were exposed to
daily cycles of 80% oxygen (20.5 h), ambient air (0.5 h), and progressive return to
80% oxygen (3 h) from postnatal day 0 (P0) to P12, and then the rats were placed
in ambient air until P18. The mother rats were rotated every two days. Body weight
of rat pups were measured everyday. At P13 and P18, rat pups were sacrificed, and
then blood was collected from the left ventricle. Retinas in left eyes were collected
for ELISA, and retinas in right eyes were fixed, flatmounted and stained with
ADPase. Concentrations of VEGF in retina and of IGF-1 in serum were measured
by ELISA. In the stained retinal flatmounts, retinal neovascularization was scored
(NV score), and avascular areas were measured as a percent of total retinal area
(%AVA) using Image J software (NIH). Statistical analyses were performed with
Mann-Whitney’s U test. P value <0.05 was considered significantly.
Results: The body weight of rats in 7-rats group increased significantly greater
than in 14-rats group from P3. The results were shown at Table 1. At P13, there
was significant difference in concentration of IGF-1 in serum and %AVA. NV did
not occur at P13. At P18, there was significant difference in concentration of VEGF
in retina and IGF-1 in serum.
Conclusions: The more amount of milk intake increased body weight gain,
concentration of VEGF in retina and of IGF-1 in serum significantly but not
affected on retinopathy in the rat model of OIR.
Commercial Relationships: Michiko Matsubara, None; Yuta Saito,
None; Takako Nakanishi-Ueda, None; Junkichi Kabayama, None; Toshihiko
Ueda, None; Yoshihiro Wada, None; Tadashi Hisamitsu, None; Ryohei Koide,
Support: None
Program Number: 2530 Poster Board Number: D908
Presentation Time: 3:45 PM - 5:30 PM
Topical Endoscopic Fundus Imaging in Murine Oxygen-Induced Retinopathy
Michael H. Davies1A, Steven Yeh2, Andrew J. Stempel1B, Kathleen Mohs1B, John F.
Payne2, Hassan T. Rahman2, Michael R. Powers1A, Justine R. Smith1B, Joao M.
Furtado1B. APediatrics and Ophthalmology, BOphthalmology, 1Casey Eye InstituteOHSU, Portland, OR; 2Ophthalmology, Emory Eye Center, Decatur, GA.
Purpose: Murine oxygen-induced retinopathy (OIR) is widely used to investigate
retinal ischemic vasculopathy. However, this model relies on the study of postmortem tissue. We aimed to visualize OIR in vivo using topical endoscopy fundus
imaging (TEFI), and correlate the findings with results of standard
histopathological evaluations.
Methods: OIR was generated by exposing 7-day old (P7) pups to 75% oxygen for
5 days, followed by room air recovery. Controls were exposed to room air only.
TEFI was performed on P12, P15, P17, P21 and P25 mice (n=3-6 mice/group)
under general anesthesia with dilated pupils using a tripod-mounted Nikon D90
camera, a 60 mm F/2.8 D lens, a 58-52 step down ring, a Storz 481C halogen light
and a Storz 1218 tele-otoscope. Retinal images of posterior pole, and where
possible, peripheries, were formatted with Nikon ViewNX2 software, and vascular
tortuosity was graded 0-4 against standard photographs by two masked examiners.
Mice were euthanized immediately following image acquisition. Right retinas were
fixed in 4% PFA, stained with fluorescently tagged isolectin to identify vessels, and
visualized by confocal microscopy. Percentage of retinal vaso-obliteration was
measured by a masked examiner. Left eyes were formalin-fixed, and 5µm paraffin
sections stained with H&E and retinal neovascularization (NV) was quantified.
Results: In agreement with the literature, compared to control mice, hyperoxiaexposed mice suffered retinal vaso-obliteration, peaking at P12 (32.4% vs. 1.5%, p
= 0.0011) and retinal NV, maximal at P17 (23.7 ± 2.6 nuclei/section vs. 0.09 ± 0.03
nuclei/section, p <0.0001). By TEFI, retinal vascular tortuosity was observed in
hyperoxia-exposed mice as early as P15 (p = 0.011). Tortuosity peaked at P17
(Grade 2.75 ± 0.08 vs. Grade 0.06 ± 0.06, p < 0.0001), correlating with retinal NV
observed in H&E histology. In addition, tortuosity was reduced, but still
significantly higher than controls, at P21 and P25 (p<0.005), in concordance with
histological evidence of reduced retinal NV.
Conclusions: We report the clinical observation of retinal vascular tortuosity in
mice, as visualized by TEFI. These data appear to correlate with histological and
whole mount results, demonstrating the validity of this technique. One advantage of
this technique is the potential to image a single animal over the time course of the
Commercial Relationships: Michael H. Davies, None; Steven Yeh,
None; Andrew J. Stempel, None; Kathleen Mohs, None; John F. Payne,
None; Hassan T. Rahman, None; Michael R. Powers, None; Justine R. Smith,
None; Joao M. Furtado, None
Support: NIH Grant EY011548 (MRP) and an unrestricted grant from Research to
Prevent Blindness
Program Number: 2531 Poster Board Number: D909
Presentation Time: 3:45 PM - 5:30 PM
Hyperoxia Causes Regression of Established Vitreous Neovascularization By
Targeting the VEGF/VEGFR-2 Survival Pathway
Steven E. Brooks1A, Wenbo Zhang1B, Hua Liu1B, Zhimin Xu1B, Harumasa Yokota2,
Robert W. Caldwell3, Akitoshi Yoshida2, Ruth B. Caldwell1B. AOphthalmology,
Cellular Biology and Anatomy, 1GHSU/Vascular Biology Center/Vision
Discovery Institute, Augusta, GA; 2Ophthalmology, Asahikawa Medical
University, Asahikawa, Japan; 3Pharmacology and Toxicology, GHSU, Augusta,
Purpose: We have previously shown that hyperoxia treatment (HT) can prevent
vitreous neovascularization (NV) while simultaneously allowing retinal
revascularization. Here we show that HT can selectively target established vitreous
NV in a mouse model of ischemic retinopathy by blocking the VEGF/VEGFR-2,
but not the VEGF/VEGFR-1, survival pathway.
Methods: P17 mice demonstrating vitreous NV in an established mouse model of
ischemic retinopathy were treated with 75% oxygen (HT), VEGF trap, or selective
VEGFR-2 blockers (VEGFR-2 inhibitor or VEGFR-2 siRNA). Intra-retinal and
vitreous NV were assessed in lectin stained retinal whole mounts using confocal
microscopy and image analysis software. Apoptosis was measured by immunolabeling for cleaved caspase-3. The rescue effects of intravitreal VEGF-A, VEGF-E
(specific for VEGFR-2), or PlGF-1 (specific for VEGFR-1) were evaluated in mice
receiving HT. VEGFR-2 expression in NV tufts and vessels was assessed in by
quantitative RT-PCR and immuno-labeling. Macrophages/microglia were identified
by Iba-1 immuno-labeling.
Results: HT reduced vitreous NV by 70% (p<0.0001) within 24 hrs, while
increasing avascular area by only 21% (p<0.05). Robust expression of cleaved
caspase-3 was noted in NV by 12hrs, and was associated with
macrophage/microglial infiltration into NV tufts. Intra-vitreal injection of VEGF-A
(p<0.02) or VEGF-E (p<0.01), but not PlGF-1, prevented the HT-induced
apoptosis. VEGFR-2 expression in vitreous NV was markedly reduced by HT
(p<0.01), but was significantly increased in non-vascular cells (p<0.05). VEGF trap
and VEGFR-2 blockers administered on P17 were also effective in causing NV
regression (p<0.05), though less effectively than HT.
Conclusions: HT selectively targets pathological NV by inhibiting VEGFR-2
activity and expression, causing rapid onset of apoptosis and
macrophage/microglial infiltration. The relatively higher sensitivity to HT of
vitreous NV compared to intraretinal NV may be due to its increased dependence
on VEGFR-2 activity for survival, a characteristic suggested by the relatively high
level of VEGFR-2 expression in pathological NV. This feature may lead to the
discovery of selective anti-angiogenic therapies, including HT, which specifically
target vitreous NV, while permitting or even enhancing physiologic vascular repair
in ischemic retinopathy.
Commercial Relationships: Steven E. Brooks, None; Wenbo Zhang,
None; Hua Liu, None; Zhimin Xu, None; Harumasa Yokota, None; Robert W.
Caldwell, None; Akitoshi Yoshida, None; Ruth B. Caldwell, None
Support: GHSU Vision Discovery Institute, NIH Grants EY11766, EY04618,
HL70215, VA Merit Award, JDRF 10-2009-575, AHA11SDG4960005
Program Number: 2532 Poster Board Number: D910
Presentation Time: 3:45 PM - 5:30 PM
A Descriptive Analysis of Surfactant Proteins in the Mouse Retina
Faizah N. Bhatti1, Genevieve S. Ball2A, Ronald Hobbs2B. 1Pediatric Neonatology
and Ophthalmology, Univ of Oklahoma Health Sciences Center, Oklahoma City,
OK; APediatric Neonatology, BOphthalmology, 2University of Oklahoma Health
Sciences Center, Oklahoma City, OK.
Purpose: In retinopathy of prematurity, research has mainly focused on angiogenic
mediators in hyperoxia/hypoxia and altered nutritional status, with less being
known about the role of inflammatory signals. The surfactant proteins A (SP-A)
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
and D (SP-D) are c-type lectins that interact with cytokines and macrophages and
are well studied especially in lung disease of prematurity. Here, we present the first
study looking at the presence and developmental pattern of SP-A and D in mouse
retinal tissue.
Methods: C57BL/6J (WT) mice were euthanized, and retinas collected at postnatal
day 0 (P0), P2, P5, P7, P14 and 6 weeks of life. SP-A and SP-D localization and
levels were evaluated by immunohistochemistry (IHC) and ELISA (expressed as
ng/ul + SEM with lung tissue for comparison. Results were analyzed via Student's
t-test with a p value of <0.05 significance. The retinas of 6 week old SP-A-/- mice
were also visualized via Optical Coherance Tomography (OCT). Observational
comparisons were made to WT mice.
Results: Retinal SP-A and D expression over the first week of life is similar to the
pattern seen in the lung. In the retina, the SP-A level is 6.6 + 0.6 at P0, decreases to
2.8 + 0.4 at P5 (p <0.001), then increases to 6.7 + 1.2 at P17. By IHC, SP-A first
appears at the outer segment and the inner neuroblastic layers. Over the next week,
it localizes towards retinal blood vessels as well as the developing inner and outer
plexiform layers. In the retina, SP-D at P0 is 1.5 + 0.1 and peaks at P5 to 5.4 + 0.7
(p<0.001), then decreasing to 2.1 + 0.2 in adulthood. IHC of SP-D shows a similar
pattern to SP-A with localization in the inner plexiform, outer plexiform and outer
segment layers in adult mice. OCT and angiography shows that SP-A-/- mice have
significantly reduced retinal thickness as compared to WT mice under basal
Conclusions: SP-A and D are both found in the retina in close proximity to the cell
layers where Mueller cell bodies and retinal blood vessels are in abundance.
Absence of SP-A leads to abnormal retinal development. The mouse pup, at birth,
has blood vessel development approximating that of a 24-week fetus. Therefore
adequate levels of SP-A and SP-D in the early stages of retinal blood development
may be important for maintaining vascular and immune homeostasis in the preterm
retina. This is being further evaluated in our lab.
Commercial Relationships: Faizah N. Bhatti, None; Genevieve S. Ball,
None; Ronald Hobbs, None
Support: NIH COBRE P20 RR017703-10
Program Number: 2533 Poster Board Number: D911
Presentation Time: 3:45 PM - 5:30 PM
MyD88 Mediated TLR2 and TLR4 Activation Leads to Expression of
Surfactant Protein A in the Mouse Retina
Ronald Hobbs1, Saad Munzar2A, Genevieve S. Ball2B, Faizah N. Bhatti2C.
Ophthalmology, Dean McGee Eye Institute, Oklahoma City, OK; APediatrics,
Pediatrics Neonatology, CPediatric Neonatology and Ophthalmology, 2University
of Oklahoma Health Sciences Center, Oklahoma City, OK.
Purpose: We have successfully shown the developmental pattern of Surfactant
Proteins A (SP-A) and D (SP-D) in the mouse retina. These proteins belong to the
superfamily of collectin molecules which are involved in innate host defense. SP-A
activity can be mediated by toll like receptor (TLR) signaling, especially by TLR2
and TLR4. MyD88 is an important intermediary between TLR2/TLR4 activation
and NF B signaling. NF B is a nuclear transcription factor for many collectin
molecules including SP-A. No data currently exists regarding surfactant protein
signaling pathways in the retina. Our objective was to study the in vivo role of
TLR2 and TLR4 activation on retinal SP-A expression using TLR ligands in the
retinas of TLR2-/-, TLR4-/- and MyD88-/- mice.
Methods: For this study 6-10 week old C57BL/6J (WT) and TLR2-/-, TLR4-/- and
MyD88-/- mice were used. Intravitreal injections of the TLR2 ligand PamC3 and the
TLR4 ligand LPS were performed with PBS as control in all the mice. After
euthanasia, retinas were harvested at 6, 12, 18 and 24 hours after injection and SPA levels were measured in whole retinal homogenate by ELISA. Results were
analyzed by Student's t-test with a p value of <0.05 significance.
Results: Intravitreal injection of LPS in WT mice resulted in a fourfold increase in
retinal SP-A levels as compared to control (p value 0.028) with a peak observed at
12 hours, while PamC3 resulted in a two-fold increase of SP-A as compared to
control (p value 0.036) with a peak at 6 hours. In MyD88-/- mice, injection with
LPS and PamC3 led to no increase in SP-A compared to control.
Conclusions: In the mouse retina, TLR2 as well as TLR4 activation leads to
increased expression of SP-A. As this is attenuated in MyD88-/- mice, we
hypothesize that this effect is mediated via TRAF6 and NF B. This needs further
investigation to examine the precise mechanisms involved. We are now in the
process studying these pathways in SP-A-/- null mice and looking to determine if
hyperoxia and stress also up regulate this anti-inflammatory
Commercial Relationships: Ronald Hobbs, None; Saad Munzar,
None; Genevieve S. Ball, None; Faizah N. Bhatti, None
Support: NIH COBRE P20 RR017703-10
Program Number: 2534 Poster Board Number: D912
Presentation Time: 3:45 PM - 5:30 PM
Prolyl Hydroxylase Inhibition during Hyperoxia Prevents Oxygen-induced
Retinopathy in the Rat 50/10 Model
George Trichonas, George Hoppe, Tamara J. Lee, Jonathan E. Sears.
Ophthalmology, Cleveland Clinic, Cleveland, OH.
Purpose: To study the effect of systemic prolyl hydroxylase inhibition (PHDi) in
the rat oxygen-induced retinopathy (OIR) model.
Methods: Litters of Sprague-Dawley rat pups and their mothers were transferred
within 10 h after birth to oxygen exposure chambers (BioSpherix) where they were
subjected to alternating 24-h periods of 50% oxygen and 10% oxygen for 14 days.
Control rats were raised simultaneously in room air. On postnatal day (P) 14, the
oxygen-exposed rats were returned to room air. OIR animals received
intraperitoneal injections of dimethyloxalylglycine (DMOG, 200µg/g), an
antagonist of α-ketoglutarate cofactor and inhibitor for PHD, on P3, P5, P7 and P9.
Control animals received intraperitoneal injections of PBS. On P14 and P21,
animals were anesthetized with Ketamine (80 mg/kg) and Xylazine (8 mg/kg) and
were perfused through the left ventricle with 0.5 ml of 0.1% Evans Blue dye. Eye
cups were dissected to prepare retinal flat-mounts which were examined under
fluorescent microscopy. Neovascularization and avascular areas were assessed on
retinal flat-mounts by computerized image analysis.
Results: Alternating hyperoxia/hypoxia in untreated rats led to peripheral as well
as central vascular obliteration on day P14. Rats that were treated with systemic
DMOG by intraperitoneal injections had less posterior ischemia and greater
peripheral vascularity than control animals treated with PBS injections. DMOG
administration had no adverse effect on retinal vasculature of rats reared in room
Conclusions: We have previously demonstrated that PHDi prevents OIR in mice
exposed to 5 days of 75% oxygen followed by 5 days of 21% oxygen. The 50/10
rat experiments demonstrate that PHDi is also effective in a 24-h alternating
hyperoxia/hypoxia model. The rat OIR model further validates the therapeutic
value of PHDi to prevent retinopathy of prematurity, because it reduces oxygeninduced vascular obliteration and retinovascular growth attenuation in prolonged
and/or alternating hyperoxia.
Commercial Relationships: George Trichonas, None; George Hoppe,
None; Tamara J. Lee, None; Jonathan E. Sears, None
Support: Research to Prevent Blindness Challenge Grant, Cleveland Clinic
Product Development Fund, Knights Templar Eye Foundation, Clarinda Ross
Macular Degeneration Fund.
Program Number: 2535 Poster Board Number: D913
Presentation Time: 3:45 PM - 5:30 PM
Capillary Rescue in Oxygen Induced Retinopathy by Inhibition of Prolyl
Hydroxylation is Associated with Transcriptional Induction of HIF-1α
through Non-inflammatory Activation of NF-κB
George Hoppe, John Au, Julieta Zutel, Jonathan E. Sears. Cole Eye Institute,
Cleveland Clinic, Cleveland, OH.
Purpose: Systemic inhibition of prolyl hydroxylase (PHD) activity prevents
oxygen-induced loss of retinal capillaries in the mouse model of retinopathy of
prematurity. PHD inhibition was not only associated with the canonical post-
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
translational stabilization of hepatic hypoxia-inducible factor (HIF), but also with
an unanticipated increase in retinal HIF mRNA. Since HIF expression requires
transcriptional activity of NF-κB, we asked whether hypoxia-mimetics are capable
of NF-κB activation.
Methods: Three structurally distinct PHD inhibitors, i.e., α-ketoglutarate analog
dimethyloxalylglycine (DMOG), triazinoindole hydrazone derivative CB1, and
phthalazine derivative hydralazine (HZ), were tested for their ability upregulate
NF-κB pathway in cultured Muller cells and hepatocytes. Phosphorylation at Ser536 and nuclear translocation of p65 subunit, phosphorylation of IKK, protein
levels of A20, COX-2 and HIF-1α were investigated by Western blotting following
subcellular fractionation. Gene products of HIF and NF-kB were assessed by
measuring erythropoietin and IL-6 in cell culture media using ELISA. Finally the
transcriptional activity of NF-κB was measured by binding of p50 to κB consensus
sequence and the activity of κB promoter was evaluated by κB reporter gene assay
using HEK293-κB-Luc stable cells. The effect of intraperitoneal (i.p.)
administration of DMOG on the levels of HIF-1α transcript in the liver, kidney,
brain and retina was assessed by quantitative RT-PCR.
Results: PHD inhibitors demonstrated wide range of potency to induce p65
phosphorylation and nuclear translocation from 3 mM (DMOG) to 1 µM (CB1).
All compounds caused rapid accumulation of HIF-1α, while NF-κB activation was
delayed by 24 hrs. Based on the ability of CB1 and HZ, but not of DMOG, to
stimulate sustained secretion of erythropoietin, it is likely that DMOG is relatively
unstable. In fact, in vivo DMOG was undetectable 3 hrs after i.p. injection in mice.
Despite robust mobilization of p65 subunit, neither activation of IKK nor induction
of inflammatory mediators IL-6 and COX-2 was induced by PHD inhibition. In
contrast, TNFα treatment activated multiple steps of the NF-κB pathway, e.g., p65
phosphorylation and nuclear accumulation, IKK phosphorylation, DNA-binding of
p50 to κB site, κB-driven transcription, and secretion of IL-6.
Conclusions: In addition to blocking oxygen-dependent HIF degradation and
transcriptional upregulation of HIF, PHD inhibitors induce p65 nuclear
translocation in Muller cells. Non-inflammatory activation of retinal NF-κB may
regulate HIF expression and vascular protection.
Commercial Relationships: George Hoppe, None; John Au, None; Julieta
Zutel, None; Jonathan E. Sears, None
Support: Research to Prevent Blindness Challenge Grant, Cleveland Clinic
Product Development Fund, Knights Templar Eye Foundation, Clarinda Ross
Macular Degeneration Fund.
Program Number: 2536 Poster Board Number: D914
Presentation Time: 3:45 PM - 5:30 PM
Association Of Runx1 With Vascular Development In The Fetal Human Eye
Gerard A. Lutty, Ines Galtier d’Auriac, D. S. McLeod. Wilmer Eye Inst, Johns
Hopkins Univ Sch of Med, Baltimore, MD.
Purpose: Runt-related transcription factor 1 (RUNX1) also known as acute
myeloid leukemia 1 protein (AML1) is a transcription factor that is essential for
definitive hematopoiesis and regulates the differentiation of hematopoietic stem
cells into mature blood. RUNX1 is required for blood and vessel development at
the hemangioblast level in Zebra fish. We have found that both the human
choriocapillaris (CC) and fetal vasculature of vitreous (FVV) develop by hemovasculogenesis, development of blood and blood vessels from hemangioblasts (1,
2). Thus we investigated the association of RUNX1 with development of these
vasculatures in fetal human eyes.
Methods: Fetal human eyes [7-21 weeks gestation(WG)] were cryopreserved and
sections incubated with antibodies against RUNX1, CXCR4, cKit, and CD31.
Analysis was performed on Zeiss meta 710 confocal microscope.
Results: RUNX1 was present in the cytoplasm of progenitors forming the FVV at
7 WG. From 10-17 WG, RUNX1 was prominently localized to the nucleus of
endothelial cells (ECs) and presumed pericytes in developing FVV. At 14-17 WG,
RUNX1 was prominent in subnuclear compartments of both cell types. RUNX1
was present in cytoplasm of some cells in the islands of progenitors in the 7 WG
developing CC, but nuclear localization was far more prominent at 10-21 WG. It
appeared that pericytes and EC in CC expressed RUNX1 as well as the
mesenchymal precursors throughout choroidal stroma. RUNX1 was also associated
with angioblasts in inner retina as they assembled by vasculogenesis. Also RUNX1
was present in the inner neuroblastic layer progenitor pool that we have previously
found to express CXCR4 and cKit. As these progenitors differentiate into ganglion
cells and EC, RUNX1 expression diminished.
Conclusions: Conclusions: RUNX1 was associated with vascular progenitors in
developing CC, FVV, and retina. Expression declined as ECs matured in the retinal
vasculature but remained prominent in EC of CC and FVV that form by hemovasculogenesis.
1. Hasegawa T, et al. Developmentof the choriocapillaris by hemovasculogenesis.
Dev Dyn 2007;236:2089-100.
2. Lutty G, et al, Developmentof the fetal vasculature of vitreous by
hemovasculogenesis. ARVO, 2006.
Commercial Relationships: Gerard A. Lutty, None; Ines Galtier d’Auriac,
None; D. S. McLeod, None
Support: NIH grants RO1EY09357 (GL), RO1EY016151 (GL), EY01765
(Wilmer), and an unrestricted grant from RPB (Wilmer)
Program Number: 2537 Poster Board Number: D915
Presentation Time: 3:45 PM - 5:30 PM
Activated Protease-activated Receptor Type 2 Reduces Vaso-obliteration And
Accelerates Normal Revascularization In A Mouse Model Of Oxygen-induced
Nicholas Sitaras1A,1B, Jean-Sebastien Joyal2, Zhuo Shao2, Mike (Przemyslaw)
Sapieha1B, Sylvain Chemtob1A,2. APharmacology, BOphthalmology, 1MaisonneuveRosemont Hosp, Montreal, QC, Canada; 2Pharmacology, Sainte-Justine Hospital,
Montreal, QC, Canada.
Purpose: Proliferative Retinopathies are characterized by an initial micro-vascular
degeneration followed by ischemia-driven aberrant pre-retinal neovascularization,
which predisposes the retina to blinding retinal detachment. Accordingly, a prompt
normal revascularization prevents the exaggerated intravitreal neovascularization.
Selective targets to accelerate revascularization of the retina are not known. We
have previously demonstrated that G protein-coupled receptor protease-activated
receptor type 2 (PAR-2) induces angiogenesis in the developing retina. Herein, we
investigate the role of PAR-2 in accelerating revascularization in oxygen-induced
retinopathy (OIR).
Methods: PAR-2 expression was measured in mouse whole retina by Western blot
and localization determined by immunohistochemistry on coronal sections. Retinal
vasculature was assessed subsequent to intra-vitreal administration of PAR-2
agonist peptide (SLIGRL, 100µM) or lentiviral-based shRNA knockdown of PAR2 (LV shPAR-2) in wild-type mice exposed to a model of OIR (75% O2 from P7P12). Expression profiles of pro- and anti-angiogenic cues were assessed in whole
retina of mice subjected to OIR treated with SLIGRL or LV shPAR-2. The RGC-5
cell line served as an ex-vivo model of retinal ganglion cells (RGC). Microvascular
growth was assessed using the aortic explant sprouting assay.
Results: Our results reveal a prominent expression of PAR-2 in RGCs. Levels of
PAR-2 significantly increased during the initial phases of VO (P8) and during
revascularization (P16) compared to normoxic controls. Treatment with SLIGRL in
vivo prior to and during hyperoxia accelerated revascularization of the retina,
which in turn decreased VO at P12. Similarly, SLIGRL treatment following
hyperoxia reduced avascular area and pathological neovascularization at P17.
Conversely, knockdown of PAR-2 using LV shPAR-2 increased VO at P12 and
P17 while augmenting avascular zones and pathological neovascularization at P17.
Activation of PAR-2 with SLIGRL in vivo reduced anti-apoptotic cues while
increasing pro-angiogenic factors. Similar gene regulation was observed in
SLIGRL-treated RGC-5. Aortic ring explants exposed to SLIGRL-primed RGC
conditioned media exhibited significant increases in vascular growth; this was
abrogated by LV shPAR-2.
Conclusions: This study highlights the role of PAR-2 in triggering
revascularization during and following the vaso-obliterative phase by modifying
pro- and anti-angiogenic cues. These data demonstrate a novel approach to curtail
vessel loss and promote normal revascularization in proliferative retinopathies
using PAR-2 agonists.
Commercial Relationships: Nicholas Sitaras, None; Jean-Sebastien Joyal,
None; Zhuo Shao, None; Mike (Przemyslaw) Sapieha, None; Sylvain Chemtob,
Support: FRSQ
Program Number: 2538 Poster Board Number: D916
Presentation Time: 3:45 PM - 5:30 PM
Ang II Subtype 2 Receptor (AT2R) Activation By Compound 21 Reduces
Endothelial Cell Migration in vitro and Neovascularization in the Oxygeninduced Retinopathy (OIR) Model
Sergio Li Calzi1A, Lynn C. Shaw1A, Tatiana Salazar1A, Colin Sumners1B, Julia
Busik2, Mohan Raizada1B, Maria Tikhonenko2, Ulrike Steckelings3, Maria B.
Grant1A. APharmacology and Therapeutics, BPhysiology and Functional Genomics,
University of Florida, Gainesville, FL; 2Physiology, Michigan State University,
East Lansing, MI; 3Center for Cardiovascular Research, Universitatsmedzin Berlin,
Berlin, Germany.
Purpose: In the Renin-Angiotensin System (RAS), angiotensin II (Ang II) binds to
the Ang II subtype 1 receptor (AT1R) to stimulate inflammation whereas the Ang II
subtype 2 receptor (AT2R) blocks the pro-inflammatory effect of AT1R and reduces
inflammatory cytokine expression by vascular cells. The involvement of the RAS
in diabetic retinopathy (DR) is suggested by increased angiotensin-converting
enzyme (ACE) serum levels in diabetics; and by the reduction of retinal
neovascularization in the OIR model in rats by treatment with inhibitors of the
AT1R. In this study, we examined the effect of AT2R activation on retinal
endothelial cell function in vitro and on neovascularization in the OIR model.
Methods: Compound 21 (C21) was used as a highly selective AT2R agonist for
both in vitro and in vivo studies. After removal from 75% oxygen on postnatal day
12, mouse pups were gavaged daily for five days with up to 0.06 mg/kg/day of
C21. Mice were perfused with FITC-conjugated dextran on postnatal day 17 to
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
visualize the retinal vasculature. One eye was taken for retinal flatmount analysis
and the second eye was taken for retinal cross section analysis of pre-retinal
neovascularization. Human retinal endothelial cells (HRECs) were used to
determine the effect of C21 on migration to VEGF using a modified Boyden
chamber assay.
Results: Oral treatment of mouse pups with C21 reduced the amount of pre-retinal
neovascularization by 26 ± 8% (p < 0.05) at a concentration of 0.06 mg/kg/day.
Retinal flatmount analysis showed a significant reduction in vaso-obliteration in
pups treated with C21 treatment. In vitro studies demonstrated that pretreatment of
HREC with C21 inhibited their migration to VEGF in a dose dependent manner.
Conclusions: C21 reduced pre-retinal neovascularization in the OIR model. C21
also reduced cell migration in HRECs. These results support the involvement of
RAS in retinal neovascularization and in particular of the protective role of AT2R
activation. C21 is a safe oral agent and may represent a viable therapeutic for
treatment in DR and retinopathy of prematurity.
Commercial Relationships: Sergio Li Calzi, None; Lynn C. Shaw,
None; Tatiana Salazar, None; Colin Sumners, None; Julia Busik, None; Mohan
Raizada, None; Maria Tikhonenko, None; Ulrike Steckelings, None; Maria B.
Grant, None
Support: NEI RO1 EY012601, NEI RO1 007739
Program Number: 2539 Poster Board Number: D917
Presentation Time: 3:45 PM - 5:30 PM
Systemic Administration of VEGF Trap Prevents Pervasive Blood Vessel
Regression in the Murine Model of Oxygen-Induced Retinopathy (OIR)
Ivan B. Lobov, Eunice Cheung, George Yancopoulos, Stanley J. Wiegand.
Regeneron Pharmaceuticals, Inc., Tarrytown, NY.
Purpose: VEGF plays a critical role in both normal and pathological angiogenesis,
but much less is known about its roles in vascular remodeling. In the retina, VEGF
inhibition prevents pathological neovascularization and leads to regression of
existing neovasculature. In the OIR model, it has been believed that the
characteristic obliteration of capillaries in the central retina is caused by a
decreased expression of VEGF. However, we have recently observed that
administration of VEGF Trap (a potent VEGF-A and PlGF blocker) does not cause
pervasive vasoobliteration, but rather induces a moderate, diffuse pruning of
immature capillaries [Lobov et al, Blood 2011]. Here we tested the effect of VEGF
Trap on oxygen-induced vasoobliteration.
Methods: Mouse pups received intraperitoneal injections of VEGF Trap (25
mg/kg) or a control hFc protein on postnatal day 8 (P8), 24 hours prior to exposure
to 75% O2 on P9. Retinas were stained with FITC-labeled GS Lectin I (Vector
Labs) to visualize the vasculature. Concanavalin A (Vector Labs) labeled with
rhodamine was infused intracardially to assess blood vessel patency. To study the
progression of blood vessel regression, retinas were collected 6, 16, 18, and 24
hours after the beginning of oxygen exposure and stained using an antibody against
cleaved Caspase3 (Cell Signaling). Changes in retinal gene expression were
evaluated by microarray.
Results: Six hours after the beginning of high oxygen exposure, capillaries of
control animals became occluded and at 16 hours showed a characteristic pattern of
synchronous apoptosis. Administration of VEGF Trap prior to exposure to
hyperoxia significantly reduced blood vessel loss. This was associated with
maintenance of blood flow crucial for survival of newly-formed microvessels.
VEGF Trap treatment significantly down-regulated the expression of Dll4. We
recently showed that inhibition of Dll4/Notch signaling similarly improves blood
flow and retinal blood vessel survival after high-oxygen exposure [Lobov, Blood
2011]. Moreover, administration of VEGF Trap upregulated the expression of
adrenomedullin, a potent vasodilator critical for maintaining capillary perfusion.
Conclusions: It has been shown that pharmacological inhibition of VEGF
ameliorates pathological neovascularization in diverse models of disease.
Paradoxically, in this preclinical study, systemic administration of VEGF Trap
reduced the loss of normal immature retinal capillaries in mice exposed to
hyperoxia. This seemingly paradoxical response appears to be mediated, at least in
part, by changes in the expression of genes involved in the oxygen-mediated
regulation of retinal blood flow, such as Dll4 and adrenomedullin.
Commercial Relationships: Ivan B. Lobov, Regeneron Pharmaceuticals, Inc.
(E); Eunice Cheung, Regeneron Pharmaceuticals, Inc. (E); George Yancopoulos,
Regeneron Pharmaceuticals, Inc. (E); Stanley J. Wiegand, Regeneron
Pharmaceuticals, Inc. (E)
Support: None
Program Number: 2540 Poster Board Number: D918
Presentation Time: 3:45 PM - 5:30 PM
Genetic Deletion Of The α2β1 Integrin Protects Against Neovascularization By
VEGF Modulation In A Mouse Model Of Oxygen-Induced Retinopathy
Megan E. Capozzi, Bobby Madamanchi, Ling Geng, Richard Friedman, Zhengzhi
Li, Mary M. Zutter. Vanderbilt University, Nashville, TN.
Purpose: The α2β1 integrin, a cell-surface collagen receptor, has been implicated
in mediating growth factor signaling in angiogenesis. We hypothesize that α2β1
integrin may interact with VEGF to produce a neoangiogenic phenotype. Therefore,
we investigated the effect of genetic manipulation of α2β1 integrin on induction of
VEGF and development of neovascularization (NV) in a mouse model of oxygeninduced retinopathy (OIR).
Methods: An OIR mouse model was used to determine the effect of α2β1 on NV.
Eyes from OIR and room air controls of α2β1 wild type (α2WT) and α2β1
knockout (α2KO) mice were enucleated and sectioned on postnatal day 18 (P18).
Localization of the α2β1 integrin was analyzed by immunohistochemistry. Retinas
were collected following OIR on P13 to measure VEGF expression by qRT-PCR
and VEGF secretion by ELISA. Primary cultures of muller cells were isolated from
α2WT and α2KO mice, treated in normoxia or hypoxia for 24 hours, and the level
of VEGF secreted into the media measured by ELISA.
Results: α2β1 integrin was strongly expressed in a pattern consistent with muller
cell distribution and moderately in endothelial cells. Expression was upregulated in
response to OIR treatment. Following OIR, on P12 the α2KO exhibited a 32%
decrease (p = 0.0002) of avascular area and on P17 caused a 49% decrease (p =
0.0069) of NV compared to WT controls. The OIR exposed α2KO exhibited a 39%
decrease (p = 0.02) of VEGF expression and a 41% decrease (p = 0.0006) of VEGF
secretion compared to the α2WT on P13. VEGF secretion was consistent between
α2WT and α2KO muller cells in normoxia. In hypoxia, VEGF secretion was
decreased by 46% (p = 0.01) in α2KO compared to α2WT muller cells.
Conclusions: This study suggests that the α2β1 integrin is an important mediator of
VEGF induction by muller cells. Additionally, genetic deletion of the α2β1 integrin
exerts a protective effect against the development of retinal NV. Therefore, α2β1
may serve as a valuable therapeutic target in treating pathological angiogenesis.
Commercial Relationships: Megan E. Capozzi, None; Bobby Madamanchi,
None; Ling Geng, None; Richard Friedman, None; Zhengzhi Li, None; Mary
M. Zutter, None
Support: NIH Grant R01CA133230-01A1; NIH Grant 2R01CA098027-07A1;
Louise B. McGavock Chair
Program Number: 2541 Poster Board Number: D919
Presentation Time: 3:45 PM - 5:30 PM
Inhibition Of β-adrenergic Receptor With Propranolol Does Not Protect
Against Oxygen-induced Retinopathy
Jing Chen1, Colman J. Hatton1, Aimee M. Juan1, Dorothy T. Pei1, Christian G.
Hurst1, Jean-Sebastian Joyal1, Dan Xu1, Ann Hellstrom2, Lois E. Smith1.
Ophthalmology, Harvard Med Sch Children's Hosp, Boston, MA; 2Department of
Ophthalmology, Institute of Neuroscience and Physiology, The Sahlgrenska
Academy at University of Gothenburg, Gothenburg, Sweden.
Purpose: Retinopathy of prematurity (ROP) is a leading cause of blindness in
children characterized by vision-threatening pathologic vessels. Propranolol, a nonselective β-adrenergic receptor blocker, has been serendipitously identified as a
new treatment for infantile hemangioma, a type of benign tumor with vascular
elements, with some severe side effects reported. Interestingly in a mouse model of
oxygen-induced ROP (OIR), propranolol was reported to be effective in protecting
against pathologic retinal neovascularization, although retinopathy was evaluated
with non-standard techniques. Based on this single report, a clinical trial (PROPROP) is currently ongoing to evaluate propranolol treatment in ROP patients.
Because of the fragility of incompletely developed premature infants the efficacy of
propranolol treatment in retinopathy needs to be evaluated thoroughly in preclinical animal models of retinopathy, which is the focus of this study.
Methods: Retinopathy was induced by exposing neonatal mice to 75% oxygen
from postnatal day (P) 7 to 12. Two doses of propranolol (2 or 20mg/kg/day) were
given daily from P13 to P16. Three routes of invention were assessed: oral gavage,
intraperitoneal injection or subcutaneous injection. At P17, per standard protocol,
retinas were flatmounted and stained with lectin to visualize vaso-obliteration (VO)
and pathologic neovascularization (NV). Retinal gene expression was analyzed
with qRT-PCR using RNA isolated from retinas of control and propranolol treated
Results: There was no difference in either VO or in pathologic NV in OIR at P17
with oral gavage of propranolol at 2mg/kg/day (equivalent to the standard human
dose), nor with intraperitoneal injection of propranolol at the same dose. A higher
dose of propranolol (20mg/kg/day) injected subcutaneously was not effective in
suppressing either VO or NV in OIR. Importantly, intraperitoneal injection of
propranolol at 20mg/kg/day was associated with a modest but significant increase
in both VO and NV in retinopathy.
Conclusions: Propranolol treatment via three routes and up to 10 times the
standard human dose fails to suppress retinopathy development in mice. These data
bring into question whether propranolol and inhibition of beta-adrenergic receptors
is a viable therapeutic approach for treating ROP.
Commercial Relationships: Jing Chen, None; Colman J. Hatton, None; Aimee
M. Juan, None; Dorothy T. Pei, None; Christian G. Hurst, None; JeanSebastian Joyal, None; Dan Xu, None; Ann Hellstrom, None; Lois E. Smith,
Support: NIH grant (EY017017, EY017017-04S1, EY017017-05), Research to
Prevent Blindness Award (LEHS), CHB Manton Center for Orphan Disease
Research Innovation Fund, Charles H. Hood Foundation (to J.C).
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2542 Poster Board Number: D920
Presentation Time: 3:45 PM - 5:30 PM
Hypoxia-induced Insulin-like Growth Factor II Contributes to Retinal
Vascularization in Ocular Development
Jeong Hun Kim1, Jin Hyoung Kim1, Sung Wook Park2, Noo Li Jeon3, Young S. Yu4.
Ophthalmology-Coll of Med, Seoul National Univ Hospital, Seoul, Republic of
Korea; 2FARB, Department of Ophthal, Seoul National University Hosp, Seoul,
Republic of Korea; 3School of Mechanical and Aerospace Engineering, Seoul
National University, Seoul, Republic of Korea; 4Ophthalmology/Coll of Med,
Seoul National Univ Hosp, Seoul, Republic of Korea.
Purpose: In ocular development, retinal physiological hypoxia in response to the
retinal metabolic activity controls retinal vascular development, which is regulated
by variable angiogenic factors. Herein, we demonstrated that hypoxia-induced IGFII could contribute to retinal vascularization in ocular development.
Methods: In developing mouse, immunohistochemistry for IGF-II as well as
immunofluorescence staining of IGF-II with vWF were performed. In human
retinal microvascular endothelial cells (HRMEC), IGF-2 and VEGF expression
were measured by RT-PCR and Western blot analysis under hypoxic condition
(1%). To investigate IGF-2 induced regulation of VEGF expression in HRMEC,
western blot analysis for VEGF, ERK-1/2, p-ERK-1/2, Akt, and p-Akt was
performed with treatment of IGF-II. In addition, VEGF expression in HRMEC was
evaluated under hypoxia with treatment of a blocking antibody to IGF-2 by RTPCR and Western blot analysis. To confirm the direct angiogenic activity of IGF-II,
angiogenesis assays of migration & tube formation was performed as well.
Results: In the developing retina, IGF-II expression appears to be predominant on
retinal vessels, which was chronologically increased and peaked during active
retinal angiogenesis similar to VEGF expression. Under hypoxic condition, IGF-II
as well as VEGF was significantly up-regulated in HRMEC. In addition, IGF-II
treatment could also increase VEGF expression in HRMEC. The VEGF expression
induced by IGF-II was mediated by ERK-1/2 activation. Moreover, IGF-II strongly
promoted angiogenic processes of migration and tube formation of HRMEC.
Conclusions: Our results provided that hypoxia-induced IGF-II may regulate
retinal vascular development not only directly by IGF-II-mediated angiogenic
activity, but also indirectly by IGF-II-induced VEGF expression. Therefore, the
potential contribution of IGF-II to pathological retinal angiogenesis should be
furthermore explored for the development of novel treatments to vaso-proliferative
Commercial Relationships: Jeong Hun Kim, None; Jin Hyoung Kim,
None; Sung Wook Park, None; Noo Li Jeon, None; Young S. Yu, None
Support: None
Program Number: 2543 Poster Board Number: D921
Presentation Time: 3:45 PM - 5:30 PM
Gene Expression Analysis Of Retinas From ω-3-PUFAs And ω-6-PUFAs Fed
Mice In Oxygen-induced Retinopathy (OIR) Model
Dan Xu1, Christopher M. Aderman1, Jing Chen1, Aimee M. Juan1, Colman J.
Hatton1, Christian G. Hurst1, Dorothy T. Pei1, Jean-Sebastian Joyal1, John Paul
SanGiovanni2, Lois E. Smith1. 1Department of Ophthalmology, Harvard Medical
School, Children's Hospital Boston, Boston, MA; 2Clinical Trials Branch, National
Eye Institute, Bethesda, MD.
Purpose: We have previously demonstrated that, in a mouse model of oxygeninduced retinopathy (OIR) that dietary ω-3-polyunsaturated fatty acids (PUFAs)
protect against pathological neovascularization (NV) after vessel loss by promoting
normal vascular regrowth thereby decreasing avascular area and decreasing
stimulus for pathological NV. To further explore the factors and signaling
pathways promoting regrowth of normal vessels in ω-3-PUFAs-fed versus ω-6PUFAs-fed mice, we performed Illumina microarray analysis on retinas at P14
after exposure to hyperoxia (P7-P12) with vessel loss and return to room air when
vessels are regrowing (P12-P17).
Methods: C57BL/6 mouse mothers at delivery were fed a defined rodent diet with
10% (w/w) safflower oil containing either 2% ω-6-PUFAs (arachidonic acid) or 2%
ω-3-PUFAs (DHA and EPA). Retinopathy was induced by exposing neonatal mice
with their nursing mother to 75% oxygen from postnatal day (P) 7 to P12 and
returning them to room air. Retinas from P14 pups were isolated. Total RNA was
extracted and prepared for microarray analysis with Illumina Mouse-ref 6 chip.
Data were acquired using BeadStudio software and analyzed with Significance
Analysis of Microarrays (SAM) program and BIOBASE.
Results: Using p<0.05 and a 1.5 fold change cutoff of gene expression, we
identified 612 up-regulated and 597 down-regulated genes in ω-3-PUFAs-fed
compared to ω-6-PUFAs-fed mice at P14. Consistent with a cyto-protective role of
ω-3-PUFAs, our Gene-Ontology (GO) analysis revealed that anti-apoptosis genes
such as Atm, Bcl2 are ~2.5 fold up-regulated by ω-3-PUFAs. On the other hand, ω3-PUFAs down-regulates key genes involved in the response to hypoxia and
oxidative stress such as Adm (8.6 fold), Vegf (3.0 fold) and Angptl4 (2.5 fold).
Conclusions: These findings suggest that ω-3-PUFAs may be responsible for
neuroprotection in retina during OIR by repressing cell apoptosis and hypoxia
responses, thereby resulting in an enhanced vascular regrowth and a less severe
pathological neovascularization.
Commercial Relationships: Dan Xu, None; Christopher M. Aderman,
None; Jing Chen, None; Aimee M. Juan, None; Colman J. Hatton,
None; Christian G. Hurst, None; Dorothy T. Pei, None; Jean-Sebastian Joyal,
None; John Paul SanGiovanni, None; Lois E. Smith, None
Support: NIH (EY017017, EY017017-04S1, EY017017-05), Research to Prevent
Blindness Senior Investigator Award (LEHS)
Program Number: 2544 Poster Board Number: D922
Presentation Time: 3:45 PM - 5:30 PM
Characterization of the Dishevelled Family Proteins in Oxygen-Induced
Colman J. Hatton1, Jing Chen2, Aimee Juan1, Christian G. Hurst1, Dorothy T. Pei1,
Andreas Stahl3, Przemyslaw Mike Sapieha4, Jean-Sebastien Joyal5, Dan Xu1, Lois
E. Smith6. 1Ophthalmology, Children's Hospital Boston, Boston, MA;
Ophthalmology, Harvard Med Sch Children's Hosp, Boston, MA; 3Univ Eye
Hospital Freiburg, Freiburg, Germany; 4Ophthalmology, University of Montreal,
Montreal, QC, Canada; 5Ophthalmology, Children's Hospital, Harvard Med Sch,
Boston, MA; 6Ophthalmology, Harvard Univ/Childrens Hospital, Boston, MA.
Purpose: Proliferative retinopathies are the leading cause of blindness in children
and working age adults. Disease severity could be ameliorated by specifically
targeting pathological neovessels while leaving normal vessels untouched. We
found recently that Wnt signaling regulates pathological revascularization in the
retina. Dishevelled (Dvl) 1, 2, and 3 are key components of the canonical Wnt
pathway relaying signals to downstream targets. In this study, we characterized
mutant mice lacking Dvls (Dvl1, Dvl2, and Dvl3) during normal retinal
development and in oxygen-induced retinopathy (OIR) to gain a better
understanding of the role of Dvls in regulating retinal blood vessel growth.
Methods: Dvl1-/-, Dvl2-/-, and Dvl3+/- mice and their littermate controls were
exposed to 75% oxygen from postnatal day (P) 7 to P12. Dvl3+/- mice were
assessed in OIR because Dvl3-/- mice do not survive long after birth. Eyes were
dissected at P17, and vaso-obliteration and pathological neovascularization (NV)
were quantified. Normal retinal development was characterized by quantifying
vascularized area during development as well as assessing adult retinal vasculature
in flatmounts and cross sections. Additionally, mRNA expression of Dvls during
retinal development was examined in wild type retinas (P1-P17) using RT-qPCR.
Results: Retinal expression levels of Dvl1 and Dvl3 in wild type mice remain
relatively unchanged from P1 to P17. In contrast, Dvl2 shows robust mRNA
expression during the first week after birth and gradually decreases as retina
matures. Despite normal vascular growth during development, after OIR, Dvl2-/mice have significantly reduced NV (6.5±1% versus 9.1±0.5%; P≤0.01) at P17 as
compared to littermate controls. In contrast, both Dvl1-/- and Dvl3+/- mice exhibit
similar levels of NV at P17 after OIR compared to their relative controls.
Conclusions: Our results demonstrate that Wnt signaling through Dvl2 contributes
to pathological retinal NV in a mouse model of OIR. The normal retinal vascular
development in Dvl2 KO mice and lack of OIR phenotype in Dvl1 and Dvl3
mutant mice may reflect redundant roles of Dvl 1, 2, and 3 in development and
Commercial Relationships: Colman J. Hatton, None; Jing Chen, None; Aimee
Juan, None; Christian G. Hurst, None; Dorothy T. Pei, None; Andreas Stahl,
None; Przemyslaw Mike Sapieha, None; Jean-Sebastien Joyal, None; Dan Xu,
None; Lois E. Smith, None
Support: CHB Manton Center for Orphan Disease Research Fund, Juvenile
Diabetes Research Foundation International, Charles H. Hood Foundation Child
Health Research Award, NIH Grant EY017017 and EY017017-04S1
Program Number: 2545 Poster Board Number: D923
Presentation Time: 3:45 PM - 5:30 PM
Opticin Suppresses Preretinal Neovascularization, But Does Not Influence
Vascular Development In The Murine Eye
Paul N. Bishop1, Marta Ugarte1, Hongbin Lu1, Shephen Henry2, Masamine
Takanosu3, Richard Mayne3, Magali M. Le Goff1. 1School of Biomedicine,
University of Manchester, Manchester, United Kingdom; 2M.D. Anderson Cancer
Centre, Houston, TX; 3Department of Cell Biology, University of Alabama,
Birmingham, AL.
Purpose: To investigate whether a lack of the extracellular matrix glycoprotein
opticin affects retinal vascular development or pathological angiogenesis in the eye.
Methods: In these experiments opticin null and wild-type mice were compared.
The hyaloid vasculature was quantified at P0 and P4 by analysing serial sections
through the entire globe. Retinal vascular development at P4 and P7 was analysed
by fluorescently-labeling the vasculature in flat mounts. The oxygen-induced
retinopathy model was used to assess the effects of a lack of opticin on ischaemiainduced retinal vascular changes and preretinal neovascularization; the results were
quantified using SWIFT_NV software.
Results: Analyses of the hyaloid and retinal vasculature showed that a lack of
opticin had no effect on hyaloid vascular regression or development of the retinal
vasculature. However, using the oxygen-induced retinopathy model, we
demonstrated that opticin knockout mice had decreased retinal vascular pruning at
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
P12, although there were no differences in the hyaloid vasculature at this time
point, but increased preretinal neovascularization at P17 compared to wild-type
Conclusions: A lack of opticin does not influence vascular development, but
opticin is anti-angiogenic and inhibits preretinal neovascularization.
Commercial Relationships: Paul N. Bishop, Patents US 7910567 B2. and US
7,358,224 B2 (P); Marta Ugarte, None; Hongbin Lu, None; Shephen Henry,
None; Masamine Takanosu, None; Richard Mayne, None; Magali M. Le Goff,
Support: The Wellcome Trust (Grant 057940); NIH Grant R37 AR30481;
Manchester NIHR Biomedical Research Centre
Program Number: 2546 Poster Board Number: D924
Presentation Time: 3:45 PM - 5:30 PM
A Multicenter Study Analyzing Weight Gain to Predict Retinopathy of
Aimee Juan1, Carolyn Wu1, Chatarina Löfqvist2, Lois E. Smith1, Deborah K.
VanderVeen1, Ann Hellström2, WINROP Consortium. 1Ophthalmology, Children's
Hospital Boston, Harvard Medical School, Boston, MA; 2Ophthalmology, Institute
of Neuroscience and Physiology, Sahlgrenska Academy, University of Gothenburg,
Gothenburg, Sweden.
Purpose: Retinopathy of prematurity (ROP) is a leading cause of preventable
childhood blindness. However, <8% of infants examined by current criteria require
treatment and diagnosis only occurs when ROP is established. We need to
differentiate early those infants at higher risk for severe ROP. WINROP (Weight,
IGF, Neonatal ROP) is a free online surveillance algorithm using longitudinal
postnatal weight measurements to better predict early, infants’ risk for developing
severe ROP. The purpose of this study was to validate WINROP as a tool to help
predict ROP in geographically and racially diverse populations of premature infants
in 10 neonatal intensive care units in the U.S. and Canada.
Methods: WINROP analysis was performed on 1706 premature infants selected by
conventional criteria for ROP screening, with median gestational age 28 weeks at
birth and median birth weight of 1016 g. ROP exams consisted of standard
binocular indirect ophthalmoscopy with scleral depression and ROP was classified
according to international standards. Weekly postnatal weight measurements were
entered into WINROP, which calculated deviations between observed and
reference values of expected weight gain velocity in infants without ROP. An alarm
signaled when the rate of weight gain decreased compared with healthy GA
matched control subjects, indicating high risk of ROP.
Results: An alarm was signaled in 1101 infants: 78 alarmed at week 0 and 1023
alarmed for poor postnatal weight gain at a median time of 3 weeks. Type 1 ROP
developed in 144 of these infants. Conversely, no alarm signaled in 605 infants. Of
these, 603 did not develop type 1 ROP. The overall sensitivity of WINROP to
detect type 1 ROP was 98.6% (144/146 infants) and the specificity was 38.7%
(603/1560 infants). The positive predictive value was 13.1% (144/1101 infants) and
the negative predictive value was 99.7% (603/605 infants).
Conclusions: This multicenter study validated the use of postnatal weight gain
analysis by WINROP to help predict very early, infants at high risk for type 1 ROP.
With early and sensitive detection of those at higher risk and 99.7% detection of
those at no or low risk, WINROP is a valuable tool to optimize patient ROP
screening and care.
Commercial Relationships: Aimee Juan, None; Carolyn Wu, None; Chatarina
Löfqvist, None; Lois E. Smith, None; Deborah K. VanderVeen, None; Ann
Hellström, None
Support: Children’s Hospital Ophthalmology Foundation
Program Number: 2547 Poster Board Number: D925
Presentation Time: 3:45 PM - 5:30 PM
Development of Persistent Hyperplastic Primary Vitreous in Ephrin-A5-/Mice
Alexander I. Son1A, Margaret A. Cooper2, Yuhai Sun1B, Norman J. Kleiman3,
Renping Zhou4. ANeuroscience, BChemical Biology, 1Rutgers Univ, State
University of NJ, Piscataway, NJ; 2Molecular Biophysics and Biochemistry, Yale
University, New Haven, CT; 3Environmental Health Sciences, Columbia
University, New York, NY; 4Chemical Biology, Rutgers University, Piscataway,
Purpose: The primary vitreous is a collection of cells appearing early in ocular
development and occupying the region which will eventually become the vitreous
humor. These cells evolve into the hyaloid vasculature, the circulatory network for
supplying nutrients and removing metabolic waste in the developing mammalian
eye. After birth, the hyaloid vasculature regresses to allow unimpeded light passage
to the retina. Failure to regress results in severe consequences, the most notable of
which is the development of the pathology Persistent Hyperplastic Primary
Vitreous (PHPV). This condition is characterized by the presence of a large
retrolental fibrous mass, sometime accompanied by other developmental ocular
defects and usually associated with visual disability and/or blindness. Although the
condition has been described for more than 50 years, the mechanisms underlying
primary vitreous regression remain poorly understood. We have recently found that
mice lacking the A-class ligand ephrin-A5 develop hallmark symptoms of PHPV.
Thus, expression of the Eph family of receptor tyrosine kinases is essential role to
the regression of the primary vitreous and hyaloid vasculature. In the present study,
we characterize the nature of PHPV in ephrin-A5-/- mice.
Methods: Wild-type and ephrin-A5-/- eyes were examined at various
developmental stages to determine the progression of PHPV. Eye tissue was
sectioned and examined by H&E staining. Protein expression and localization was
determined through immunohistochemistry. Relative levels of Eph receptors were
determined by RT-PCR.
Results: Ephrin-A5-/- mice develop a large pigmented mass attached to the retina
posterior to the lens. In contrast to wild-type eyes, where cells of the primary
vitreous regress by birth, primary vitreous cells in the ephrin-A5-/- mice persist
throughout their lifetime. In these animals a hyperplastic mass consisting of neural
crest-derived pigmented cells is noted encapsulating the hyaloid vasculature. These
cells are mitotically active in the adult ephrin-A5-/- mouse. Eph receptor expression
is observed throughout the mass at postnatal stages. Ephrin-A5 expression is
observed throughout the eye but not within the developing primary vitreous.
Conclusions: These findings identify ephrin-A5 to be essential to the regression of
the primary vitreous in a non-cell-autonomous manner. Absence of ephrin-A5
results in the failed regression of the primary vitreous and resultant continued
presence of the hyaloid vasculature. Loss of ephrin-A5 expression may also
contribute to associated or subsequent ocular pathologies.
Commercial Relationships: Alexander I. Son, None; Margaret A. Cooper,
None; Yuhai Sun, None; Norman J. Kleiman, None; Renping Zhou, None
Support: NEI Grant R01EY019012 and NIA Grant F31AG032806
Program Number: 2548 Poster Board Number: D926
Presentation Time: 3:45 PM - 5:30 PM
Pathological Retinal Neovascularization In Oxygen-induced Retinopathy Is
Increased In Timp3-/- Mice, And Inhibited By Intravitreal Injection Of
Nina J. Jonsson1,2, Gisela Weskamp2, Krzysztof Glomski3, Steven L. Swendeman2,
Rama Khokha4, Carl P. Blobel2,3. 1Department of Ophthalmology, Weill Cornell
Medical College, New York, NY; 2Arthritis and Tissue Degeneration Program,
Hospital for Special Surgery, New York, NY; 3Biochemistry, Cell and Molecular
Biology Program, Weill Medical College of Cornell University, New York, NY;
Department of Medical Biophysics, Ontario Cancer Institute, University of
Toronto, Toronto, ON, Canada.
Purpose: Pathological neovascularization is causal for the development of
proliferative retinopathies. Previous studies have shown that pathological
neovascularization is reduced by inactivation of ADAM17, which is important for
EGFR signaling by cleaving and activating several EGFR-ligands. The tissue
inhibitor of metalloproteinases-3 (TIMP-3) is a natural inhibitor of ADAM17. In
this study we investigated whether intravitreal injection of the EGFR-inhibitor
Erlotinib (Tarceva®), an EGFR antagonist, inhibits pathological neovascularization
after oxygen-induced retinopathy (OIR). Moreover, we tested how the lack of
TIMP3 affected retinal neovascularization in mice following OIR.
Methods: Wild-type mice and Timp-3-/- mice underwent OIR (75% oxygen for 5
days starting at postnatal day 7 (P7), followed by 21% oxygen for additional 5
days). On P12 wild-type mice underwent a single intravitreal injection (0.5µl) of
Erlotinib (20µM) or carrier control. The central avascular area and the development
of neovascular tufts were measured at P17.
Results: Mice injected with Erlotinib developed fewer neovascular tufts above the
internal limiting membrane when compared to the untreated eye of a littermate, but
vessel re-growth into the avascular area was not affected. Timp-3-/- mice showed
greater revascularization and development of more neovascular tufts above the
internal limiting membrane.
Conclusions: These studies demonstrate that Erlotinib is able to inhibit
pathological neovascularization in mouse retina, most likely due to inactivation of
the EGFR. These studies also suggest that TIMP-3 is a natural negative regulator of
pathological neovascularization. Erlotinib, and potentially also TIMP3, could be
used as anti-angiogenic compounds to prevent proliferative retinopathies.
Commercial Relationships: Nina J. Jonsson, None; Gisela Weskamp,
None; Krzysztof Glomski, None; Steven L. Swendeman, None; Rama Khokha,
None; Carl P. Blobel, None
Support: Research grant, Dr. Werner Jackstaedt Foundation, Wuppertal Germany
(NJJ); GM64750 (CPB, GW, SLS); NHLBI, T32 GM007739-31S1 (KG)
Program Number: 2549 Poster Board Number: D927
Presentation Time: 3:45 PM - 5:30 PM
Birth Weight Is The Most Important Predictor For Abnormal Retinal
Vascularisation In Moderately To Late Preterm Infants
Marita A. Gronlund1, Kerstin Allvin2, Jovanna Dahlgren2, Ann Hellström1.
Pediatric Ophthalmology, Inst Neurosci & Phys/Ophthal, Goteborg, Sweden;
Pediatrics, Inst of Clinical Science, The Sahlgrenska Academy at the University of
Gothenburg, Goteborg, Sweden.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Purpose: The aim of this prospective study was to find predictors of abnormal
retinal vascularisation in moderately to late preterm newborn infants considered
having no risk of developing retinopathy of prematurity (ROP).
Methods: Seventy-eight infants (34 girls, 44 boys), 27% born small for gestational
age (SGA) were recruited from an ongoing longitudinal study of otherwise healthy
premature children born with a gestational age (GA) of 32.0 to 36.9 weeks. Retinal
vessel morphology was evaluated manually according to a standardized protocol by
one of the authors (AH) during neonatal life (mean postnatal age: 7 days (range 128 days). Insulin-like growth factor-I (IGF-I) levels were analyzed in umbilical
cord blood.
Results: Totally 21/78 infants (27%) had abnormal retinal vessel morphology such
as increased vascular tortuosity (n=3), few vascular branching points (n=12),
increased number of vascular branching points (n=1), and narrow arterioles and/or
venules (n=13). They had significantly lower mean ± SD birth weight (1850 ± 430
versus 2410 ± 450 gram, p<0.0001; -2.0 ± 1.5 versus -0.6 ± 1.1 standard deviation
scores (SDS), p=0.0001), birth length (43.2 ± 2.2 versus 45.9 ± 2.5 cm, p<0.0001; 1.6 ± 1.2 versus -0.5 ± 1.4 SDS, p=0.0007), head circumference at birth (30.7 ± 1.6
cm versus 32.1 ± 1.8 cm, p<0.01), with lower GA (33.9 ± 0.9 versus 34.9 ± 1.3,
p=0.004) and IGF-I levels (24.6 ± 17.0 versus 46.7 ± 21.5 µg/L, p<0.0001) as well
as a higher percentage of SGA infants (57.1% versus 15.8%, p=0.001) and
maternal hypertension or preeclampsia (47.6% versus 19.3%, p=0.03). Stepwise
logistic regression shows that birth weight is the strongest predictor and no other
variables contribute significantly to the model explanation.
Conclusions: In this population of moderately to late preterm newborns, birth size,
and especially birth weight, seems to affect the vascular system, which could result
in a lower threshold for the development of vascular disease later in life.
Commercial Relationships: Marita A. Gronlund, None; Kerstin Allvin,
None; Jovanna Dahlgren, None; Ann Hellström, None
Support: The Swedish research Council (552-7238), the Swedish Society of
Medicine, Gothenburg Medical Society, Agreement concerning research and
education of doctors (ALFGBG-11626 and ALFGBG-11869).
Program Number: 2550 Poster Board Number: D928
Presentation Time: 3:45 PM - 5:30 PM
The Effects of the ApoE Genotype on Murine Retinal Vasculature in
Development and Aging
Idit Maharshak1,2, Tami Livnat3, Yael Nisgav3, Mordechai Rosner4, Arieh S.
Solomon4, Dov Weinberger5,2, Carol A. Colton6, Daniel M. Michaelson1.
Department of Neurobiology, George S. Wise Faculty of Life Sciences, Tel Aviv
University, Tel Aviv, Israel; 2Department of Ophthalmology, Sackler School of
Medicine, Tel-Aviv University, Tel-Aviv, Israel; 3Laboratory of Eye Research,
Felsenstein Medical Research Center, Rabin Medical Center, Petach Tikva, Israel;
Goldschleger Eye Institute, Sackler School of Medicine, Tel-Aviv University, TelHashomer, Israel; 5Department of Ophthalmology, Rabin Medical Center, Petach
Tikva, Israel; 6Division of Neurology, Duke University Medical Center,
Department of Medicine, Durham, NC.
Purpose: ApoE4, the most prevalent genetic risk factor of Alzheimer’s disease, is
protective for age related macular degeneration, the most prevalent cause of
blindness in the older population in industrial
countries. Our hypothesis suggests that apoE4 affects vascular plastic changes in
the retina during development and aging. The objective of this study is to test this
hypothesis and to investigate
the mechanisms of this effect.
Methods: Retinal whole mounts of neonatal, 3 and 8 months old human apoE4 and
apoE3 targeted replacement mice were stained for endothelial cells. Confocal
images of the vasculature were analyzed. The Oxygen Induced Retinopathy (OIR)
model was applied to neonatal apoE4 and apoE3 mice (i.e. exposure to hyperoxia
on post-natal days 7 to 12) which were sacrificed at different time points. Paraffin
cross-sections of the eyes were analyzed for neovascularization. Quantitative RTPCR analysis of mRNAs of VEGF ligands and receptors was performed on the OIR
Results: 1. Neonatal vascular development. We found a higher vascular density
and an increased number of vascular buds in apoE4 mice compared to apoE3 mice
(p=0.06 and p=0.002, respectively). These differences were transient and did not
show up in adult mice. 2. Neonatal OIR model. This revealed a higher number of
neovascular tufts in the apoE4 Vs. the apoE3 mice (p=0.03). The hyperoxia treated
mice in both groups had elevated VEGFA, VEGFB, VEGFR1 and VEGFR2
expression levels compared to sham mice, which were however similar in the
apoE4 and apoE3 mice. Interestingly, in the non treated control mice the expression
of VEGFB in the apoE4 mice was higher than that of the corresponding apoE3
mice . This effect was transient and was maximal on post-natal day 7 (p=0.03). 3.
Retinal vasculature in aging. We found an age dependent decrease in retinal
vascular density in both groups with a significantly more pronounced decrease in
the apoeE4 mice compared to the apoE3 mice (p=0.01).
Conclusions: 1. The ApoE4 genotype affects retinal vascular development. This is
associated with a transient increase in vascular buds, vascular density, and
increased expression of VEGFB. 2. The OIR model revealed a similar elevation in
VEGF ligands and receptors in both groups of treated mice, with an increased
neovascularization in apoE4 mice, suggesting that these effects of apoE4 are not
mediated by VEGF. 3. Whereas the density of the retinal vasculature of young
adult apoE4 and apoE3 mice is similar, there is a significant reduction in vascular
density in aged apoE4 mice compared to corresponding aged apoE3 mice.
Commercial Relationships: Idit Maharshak, None; Tami Livnat, None; Yael
Nisgav, None; Mordechai Rosner, None; Arieh S. Solomon, None; Dov
Weinberger, None; Carol A. Colton, None; Daniel M. Michaelson, None
Support: None
Program Number: 2551 Poster Board Number: D929
Presentation Time: 3:45 PM - 5:30 PM
Norrin Expression In The Endothelial Cell In Developing Retina
Hanjae Lee1,2, Esther Yang2, Dong Hyun Jo3,2, Young Suk Yu3,2, Jeong Hun Kim3,2.
Brown University, Providence, RI; 2Fight against Angiogenesis-Related Blindness
(FARB) Laboratory, Clinical Research Institute, Seoul National University
Hospital, Seoul, Republic of Korea; 3Department of Ophthalmology, College of
Medicine, Seoul National University, Seoul, Republic of Korea.
Purpose: To investigate the Norrin expression during retinal angiogenesis and
trace the primary source of Norrin to find out whether a specific cell group is
involved in the process, and if so, how these cells might be implicated with the
activation of the classical wnt pathway.
Methods: Using the developing retina of postnatal 4, 8, 12, 16 and 28 mouse
(C57BL/6), we first examined the expression of Norrin through
immunohistochemistry. Then, to confirm the primary source of Norrin,
immunofluorescence was performed for Norrin and Collagen Type IV (EC
Results: We confirm, for the first time, that Norrin protein is expressed from the
endothelial cells (ECs) of the retinal blood vessels. Through
immunohistochemistry, it was observed that Norrin expression coincided with the
vascularization of the developing retina. In the early stage of the development,
during which retinal angiogenesis mainly occurs in the ganglion cell layer (GCL),
the Norrin expression was also limited to the GCL. In the later stage, as more
ocular capillaries formed in the deeper layer of the retina, more Norrin was
expressed in the deeper layer as well. From this pattern and shape of Norrin
expression in the developing retina, ECs were hypothesized to be the primary
source of Norrin. This was confirmed through double immunofluorescence of
Norrin and Collagen Type IV.
Conclusions: Since Norrin is crucial in the normal development and maintenance
of ocular capillaries, our finding suggests the Norrin’s involvement in the self
generative and protective mechanism of the ECs, which themselves seem to secret
Norrin and activate the wnt pathway.
Commercial Relationships: Hanjae Lee, None; Esther Yang, None; Dong
Hyun Jo, None; Young Suk Yu, None; Jeong Hun Kim, None
Support: None
Program Number: 2552 Poster Board Number: D930
Presentation Time: 3:45 PM - 5:30 PM
Nrf2 Modulates Retinal Revascularization And Pathologic Angiogenesis In
Oxygen-induced Retinopathy
Yanhong Wei1, Junsong Gong1, Zhenhua Xu1, Rajesh Thimmulappa2, Shyam
Biswal2, Elia Duh1. 1Ophthalmology, Johns Hopkins University, Baltimore, MD;
Environmental Health Sciences, Johns Hopkins University, Bloomberg School of
Public Health, Baltimore, MD.
Purpose: The oxygen-induced retinopathy (OIR) model has yielded significant
insights into the regulation of physiologic retinal revascularization, but much
remains unknown regarding molecules that promotes revascularization in this
condition. Nrf2 has a well-known cytoprotective role in many tissues including the
retina. The purpose of this study is to investigate the possible role of Nrf2 in
oxygen-induced retinopathy using Nrf2-deficient mice.
Methods: Nrf2 knockout mice (Nrf2-/-) and corresponding wild-type control mice
were subjected hyperoxia from postnatal day 7 (P7) to 12, followed by return to
room air. Retinal flatmounts were prepared at P12 and P17, and analyzed for
avascular retinal area as well as retinal neovascularization. Nrf2 translocation was
evaluated beginning at various time-points by immunoblotting of nuclear and
cytosolic retinal extracts. GSH levels were measured from P12 to P17. Real-time
PCR was used to evaluate expression of multiple pro-inflammatory genes. The
aortic ring assay, using segments from knockout and wild-type mice, was employed
as a complementary approach to assess vascularization.
Results: Nrf2 knockout mice exhibited significantly increased avascular retina and
pathologic retinal neovascularization at P17 compared to wild-type. Nrf2
translocation in wild-type mice was detected as early as P12 + 2 hours. GSH levels
were initially reduced in both wild-type and knockout mice. GSH levels were
eventually replenished in wild-type but not knockout mice. Nrf2 knockout mice
exhibited accentuation of pro-inflammatory gene expression, including IL-1β.
Similar to OIR retinas, aortic ring segments from Nrf2 knockout mice exhibited
decreased vascularization compared to wild-type.
Conclusions: These studies indicate that Nrf2 is an important modulator of retinal
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
revascularization and pathologic neovascularization in oxygen-induced retinopathy,
regulating both oxidative stress and pro-inflammatory gene expression.
Commercial Relationships: Yanhong Wei, None; Junsong Gong,
None; Zhenhua Xu, None; Rajesh Thimmulappa, None; Shyam Biswal,
None; Elia Duh, None
Support: Research to Prevent Blindness
Program Number: 2553 Poster Board Number: D931
Presentation Time: 3:45 PM - 5:30 PM
Hyaluronic Acid as a Contributing Factor to Epithelial-Mesenchymal
Transition in Retinal Pigment Epithelial Cells and Angiogenesis
Eri Takahashi, Hidenobu Tanihara. Ophthalmology, Kumamoto University
Graduate School of Medical Sciences, Kumamoto, Japan.
Purpose: To elucidate potential role of hyaluronic acid (HA) in epithelialmesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells and
Methods: ARPE-19 cells were cultured and stimulated with tumor necrosis factorα (TNF-α) with or without hyaluronidase (HAase) treatments. Morphological
changes were photographed by phase-contrast microscopy. Status of adherens
junctions was observed by immunofluorescence. Endothelial cell-attachment assay
was performed between human umbilical vein endothelial cells (HUVECs) and
ARPE-19 cells.
Results: In cultured ARPE-19 cells, EMT-associated fibrotic changes resulted
from interplay between TNF-α and transforming growth factor-β (TGF-β)
signaling, as reported in our previous study (Takahashi et al., J Biol Chem, 2010).
Immunostaining experiments demonstrated abundant expression of HA in the TNFα-induced cell aggregations (tentatively designated as EMT-associated fibrotic foci,
EAFFs) in ARPE-19 cells. After the occurrence of EAFFs, additional experiments
with HAase treatment and removal of TNF-α from the culture media induced the
reverse changes in cell types, mesenchymal to epithelial transition (MET). In this
experimental condition, the number of TNF-α-induced EAFFs was significantly
reduced, and cadherin-madiated cell-cell interaction was re-established. Moreover,
in further experiments by using co-cultured ARPE-19 with HUVECs, our studies
showed that vascular endothelial cells could adhere onto the cultured RPE cells
predominantly in TNF-α-induced HA deposits.
Conclusions: It is well known that, in chronic inflammatory lesions, inflammatory
cells migrate, and produce numerous cytokines, contributing to the occurrence of
EMT. Subsequently, EMT elicits over-production of extracellular matrix in the
msenchymal cells (originated from epithelial cells), resulting in organ fibrosis. Our
results showed that HA may play not only a key role of maintenance of EMT in
RPE cells, but also can be a critical contributing factor to regulate RPE-associated
angiogenesis. Our hypothesis implies that regulation of HA-associated phenomena
may allow us to create new strategy for the treatment of choroidal
neovascularization and other inflammatory disorders in the outer retina.
Commercial Relationships: Eri Takahashi, None; Hidenobu Tanihara, None
Support: None
Program Number: 2554 Poster Board Number: D932
Presentation Time: 3:45 PM - 5:30 PM
TMP Prevents Retinal Neovascularization and Imparts Neuroprotection in an
Oxygen-Induced Retinopathy Model
Huanjiao Zhou, Yungang Ding, Shiqing Li, Gang Sun, Cheng Yang, Xifang Zhang,
Liqing Wei, Jian Ge, Xiaoling Liang. State Key Laboratory of Ophthalmology,
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
Purpose: To evaluate the effects of Tetramethylpyrazine (TMP) on retinal
pathological neovascularization and neuroprotection in an oxygen-induced
retinopathy (OIR) model.
Methods: Neonatal C57BL/6J mice were subjected to 75% oxygen from postnatal
day (P) 7 to P12 and then were abruptly placed in room air. TMP (200mg/kg) or
normal saline was given daily from P12, and mice were sacrificed at P14, P15 or
P17. Retina whole-mount and immunofluorescence were used to assess the effects
of TMP on retinal neurovascular repair.
Results: Administration of TMP during ischemic phase of the retinopathy
effectively prevented pathological intravitreal neovascular tufts (P<0.01), and
decreased the avascular area of the retina (P<0.05). These morphologic changes
were associated with the protective role of TMP in markedly enhancing the
formation of endothelial tip cells at the edges of the repairing capillary networks
and preserving the astrocytic template in the avascular area. TMP also reduced the
reactive expression of GFAP in Müller cells. In the avascular area of TMP-treated
retinas, there was a less obvious loss of amacrine cell bodies and disorganized
distinct bands; the number of both rod bipolar and horizontal cell bodies, as well as
the density of their synaptic terminals in the outer plexiform layer, was greater than
in OIR control mice. Moreover, TMP decreased the loss of alignment of Müller cell
bodies and distortion of processes observed in OIR control mice.
Conclusions: TMP improved neurovascular recovery by preventing retinal
pathological neovascularization, accelerating physiological revascularization, and
protecting retinal astroglias and neurons from ischemia-induced cell death.
Commercial Relationships: Huanjiao Zhou, None; Yungang Ding,
None; Shiqing Li, None; Gang Sun, None; Cheng Yang, None; Xifang Zhang,
None; Liqing Wei, None; Jian Ge, None; Xiaoling Liang, None
Support: None
Program Number: 2555 Poster Board Number: D933
Presentation Time: 3:45 PM - 5:30 PM
Tweak/fn14 Pathway Is A Novel Mediator Of Neovascularization During
Ischemic Retinopathy
Hossein Ameri1A, Jun Wang1A, Ronald G. Tilton1A,1B, Bernard F. Godley1A, Wenbo
Zhang1A. AOphthalmology & Visual Sciences, BDivision of Endocrinology and
Stark Diabetes Center, Department of Internal Medicine, 1University of Texas
Medical Branch, Galveston, TX.
Purpose: Retinal neovascularization is a major cause of blindness in proliferative
diabetic retinopathy and retinopathy of prematurity. The interaction between tumor
necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor
Fn14 is implicated in angiogenesis but the specific role of TWEAK/Fn14 in retinal
diseases is completely unknown. The goal of this study was to test whether
TWEAK/Fn14 is required for retinal neovascularization during ischemic
Methods: Oxygen-induced ischemic retinopathy (OIR) was produced by
maintaining C57BL/6 wild type neonatal mice in 75% oxygen from postnatal day
(P)7 to P12 and room air from P12 to P17. Control mice were kept in room air.
Retinas were prepared for analysis of mRNA for TWEAK and Fn14 by quantitative
PCR. The interaction of TWEAK and Fn14 was blocked by intravitreal injection of
a soluble Fn14-Fc decoy receptor (2.5 µg/eye) at P12. The retinal vasculature was
examined at P17 by fluorescence microscopy after labeling retinal whole mounts
with isolectin-B4.
Results: At P12 after pups were exposed to hyperoxia for 5 days, there was no
change in retinal levels of Fn14 and TWEAK mRNA vs control retinas. Fn14
mRNA remained constant in control mice from P12 to P17. However, the level of
Fn14 was markedly increased at P13 in OIR mice and maintained at high level (~ 3
fold vs control) until P17. TWEAK mRNA was increased in control mice from P12
to P17 but OIR did not increase it further. Treatment with Fn14-Fc decoy receptor
substantially reduced retinal neovascularization during ischemic retinopathy.
Conclusions: These findings suggest that the TWEAK/Fn14 pathway is
upregulated and is critically involved in the initiation and progression of retinal
neovascularization during ischemic retinopathy.
Commercial Relationships: Hossein Ameri, None; Jun Wang, None; Ronald
G. Tilton, None; Bernard F. Godley, None; Wenbo Zhang, None
Support: AHA11SDG4960005 and JDRF 10-2009-575 (W.Z.); RPB (B.F.G. &
H.A.); NIH DK079053 (R.G.T.)
Program Number: 2556 Poster Board Number: D934
Presentation Time: 3:45 PM - 5:30 PM
The Effect Of Bh4 Deficiency In Ischemic Retinopathy
Denise M. McDonald1, Kevin Edgar1, Nuria Matesanz1, Keith Channon2, Tom A.
Gardiner1. 1Vision and Vascular Science, Queens University Belfast, Belfast,
United Kingdom; 2Cardiovascular Medicine, Oxford University, Oxford, United
Purpose: Nitric oxide (NO) can have opposing functions in ischemic retinopathy
as it can either impede or promote vascular recovery. These conflicting roles are
believed to be dependent upon the enzymatic and cellular source of NO production;
endothelial nitric oxide synthase (eNOS) in the endothelium or inducible NOS
(iNOS), upregulated in reponse to hypoxia in Muller and microglial cells. All NOS
isoforms require the cofactor tetrahydrobiopterin (BH4) for full activity and here
using a murine model partially deficient in BH4 (hph-1) we investigated the role of
BH4 in ischemic retinopathy.
Methods: Hph-1 mice and littermate controls were subject to oxygen-induced
retinopathy (OIR) by exposure to 75% oxygen (postnatal days 7-12) and retinas
collected at various time points following return to room air. Retinal cell death in
the acute hypoxic phase (P13; high iNOS expression) was determined by TUNEL
staining. Vascular growth in the proliferative phase of OIR (P17) was quantified by
B simplicifolia isolectin staining of retinal flat mounts. Vascular growth was also
determined in aortic explants cultured in Matrigel.
Results: At P13 there was a significant decrease in the number of TUNEL positive
apoptotic cells in the inner nuclear layer of the ischemic retina of the hph-1 group.
In parallel, this group also demonstrated reduced retinal revascularisation and
neovascularisation at P17. Accordingly, aortic explants also exhibited reduced tube
formation reversible upon BH4 supplementation.
Conclusions: Collectively, our results demonstrate that BH4 deficiency attenuates
angiogenic drive and vascular growth and also reduces hypoxia induced retinal
Khoo et al Circulation 2005 111:2126-33.
Commercial Relationships: Denise M. McDonald, None; Kevin Edgar,
None; Nuria Matesanz, None; Keith Channon, None; Tom A. Gardiner, None
Support: Wellcome Trust UK; Action Medical Research UK
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2557 Poster Board Number: D935
Presentation Time: 3:45 PM - 5:30 PM
Altered Retinal Vasculature in Hyperhomocysteinemic Mice
Amany M. Tawfik1A, Srinivas Sonne1B, Cory Williams1A, Mohamed Al-Shabrawey1C,
Vadivel Ganapathy1B, Sylvia B. Smith1A. ACellular Biology and Anatomy,
Biochemistry, COral Biology and Anatomy, 1Georgia Health Sciences Univ,
Augusta, GA.
Purpose: Homocysteine (Hcy) is a non-proteinogenic amino acid whose elevation
has been implicated in human visual disorders. Recently, we investigated the retinal
phenotype of mice with moderate/severe hyperhomocysteinemia due to
deficiency/absence, respectively, of the gene encoding cystathionine-beta-synthase
(CBS). We reported marked retinal disruption, ganglion cell loss, overstimulation
of the NMDA receptor, optic nerve mitochondrial dysfunction and ERG defects in
cbs-/- mice and a delayed morphological/functional phenotype in cbs+/- mice
(Ganapathy et al, 2009, 2011; Yu et al, 2011). Excess Hcy is an independent risk
factor for human systemic cardiovascular diseases such as hypertension; however it
is not known whether excess Hcy alters retinal vasculature. We used cbs mutant
mice to address this.
Methods: Retinas of cbs+/+ (n =21), cbs +/- (n = 34), cbs -/- (n =16) mice (age 2 wks)
were harvested for wholemount preparation or cryosections and subjected to
immunofluorescence microscopy to (1) visualize vessels via the endothelial cell
marker isolectin-B4; to detect (2) angiogenesis (VEGF and CD 105 (endoglin)), (3)
activated glial cells (GFAP), (4) inflammation (TNF-α) and (5) superoxide
production (DHE). Retinal wholemounts were examined by LSCM; cryosections
were evaluated using a Zeiss Axioplan-2 microscope and the AxioVision program.
Results: Retinal vasculature of cbs+/- mice was similar to WT mice, consistent with
a normal phenotype at this age (Ganapathy et al, 2009). However, cbs-/- mice
demonstrated vascular patterns consistent with ischemia: isolectin B4 labeling
revealed a marked capillary-free zone in the central retina and new vessels with
capillary tufts in the mid-peripheral area. This altered vascular pattern was
associated with increased VEGF and CD105 levels. In retinal cryosections, marked
increase in GFAP, consistent with glial cell activation was observed in cbs -/- mice,
as were increased levels of TNFα and superoxide.
Conclusions: The findings suggest that severe elevation of Hcy, as seen in the cbs/mouse, is accompanied by alterations in retinal vasculature. The retinal disruption
and decreased visual function reported previously in cbs-/- mice may reflect
vasculopathy as well as the neuropathy.
Commercial Relationships: Amany M. Tawfik, None; Srinivas Sonne,
None; Cory Williams, None; Mohamed Al-Shabrawey, None; Vadivel
Ganapathy, None; Sylvia B. Smith, None
Support: National Eye Institute,NIH Grant EY012830
Program Number: 2558 Poster Board Number: D936
Presentation Time: 3:45 PM - 5:30 PM
A Viable Mouse PDGFR-Beta Mutant Exhibits CNS-restricted Pericyte
Deficiency And Blood Brain Barrier Defects
Shalini Jadeja, Margaret Keighren, Fay Cooper, Ian J. Jackson. MRC Human
Genetics Unit, MRC IGMM at the University of Edinburgh, Edinburgh, United
Purpose: To characterize the mouse mutant Pedv/128, a partial loss of function of
PdgfrB, as a possible model for Diabetic Retinopathy and as a tool for investigating
the role of pericytes in maintaining a normal retinal vasculature.
Methods: The mutant line Pedv/128 was identified during a recessive ENU
mutagenesis screen at MRC Harwell, with blood in the eyes and was named redeye.
Haplotype mapping followed by exome sequencing identified the gene mutated.
Subsequently, detailed histological analysis on eye sections and
immunohistochemistry on wholemount retinas were carried out to determine the
effect of the mutation on the retinal vasculature. Vascular permeability assays were
also performed to determine the extent of the vessel leakage.
Results: A donor splice site mutation was identified in Pdgfrb which caused partial
loss of normal splicing resulting in a frameshift and premature termination.
Consequently there is a substantial reduction in expression of the normal Pdgfrb
transcript. PDGF is an important regulator of embryological development, cell
proliferation, migration and survival. PDGFB/PDGFRΒ signalling is required for
pericyte recruitment to developing blood vessels in order to confer vessel stability
and more recently has been implicated in the formation of the Blood Brain Barrier
(BBB). We find that that the redeye strain exhibits vascular leakage due to a defect
in pericyte recruitment, that is confined to the central nervous system (CNS).
Consequently there is defective vessel patterning and vessel tortuousity, reduced
basement membrane deposition as well as changes in the expression of BBB
Conclusions: Loss of function mutants of Pdgfrb are usually embryonic lethal due
to severe haemorrhaging. Unusually, the redeye mutant mice are viable, with only
the CNS vasculature affected. They are therefore a better model for diseases
involving pericyte deficiencies such as Diabetic Retinopathy than those currently
being used. In addition, the defect in BBB development makes them a useful tool
for dissecting the role of pericytes in BBB formation and vascular development.
Commercial Relationships: Shalini Jadeja, None; Margaret Keighren,
None; Fay Cooper, None; Ian J. Jackson, None
Support: None
292 Life and Death of Photoreceptors
Monday, May 7, 2012, 3:45 PM - 5:30 PM
Hall B/C Poster Session
Program #/Board # Range: 2559-2592/D937-D970
Organizing Section: Retinal Cell Biology
Program Number: 2559 Poster Board Number: D937
Presentation Time: 3:45 PM - 5:30 PM
An Inexpensive Led Light Box For Light Damage In Rodents
Micah A. Chrenek1, Tiffany L. Liao1, Jana T. Sellers1, Jeffrey H. Boatright2, J M.
Nickerson3. 1Ophthalmology, Emory University, Atlanta, GA; 2Ophthalmology,
Emory University School of Med, Atlanta, GA; 3Ophthalmology, Emory Univ,
Atlanta, GA.
Purpose: We wished to develop a light box that alleviates some of the problems
associated with the classic light chambers used for light induced retinal
degeneration in mice. This study demonstrates the use of a low cost LED light box
for conducting light damage on single animals.
Methods: Fancier 500-A LED lamps were chosen because they can be modified
slightly to fit on clear polycarbonate model 750 cages from Lab Supply Inc
(standard mouse housing cages) and because they have can be adjusted in intensity
by turning on/off banks of lights and adjusting the intensity of the LEDs over a
wide range with a dial. We used white and black acrylic paint to paint the outside
of the cages to obtain a highly reflective interior of the cage. The light level in
boxes with bedding can be adjusted from 500-65000 lux. Retinal light damage was
assessed in cyclically reared Balb-C mice from Charles River and The Jackson
Laboratory, and C57BL/6 mice from Charles River. The C57BL/6 mice were
treated with 1% atropine eye drops 30 min prior to light damage to dilate their
pupils. Comparisons between a “classic” light chamber designed by Dan
Organisciak (IOVS 26:1580-8, 1985) and our LED light boxes were done with the
Balb-C mice. Measurements of visual outcome were done using ERG analysis 1
week and 3 weeks following light damage. Histology was performed on eyes after
ERGs to measure morphological changes in the retina including ONL nuclei
counts. We were also able to induce significant light damage in C57BL/6J mice
treated with atropine to dilate their pupils using 40000 lux for 6 hours.
Results: The cost per LED light box ranges from $215-265 depending on the sale
price of the LED lamps, as compared to approximately $4250 to have a custom
“classic” light box built (cost for manufacture in 2006). We compared the amount
of light damage using equivalent light intensity (8400 lux for 4 hours) using the
classic model and the LED model and found that the LED model caused 80% as
much damage as measured by ERG amplitudes 1 week following light damage.
Using single Balb-C mice per box and 10000 lux for 4 hours in the LED light box,
we found complete abrogation of the ERG response.
Conclusions: The LED boxes offer significant advantages (lower cost, much
higher light output, standard caging, reduced variability) over prior light damage
Commercial Relationships: Micah A. Chrenek, None; Tiffany L. Liao,
None; Jana T. Sellers, None; Jeffrey H. Boatright, None; J. M. Nickerson, None
Support: NIH R01EY016470, NIH R01EY014026, NIH R24EY017045, NIH
P30EY06360, The Abraham and Phyllis Katz Foundation, RPB
Program Number: 2560 Poster Board Number: D938
Presentation Time: 3:45 PM - 5:30 PM
Prevention of Retinal Light Damage by the Trace Element Zinc and the
Natural Antioxidant Rosemary
Daniel T. Organisciak1, Ruth M. Darrow1, Christine M. Rapp1, Rekha
Rangarajan2, John C. Lang2. 1Biochemistry & Molecular Biology, Wright State
University, Dayton, OH; 2Alcon Research Ltd., Fort Worth, TX.
Purpose: To assess protective mechanisms and potential synergistic effects
between a natural antioxidant and an essential trace element in an animal model of
light-induced retinal degeneration.
Methods: Weanling male Sprague-Dawley rats were maintained in darkness until
P60 and then exposed to intense visible light for 4 hours, beginning at 9 am. One
hour before light exposure some animals were injected 1X IP with various doses of
zinc oxide, or a Tween detergent extract of Rosemary powder. Other rats received
combined doses of zinc plus Rosemary prior to light treatment. Following light
exposure all animals were returned to the dark environment for 2 weeks to allow
for removal of dead photoreceptor cells and recovery of surviving photoreceptors.
To determine the extent of visual cell survival photoreceptor cell DNA and
rhodopsin levels were measured. Retinal histology and western analysis were
performed to confirm antioxidant efficacy and to assess changes in protein levels.
Results: Dose response curves for zinc oxide and Rosemary powder indicate that
each is effective in preventing retinal light damage. Compared to vehicle injected
rats, over 50% visual cell recovery occurred with zinc oxide at the AREDS
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
formulation recommended dose (1.3 mg/kg). Detergent extracts of Rosemary
powder (17 mg/kg) were equally effective. Zinc combined with Rosemary extract
was effective at one-half the dose of the individual compounds. Retinal histology
confirmed the beneficial effects of combined treatment. By western analysis,
immunoreactivity for heme oxygenase and carboxyethylpyrrole, were reduced by
either zinc or Rosemary treatment.
Conclusions: The protective efficacy of zinc combined with Rosemary indicates
that their effects on photoreceptor cell survival are additive. The levels of two
protein markers of oxidative stress were decreased by treatment with either zinc or
Rosemary, suggesting that their mechanisms of action may also be complementary.
The antioxidants present in Rosemary may also reduce potential toxicity associated
with high doses of zinc.
Commercial Relationships: Daniel T. Organisciak, Alcon Research Ltd.
(F); Ruth M. Darrow, None; Christine M. Rapp, None; Rekha Rangarajan,
Alcon Research Ltd. (E); John C. Lang, Alcon Research Ltd. (E)
Support: Alcon Ltd. Fort Worth TX and Ohio Lions Research Foundation
Program Number: 2561 Poster Board Number: D939
Presentation Time: 3:45 PM - 5:30 PM
Role of Endoplasmic Reticulum (ER) Stress Following Light Exposure in the
Canine T4R RHO Mutant Model
Stefania Marsili, Jeremy Gingrich, Gustavo D. Aguirre, William A. Beltran.
Clinical Studies, Univ of Pennsylvania, Sch Vet Med, Philadelphia, PA.
Purpose: The canine T4R RHO model of autosomal dominant retinitis pigmentosa
shows extreme sensitivity to light. Brief exposure to light levels commonly used in
clinical settings leads to acute death of rod photoreceptors that can be detected 6
hours after exposure. The purpose of this study is to assess the role of the unfolded
protein response (UPR) induced endoplasmic reticulum stress in light induced
retinal degeneration in the T4R RHO model.
Methods: Three heterozygous mutant (RHOT4R/+), and three wild type (RHO+/+)
dogs (ages: 12-24 weeks) were used. Following overnight dark adaptation, left eyes
were exposed to bright white light (corneal irradiance: 1mW/cm2) for 60s; right
eyes were shielded and used as negative control. Six hours following light
exposure, neuroretinas were collected under dim red light, snap-frozen in liquid
nitrogen, and subsequently processed for Western Blot analysis. Twenty or forty
micrograms of whole retinal tissue lysates were used to detect the levels of the
following stress-induced proteins: HSP70, HSF1, BIP/Grp78, PDI, calnexin, ATF4.
Beta-actin or α-tubulin were used as internal controls for normalization.
Results: No differences in protein expression levels of ER-resident (BIP/Grp78,
PDI, calnexin) and cytoplasmic (HSP70, HSF1) chaperones were found between
exposed and shielded retinas. Additionally, activation of the PERK-ATF4 ER stress
signaling pathway was not observed at the time point studied.
Conclusions: Preliminary results do not indicate the involvement of a UPR 6 hours
post light exposure in the T4R RHO mutant canine retina. On-going studies are
evaluating the role of the two other ER stress pathways downstream of the ATF6
and IRE1 ER sensors. In addition, the occurrence of ER stress is being examined at
earlier and later time points following light exposure.
Commercial Relationships: Stefania Marsili, None; Jeremy Gingrich,
None; Gustavo D. Aguirre, None; William A. Beltran, None
Support: EY-06855, -17549, R24EY021126, 2PNEY018241, Foundation Fighting
Program Number: 2562 Poster Board Number: D940
Presentation Time: 3:45 PM - 5:30 PM
Light-induced Retinal Damage: Role Of Dietary Zinc And Zinc Binding
Christian Sheline, Shi Bai. Ophthalmology & Neurosci Ctr, LSU Health Sciences
Center, New Orleans, LA.
Purpose: Intense visible light induces severe damage (LD) to retina and serves as a
model of human oxidation-induced, light-potentiated retinal degenerative diseases
such as AMD. Our previous study on LD suggests the involvement of zinc toxicity
in the death of photoreceptor and RPE cells after intense light. However, the
AREDS trial shows that chronic dietary Zn2+ supplementation (ZnE) delays the
progression of AMD. Zn2+ is present physiologically in layers of the retina: in
particular in the rod segments bound to rhodopsin, the outer plexiform layer, and
RPE cells. The loosely bound, histochemically reactive Zn2+ plays important roles
in the visual cycle, dark/light adaptation, neuro-transmission, and intracellular
metabolism. So we examined the effects of manipulating zinc levels on retinal zinc
binding protein levels (ZBP), and light-induced Zn2+ accumulation and damage in
rodents, by both reducing and increasing dietary zinc.
Methods: Sprague Dawley or CD1 albino rats or mice were fed for 3 wk with a 1
ppm zinc diet (ZnR), a 60 ppm zinc diet (ZnN, normal), or a 200 ppm zinc diet
(ZnE) and rats were exposed to 18 kLux of white light for 4 h. Retinal extracts
from mice were purified by Zn2+ affinity, and analyzed by 2D gels. ZBP’s were
identified by LC-MS.
Results: Both a ZnR and a ZnE diet attenuated Zn2+ accumulation and
photoreceptor degeneration after LD compared to ZnN. ZnE increases levels of a
group of low MW ZBP’s. Three groups of retinal ZBP proteins were identified by
proteomics and westerns: visual cycle proteins, energy metabolic enzymes, and
chaperone proteins.
Conclusions: Zn2+ homeostasis and ZBP’s are important in the visual cycle, stress,
LD, and retinal health and disease. Dietary Zn2+ manipulations which chronically
increase ZBP’s, or acutely reduce the retinal Zn2+ accumulation will attenuate
retinal injury in rodents, and may explain the efficacy of zinc supplementation on
AMD in humans.
Commercial Relationships: Christian Sheline, None; Shi Bai, None
Support: DK073446
Program Number: 2563 Poster Board Number: D941
Presentation Time: 3:45 PM - 5:30 PM
Microarray Analysis Following Murine Retinal Light Damage Reveals
Changes in Iron Regulatory Genes, Complement, and Antioxidant Genes in
the Neural Retina and Isolated RPE
Majda Hadziahmetovic1, Usha Kumar2, Ying Song1, Steven Grieco1, Yafeng Li1,
Delu Song1, Joshua L. Dunaief1. 1FM Kirby Ctr, Scheie Eye Institute Univ of Penn,
Philadelphia, PA; 2Drexel University, Philadelphia, PA.
Purpose: Light damage is an established model of retinal degeneration. The
purpose of our study was to investigate light damage induced transcript changes
within the neurosensory retina (NSR) and isolated RPE cells. Similar studies have
been conducted previously, but usually limited to the NSR and only a portion of the
genome. Herein we were able to query most of the genome in not just the neural
retina but also isolated RPE cells.
Methods: Mice (n = 4), were exposed to 10,000 lux cool white fluorescent light for
18 hours. The control group (n = 4) was kept on a regular 12-hour light-dark cycle.
Mice were sacrificed 4 hours after photic injury. NSR and isolated RPE were
collected, and RNA was isolated. DNA microarray hybridization was conducted as
described in the Affymetrix GeneChip Expression Analysis Technical Manual.
Microarray analysis was performed using probe intensity data derived from the
Mouse Gene 1.0 ST Array. For the genes of interest, confirmation of gene
expression was done using quantitative real-time PCR. Immunofluorescence
assessed protein levels and localization.
Results: Sixteen iron regulatory genes were significantly changed in the light
exposed NSR and 20 genes in the RPE. Several of these gene expression changes
could cause an iron-overloaded state. For example, the iron importer transferrin
receptor was upregulated in both light exposed NSR and RPE. Consistent with this,
there was stronger transferrin receptor immunoreactivity in the light exposed
retinas. In the oxidative-stress category, we identified 4 genes significantly changed
in the light exposed NSR and 6 genes in the RPE. In the complement category,
there were 5 genes in both the NSR and RPE that were significantly changed.
Conclusions: We introduce the concept of a photo-oxidative stress induced vicious
cycle of increased iron uptake followed by iron-exacerbated oxidative stress. Our
findings provide further insight into pathways by which excessive light exposure
may promote dangerous spiral of retinal oxidative stress.
Commercial Relationships: Majda Hadziahmetovic, None; Usha Kumar,
None; Ying Song, None; Steven Grieco, None; Yafeng Li, None; Delu Song,
None; Joshua L. Dunaief, None
Support: NIH EY015240, Research to Prevent Blindness, the F.M. Kirby
Foundation, the Arnold and Mabel Beckman Initiative for Macular Research, the
Paul and Evanina Bell Mackall Foundation Trust
Program Number: 2564 Poster Board Number: D942
Presentation Time: 3:45 PM - 5:30 PM
Chronic Photo-oxidative Stress And Subsequent Mcp-1 Activation As
Causative Factors For Age-related Macular Degeneration
Mihoko Suzuki1, Motohiro Kamei1, Motokazu Tsujikawa1, Ping Xie1, Zhao-Jiang
Du1, Hiroyuki Itabe2, Nagakazu Matsumura1, Kohji Nishida1. 1Ophthalmology,
Osaka University Graduate School of Medicine, Suita, Japan; 2Biological
Chemistry, Showa University, Shinagawa-ku, Japan.
Purpose: We previously reported that mild light irradiation induces phospholipids
oxidation and, subsequently, an increase of MCP-1 secretion and accumulation of
macrophages. The purpose of this study is to investigate whether chronic photooxidative stress is involved in the pathogenesis of age-related macular degeneration
(AMD) as an environmental factor.
Methods: Freely moving 12-month-old C57BL/6 wild mice were subjected to lowlevel blue-light irradiation (500 lux) every 2 days for 4 to 6 months. We performed
fundus examination, fluorescein angiography, and electron microscopic and
immunohistochemical examination. We also examined the difference of oxidized
phospholipids and monocyte chemoattractant protein-1(MCP-1) by real-time PCR,
ELISA and immunohistochemistry in acute blue-light irradiated mice (1000 lux)
with daily injected antioxidant, α-Phenyl-N-tert-butylnitrone (PBN) treatment.
Results: Choroidal neovascularization (CNV) was observed in 4 of 8 eyes (50%) in
long term light irradiated mice, while the non-irradiated control mice showed no
CNV (0%). Fluorescein angiography and histopathologic examinations revealed
that the CNV was the occult-dominant type with a proliferated vascular tissue
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
between the RPE and Bruch's membrane. Macrophages accumulation around the
CNV and engulfing the photoreceptor outer segments, including the oxidized
phospholipids, were revealed by the electron microscopic and
immunohistochemical examinations. The increased oxidized phopholipids and
MCP-1 mRNA and protein (p<0.001) after light irradiation were significantly
decreased by the PBN treatment (p<0.05).
Conclusions: The present study suggests that mild chronic photo-oxidative stress
may be involved in AMD pathogenesis via phospholipids oxidation and MCP-1
Commercial Relationships: Mihoko Suzuki, None; Motohiro Kamei,
None; Motokazu Tsujikawa, None; Ping Xie, None; Zhao-Jiang Du,
None; Hiroyuki Itabe, None; Nagakazu Matsumura, None; Kohji Nishida,
Support: Grant-in-Aid for Scientific Research from the Ministry of Education
Program Number: 2565 Poster Board Number: D943
Presentation Time: 3:45 PM - 5:30 PM
Progesterone Protects Photoreceptors From Light-induced Damage
Marisa A. Cubilla, Tomas P. Bachor, Vicente Bermudez, Melisa D. Marquioni,
Angela M. Suburo. Cell and Molecular Medicine, Ciencias Biomedicas Univ
Austral, Pilar, Argentina.
Purpose: We have previously shown that mifepristone (MFP), a blocker of
progesterone (PRG) and glucocorticoid (GC) receptors (GRs), increases retinal
light-induced damage. Although phototoxic lesions can be prevented by
dexamethasone (DEX), a pure GR agonist, PRG receptors could also be involved.
Therefore, we tested the effect of this steroid on light-exposed retinas.
Methods: Experimental work was done according to the ARVO Statement for the
use of animals. Male Balb-c mice (35-45 days-old), bred under standard
illumination conditions (12:12 h light: dark; < 60 lux), remained in complete
darkness for 24 h. Control mice returned to standard illumination, whereas
experimental animals were exposed to 1,500 lux. These mice received progesterone
(1 mg/kg/day) or its vehicle. The following parameters were evaluated at 2-4 days
of exposure: preservation of the outer nuclear layer (ONL) in neutral red-stained
cryosections; DNA fragmentation in the ONL by TUNEL; and rhodopsin (RHO)
and cleaved caspase-3 (CC-3) by Western blot.
Results: After 2 days of light exposure, the ONL showed numerous TUNEL+
nuclei, and retinal extracts showed a reduction of RHO levels and the appearance of
CC-3 protein. In exposed animals receiving PRG, the ONL had no TUNEL+ nuclei,
and extracts did not contain CC-3 protein. RHO was equal or higher than in
controls. No structural damage could be discerned after 2 day exposure, therefore,
we used 4-day exposed retinas to evaluate changes in the number of rows in the
ONL. In animals receiving VHC injections, photoreceptor nuclei were missing in
about 50% of the retinal area. Largest damage appeared in the dorsotemporal
quadrant, close to the optic nerve head. No abnormalities of ONL could be detected
in PRG-treated mice
Conclusions: Progesterone prevented light-induced damage in mice retina exposed
to 1,500 lux. The PRG (1 mg/kg) protective effect was equal or better than the
protection provided by DEX (4 mg/kg, Cubilla et al., in preparation)
Commercial Relationships: Marisa A. Cubilla, None; Tomas P. Bachor,
None; Vicente Bermudez, None; Melisa D. Marquioni, None; Angela M.
Suburo, None
Support: PICTO Austral 2008-0090
Program Number: 2566 Poster Board Number: D944
Presentation Time: 3:45 PM - 5:30 PM
Phototoxic Retinal Damage From Industry Standard Light In Albino Rats In
Carcinogenicity Study With 1,4-Benzenedicarboxylic Acid, Bis (2-ethylhexyl)
Vivian Lee1, Leandro B. Teixeira2, James Deyo3, Daniel M. Albert4A, Richard R.
Dubielzig4B. 1Department of Ophthalmology and Visual Sciences, University of
Wisconsin, Madison, WI; 2Pathobiological Science, UW-Madison Sch of Vet Med,
Madison, WI; 3Toxicology, Eastman Chemical Company, Kingsport, TN;
Ophthal/Vis Sciences, BPathobiol Sciences, 4Univ of Wisconsin-Madison,
Madison, WI.
Purpose: To report the retinal changes in albino rats induced from industry
standard light exposure, and report these changes in the context of a toxicity study
with compound, 1,4-Benzenedicarboxylic acid, bis (2-ethylhexyl) ester (DEHT).
Methods: F-344 albino rats were maintained under standard conditions to assess
the carcinogenicity of Eastman Plasticizer 168 [1,4-Benzenedicarboxylic acid, bis
(2-ethylhexyl) ester (DEHT)]. The rats were exposed to industry standard ambient
light for 2 years. DEHT was administered in the diet for 104 weeks at dietary levels
of 1500, 6000, and 12000 ppm. At the end of the 2-year period, the eyes were
removed and processed for routine histologic evaluation. Each eye was
independently and blindly examined for histopathological changes in the retina.
Results: Overall achieved dosages of DEHT were 79, 324, and 666mg/kg/day for
males and 102, 418, 901 mg/kg/day for females. Degeneration of the outer nuclear
layer of the retina was noted in some degree in all of the eyes, including the control
group. Terminal males and females given 6000 and 12000 ppm, however, exhibited
an increase in the severity of degenerative changes over terminal control animals.
The retina of the female rats also appeared to be more severely affected than the
male rats even though the female rats only achieved slightly higher doses of DEHT.
Conclusions: Previous investigators have reported the phototoxic effects on the
retina of albino rats that have been exposed to intense light levels for short periods
of time as a model for age related macular degeneration. In this study, albino rats
demonstrated microscopic changes that are morphologically consistent with photoinduced retinal degeneration resulting from exposure to industry standard
illumination. These changes occurred not only in the experiment group but also in
the control group. DEHT appeared to potentiate these changes, exhibiting a dose
dependent effect on the retinal damage. On subanalysis, gender appeared to have an
effect on the phototoxic retinal changes, where female rats exhibited increased
degenerative changes over male rats. Increased survivability for high dose females
may have contributed to this difference. Therefore, it is highly recommended that
studies conducted on albino rats exposed to industry standard illumination for
extended periods of time be aware of the retinal changes that may occur, as well as
to perform subanalyses with time matched controls and genders since these factors
may alter the conclusions of future studies.
Commercial Relationships: Vivian Lee, None; Leandro B. Teixeira, None;
James Deyo, Senior Associate (E); Daniel M. Albert, None; Richard R.
Dubielzig, None
Support: None
Program Number: 2567 Poster Board Number: D945
Presentation Time: 3:45 PM - 5:30 PM
Oxidative Stress In Photoreceptors After Blue Light Exposure And Rescue By
Wide-range Long-wavelength Light
Richard H. Funk1A,2, Ulrike Schumann1A, Marius Ader3, Lilla Knels1B, Cora
Roehlecke1A. AInst of Anatomy, BDepartment of Anatomy, 1TU Dresden, Dresden,
Germany; 2CRTD / DFG-Center for Regenerative Therapies Dresden - Cluster of
Excellence, Dresden, Germany; 3Cntr for Regenerative Therapies Dresden,
Dresden, Germany.
Purpose: Exposure of the retina to shorter wavelengths of light can lead to
oxidative stress and severe damage in photoreceptors. In the present study, we
focused on the effects of blue light irradiation on retinal explant cultures and
possible rescue of wide-range long-wavelength light on photoreceptor morphology
as well as metabolic function.
Methods: Murine retinal explant cultures were exposed to visible blue light (405
nm) as well as wide-range long-wavelength light (670 - 1250 nm). Live-dead kit
was used to determine the viability of retinal (photoreceptor) cells. ROS production
in the retinas was determined by dihydroethidium and CM-H2DCFDA,
respectively. Mitochondrial membrane potential was estimated by JC-1 staining.
The presence of the stress related proteins nicotinamide adenine dinucleotide
phosphate (NADPH) oxidase (Nox)-2 and 4-hydroxy-2-nonenal (4-HNE) was
assessed by immunohistochemistry. Morphological alterations of photoreceptor
outer segments were studied by live imaging microscopy, transmission and
scanning electron microscopy.
Results: Already after 30 min of blue light exposure, live retinal explants displayed
an increase in ROS production in photoreceptor cells; 3 h of blue light exposure
caused a disorganization of the normally neatly stacked outer segments of living
photoreceptors. Additionally, breaks in the cell membrane that surrounds the outer
segments were observed. The outer segments appeared tortuous, and the lamellar
structures became disrupted. Nox-2, a protein within the cell membrane and a
major source for ROS, and 4-HNE (produced by lipid peroxidation after oxidative
stress), were increased in the outer segments. Wide-range long-wavelength light
exposure protected photoreceptors by reducing stress induced cell death.
Conclusions: In vitro blue light irradiation elicited very early morphological and
metabolic changes that can lead to photoreceptor apoptosis. The molecular
mechanisms of mitochondrial rescue by wide-range long-wavelength light are
under investigation.
Commercial Relationships: Richard H. Funk, None; Ulrike Schumann,
None; Marius Ader, None; Lilla Knels, None; Cora Roehlecke, None
Support: None
Program Number: 2568 Poster Board Number: D946
Presentation Time: 3:45 PM - 5:30 PM
Retinal Cell Damage by Exposure to Short-wavelength Light
Toshio Narimatsu1,2, Shunsuke Kubota3, Takunori Ogawa1, Seiji Miyake1,2,
Norihiro Nagai1,2, Yoko Ozawa1,2, Kazuo Tsubota2. 1Laboratory of Retinal Cell
Biology, Keio University School of Med, Shinjuku-ku, Japan; 2Ophthalmology,
Keio Univ School of Medicine, Shinjuku-ku, Japan; 3Ophthalmology & Visual
Science, Apte Lab, Washington University in St.Louis, St.Louis, MO.
Purpose: Excessive light causes retinal cell death by apoptosis. Because of the
difference in light energy level, the severity of retinal damage may be altered by the
wavelength involved in the exposed light. However, the details are not fully
understood. In this study, we analyzed the levels of the retinal light damage caused
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
by each wavelength using the filters that block either ultraviolet (UV) light, or UV
and blue light.
Methods: BALB/c male mice (6 to 8 weeks old) were purchased and kept under
dim cyclic light (5 lux, 12 h on/off). The mice were dark-adapted by keeping them
in complete darkness for 12 hours, before exposure to a white fluorescence lamp
for 3 hours (5000 lux). The mice were separated into 3 groups, and exposed to the
light through different characteristic filters; control filter, UV light filter, UV-andblue light filter. The intensity of the light through the filters was confirmed to be
equal in each group. The retinal cell damage was evaluated by analyzing the
apoptosis with TUNEL assay (2 days after light exposure).
Results: The number of TUNEL positive cells was significantly decreased both in
the UV light filter group and the UV-and-blue light filter group compared to
control group. Interestingly, UV-and-blue light filter group showed significantly
smaller number of apoptotic cells than UV light filter group.
Conclusions: Blue light itself, not only UV light, caused significant retinal cell
damage by inducing apoptosis. Blocking blue light in addition to UV light was
more effective to protect retinal tissue against apoptosis.
Commercial Relationships: Toshio Narimatsu, None; Shunsuke Kubota,
None; Takunori Ogawa, None; Seiji Miyake, None; Norihiro Nagai, None;
Yoko Ozawa, JIN CO.,LTD. (R); Kazuo Tsubota, None
Support: None
Program Number: 2569 Poster Board Number: D947
Presentation Time: 3:45 PM - 5:30 PM
Fas-mediated Death of Photoreceptors in A Phototoxicity Model
Qing Chang1, Marcus Peter2, Michael A. Grassi1. 1Ophthalmology & Visual Sci,
Univ of Illinois Eye & Ear Infirmary, Chicago, IL; 2Hematology & Oncology,
Northwestern University, Chicago, IL.
Purpose: Death of photoreceptors is the pathological endpoint that leads to vision
loss in various retinal degenerative diseases. Previously, we demonstrated that the
prototype death receptor Fas was expressed in photoreceptors of human retina.
Activation of Fas by the agonist or Fas ligand (FasL) in a surrogate photoreceptor
culture model induced apoptosis. In this study, we investigated the role of Fas
signaling in white light (WL)-induced cell death in photoreceptors.
Methods: Mouse-derived photoreceptor cells (661W) pre-treated with 9-cis retinal
were exposed to WL with/without pharmacological manipulation of Fas signaling.
Cells under continuous darkness were treated as negative control. Apoptosis and
necrosis were examined by flow cytometry using Alexa Fluor 488 annexin V and
propidium iodide labeling. Fractionated cellular extracts were prepared to detect
protein expression by western blotting. Phosphorylation of the receptor interacting
protein 1 (RIP1) kinase, a key regulator in the necrotic death pathway, was detected
by immunoprecipitation using anti-phosphoserine antibody. Cell-Tracker dyelabeled live cells under maintenance of continuous darkness were incubated with
the conditioned medium or cell suspensions from WL-exposed cells to detect the
likelihood of apoptosis induction.
Results: WL induced both necrotic and apoptotic death in 661W cells, which
exhibited death kinetics that correlated with the duration of light exposure.
Administration of the anti-FasL neutralizing antibody or caspase 8 inhibitor zIETD substantially inhibited apoptosis. However, it simultaneously induced
necroptosis, which could be blocked by the RIP1 kinase inhibitor, Necrostatin-1.
WL exposure plus co-treatment with z-IETD caused hyper-phosphorylation of
RIP1 kinase. WL exposure did not up-regulate expression of FasL or Fas. CellTracker dye-labeled live cells when incubated with the conditioned medium or cell
suspensions from WL-exposed cells induced apoptosis, which could be partially
prevented by the anti-FasL neutralizing antibody.
Conclusions: The canonical Fas-mediated apoptotic death pathway is activated in
photoreceptors under light stress. Although blockade of this pathway will prevent
apoptosis, it would not improve the overall photoreceptor survival due to a
compensatory activation of necroptosis. Prevention of photoreceptor loss from
retinal photo-oxidative stress may benefit from targeting both Fas and R1P1. In
addition to FasL, light stress also induces secretion of cytotoxic soluble factors that
can contribute to the death of photoreceptors by apoptosis through paracrine
Commercial Relationships: Qing Chang, None; Marcus Peter, None; Michael
A. Grassi, None
Support: Foundation Fighting Blindness, Hope for Vision, Research to Prevent
Program Number: 2570 Poster Board Number: D948
Presentation Time: 3:45 PM - 5:30 PM
Acute Psychosocial Stress Protects Against Light-induced Damage Of
Photoreceptors Via A Corticosterone-mediated Activation Of The Akt
Tembei K. Forkwa1A, Stefan Reber1B, Inga Neumann1B, Ernst Tamm1A, Andreas
Ohlmann1A. AHuman Anatomy and Embryology, BDepartment of Behavioral and
Molecular Neuroendocrinology, 1University of Regensburg, Regensburg, Germany.
Purpose: Glucocorticoids have repeatedly been shown to be neuroprotective in
animal models of hereditary retinal degenerations and glaucoma by inhibiting the
apoptotic death of photoreceptors or retinal ganglion cells. Glucocorticoid release is
one of the hallmarks of the classical stress response. To analyze if psychosocial
stress has protective effects on retinal neurons, mice were stressed by subordinate
colony housing (CSC) followed by a light-induced damage of photoreceptors. In
addition, the roles of the HPA axis, Müller cell gliosis and AKT pathway activation
in stress-mediated neuroprotection was investigated.
Methods: Psychosocial stress was induced via subordinate colony housing (CSC)
either for 10 hrs (acute stress) or 19 days (chronic stress). To induce photoreceptor
degeneration Balb/c mice were exposed to 5000 lux white fluorescent light.
Apoptosis and loss of photoreceptors were assessed by TUNEL staining and/or
light microscopy. Corticosterone levels were measured by ELISA. To investigate
the role of the HPA axis, adrenalectomy was performed. Retinal expression of
neuroprotective growth factors and phosphorylation of AKT was analyzed by realtime RT-PCR and western blotting.
Results: Acute psychosocial stress due to 10 hrs CSC housing protected
photoreceptors from light induced damage when compared to single-housed
controls, an effect that was lost after 19 days CSC housing. Injection of
corticosterone (52mg/kg ip) was as effective in protecting photoreceptors from
damage as was 10 hrs CSC housing. As opposed to those of sham-operated mice,
photoreceptors of adrenalectomized mice were not protected from light damage
after 10 hrs CSC, but showed an extremely high susceptibility to light damage.
Retinal mRNA expression of neuroprotective factors (LIF, EDN-2, FGF-2, CNTF)
was not changed after 10 hrs CSC or corticosterone injection when compared to
SHC and vehicle-injected controls, respectively, both before and after light
damage. After 10 hrs CSC or corticosterone injection, a significant higher level of
Akt phoshorylation was observed when compared to single-housed controls or
vehicle-injected animals. The same was true when sham-operated mice were
compared with adrenalectomized mice.
Conclusions: Acute psychosocial stress protects photoreceptors against lightinduced damage. This effect is most likely mediated via an increase in plasma
corticosterone which in turn activates the AKT signalling pathway.
Commercial Relationships: Tembei K. Forkwa, None; Stefan Reber,
None; Inga Neumann, None; Ernst Tamm, None; Andreas Ohlmann, None
Support: None
Program Number: 2571 Poster Board Number: D949
Presentation Time: 3:45 PM - 5:30 PM
Endothelin Receptor A Antagonist Enhances Light-induced Damage In The
Mouse Retina
Vicente Bermudez, Marisa A. Cubilla, Tomas P. Bachor, Maximiliano Olivera,
Angela M. Suburo. Cell and Molecular Medicine, Universidad Austral - FCB, Pilar,
Purpose: Exposure to excessive white light induces retinal degeneration or
exacerbates the rate of photoreceptor apoptosis in several retinal diseases. In
previous studies we demonstrated that stimulation of the endothelinergic pathways
could intervene in photoreceptor death control. In this work we analyzed whether
the endothelinergic antagonists Tezosentan (ETRA and ETRB) and Clazosentan
(ETRA) could modify the course of light-induced degeneration.
Methods: Experimental work was done according to the ARVO Statement for the
use of animals. Male Balb-c mice (5-7 weeks old), bred under standard illumination
conditions (12:12 h light: dark; < 60 lux), remained in complete darkness for 24 h.
Mice were separated in groups with standard illumination (< 60 lux) and groups
exposed to intense light (1500 lux) for 4 days (n = 3 per group). During the
exposure period, animals received saline solution, Tezosentan (10 mg/kg/day) or
Clazosentan (10 mg/kg/day). Afterwards they were allowed to recover, without
treatment under standard illumination for another 6 days. Mice were then
anesthetized and their eyes enucleated. Seriate retina cryosections were stained
with neutral red, and thickness of outer nuclear layer (ONL) was measured to
determine the level of light-induced damage.
Results: Damage to the retina was higher in the temporal than in the nasal retina,
and the largest attrition retina occurred around the optic nerve head. In Tezosentantreated mice, damage was not significantly different from that in control animals.
By contrast, the retina of mice receiving Clazosentan during light exposure was
more severely damaged than those receiving saline or Tezosentan.
Conclusions: Our present findings indicate that inhibition of ETRA signaling
increased vulnerability of photoreceptors to light-induced damage. ETRA are found
in photoreceptor terminals (Torbidoni et al., 2005) and the RPE (Narayan et al.,
2003). Activation of these receptors, either by ET-1 or ET-2, would be involved in
photoreceptor survival. Retinal damage observed after Tezosentan treatment was
similar to that of controls, suggesting that blockade of both receptors cancelled the
pro-apoptotic effect of ETRA inhibition.
Commercial Relationships: Vicente Bermudez, None; Marisa A. Cubilla,
None; Tomas P. Bachor, None; Maximiliano Olivera, None; Angela M. Suburo,
Support: PICTO Austral 2008-0090
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2572 Poster Board Number: D950
Presentation Time: 3:45 PM - 5:30 PM
Estrogen Reduces Photoreceptor Cell Loss In A Light-induced Photoreceptor
Degeneration Mouse Model
Mausumi Bandyopadhyay, Elizabeth O'Quinn, Lara Seidman, Baerbel Rohrer.
Ophthalmology, MUSC, Charleston, SC.
Purpose: Females are more susceptible to age related macular degeneration than
males. The purpose of this study is to evaluate the effects of estrogen and estrogen
receptor beta (ERβ) on photoreceptor cell structure and function in an experimental
model for light-induced photoreceptor degeneration, the light damage mouse
Methods: 3-5 months old Balb/c mice were exposed to constant light of ~150 lux.
Mice were divided into four groups and treated via IP injections. The first group
received 0.2 mg/kg body weight 17β-estradiol each day for 10 days, the second
group received 25 µg/kg body weight ERβ inhibitor [4-(2-phenyl-5,7bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl)phenol, PHTPP] every alternate
day; the third group received combination of 17β-estradiol and PHTPP and the
fourth group received vehicle. Rod cell function was assessed using
electroretinogram (ERG) recordings in dark-adapted animals. Optokinetic
Response (OKR) measurements were used to assess cone photoreceptor function.
Tissues were collected for histological and biochemical studies.
Rod photoreceptor survival was assessed by ERG and by determining rows of
photoreceptor in the outer nuclear layer (ONL). Our results showed that, estrogen
treatment significantly (P<0.001) increased rows of photoreceptor in ONL than
vehicle treated control; whereas the ERβ inhibitor treatment reduced (P<0.001)
photoreceptor cell numbers. Co-administering PHTPP and estrogen eliminated the
protective effect of estrogen. ERG analysis showed good correlation between
protection of structure and function; the rod photoreceptor-driven a- wave response
was increased in the estrogen treated group, while ERβ inhibitor treated group
showed a significant decrease (P<0.01) when compared to the control mice.
Likewise, estrogen treatment was found to increase cone cell function (P<0.01) as
demonstrated by OKR analysis; and immunohistochemistry indicated lower cone
opsin level in the ERβ inhibitor, combination and vehicle treated mice whereas
estrogen treatment has been found to improve cone opsin protein level.
Conclusions: We found that estrogen treatment reduced photoreceptor cell damage
in this light-induced photoreceptor degeneration mouse model. The estrogen
receptor inhibitor experiments suggest that the protective effect is mediated via
Commercial Relationships: Mausumi Bandyopadhyay, None; Elizabeth
O'Quinn, None; Lara Seidman, None; Baerbel Rohrer, None
Support: None
Program Number: 2573 Poster Board Number: D951
Presentation Time: 3:45 PM - 5:30 PM
Mitochondrial Dysfunction in Transgenic Mice Overexpressing iNOS in
Guey Shuang Wu1, Narsing A. Rao2. 1Doheny Eye Institute, Univ. of Southern
Califronia, Los Angeles, CA; 2Dohney Eye Institute, Univ of Southern Califronia,
Los Angeles, CA.
Purpose: Unlike its constitutive isoforms, inducible nitric oxide synthase (iNOS) is
generated for the pathologic cause by the immune system activator in inflammatory
diseases. In this study, transgenic mice with iNOS overexpression in
photoreceptors were generated to evaluate the effect of iNOS itself, devoid of its
associated immune response contributions. Such an endeavor to pinpoint the exact
role of iNOS using well-defined transgenic mice has not previously been
Methods: Opsin/iNOS transgene for pronuclei injection was generated by cloning
4.4 kb Acc65I/Xhol mouse opsin promoter into the Acc65I/Sall sites of
pCMVSport6-iNOS. A line of homozygotes and two lines of heterozygotes were
generated. Presence of iNOS was confirmed and the iNOS-derived oxidative and
nitrosative damages were detected by protein nitration using Western and confocal
localization and by apoptosis using TUNEL.
Results: The iNOS insertion was demonstrated by a single 130 kDa iNOS band in
Western analysis and was also found to localize specifically to the inner segments
(IS) and outer plexiform layer (OPL). Under basal conditions, the cellular toxicity
of sustained iNOS expression was detected by the formation of nitrosative product,
namely, tyrosine-nitrated proteins, the most significant of all was nitrated
mitochondrial cytochrome c in IS, OPL and retinal pigment epithelium. Marked
apoptotic cells were detected in the outer nuclear layer.
Conclusions: This study generated a pathologic phenotype with sustained iNOS
expression in photoreceptors delivering high output of nitric oxide and
peroxynitrite. The endogenous iNOS without any immunological stimulation
caused marked nitration/release of mitochondrial cytochrome c and apoptosis in
photoreceptors. Thus, manipulation of pathways leading to iNOS upregulation, or
an effective neutralization of iNOS can be of significant therapeutic benefit in
photoreceptor degeneration mediated by mitochondrial oxidative stress.
Commercial Relationships: Guey Shuang Wu, None; Narsing A. Rao, None
Support: NIH Grants EY017347 and EY03040
Program Number: 2574 Poster Board Number: D952
Presentation Time: 3:45 PM - 5:30 PM
Resveratrol Protects Photoreceptors from Cells Death after Experimental
Retinal Detachment
Wei Huang, Guorong Li, Jianming Qiu, Iris Navarro, Pedro Gonzalez, Pratap
Challa. Duke Eye Center, Duke University Medical Center, Durham, NC.
Purpose: Photoreceptor degeneration is a major cause of visual loss in various
retinal diseases, including retinal detachment (RD) and neovascular AMD,
pharmacologic inhibition of photoreceptor cell death may prevent this outcome.
This study tests whether systemic administration of Resveratrol can protect
photoreceptors from cell death after experimental retinal detachment in rodents.
Methods: Retinal detachment was created in rats by subretinal injection of
hyaluronic acid. The animals were treated daily with vehicle, Resveratrol
(20mg/kg) intraperitoneal (IP) injection only or Resveratrol intraperitoneal
injection (20mg/kg) with subretinal injection (25 µM). Photoreceptor degeneration
was assessed by counting the number of apoptotic cells with TdT-dUTP terminal
nick-end labeling (TUNEL) 3 days after RD. Changes in expression of FoxO1A,
FoxO3A, and FoxO4 were analyzed by western blot. Caspase 3 and 9 levels were
studied as well.
Results: Three days after detachment, Resveratrol decreased the number of
TUNEL-positive cells in both Resveratrol treatment groups by 71% and 74%
compared to vehicle in the IP and IP with subretinal injection, respectively. Retinal
detachment increased the expression of FoxO1A, FoxO3A, and FoxO4. Resveratrol
treatment significantly increases FoxO4 expression in retinal detachment eyes. The
increase in activity of caspase 3 and 9 in retinal detachment was partially inhibited
by Resveratrol.
Conclusions: Systemic administration of Resveratrol preserved photoreceptors
after retinal detachment, and was associated with activation of FoxO family
expression and decreased caspase activity. Resveratrol has great potential to be
used as a novel therapeutic agent for preventing vision loss in diseases that are
characterized by photoreceptor detachment.
Commercial Relationships: Wei Huang, None; Guorong Li, None; Jianming
Qiu, None; Iris Navarro, None; Pedro Gonzalez, None; Pratap Challa, None
Support: Duke Eye Center Small Grant, NIH P30 EY-005722, RPB
Program Number: 2575 Poster Board Number: D953
Presentation Time: 3:45 PM - 5:30 PM
Norrin Protects Photoreceptors From Apoptotic Cell Death
Barbara M. Braunger1, Andreas Ohlmann1, Marcus Koch1, Naoyuki Tanimoto2,
Ying Yang3, Ales Cvekl3, Michael Bösl4, Mathias W. Seeliger2, Ernst R. Tamm1.
Anatomy, University of Regensburg, Regensburg, Germany; 2Division of Ocular
Neurodegeneration, Ctr Ophthalmology Inst Ophthalmic Res, Tuebingen,
Germany; 3Ophthalmology & Vis Sci & Genetics, Albert Einstein Coll of
Medicine, Bronx, NY; 4Max Planck Institute of Neurobiology, Martinsried,
Purpose: Norrin is a signaling molecule, which binds to Fz4 and activates
canonical Wnt/beta catenin signaling. We were interested to learn, if
overexpression of Norrin in the retina of mice is a mechanism to protect
photoreceptors from light-induced damage.
Methods: Transgenic Rpe65-Norrin mice which overexpress Norrin in the retinal
pigment epithelium were generated, characterized, exposed to light damage and
analyzed by Western blotting, real time RT-PCR, semithin sectioning,
immunohistochemistry and electroretinography (ERG). TOP-Gal Wnt pathway
reporter mice were used to detect activation of canonical Wnt signaling.
Results: Rpe65-Norrin mice do not express an obvious retinal phenotype, but show
high amounts of transgenic Norrin in the outer retina and strong activation of
Wnt/beta-catenin signaling. 30 h after light damage, significantly fewer TUNELpositive cells were detected in the outer nuclear layer (ONL) of Rpe65-Norrin mice
as compared to wild-type littermates. 7 and 14 days after light damage, the ONL
was significantly thinner in control littermates than in Rpe65-Norrin mice.
Structural changes correlated with functional changes as detected by ERG. Light
damage was enhanced after blocking Wnt-signaling with Dickkopf-1. Untreated
Rpe65-Norrin mice showed a significant upregulation of mRNA for GFAP and
endothelin-2 , while light damage induced a significant upregulation of BDNF and
LEDGF expression. Inhibition of endothelin-2 signaling in Rpe65-Norrin mice
caused a significant increase in photoreceptor apoptosis 30h after light damage and
prevented the increase in BDNF expression.
Conclusions: Norrin protects photoreceptors from light-induced apoptotic cell
death indicating a neuroprotective role of Norrin in a signaling network that
involves the canonical Wnt-pathway, endothelin-2 and upregulation of BDNF.
Commercial Relationships: Barbara M. Braunger, None; Andreas Ohlmann,
None; Marcus Koch, None; Naoyuki Tanimoto, None; Ying Yang, None; Ales
Cvekl, None; Michael Bösl, None; Mathias W. Seeliger, None; Ernst R. Tamm,
Support: DFG Research Unit (Forschergruppe) FOR1075
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2576 Poster Board Number: D954
Presentation Time: 3:45 PM - 5:30 PM
Anatomical Evidence of Photoreceptor Degeneration in P23H Mutant
Rhodopsin Transgenic Hybrid Swine
Patrick A. Scott1, Wei Wang1, Jason W. Ross2, Henry J. Kaplan1. 1Ophthalmology
& Visual Sciences, University of Louisville, Louisville, KY; 2Animal Science,
Iowa State University, Ames, IA.
Purpose: Retinitis pigmentosa (RP) is an inherited retinal degeneration that causes
progressive loss of rod photoreceptors (PR), affecting cones later in the disease.
While rodent models of retinal degeneration have bettered our understanding of
inherited retinal disease, differences in eye size, retinal anatomy and physiology,
render many of these models ill-suited for studying retinal intervention therapies
intended for man. Because the swine eye closely resembles the human eye, our
group has created transgenic hybrid swine with the most common autosomal
dominant rhodopsin mutation, Proline-23-Histidine (P23H). We investigated
whether the P23H rhodopsin mutation induces PR degeneration in swine eyes, as
well as the progression and topographical distribution of PR degeneration in the
P23H transgenic hybrid swine.
Methods: P23H swine and age-matched wild type (wt) controls were euthanized at
post-natal days (D) 3, 14, 25, 30, 60, 90, and 120. Eyes were enucleated and fixed
in 4% paraformaldehyde in PO4 buffer. Each group contained 1 wt eye and at least
1 transgenic eye. Posterior eyecups containing the retina were bisected along the
vertical and horizontal meridians and processed for light microscopy. To quantify
PR degeneration, rows of nuclei in the outer nuclear layer (ONL) and inner nuclear
layer (INL) were counted at specific locations along the vertical and horizontal
Results: With the exception of D3, significant reductions in the mean number of
rows of nuclei in the ONL were found in all P23H pigs, and ONL reduction
appears to be related temporally. D90 and D120 P23H pigs were most severely
affected, with only a single row of nuclei remaining in the ONL. The INL was
minimally disturbed, with small but significant increases in the number of nuclei in
D90 and D120 P23H pigs, as compared to wt.
Conclusions: P23H transgenic hybrid swine exhibit morphological changes to rod
photoreceptors that are evident shortly after birth, followed by progressive dropout
of rods across the retina. This may be a useful model for development of
intervention therapies that can eventually be applied to humans with PR
Commercial Relationships: Patrick A. Scott, None; Wei Wang, None; Jason
W. Ross, None; Henry J. Kaplan, None
Support: NEI R21 EY020647; Research to Prevent Blindness, Inc. NYC, NY; KY
Challenge Research Trust Fund (HJK); KY Science and Engineering Foundation
Program Number: 2577 Poster Board Number: D955
Presentation Time: 3:45 PM - 5:30 PM
Characterization of Cell Death Mechanisms in Cone and Rod Photoreceptors
Caused by the Conditional Loss of Ran-binding Protein-2 (RanBP2) in M-cone
Kyoung-in Cho1, MdEmdadul Haque1, Minzhong Yu2,3, Indulekha C. Lalitha1,
Nomingerel Tserentsoodol1, Neal S. Peachey2,3, Paulo A. Ferreira1.
Ophthalmology, Duke University Medical Center, Durham, NC; 2Cole Eye
Institute, Cleveland Clinic Foundation, Cleveland, OH; 3Research Service, Louis
Stokes VA Medical Center, Cleveland, OH.
Purpose: Photoreceptor cell death underlies severe visual impairments shared by
numerous retinal diseases such as Retinitis Pigmentosa (RP) and age-related
macular degeneration (AMD). Hence, the identification of molecular pathways
triggering the death of photoreceptor neurons is crucial for the understanding of
disease pathogenesis and development of novel therapies. The pleiotropic protein,
RANBP2, is implicated in molecular processes, such as nucleocytoplasmic
trafficking, oxidative stress signaling, proteostasis and lipid homeostasis, and
mitochondria dynamics. Previously, we found that conditional ablation of RanBP2
in M-cone photoreceptors promotes the rampant death of these neurons within 2weeks of cre-mediated RanBP2 excision. This study aims at testing the hypothesis
that conditional loss of RanBP2 expression in M-cones promotes novel molecular
mechanisms underlying cone degeneration.
Methods: The retinas of mice with a loxP-flanked RANBP2 gene and expressing
Cre under control of the human L/M opsin promoter were analyzed by
immunohistochemistry, electron microscopy, electroretinogram recordings (ERG)
of rod and cone responses, and biochemical assays.
Results: Analyses of flat mounts and cross-sections of retinas indicate that loss of
RANBP2 selectively in M-cones causes the rapid degeneration of M-cones by the
activation of caspases 3/7, but not of other six caspases, yet without producing
TUNEL-positive cells. Light microscopy and ultrastructural analyses of M-cones
showed that the degeneration was accompanied first by the disintegration of outer
segments followed by apparent vacuolization of cell bodies and inner fibers.
Remarkably, these effects were accompanied by the formation of interstitial spaces
between the dysmorphic cones and immediate neighboring rods with limited
disintegration of the plasma membrane and morphological changes, and apparent
TUNEL-positive rods. We identified a novel retinal metalloproteinase, which is
strongly up-regulated in the retina of mice with RANBP2 ablation selectively in Mcones. ERGs of dark-adapted b- and a-waves of rods indicate a small decrease of
the amplitude of these waves throughout the progression of M-cone degeneration,
whereas deficits of the amplitude of cone ERGs were clear by P15 and thereafter
decreased rapidly with almost no electrophysiological responses retained by P22.
Conclusions: The data support that the loss of RANBP2 in M-cones triggers a noncanonical programmed cell death pathway in M-cones, whereas neighboring rods
undergo non-autonomous cell death or impairment as manifested by a decrease in
ERG amplitudes and morphological changes.
Commercial Relationships: Kyoung-in Cho, None; MdEmdadul Haque,
None; Minzhong Yu, None; Indulekha C. Lalitha, None; Nomingerel
Tserentsoodol, None; Neal S. Peachey, None; Paulo A. Ferreira, None
Support: NIH grants EY019492 & GM083165, RPB
Program Number: 2578 Poster Board Number: D956
Presentation Time: 3:45 PM - 5:30 PM
Long-term effects of Environmental Enrichment (EE) on a mouse model of
Retinitis Pigmentosa
Ilaria Barone1, Elena Novelli2, Enrica Strettoi1. 1Italian National Research Council,
CNR -Neuroscience Institute, Pisa, Italy; 2GB-Bietti Foundation for
Ophthalmology, Rome, Italy.
Purpose: In mouse models of typical Retinitis Pigmentosa (RP), rod death is
accompanied and followed by secondary degeneration of cones. Enviromental
Enrichment (EE) consists in the exposure to a combination of complex motor,
inanimate and social stimuli. EE is an experimental paradigm that prolongs
neuronal survival, slows down neuronal degeneration, enhances plasticity and
neurotrophic factor production throughout the CNS. In previous studied we showed
that EE delays photoreceptor death and preserves visual structure and function in
rd10 mutant mice (a model of RP) up to 90 days of age. Here, we assess the effects
of EE on the same mutants, kept in the same environment, up to 1 year of age. We
focus on the morphology and survival of cones and on visual function in photopic
Methods: Animals were treated in accordance with ARVO and institutional
guidelines. rd10 mice (homozygous for the rod-specific phosphodiesterase
mutation, C57BL/6J-Pde6brd10/J mice), were conceived, born and maintained in
enriched conditions up to 1 year of age. Retinal morphology, photoreceptor
survival and visual behaviour were assessed on a group of 4 mice at 1 year of age,
and compared to those of age-matched rd10 mice, kept in standard laboratory
conditions(ST). Morphological studies were done with retinal cell type specific
antibodies, confocal microscopy and morphometric analysis of retinal whole
mounts and histological sections. Visual acuity was assessed in photopic conditions
by means of a visual water maze adapted by Prusky.
Results: Albeit a continue decline in the number of cones with time, EE rd10 mice
of 1 year show almost three times as many surviving cones than ST controls
(34,000±4,000 vs 12,700± 1,800; t-test; **p=0.003). The morphology of individual
cones in EE mice is better preserved as well, as shown by more numerous and
longer outer segments. Behaviour-based tests show that the same EE mice preserve
a residual acuity of 0.138 cycles/degree. On the contrary, virtually all age-matched
rd10 ST controls are uncapable to perform the visual task (0.138±0 vs 0.063±0.014
t-test *p=0.029).
Conclusions: Early and prolonged exposure of RP mutant mice to Environmental
Enrichment preserves cones even in the long run. A minimal visual acuity is also
maintained. This experimental paradigm migth be exploited for developing non
invasive strategies to delay vision loss in RP individuals, also in perspective of
treatments requiring cone viability.
Commercial Relationships: Ilaria Barone, None; Elena Novelli, None; Enrica
Strettoi, None
Support: NIH GRANT R01 EY12654, Italian Natl Res Council (CNR);Velux
Foundation Proj.#691, G.B. Bietti Foundation for Ophthalmology.
Program Number: 2579 Poster Board Number: D957
Presentation Time: 3:45 PM - 5:30 PM
The USH1G Protein SANS Collaborates With Cytoplasmic Dynein Motor
Components In Mammalian Photoreceptor Cells
Nasrin Sorusch1, Nora Overlack1, Andrea Kunz1, Erwin van Wijk2A, Katharina
Bauss1, Tina Maerker1, Ronald Roepman2B, Hannie Kremer2A, Uwe Wolfrum1.
Inst. f. Zoology, Cell & Matrix Biology, Johannes Gutenberg-University Mainz,
Mainz, Germany; ADept. of Otorhinolaryngology, BDept of Human Genetics,
Radboud Univ Nijmegen Med Ctr, Nijmegen, The Netherlands.
Purpose: Human Usher syndrome (USH) is the most common form of combined
deaf-blindness. Usher type I (USH1), the most severe form, is characterized by
profound congenital deafness, constant vestibular dysfunction and pre-pubertal
onset of retinitis pigmentosa. The USH1G protein SANS (scaffold protein
containing ankyrin repeats and SAM domain) is associated with microtubules and
mediates a transport related periciliary protein network in photoreceptor cells. The
present work was designed to enlighten the function of SANS in intracellular
transport in photoreceptor cells.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Methods: Identification and validation of protein-protein interactions via yeast-2hybrid (Y2H) screen of retinal cDNA library, GST-pull down, co-transfection
assays, microtubule spin-down and microtubule destabilization assays.
Investigation of protein localization in mouse and human retina by indirect
immunofluorescence and immunoelectron microscopy.
Results: In our Y2H screen we identified the interaction between SANS and
dynactin-1 (p150Glued), a subunit of the dynactin complex and cargo linker to the
cytoplasmic dynein motor. This interaction was validated in independent
interaction assays in vitro and in cell culture. Furthermore, we demonstrated the
integration of SANS into dynein transport complex by pull down of SANS with
GST-fused cytoplasmic dynein intermediate chain (cDIC74). Correlative
immunofluorescence and immunoelectron microscopy revealed co-localization of
p150Glued, basic dynein components and SANS at microtubule tracks of
photoreceptor inner segments.
Conclusions: The present data strengthen our hypothesis that SANS participates in
cargo transport to its ciliary destination. Here we demonstrate the integration of
SANS into cytoplasmic dynein-dynactin complexes, essential for microtubule
based transport in photoreceptor cells. Defects in these transport mechanisms may
lead to retinal degeneration as characteristic for USH1G patients.
Commercial Relationships: Nasrin Sorusch, None; Nora Overlack,
None; Andrea Kunz, None; Erwin van Wijk, None; Katharina Bauss,
None; Tina Maerker, None; Ronald Roepman, None; Hannie Kremer,
None; Uwe Wolfrum, None
Support: FcB, DFG, FAUN, Pro Retina, EU-FP7 Syscilia
Program Number: 2580 Poster Board Number: D958
Presentation Time: 3:45 PM - 5:30 PM
Rod And Cone Photoreceptors Die By Different Cell-death Mechanisms In
The Pde6c Mutant Zebrafish Model Of Achromatopsia
ZEINABSADAT Mohammadi, Jiang P. Zhang, Kevin Gregory-Evans, Joanne A.
Matsubara, Orson L. Moritz, Cheryl Y. Gregory-Evans. OPHTHALMOLOGY
Purpose: Achromatopsia is an autosomal recessive trait characterized by loss of
colour discrimination, photophobia and poor visual acuity. Mutations in CNGA3,
CNGB3, GNAT2 and PDE6C account for 90% of cases. Due to this genetic
heterogeneity, mutation-independent treatment strategies aimed at preventing cell
death are very attractive for such photoreceptor disorders. The aim of this study
was to determine the mechanism of photoreceptor cell death in the pde6c zebrafish
model of achromatopsia.
Methods: Cell death in pde6c-/- mutant zebrafish model was analysed by histology,
TUNEL staining and immunohistochemistry for the following cell death-associated
markers: cGMP, cleaved caspase-3, cleaved poly-ADP-ribose polymerase (PARP),
calpain, RIP1 and RIP3. Necrostatin-1 small molecule drug treatment was used in
vivo to inhibit RIP1 activity.
Results: Although cone photoreceptors are formed initially in the retina, rapid cell
death occurs between 3-4 days post-fertilization (dpf). At this stage of
development, TUNEL-positive cells were detected in the mutant retina yet the
expression of activated caspase-3 was not elevated in the photoreceptors. At 4 dpf
in homozygous pde6c mutant zebrafish immunohistochemistry demonstrated that
rod photoreceptors cells were immunopositive for PARP and calpain, whereas cone
photoreceptors expressed high levels of RIP1 and RIP3, demonstrating activation
of the caspase-independent cell death pathways in this retinal degeneration model.
Treatment with necrostatin-1 delayed cone degeneration by several days.
Conclusions: Collectively, these results show in the pde6c mutant retina that cone
and rod photoreceptors die by different caspase-independent mechanisms. Elevated
levels of RIP1and RIP3 suggest that the necroptosis cell death pathway is activated
in cone photoreceptors. Surprisingly though, elevation of PARP and calpain in rod
photoreceptors implies an alternative cell death pathway causing the bystander rod
cell death. This suggests that a combinatorial approach of inhibitors for necroptosis
and PARP might be needed in studies to treat this disease. Bystander cell death is
an important feature of many retinal degenerations and so combinatorial
approaches may evolve as an important principle in treatment.
Commercial Relationships: ZEINABSADAT Mohammadi, None; Jiang P.
Zhang, None; Kevin Gregory-Evans, None; Joanne A. Matsubara,
None; Orson L. Moritz, None; Cheryl Y. Gregory-Evans, None
Support: Canadian Institutes of Health Research (222728)
Program Number: 2581 Poster Board Number: D959
Presentation Time: 3:45 PM - 5:30 PM
Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) Protects Rod
and Cone Photoreceptors from Degeneration in Transgenic Rats Carrying the
S334ter Rhodopsin Mutation
Rong Wen, Lingyu Luo, Dequang Huang, Xin Xia, Zhengying Wang, Pingping
Chen, Yiwen Li. Bascom Palmer Eye Inst, University of Miami, Miami, FL.
Purpose: Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a
member of a novel evolutionarily conserved protein family with neurotrophic
capabilities. It was identified from the conditional medium of a rat type-1 astrocyte
cell line, the ventral mesencephalic cell line 1 (VMCL1) to be a factor that
promotes the survival of cultured embrayonic dopaminergic neurons. It also
significantly reduces the infarction in the ischemic cortex in a rat model of stroke
and promotes the survival of cultured heart muscle cells. In the present study, we
examined the neuroprotective effects of MANF on photoreceptors.
Methods: Recombinant human MANF was expressed in E. coli and purified. To
examine the neuroprotective effect of MANF on rod photoreceptors, recombinant
human MANF (6 µg) was intravitreally injected to the left eyes of transgenic rat
S334ter rats at PD9, and the right eyes were injected with PBS as controls. Eyes
were collected at PD21 examined by light microscopy. To examine effect of
MANF on cone photoreceptors, MANF (6 µg) was injected intravitreally to the left
eyes of the transgenic rats at PD20, and the right eyes were injected with PBS as
controls. Retinas were collected at PD30 and cone outer segments were stained
with Alexa 488 conjugated peanut agglutinin (PNA). Flat-mounted retinas were
examined by confocal microscopy.
Results: MANF significant protected rod photoreceptors. In MANF-treated retinas,
three to four rows of nuclei remained in the outer nuclear layer (ONL), as compare
to only one row of nuclei in PBS-treated fellow eyes. The thickness of ONL in
MANF-treated retinas (superior region) (17.47±3.96 µm, mean±SD, n=5) is
significantly greater than that in PBS-treated retinas (7.07±1.12, n=5) (P < 0.001,
student t-test). Loss of cone outer segments was characterized by many PNAnegative areas distributed across the retina in PBS treated retinas, as reported
previously,. In RdCVF treated eye, however, the PNA negative areas became much
smaller or in many cases completely disappeared. Quantitative analysis showed that
PNA-positive cells are significantly more in MANF treated retinas (569.5±46.5/0.1
mm2, mean±SD, n=6) than in PBS-treated retinas (398.7±25.4, n=6) (P< 0.001,
student t-test).
Conclusions: Intravitreal injection of recombinant human MANF protein protects
both rod and cone from degeneration.
Commercial Relationships: Rong Wen, None; Lingyu Luo, None; Dequang
Huang, None; Xin Xia, None; Zhengying Wang, None; Pingping Chen,
None; Yiwen Li, None
Support: NIH Grant R01EY018586, P30EY14801; Hope for Vision, the James
and Esther King Biomedical Research Program of the State of Florida JEK 08-KN09; the Department of Defense Grant W81XWH-09-1-0674; RPB.
Program Number: 2582 Poster Board Number: D960
Presentation Time: 3:45 PM - 5:30 PM
Genetic Ablation of DHDDS in Photoreceptors in Mouse
Ying Li, Zhengying Wang, Pingping Chen, Lauren N. Correa, Byron L. Lam, Yiwen
Li, Rong Wen. Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: We have recently identified the K42E mutation in DHDDS
(dehydrodolichol diphosphate synthase) as the cause of retinitis pigmentosa (RP)
phenotype in a single family with non-syndromic recessive RP. To further study
dehydrodolichol diphosphate synthase in the retina, we genetically ablated DHDDS
in mouse.
Methods: Mouse embryonic stem (ES) cells containing the DHDDStm1a allele
(clone Dhdds_D05) were obtained from UC Davis KOMP Repository. This allele
has a trapping cassette “SA-βgeo-pA” (splice acceptor-beta-geo-polyA) flanked by
flippase recombinase (Flp) target FRT sites upstream of exon4, resulting in
truncation of the endogenous transcript and thus creating a constitutive null
mutation. The cassette also tags the gene with a lacZ reporter. The FRT flanked
region can be removed by Flp, but exon4, a critical exon, remains floxed. Exon4
can be further removed by the Cre recombinase to achieve conditional knock-out.
Results: The ES cells were injected into blastocysts and 5 chimeric mice were
born. Crossing the chimeric mice with wild type mice produced two F1 mice
carrying the DHDDStm1a allele. Intercrossing DHDDStm1a/+ mice produced more
DHDDStm1a/+. However, no homozygous DHDDStm1a (DHDDS-/-) mice were
obtained even after extensive crossing. The absence of homozygous DHDDStm1a
may suggest that DHDDS-/- is lethal. Morphological analysis of retinas from
DHDDStm1a/+ mice showed normal development of photoreceptors. Strong β-gal
staining was found in the photoreceptors as well as in cells in the inner nuclear
layer and retinal; ganglion cells. Functional analysis indicates normal ERGs from
DHDDStm1a/+ mice. Some DHDDStm1a/+ mice were crossed with the Rosa26-FLP1
mice that express Flp under the control of Rosa26 to remove the “SA-βgeo-pA”
cassette to create DHDDSfl/fl mice, which express wild-type DHDDS. The
DHDDSfl/fl are been crossed with mice expressing Cre recombinase under the
control of the long-mouse opsin promoter (LMOP-Cre mice) to create DHDDS
conditional knock-out mice in which DHDDS is ablated only in photoreceptors.
Conclusions: We have successfully ablated DHDDS in mouse. DHDD+/- is
phenotypically normal but DHDDS-/- is lethal. DHDDSfl/fl mice have been created
for conditional knock-out of DHDDS in photoreceptors.
Commercial Relationships: Ying Li, None; Zhengying Wang, None; Pingping
Chen, None; Lauren N. Correa, None; Byron L. Lam, None; Yiwen Li,
None; Rong Wen, None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Support: NIH Grant R01EY018586, P30EY14801; Hope for Vision, the James
and Esther King Biomedical Research Program of the State of Florida JEK 08-KN09; the Department of Defense Grant W81XWH-09-1-0674; RPB.
Program Number: 2583 Poster Board Number: D961
Presentation Time: 3:45 PM - 5:30 PM
Cell Death Pathways Activated In Rod Photoreceptors In Different Models Of
Retinitis Pigmentosa
Valeria Marigo, Antonella Comitato, Paolo Morandi. Biomedical Sciences, Univ
of Modena and Reggio Emilia, Modena, Italy.
Purpose: Retinitis pigmentosa (RP) is a genetic degenerative disease causing
blindness in later life. About 50 genes have been linked to this hereditary disease.
Heterogeneity of such magnitude in RP represents a major impediment to the
development therapies, therefore mutation-independent approaches to target
common molecular mechanisms activated during the degenerative process should
be exploited. We molecularly characterized mitochondrial and ER pathways
activated during rod cell death in three murine models of RP and developed
molecules to interfere with their function.
Methods: We studied the rd1, the Rho-/- and the P23H transgenic mouse models
with molecular biology, immunocytochemical and biochemical approaches. We
analyzed Aif, caspase-3 and caspase-7, Bax and Bak, calpain, cathepsin and ER
stress markers. We performed in vitro interferences with shRNAs and in vivo
treatments with Calpain, Cathepsin D and Bax inhibiting substances.
Results: We found that calpains play a key role in the activation of Aif, Bax and
cell death in the rd1 retina. shRNA experiments down-regulating either calpain 1 or
calpain 2 allowed to define the different contributions of these two proteases. By
reduction of Aif expression we confirmed the important role of Aif in apoptosis in
the rd1 retina. Aif appears also to be important in the other forms of retinal
degeneration. Otherwise, ER stress pathways do not appear to be activated in all
models analyzed.
Conclusions: The option of exploiting cell death as a therapeutic target is complex.
Nevertheless the molecular understanding of the factors activated during
degeneration and the identification of common activators are the first step toward
this goal. Our study identifies calpains and Aif as a key factors triggering
photoreceptor cell demise in the retina of different murine models of RP. The
efficacy of interfering molecules targeting the different factors activated during the
apoptotic cascade opens new perspectives for designing therapeutic approaches to
rescue photoreceptor cell death in this disease.
Commercial Relationships: Valeria Marigo, None; Antonella Comitato,
None; Paolo Morandi, None
Support: Italian Telethon Foundation Grant GGP11210A; EU grant E-RARE
Program Number: 2584 Poster Board Number: D962
Presentation Time: 3:45 PM - 5:30 PM
Combined Local and Global Cellular Therapies for Retinal Degeneration
Shaomei Wang1, Bin Lu1, Sergey Girman1, Muhammad U. Jawad1, David M.
Gamm2, Catherine W. Morgans3, Brandon Shelley1, Clive Svendsen1. 1Regenerative
Medicine Institute, Cedars-Sinai Medical Center, Los Angeles, CA;
Ophthalmology and Visual Sciences, Univ of Wisconsin-Madison, Madison, WI;
Casey Eye Institute, Oregon Health & Science Univ, Portland, OR.
Purpose: Cell-based therapy has been shown to be effective in limiting retinal
degeneration in multiple animal models. Prior to extensive photoreceptor loss,
preservation of host retinal anatomy and function is the primary therapeutic goal.
Our previous studies have shown that human neural progenitor cells derived from
prenatal cortex (hNPCctx) can survive, migrate and integrate into the host retina and
offer significant preservation of vision. We have also shown that bone marrow
derived mesenchymal stem cells (MSCs) can exert extensive protection of
photoreceptors when delivered systemically. Here, we study whether combined
subretinal (hNPCctx) and systemic (MSCs) delivery enhance the efficacy on
preserving vision in the Royal College Surgeon (RCS) rats, a well-studied model
for retinal degeneration. We hypothesize that hNPCctx will offer great local
preservation of vision, while MSCs will foster a global environment that is
favorable for hNPCctx survival and integration.
Methods: hNPCctx, 30,000 cells/eye were injected into the subretinal space of the
Royal College Surgeon (RCS) rats at P21, one million MSCs isolated from RCS
rats with our published protocol, were injected intravenously into the RCS rats at
P22. Control animals received PBS subretinal injection and untreated. All Animals
were maintained under immunosuppression (cyclosporine A in drinking water)
throughout the experimental period. Animals were examined at several time points
Results: Combined treatments showed an extensive preservation of photoreceptors
and visual function. There were 8-10 layers of photoreceptors in treated eyes
compared with a single layer of photoreceptors in the control groups. Visual
function, tested by optokinetic responses (OKR) and luminance threshold
recordings from the superior colliculus, were also extensively preserved compared
with controls
Conclusions: Combined hNPCctx and MSC cellular therapies offer extensive
preservation of both photoreceptors and visual function. hNPCctx and MSCs have
been widely used in research and clinical trials. This new approach may offer the
realistic likelihood of translation into the clinic to treat retinal degeneration.
Commercial Relationships: Shaomei Wang, None; Bin Lu, None; Sergey
Girman, None; Muhammad U. Jawad, None; David M. Gamm,
None; Catherine W. Morgans, None; Brandon Shelley, None; Clive Svendsen,
Support: EY020488, CSMC
Program Number: 2585 Poster Board Number: D963
Presentation Time: 3:45 PM - 5:30 PM
Systemic Treatment with 7,8,3'-Trihydroxyflavone Activates TrkB Receptors
and Protects Photoreceptor Cell Function in Mouse Models of Retinal
Stephanie L. Foster1A, Allia K. Lindsay1A, Sheree S. Mosley1A, Cristina Kendall1A,
Micah A. Chrenek1A, P M. Iuvone2, Keqiang Ye1B, Jeffrey H. Boatright3.
Ophthalmology, BPathology and Laboratory Medicine, 1Emory Univ School of
Med, Atlanta, GA; 2Ophthalmology, Emory University Sch of Med, Atlanta, GA;
Ophthalmology, Emory University School of Med, Atlanta, GA.
Purpose: The purpose of this study is to test whether the flavonoid derivative
7,8,3’-trihydroxyflavone (THF), an agonist of the brain-derived neurotrophic factor
(BDNF) tropomyosin-related kinase B (TrkB) receptor, is protective in models of
retinal degeneration.
Methods: Systemic THF-treatment was assessed in light-induced retinal
degeneration (LIRD) mice, an environmental stress model, and Pde6brd10 (rd10)
mice, a model of retinitis pigmentosa. BALB/C mice were injected
intraperitoneally with vehicle (PBS) or vehicle containing THF (37.5 mg/kg), darkadapted overnight, injected once more, and exposed to 3 hours of bright (8,000 lux)
or dim (200 lux) light. Visual function was assessed 3 days later by
electroretinography (ERG). Immunoblots were performed on protein extracts from
retina and hippocampal tissue with antibodies for total and activated
(phosphorylated) TrkB as well as total and activated AKT (protein kinase B; PKB),
a pathway target of activated TrkB. The rd10 mice were injected daily with THF or
vehicle from postnatal (P) day 6 until P24, at which point visual function was
assessed by ERG.
Results: LIRD mice were classified by light exposure (dim or bright) and treatment
type (vehicle or THF). Bright light exposure significantly suppressed ERG a- and
b-wave mean amplitudes in vehicle mice by 70 and 75 percent respectively,
compared to dim vehicle. This loss of function was prevented with THF treatment.
Mean a-wave amplitudes (in microvolts ± SEM) were: dim vehicle= 320±64; bright
vehicle=96±64; bright THF=352±32. Mean b-wave amplitudes were: dim vehicle:
640±64, bright vehicle 160±96; bright THF=672±128 (n=7 per group, P<0.05).
Statistical analysis was by ANOVA-SNK for LIRD data and Student's t-test for
rd10 data. Immunoblots showed that the ratio of p-TrkB to total TrkB increased
over threefold in retina and hippocampal tissue from THF mice, and remained
unchanged for vehicle mice. THF-treated rd10 mice (n=4) exhibited significantly
greater ERG a-wave mean amplitudes as compared to vehicle-treated mice (n=3,
Conclusions: Systemic treatment with THF is protective of photoreceptor function
in genetic and environmental stress models of retinal degeneration. This is
accompanied by TrkB and AKT activation in the retina and brain, suggesting that
THF treatment resulted in TrkB activation and in turn activation of the AKT
signaling pathway.
Commercial Relationships: Stephanie L. Foster, None; Allia K. Lindsay,
None; Sheree S. Mosley, None; Cristina Kendall, None; Micah A. Chrenek,
None; P. M. Iuvone, None; Keqiang Ye, None; Jeffrey H. Boatright, None
Support: NIH R01EY014026, NIH R01DC010204, NIH R01EY004864, NIH
R24EY017045, NIH P30EY06360, The Abraham and Phyllis Katz Foundation,
Program Number: 2586 Poster Board Number: D964
Presentation Time: 3:45 PM - 5:30 PM
Phosphorylation Regulates the Function of the Kinesin-2 Motor KIF17 in
Cone Photoreceptors
Jason R. Bader, Joseph C. Besharse. Cell Biology, Neurobiology, and Anatomy,
Medical College of Wisconsin, Milwaukee, WI.
Purpose: Multiple proteins are targeted to photoreceptor outer segments (OSs)
where they function in the vision pathway, and intraflagellar transport (IFT), a
highly conserved bidirectional transport pathway occurring along the microtubulebased ciliary axoneme which has been implicated in trafficking. The canonical
anterograde motor for IFT is the heterotrimeric kinesin II or KIF3 complex.
Previous work from our laboratory has demonstrated a requirement for an
additional kinesin 2 family motor, the homodimeric KIF17. The mechanism used to
coordinate KIF17 and the KIF3 complex is not clear. However, we have identified
a role for phosphorylation in regulating KIF17-based photoreceptor IFT.
Methods: Zebrafish expressing GFP-tagged WT, phosphomimetic, or non-
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
phosphorylatable KIF17 were analyzed by live cell imaging and
immunofluorescence microscopy. These constructs were also introduced into
mammalian cells and the impact was assessed by immunoflourescence.
Biochemical assays including immunoprecipitation and sucrose density
centrifugation were used to test the impact of KIF17 phosphorylation on the motorcargo interaction.
Results: Analysis utilizing non-phosphorylatable and phosphomimetic KIF17
mutants revealed that phosphorylation modulates KIF17’s translocation to the distal
tip of zebrafish cone OSs. Consistent with this, phosphorylation impacts KIF17
primary cilium distal tip accumulation in cultured cells. Biochemical analysis
revealed that phosphorylation of the KIF17 tail domain regulates the interaction
between the motor and the IFT complex. Real time imaging revealed that KIF17
moves at rates predicted for microtubule based motility in the distal domain of the
cone OS.
Conclusions: IFT is essential for photoreceptor development and maintenance.
These results demonstrate that KIF17 translocates to the distal region of cone OSs
and this activity is dependent on phosphorylation. Phosphorylation also impacts the
distal tip accumulation of KIF17 in kidney epithelial cell primary cilia, suggesting a
conserved mechanism of KIF17 regulation. Furthermore, phosphorylation
modulates KIF17’s interaction with the IFT complex. Overall, these findings reveal
a new mechanism used to coordinate KIF17 activity in photoreceptor OSs, which is
essential for photoreceptor maintenance.
Commercial Relationships: Jason R. Bader, None; Joseph C. Besharse, None
Support: NIH Grant EY03222
Program Number: 2587 Poster Board Number: D965
Presentation Time: 3:45 PM - 5:30 PM
Activation Of AMPK Protects Photoreceptors From Light Damage By
Preventing Oxidative Stress, And Requires Signaling Through GP130 And
Lei Xu, John D. Ash. Ophthalmology, Univ of Florida, Gainesville, FL.
Purpose: AMPK (AMP-activated protein kinase pathway) is a serine-threonine
kinase that is activated by reductions in cellular ATP levels. We have previously
reported that metformin, an agonist of AMPK, can protect photoreceptors from
light damage. The goal of this study was to determine the protective mechanisms.
Methods: Metformin, an agonist of AMPK was injected daily for 7 days into
BALB/cj albino mice (Jackson Laboratories, Bar Harbor, ME, U.S.A.), or gp130
knockout mice. Mice were then exposed to damaging light (4000 lux for 4 hours)
following the intravitreal or subcutaneous injection of metformin. All procedures
were in accord with the ARVO Statement for the Use of Animals in Ophthalmic
and Vision Research. To verify neuroprotective function of metformin, visual
function was measured by electroretinogram (ERG) and photoreceptor protection
was measured by counting the rows of photoreceptors in histological sections. To
investigate the mechanism we assessed activation of phosphorylated AMPK and
PGC-1 alpha by Western blots. Real time PCR was used to measure gene
expression. Mitochondrial proteomics was done by MS analysis.
Results: Both systemic and intravitreal injections of Metformin led to activation of
AMPK in the retina, and prevented light induced oxidative stress including DNA
and protein oxidation. The protection was partially lost in mice lacking gp130 or
Conclusions: We have demonstrated that metformin can prevent oxidative stress
from light damage by a mechanism that includes increased mitochondrial DNA
content and mitochondrial gene expression. The protection partially required the
activity of the protective pathway induced by gp130 and STAT3. These results
suggest that gene expression induced by STAT3 activation can interact with targets
of AMPK activation to promote photoreceptor survival.
Commercial Relationships: Lei Xu, None; John D. Ash, None
Support: Funding support to JDA include NIH R01EY016459-06, Foundation
Fighting Blindness, and Lew Wasserman Merit award and unrestricted
departmental support from Research to Prevent Blindness, Inc.
Program Number: 2588 Poster Board Number: D966
Presentation Time: 3:45 PM - 5:30 PM
Alu Rna-induced Cytotoxicity In Age-related Macular Degeneration Is
Mediated By Myd88, But Not By A Variety Of Rna Sensors
Yoshio Hirano, Valeria Tarallo, Bradley Gelfand, Sami Dridi, Younghee Kim,
Nagaraj Kerur, Hiroki Kaneko, Benjamin Fowler, Sasha Bogdanovich,
Jayakrishna Ambati. Ophthalmology and Visual Sciences, University of Kentucky,
Lexington, KY.
Purpose: We previously reported that a dramatic and specific reduction of the
RNase DICER1 results in accumulation of Alu RNA transcripts in the RPE of
human eyes with geographic atrophy (Kaneko et al., 2011). Still, the precise
mechanisms of Alu RNA-induced cytotoxicity are unknown. Numerous innate
immune receptors have been identified in the RPE (Kumar et al., 2004), and a
variety of exogenous substances can induce retinal inflammation (Kleinman et al.,
2011). However, it is not known whether this surveillance machinery recognizes or
responds to host endogenous RNAs. In this study, we determined whether innate
immune machinery would also recognize cytotoxic Alu RNA in age-related
macular degeneration.
Methods: Subretinal administration of a plasmid coding for Alu RNA (pAlu) was
tested in mice deficient in toll-like receptor-3 (TLR3), TLR4, toll / IL-1 receptor
domain-containing adapter inducing IFN-β (TRIF, encoded by Ticam1), TLR7,
melanoma-differentiation-associated gene 5 (MDA5), protein kinase R (PKR,
encoded by Prkr), mitochondrial antiviral signaling protein (MAVS), or in
Unc93b1 mutant mice to confirm whether Alu RNA is mediated via a TLR
signaling or other dsRNA sensors. Furthermore, a custom TLR ligand screen was
performed by InvivoGen using HEK293 cells over-expressing individual TLR
family members coupled with an AP-1/NF-κB reporter system.
Results: Alu RNA induced RPE degeneration in Tlr3-/-, Tlr4-/-, Ticam1-/-, Tlr7-/-,
and Unc93b1 mutant mice just as in wild-type mice, indicating that Alu RNA does
not activate these nucleic acid sensing TLRs in a redundant fashion. Moreover, two
different in vitro transcribed Alu RNAs did not activate TLR-2, 3, 4, 5, 7, 8, or 9 in
a reporter assay system based in human embryonic kidney 293 cells expressing
each of the TLRs. pAlu also induced RPE degeneration in Mda5-/-, Prkr-/-, and
Mavs-/- mice, indicating that Alu RNA does not activate other RNA sensors.
However, neither Alu RNA nor two different pAlu plasmids induced RPE
degeneration in mice deficient in myeloid differentiation factor 88 (MyD88) mice.
Conclusions: These results provide a novel mechanism of Alu RNA-induced RPE
degeneration mediated by MyD88, but not by a wide range of canonical RNA
Commercial Relationships: Yoshio Hirano, None; Valeria Tarallo,
None; Bradley Gelfand, None; Sami Dridi, None; Younghee Kim,
None; Nagaraj Kerur, None; Hiroki Kaneko, None; Benjamin Fowler,
None; Sasha Bogdanovich, None; Jayakrishna Ambati, None
Support: None
Program Number: 2589 Poster Board Number: D967
Presentation Time: 3:45 PM - 5:30 PM
Zinc Modulates The Genetic Signature Of The Retina In Response To Acute
Light Exposure
John C. Lang1, Alison Ziesel2, Daniel T. Organisciak3, Christine Rapp3, Ruth
Darrow3, Rekha Rangarajan1, Paul Wong2. 1Consumer Products R&D, Alcon
Research Ltd, Fort Worth, TX; 2Ophthalmology, Emory University, Atlanta, GA;
Biochemistry and Molecular Biology, Wright State University, Dayton, OH.
Purpose: Zinc is a component in an AREDS formulation for AMD treatment and
effectively protects against acute light induced retinal degeneration, LIRD
(Organisciak et al., ARVO 2011: 4423). How zinc mediates this effect is not
understood. Gene profiling studies were performed to examine if zinc directly
mediates changes in retinal gene expression.
Methods: Sprague Dawley rats were exposed to 4 treatment conditions (vehicle
(VEH) or zinc oxide (ZN, 5.2 mg/kg), with (LT) or without (NOLT) 4 h intense
light exposure (1200 lux; 490-580 nm)). A 4 h light exposure defines an early time
point in the LIRD process. The ZN dose used is known to provide protection from
LIRD. Retinas were taken 5 h after VEH or ZN treatment and flash frozen. Total
RNA was extracted and RNA samples were used to initiate screens of Illumina rat
gene expression arrays (Illumina, San Diego, CA). Each array allows the screening
of 22,525 gene marker loci. 3 arrays were screened per treatment condition using
independent samples. Expression data was analyzed with Illumina Genome/ Bead
Studio software, lumi and limma. Additional informatic analyses of gene lists
derived from these analyses were performed with IPA software (Ingenuity Systems,
Redwood City, CA).
Results: Three differential screens were run (1: ZN NOLT X VEH NOLT; 2: ZN
LT X ZN NOLT; 3:VEH LT X VEH NOLT). The gene profiles between the two
NOLT treatments are similar, with only 9 genes screened proving to be differential.
871 and 662 differential expressed annotated genes were identified in screen 2 and
3 respectively. Among these genes, 401 define light induced changes (genes with
the same response to LT regardless of a ZN or VEH pretreatment) and 730 genes
that define zinc influenced changes.
Conclusions: Zinc does not seem to have much of a direct effect on retinal gene
expression in the absence of intense light. It does however seem to modulate the
retinal response to light exposure and in this way has an unexpected hand in
changing the genetic signature. 535 of the ZN modulated (directly or indirectly)
genes identified were annotated in the Ingenuity biofunction/ontology database,
338 of these (63%) fall into a network of cell death, cell growth, and cell cycle
related genes and it may be that the subtle augmentation of these networks is key to
the protection from LIRD.
Commercial Relationships: John C. Lang, Alcon Research, Ltd. (E); Alison
Ziesel, None; Daniel T. Organisciak, Alcon Research, Ltd; Ohio Lions Research
Foundation (F); Christine Rapp, None; Ruth Darrow, None; Rekha
Rangarajan, Alcon Research, Ltd. (E); Paul Wong, Alcon Research Ltd.;
Research to Prevent Blindness; Fraser Parker Foundation; NIH NEI P30EY006360
Support: None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2590 Poster Board Number: D968
Presentation Time: 3:45 PM - 5:30 PM
Cone Photoreceptors Are Vulnerable To Diabetes-induced Rod Photoreceptor
Ke Shi1A,2, Yun-Zheng Le1B,3. AMedicine and Harold Hamm Diabetes Center,
Medicine and Cell Biology and Harold Hamm Oklahoma Diabetes Center,
University of Oklahoma Health Sciences Center, Oklahoma City, OK;
Ophthalmology,The Second Affiliated Hospital, Nanchang University, Nanchang,
China; 3Dean A. McGee Eye Institute, Oklahoma City, OK.
Purpose: Diabetic retinopathy (DR) is a leading cause of blindness in the United
States and is considered as a microvascular complication in diabetic retina.
However, emerging evidences suggest that the loss of retinal neuron function and
the death of retinal neurons are involved in DR. To determine whether the diabetesinduced rod photoreceptor death affects cone photoreceptor survival in DR, we
used rod-specific Bcl-x knockout (KO) mice that had been shown to have impaired
stress-induced survival previously and determined the effect of accelerated rod
death on cone survival in diabetic conditional Bcl-x KO mice.
Methods: Diabetes was induced with streptozotocin. Retinal function was
measured with electroretinography (ERG) and retinal morphology was assessed
with hematoxylin & eosin (H&E) stained sections. Cone density was evaluated
with immunohistochemistry.
Results: Thirty two weeks after inducing diabetes, rod-specific Bcl-x KO mice
demonstrated accelerated loss of outer nuclear layer (ONL) thickness and rod
photoreceptor function, compared with that of wildtype animals. Interestingly, cone
photoreceptor function, as measured by photopic ERG, was significantly reduced,
compared with wildtype controls. Immunohistochemical analysis of S-opsin and
M-opsin, markers of cone photoreceptors, showed that cone density was also
significantly reduced in diabetic rod-specific Bcl-x KO mice, compared with
wildtype animals. Investigating the underlying mechanism for our observation is in
Conclusions: Our data showed that BCL-xL, a PI3K-AKT survival pathway
downstream target, is involved in rod photoreceptor protection under diabetic
condition. Our results also suggest that cone photoreceptors are vulnerable to
diabetes-induced rod photoreceptor death.
Commercial Relationships: Ke Shi, None; Yun-Zheng Le, None
Support: NIH grants R01EY020900 and P20RR024215, ADA grant 1-10-BS-94,
OCAST contract HR09-058
Program Number: 2591 Poster Board Number: D969
Presentation Time: 3:45 PM - 5:30 PM
X-linked RP Caused by RPGR Mutations: Natural History of the Human
Disease and an Rpgr-mutant Rodent Model
Wei Chieh Huang1A, Alejandro J. Roman1A, Artur V. Cideciyan1A, Dina Y.
Gewaily1A, Maria P. Limberis1B, Peter Bell1B, James W. Wilson1B, Alan F. Wright2,
Anand Swaroop3, Samuel G. Jacobson1A. AOphthalmology, Scheie Eye Institute,
Pathology & Laboratory Medicine, 1University of Pennsylvania, Philadelphia, PA;
MRC Human Genetics Unit, Edinburgh, United Kingdom; 3NeurobiologyNeurodegeneration & Repair Laboratory, National Eye Institute, Bethesda, MD.
Purpose: To investigate the retinal disease due to mutations in the Retinitis
Pigmentosa GTPase Regulator (RPGR) gene in human patients and a mouse model.
Methods: XLRP patients with RPGR mutations (n=20; ages at first visit 8-39 yrs)
were followed longitudinally with clinical examinations and rod (R) and cone (C)
perimetry. Rpgr-ko mice (ages 4-13 mos) were studied with SD-OCT and
electroretinography (ERG). Matched controls were compared with the mutants. A
subset of mice had retinal histopathology.
Results: Different patterns of rod and cone dysfunction were present in the
youngest patients studied (8-12 yrs). There could be detectable central R and C
sensitivity, midperipheral losses, with or without peripheral R and C islands; Conly central function, midperipheral losses, and peripheral R and C function; or Conly function across the field. Over years to decades, these patterns could progress
to C-only central islands without peripheral function. Less commonly, patients had
central R and C scotomas but preserved, albeit abnormal, peripheral R and C
function. Rpgr-ko mice had R and C ERGs that were reduced at all ages studied. At
central and inferior retinal regions, mean ONL thickness was reduced by as much
as 15% at 5 mos and thinned to ~50% of controls by 13 mos. Superior retina had
relatively slower degeneration. There were disorganized photoreceptor inner and
outer segments (IS/OS) at all stages studied.
Conclusions: RPGR mutations lead to major and progressive loss of rod and cone
vision in human XLRP but show different patterns of residual photoreceptor
disease expression. Rpgr-ko mice showed onset of degeneration at young ages and
progressive rod and cone disease, as in the patients. From these results, ages for
pre-clinical treatment in this model can be determined and realistic approaches to
human therapy devised.
Commercial Relationships: Wei Chieh Huang, None; Alejandro J. Roman,
None; Artur V. Cideciyan, None; Dina Y. Gewaily, None; Maria P. Limberis,
None; Peter Bell, None; James W. Wilson, None; Alan F. Wright, None; Anand
Swaroop, None; Samuel G. Jacobson, None
Support: Macula Vision Research Foundation, Hope for Vision, Chatlos
Foundation, RPB, NEI intramural program.
Program Number: 2592 Poster Board Number: D970
Presentation Time: 3:45 PM - 5:30 PM
Identification of Retina-Specific Dynamin-1 Protein Complexes
Lindsey A. Ebke1, Gregory H. Grossman1, Gayle J. Pauer1, Craig D. Beight1,
Geeng-Fu Jang1, John W. Crabb1,2, Stephanie A. Hagstrom1,2. 1Cole Eye Institute,
Cleveland Clinic, Cleveland, OH; 2Ophthalmology, Cleveland Clinic Lerner
College of Medicine of Case Western Reserve University, Cleveland, OH.
Purpose: Dynamin-1 is a highly-expressed neuronal-specific GTPase implicated in
endocytosis and vesicle movement at presynaptic terminals of the brain; however,
its role in the retina is unknown. We previously identified a retinal-specific isoform
of Dynamin-1 that localizes to photoreceptor synaptic terminals and binds Tulp1, a
photoreceptor-specific protein thought to be involved in vesicle movement. To
further investigate this protein complex, we sought to identify additional Dynamin1-binding partners in retinal tissue.
Methods: Immunoprecipitation and Western blot experiments were performed
with bovine and mouse retinal homogenates using a Dynamin-1 antibody raised
against the epitope exclusive to the retinal-specific isoform. Immunoprecipitation
products were separated by SDS-PAGE, protein bands were excised and in-gel
digested with trypsin and identified by LC MS/MS. Identified proteins were
localized in mouse retinal sections by immunohistochemistry.
Results: We identified several canonical endocytic proteins by
immunoprecipitation, including alpha-tubulin and clathrin. Most notably, we
identified Dynamin-3, a protein thought to mediate vesicle cycling, in both bovine
and murine retinal homogenates. Western blot analysis indicates that Dynamin-3 is
expressed in the retina, and immunohistochemistry shows that it co-localizes with
Dynamin-1 and Tulp1 in photoreceptor synaptic terminals. Reciprocal
immunoprecipitation, immunohistochemistry and proximity ligand analyses are in
progress to verify and further evaluate these interactions.
Conclusions: Our data indicate that Dynamin-1 is likely involved in the endocytic
pathway of the retina. Dynamin-3 is expressed in photoreceptor cells and may
interact with the Dynamin-1/Tulp1 complex in the movement of vesicles in
synaptic terminals. Further investigation into the nature of the interactions of these
proteins will be carried out.
Commercial Relationships: Lindsey A. Ebke, None; Gregory H. Grossman,
None; Gayle J. Pauer, None; Craig D. Beight, None; Geeng-Fu Jang,
None; John W. Crabb, None; Stephanie A. Hagstrom, None
Support: NIH Grant EY16072, Foundation Fighting Blindness, and Research to
Prevent Blindness
301 Differentiation of Stem Cells In Vivo and In Vitro
Tuesday, May 8, 2012, 8:30 AM - 10:15 AM
Floridian A Paper Session
Program #/Board # Range: 2691-2697
Organizing Section: Retinal Cell Biology
Program Number: 2691
Presentation Time: 8:30 AM - 8:45 AM
Use of an iPS Reporter Cell Line Expressing RPE-Specific GFP For
Improving iPS Cell to RPE Differentiation
Kapil Bharti1A, Janine Davis1B, Barbara Corneo2, Sally Temple2, Sheldon Miller1B.
NINDS, BNational Eye Institute, 1National Institutes of Health, Bethesda, MD;
Neural Stem Cell Institute, Rensselaer, NY.
Purpose: The use of induced pluripotent stem (iPS) cell-derived retinal pigment
epithelium (RPE) for cell-based therapy requires growing cells using current Good
Manufacturing Protocols. The currently available RPE differentiation protocols are
not optimized for this purpose. The aim of this study is to develop and characterize
an iPS reporter cell line for optimizing RPE differentiation.
Methods: A lentivirus construct containing a constitutively expressed red
fluorescent protein (RFP) and an RPE-specific green fluorescent protein (GFP) was
transduced into a fibroblast- derived iPS cell line. Four independently transduced
RFP-positive colonies were differentiated into RPE using existing protocols and
GFP expression was monitored. The ability of GFP-positive cells to differentiate
into RPE cells was assessed by gene expression, imaging, FACS, and physiology
Results: GFP-positive cells were observed in differentiating iPS cell cultures 4-7
days after addition of ACTIVIN A. The GFP expression peaked at around 10-12
days when GFP-positive cells started to attain an epithelial morphology and initiate
pigmentation. GFP positive-and negative-cells were separated by FACS and
analyzed for RPE-specific and fibroblast gene expression using qRT-PCR analysis.
As compared to the GFP-negative cells, GFP-positive cells showed several fold
higher expression of early RPE transcription factors MITF, PAX6, OTX2, and
TCFEC, pigmentation enzymes including TYROSINASE, TYRP, DCT, PMEL17,
and OCA2, junctional protein CLAUDIN19, channel proteins TRPM1 and
TRPM3, and the visual cycle protein RLBP1. In comparison, the expression of
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
fibroblast genes, such as SNAIL1 and 2, was much lower in GFP-positive cells.
This reporter line was further used to optimize the differentiation process and
obtain significantly higher number of pigmented cells from iPS cells.
Conclusions: Expression and imaging analyses of the GFP-positive versus GFPnegative cells confirmed the specificity of the GFP reporter line. GFP expression
marks iPS cells that initiate an RPE-differentiation program. This reporter line will
be useful for several applications including high throughput screens to optimize iPS
cell to RPE differentiation, genomic and proteomic analysis of differentiating RPE
cells, and in tracking of RPE cells transplanted in pre-clinical animal models.
Commercial Relationships: Kapil Bharti, None; Janine Davis, None; Barbara
Corneo, None; Sally Temple, None; Sheldon Miller, None
Support: NIH Intramural grant
Program Number: 2692
Presentation Time: 8:45 AM - 9:00 AM
Retinal Laminae Formation and Synapse Development in Optic Vesicle-like
Structures Isolated from Human Blood-derived iPS Cells
Joe Phillips1A, Kyle A. Wallace1A, Sarah Dickerson2, Michael Miller2, Amelia D.
Gerner1A, Jessica M. Martin1A, Lynda S. Wright1A, Elizabeth E. Capowski1A, Enio
T. Perez1A, David M. Gamm1B. AWaisman Center, BDepartment of Ophthalmology,
Visual Sciences, and Eye Research Institute, 1University of Wisconsin, Madison,
WI; 2Cellular Dynamics International, Madison, WI.
Purpose: We sought to determine if human induced pluripotent stem cells (iPSCs)
derived from peripheral blood could generate optic vesicle-like structures (OVs)
with the capacity to stratify and express markers of intercellular communication.
Methods: Activated T-lymphocytes from a routine peripheral blood sample were
reprogrammed by retroviral transduction to iPSCs. The T-lymphocyte-derived
iPSCs (TiPSCs) were characterized for pluripotency and differentiated to OVs
containing a highly enriched population of neuroretinal progenitor cells (NRPCs).
TiPSC-OVs were then manually isolated, pooled, and cultured en masse to more
mature stages of retinogenesis. Throughout the stepwise differentiation process,
changes in anterior neural, retinal, and synaptic marker expression were monitored
by PCR, immunocytochemistry, and/or flow cytometry.
Results: TiPSCs generated OVs in abundance, which contained a near
homogenous population of proliferating NRPCs. These NRPCs differentiated into
multiple neuroretinal cell types, similar to OV cultures from human embryonic
stem cells and fibroblast-derived iPSCs. In addition, portions of some TiPSC-OVs
maintained a distinctive neuroepithelial appearance and spontaneously formed
primitive laminae, reminiscent of the developing retina. Retinal progeny from
TiPSC-OV cultures upregulated numerous genes and expressed proteins critical for
synaptogenesis and gap junction formation, concomitant with the emergence of glia
and the expression of thrombospondins in culture.
Conclusions: We demonstrate for the first time that human blood-derived iPSCs
can generate retinal cell types, providing a highly convenient donor cell source for
iPSC-based retinal studies. We also show that cultured human iPSC-OVs have the
capacity to self-assemble into rudimentary neuroretinal structures and express
markers indicative of chemical and electrical synapses.
Commercial Relationships: Joe Phillips, None; Kyle A. Wallace, None; Sarah
Dickerson, CDI (E); Michael Miller, CDI (E); Amelia D. Gerner, None; Jessica
M. Martin, None; Lynda S. Wright, None; Elizabeth E. Capowski, None; Enio
T. Perez, None; David M. Gamm, None
Support: FFB Wynn-Gund Translational Research Acceleration Award; NIH
R01EY21218; P30HD03352; Retina Research Foundation; UW-ICTR NIH
1UL1RR025011; UW Eye Research Institute; E. Matilda Ziegler Foundation
Program Number: 2693
Presentation Time: 9:00 AM - 9:15 AM
Targeting Human Mak Mutant iPSCs For In Vitro Gene Replacement
Budd A. Tucker, Kristin R. Anfinson, Jeanean L. Andorf, Luan M. Streb, Todd
Scheetz, Robert F. Mullins, Edwin M. Stone. Ophthalmology, Inst for Vision Rsrch,
Univ of Iowa, Iowa City, IA.
Purpose: The gene male germ cell associated kinase (MAK) encodes a protein
involved in regulation of photoreceptor cilia length. By combining next generation
whole exome sequencing and induced pluripotent stem cell (iPSC) technology we
recently found that a mutation in exon 9 of the MAK gene resulted in a loss of
normal MAK transcript and development of human autosomal recessive retinitis
pigmentosa. The purpose of this study was to determine if a lentiviral vector
designed to drive expression of a retinal MAK transcript could be used to treat
mutant human iPSCs and restore proper MAK expression and photoreceptor
structure post-differentiation.
Methods: iPSCs were generated via viral transduction of human keratinocytes
obtained from patients with MAK-associated retinitis pigmentosa using the
transcription factors Oct4, Sox2, C-Myc and KLF4. iPSC potency was analyzed via
ICC, WB, teratoma formation and embryoid body generation.
Immunocytochemistry and rt-PCR analysis were used to detect transgene
expression post-viral transduction.
Results: Three separate patient specific lines of MAK mutant RP iPSCs each
harboring the previously identified ALU insertion (Tucker et. al. 2011) were
generated. Each of these lines were expanded in feeder free conditions and
determined to be pluripotent based on immunocytochemical and rt-PCR
pluripotency marker expression (Nanog, SSEA3, SSEA4, Tra-1-60, and Tra-1-81)
and ability to generate tissues specific to all three germ layers (determined via
teratoma and embryoid body formation). Full-length retinal MAK cDNA,
generated via rt-PCR amplification from total human retinal RNA, was TA cloned
and subsequently ligated into a lentiviral vector containing a GFP reporter driven
under control of the EF1α promoter. MAK virus driven gfp expression could be
detected at 1-week post-iPSC viral transduction. Restoration of the normal retinal
MAK transcript could be detected via rt-PCR at this time.
Conclusions: We have successfully developed a MAK lentiviral based gene
replacement strategy that has been validated in human iPSCs. This study
demonstrates that in addition to their utility for cell replacement therapy, human
iPSCs are also a valuable tool for analysis of the pathogenic mechanism of specific
mutations and in vitro evaluation of gene replacement therapy.
Commercial Relationships: Budd A. Tucker, None; Kristin R. Anfinson,
None; Jeanean L. Andorf, None; Luan M. Streb, None; Todd Scheetz,
None; Robert F. Mullins, None; Edwin M. Stone, None
Support: NIH 1-DP2-OD007483-01; Grousbeck Family Foundation; Corley
Research Fund; Foundation Fighting Blindness
Program Number: 2694
Presentation Time: 9:15 AM - 9:30 AM
A Bioengineered Delivery System For The Transplantation Of Mouse Retinal
Stem Cell-derived Rod Photoreceptors Encourages Cell Survival And
Brian G. Ballios1A, Laura Clarke1A, Molly S. Shoichet1A,1B, Derek van der
Kooy1A,1C. AInstitute of Medical Science, BDepartment of Chemical Engineering
and Applied Chemistry, CDepartment of Molecular Genetics, 1University of
Toronto, Toronto, ON, Canada.
Purpose: Adult retinal stem cell (RSCs) derived from the ciliary epithelium of
mice can give rise to all retinal cell types. RSC-derived photoreceptors have
demonstrated functional recovery in mouse models of disease. The potential of
RSC-derived rods in adult mouse transplantation models has been limited by poor
cell distribution, survival and integration into host tissue. An injectable and
biodegradable hydrogel material, a blend of hyaluronan and methylcellulose
(HAMC), has shown promise in overcoming the cell distribution barrier. Here we
report a mechanism through which HAMC directly supports the survival and
integration of post-mitotic RSC-derived rods in vitro and in vivo.
Methods: RSC-derived rods were pre-differentiated on laminin substrate in the
presence of taurine/retinoic acid, which can increase the percentage of rods
differentiating in clonal RSC colonies to over 95% of the population. At 28 days,
induction factors were removed and cells were exposed to either HAMC, HA-only,
MC-only or serum free media (SFM) for 7 days. Survival was assessed with
ethidium homodimer and phenotype by immunocytochemical (ICC) staining for
rhodopsin. RSC-derived rods were harvested at various pre-differentiation time
points and injected subretinally into adult mice. Cell distribution was analyzed
between neural retina, subretinal space, and retinal pigment epithelium (RPE) in the
HAMC v. saline vehicles, with or without addition of DL-alpha-aminoadipic acid
Results: Post-mitotic RSC-derived rod photoreceptors showed significantly
improved survival in HA-containing mixtures (HAMC, HA-only: 90%) compared
to those without (MC-only, SFM: 60%), with no change in rhodopsin expression.
Activated caspase-3 levels showed that the pro-survival effect was through
inhibition of apoptosis. ICC and Q-PCR analysis revealed the expression of the
hyaluronan receptor CD44 on RSC-derived rods. The binding of HA to CD44 is
known to influence cellular migration and survival. CD44-/- RSC-derived rods did
not exhibit the pro-survival effect seen with HA-containing mixtures,
demonstrating the specificity of this interaction. Transplantation of wild-type RSCderived rods in HAMC+AAA results in enhanced survival and integration into
neural retina, which is lost with CD44-/- cells.
Conclusions: HAMC directly influences RSC-derived rod survival in vitro and in
vivo through the CD44 receptor. While glial barriers present a ceiling to cell
integration numbers, cell survival in vivo is a critical pre-requisite for transplant
Commercial Relationships: Brian G. Ballios, None; Laura Clarke,
None; Molly S. Shoichet, None; Derek van der Kooy, None
Support: Canadian Institutes of Health Research (CIHR), Foundation Fighting
Blindness (FFB)
Program Number: 2695
Presentation Time: 9:30 AM - 9:45 AM
Towards Photoreceptor Replacement Therapy: Identifying The Cell Surface
Marker Profile Of Photoreceptor Precursors
Jorn Lakowski1, Yating Han1, Anai A. Gonzalez-Cordero2, Emma West2, Robin R.
Ali2, Rachael A. Pearson2, Jane C. Sowden1. 1Developmental Biology Unit, UCL
Institute of Child Health, London, United Kingdom; 2Div of Molecular Therapy,
UCL Institute of Ophthalmology, London, United Kingdom.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Purpose: Loss of photoreceptors due to retinal degeneration is a major cause of
blindness in the developed world. While no effective treatment is currently
available, cell replacement therapy, using in vitro generated photoreceptor
precursor cells, may be a feasible future treatment. Safety and efficacy of this
approach depends on stringent selection and purification of optimal donor cells
from stem cell cultures. As genetic labeling of donor cells is not desirable for
therapy, we developed cell selection strategies using cell surface antigens.
Previously, we demonstrated that CD73/CD24 double positive rod precursors
derived from the developing retina integrate efficiently into the normal and
diseased mouse eye after sub-retinal transplantation. Here, we developed a cell
selection protocol for the isolation of correctly staged rod precursors from
differentiated mouse embryonic stem cell (mESC) cultures. In addition, we
assessed the deployment of the same cell surface markers for human cells.
Methods: The developmental expression profiles of 34 highly expressed cell
surface antigens identified by microarray analysis of Nrl.GFP expressing rod
precursors were analysed using semi-quantitative PCR and flow cytometry.
Markers for positive and negative selection of post-mitotic, yet immature, rod
precursors were tested on postnatal mouse retinal cells and differentiated mESCs.
Cell surface marker expression in human retinal cells was established using
immunohistochemistry and flow-cytometry.
Results: A panel of 8 cell surface markers was identified that in combination
facilitates the isolation of post-mitotic rod precursor from differentiated mESCs.
CD73, CD24, CD133 and CD47 were transiently co-expressed during retinal
development and were useful for the positive selection of rod precursors from the
developing retina and differentiated mESC. Removal of retinal progenitors and
other retinal cell types was accomplished by negative selection via CD15, CD117,
CD44 and CD138 expression.
Conclusions: Correctly staged photoreceptor precursors for the purpose of retinal
therapy can be isolated from complex mixtures of cells, such as differentiated
mESC, via cell surface marker expression. In addition to positive selection of donor
cells, removal of undesirable or mitotically active retinal cells can be accomplished
through cell surface marker-mediated cell depletion.
Commercial Relationships: Jorn Lakowski, None; Yating Han, None; Anai A.
Gonzalez-Cordero, None; Emma West, None; Robin R. Ali, None; Rachael A.
Pearson, None; Jane C. Sowden, None
Support: Medical Research Council UK (G03000341 and G0901550), Fight For
Sight, Child Health Research Appeal Trust, NIHR
Program Number: 2696
Presentation Time: 9:45 AM - 10:00 AM
Light Responsiveness Of RPE Derived From Human Embryonic Stem Cells
Lei Zheng1, Amanda-Jayne F. Carr2, Ma'ayan Semo2, Li Li Chen2, Mark Hankins3,
Pete J. Coffey2, Anthony A. Vugler2. 1Department of Biological Sciences, Hefei
University of Technology, Hefei, China; 2Ocular Biology and Therapeutics, UCL
Institute of Ophthalmology, London, United Kingdom; 3Nuffield Department of
Clinical Neurosciences, University of Oxford, Oxford, United Kingdom.
Purpose: Light is a potentially important stimulus to consider when generating
retinal cell types from pluripotent human cells in vitro. Here we examine the ability
of human embryonic stem cell-derived RPE (HESC-RPE) to respond to light at
both the electrophysiological and molecular level.
Methods: Low passage cultures of HESC-RPE were examined for their ability to
respond to light using several different approaches including Multi-Electrode Array
(MEA), patch clamping, calcium imaging, quantitative RTPCR and western blot.
The light delivered to stimulate HESC-RPE was either white (broad spectrum) or
Results: Following exposure of HESC-RPE to white light, MEA recordings
showed a clear spiking activity, which occurred within 1 second of stimulus onset.
A similar response was also observed using calcium imaging to analyse light
responses in individual cells. The ability of isolated HESC-RPE to respond
intrinsically to light was confirmed by patch clamp recordings, which revealed a
slow inward current in response to a 10 second pulse of 420nm blue light. On a
molecular level, HESC-RPE exposed to 30 minutes of blue light and sampled by
RTPCR after a further 30 minutes in darkness showed modulation of RPE
associated genes (relative to a dark control group), including an enhancement of
LRAT, IRBP and PER1 expression. Interestingly, exposure of HESC-RPE to
longer wavelengths of light for 22hrs in the cell culture incubator resulted in a
marked increase in a 65kDa band detected by western blot using the RPE65
Conclusions: Here, we demonstrate for the first time that HESC-RPE are
intrinsically light responsive. This stimulus can modulate both membrane potential
and gene expression in HESC-RPE and as such, we suggest that light is an
important variable to control during the culture of both HESC-RPE and other
potentially light-responsive retinal cell types derived from pluripotent stem cells.
Commercial Relationships: Lei Zheng, None; Amanda-Jayne F. Carr,
None; Ma'ayan Semo, None; Li Li Chen, None; Mark Hankins, None; Pete J.
Coffey, None; Anthony A. Vugler, None
Support: The London Project to Cure Blindness and BBSRC
Program Number: 2697
Presentation Time: 10:00 AM - 10:15 AM
Physiological Differences Between iPSC-RPE and Human Fetal RPE
Tarun Bansal1, Qin Wan1, Rong Li1, Patricia Lederman2, Barbara Corneo2, Sally
Temple2, Sunita D'Souza3, Kapil Bharti4, Sheldon S. Miller1. 1SERPD, National
Eye Institute, NIH, Bethesda, MD; 2Neural Stem Cell Institute, Rensselaer, NY;
Mount Sinai School of Medicine, New York, NY; 4MDS, National Institute of
Neurological Disorders and Stroke, NIH, Bethesda, MD.
Purpose: Pluripotent stem cell-derived retinal pigment epithelium (RPE) has been
used as a potential therapeutic intervention for people suffering from degenerative
eye diseases. However, the functional properties of these RPE cells have not been
fully authenticated. In this study, we compared the physiology of induced
pluripotent stem (iPS) cell-derived RPE with previously well-characterized
cultured human fetal RPE (hfRPE).
Methods: Human dermal fibroblasts derived iPS cell lines (HDF2/9) were
differentiated using existing protocols in the lab of Sally Temple (NSCI/NY).
Confluent monolayers of hfRPE/iPSC-RPE grown on Transwells were used for
imaging and physiology experiments. We used pH- and Ca2+-sensitive fluorescence
dyes (BCECF and Fura-2) to monitor intracellular pH and Ca2+ activity, while
simultaneously recording transepithelial potential (TEP) and resistance (RT).
Intracellular microelectrodes were used to measure apical/basolateral membrane
potentials (VA/VB), the ratio of apical to basolateral membrane resistance (RA/RB),
and RT.
Results: Intracellular recordings showed that iPSC-RPE had a similar resting VA
and VB as the hfRPE (≈ -55 mV, n=12); however, when extracellular K+ was
altered from 5 to 1 mM (mimicking the transition from dark to light), ∆VA, ∆VB,
and ∆TEP values in hfRPE were significantly different than iPSC-RPE (∆VA =
23.2 ± 7.5 mV for hfRPE and 10.9 ± 1.6 mV for HDF9-RPE; n=6, p < 0.005).
Intracellular Ca2+ imaging revealed that HDF2-RPE showed an impaired function
of SERC ATPase, as compared to hfRPE or HDF9-RPE. All three cell lines
showed the presence of apical membrane Na/H exchangers, apical P2Y2 receptors,
differential apical/basolateral CO2 permeability, and basolateral membrane
Cl/HCO3 exchangers. Using immunofluorescence we identified several proteins
whose polarization is identical in these cell types (EZRIN, ALDH1A3, and
BEST1). In contrast, several other proteins showed differences in localization
Conclusions: Our results suggest that a thorough functional authentication of
iPSC-RPE would be prudent before such cells are used for transplantation. We seek
to provide a set of standards that will be useful for comparison of native human
RPE and stem cell-derived RPE.
Commercial Relationships: Tarun Bansal, None; Qin Wan, None; Rong Li,
None; Patricia Lederman, None; Barbara Corneo, None; Sally Temple,
None; Sunita D'Souza, None; Kapil Bharti, None; Sheldon S. Miller, None
Support: NEI Intramural Research Program
319 Retinal and Choroidal Neovascularization
Tuesday, May 8, 2012, 8:30 AM - 10:15 AM
Hall B/C Poster Session
Program #/Board # Range: 2988-3025/D837-D874
Organizing Section: Retinal Cell Biology
Program Number: 2988 Poster Board Number: D837
Presentation Time: 8:30 AM - 10:15 AM
The Role of VEGF-A Overexpression and Bruch’s Membrane Defects in
Choroidal Neovascularization: A Computer Simulation Study
Abbas Shirinifard, James A. Glazier. Physics, Biocomplexity Institute,
Bloomington, IN.
Purpose: Choroidal neovascularization (CNV) of the macular area of the retina is
the major cause of severe vision loss in patients with age-related macular
degeneration (AMD). The factors determining both CNV initiation and progression
are poorly understood. Controlled experiments on choroidal neovascularization
(CNV) are difficult because no animal model exhibits the full range of age-related
CNV pathologies and in vitro experiments fail to reproduce CNV or to form
drusen. We have developed computational models which allow us to control the
identity and degree of activity of specific biological mechanisms without
confounding crosstalk or quantitative uncertainties to evaluate their contribution to
the initiation and progression of CNV.
Methods: Our 3D multi-scale multi-cell model includes key retinal components,
mechanisms including cell-cell, cell-extracellular matrix (ECM) and ECM-ECM
adhesion, and major angiogenesis-related processes, including BrM breakdown,
hypoxic signaling via upregulation of vascular endothelial growth factor (VEGF),
and VEGF and oxygen transport.
Results: We used our model to simulate the effects of VEGF overexpression in the
RPE and the effects of defects in BrM on CNV initiation and progression. Our key
findings are: 1) Sub-RPE CNV only forms when attachment of the basement
membrane of the RPE to BrM fails. 2) VEGF overexpression increases risk of
initiating sub-retinal CNV, but it does not promote sub-RPE CNV. 3) Holes in BrM
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
larger than about 40 µm, disrupt RPE integrity, leading to sub-retinal CNV, but not
sub-RPE CNV.
Conclusions: Our findings suggest that 1) age-related changes, like lipid
accumulation in BrM which interferes with adhesion of the basement membrane of
the RPE to BrM, may be key factors in initiation of sub-RPE CNV in AMD, 2)
VEGF overexpression and severe BrM defects may promote progression of subRPE CNV to sub-retinal CNV in AMD.
Commercial Relationships: Abbas Shirinifard, None; James A. Glazier, None
Support: EPA grant “The Texas-Indiana Virtual STAR Center" and NIH grants
Program Number: 2989 Poster Board Number: D838
Presentation Time: 8:30 AM - 10:15 AM
Histological Changes After Photodynamic Therapy (PDT) Combined With
Anti-vascular Endothelial Growth Factor (VEGF) Therapy In The Mouse
Misa Suzuki1A,1B, Shunsuke Kubota2,1A, Manabu Hirasawa1A, Seiji Miyake1A,
Kousuke Noda3, Kazuo Tsubota1B, Kazuaki Kadonosono4, Susumu Ishida3, Yoko
Ozawa1B,1B. ALaboratory of Retinal Cell Biology, BDepartment of Ophthalmology,
Keio University School of Medicine, Tokyo, Japan; 2Apte Lab Ophthalmology &
Visual Science, Washington University, St.Louis, MO; 3Department of
Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo,
Japan; 4Department of Ophthalmology, Yokohama City University Medical Center,
Yokohama, Japan.
Purpose: Photodynamic therapy (PDT) and/or anti-vascular endothelial growth
factor (VEGF) drugs constitute current treatments targeting choroidal
neovascularization in age-related macular degeneration. Concern that PDT might
induce VEGF and exacerbate the disease has led us to current practice of using
anti-VEGF drugs with PDT simultaneously. However, the underlying molecular
mechanisms of these therapies are not well understood. The influence of the
combined therapy still remains obscure. In this study, we analyze histological
influence of PDT simultaneously combined with anti-VEGF therapy.
Methods: Six week-old C57/B6 male mice were prepared. Verteporfin was
injected from their tail veins and the intact retinas were exposed to the laser to
activate the drug 15 minutes after the injection. Histological changes were
evaluated by hematoxylin eosin (HE) staining, TUNEL assay to detect apoptosis,
and immunohistochemistry. Measurement of the VEGF level in the retina or retinal
pigment epithelium (RPE)-choroid complex was performed by ELISA in each time
point after PDT. A VEGF inhibitor, VEGFRc/fusion protein, or control vehicle was
injected intravitreally immediately after PDT.
Results: HE staining and TUNEL assay showed no obvious histological change
after twenty-second irradiation during PDT. However, immunohistochemical
analysis showed that glial fibrillary acidic protein, which represents reactivation of
Müller glial cells, is upregulated in whole area of the retina after PDT. Level of
VEGF protein in the retina was upregulated immediately and transiently after PDT,
whereas in the RPE-choroid complex, it was reduced, transiently. VEGF
suppression after PDT resulted in apoptotic destruction of the RPE as well as
photoreceptor cell layer only in the irradiated area during PDT.
Conclusions: Reactivation of Müller glial cells was induced in the whole retina
after PDT, although overt tissue damage was not detected morphologically.
Immediately after PDT, VEGF was required to protect the irradiated tissue
including photoreceptor cells and RPE.
Commercial Relationships: Misa Suzuki, None; Shunsuke Kubota,
None; Manabu Hirasawa, None; Seiji Miyake, None; Kousuke Noda,
None; Kazuo Tsubota, None; Kazuaki Kadonosono, None; Susumu Ishida,
None; Yoko Ozawa, Novartis (F)
Support: Japan Society for the Promotion of Science and the Ministry of
Education, Science, Sports and Culture of Japan.
Program Number: 2990 Poster Board Number: D839
Presentation Time: 8:30 AM - 10:15 AM
In-vivo Imaging Of A Fluorescent Probe Linked To Bevacizumab
Alexander R. Cunea1, Johanna Meyer1, Pia Welker2, Kai Licha2, Dagmar SonntagBensch1, Steffen Schmitz-Valckenberg1, Frank Holz1. 1Department of
Ophthalmology, University of Bonn, Bonn, Germany; 2Mivenion GmbH, Berlin,
Purpose: To investigate a fluorescent molecular probe linked to a humanized
monoclonal antibody against vascular endothelial growth factor (VEGF) for in vivo
imaging of VEGF within the retina.
Methods: Bevacizumab was covalently attached to a near-infrared indocyanine dye
yielding a soluble conjugate. Its binding properties were assessed by an in vitro
proliferation assay. Retinal laser lesions were placed around the optic nerve head in
Dark Agouti rats. The dye conjugate was intraperitoneally, intravenously or
intravitreally injected. Its uptake was recorded in vivo using confocal scanning
laser ophthalmoscopy (cSLO) at pre-defined time intervals for up to 60 days
Results: No significant difference of cellular activity could be found between the
conjugated and the unconjugated antibody in the proliferation assay in vitro (71%,
75% respectively; control=100%). Following intraperitoneal injection, no
fluorescence was visible on the same day, while one day later a strong signal
became present in the retinal vasculature and within roundish laser lesions.
Furthermore, multiple hyperfluorescent spots were visible. By contrast, strong
fluorescence became visible immediately after intravenous and intravitreal
injection. With all three different modes of application, signal intensity
continuously decreased after injection. Few fluorescent spots were still visible on
day 60.
Conclusions: Pharmacokinetics of fluorescent-labeled bevacizumab can be
investigated in vivo following intraperitoneal, intravenous and intravitreal
injection. Following extensive investigations, the ability of monitoring VEGF
spatially and temporally in vivo may be applicable in patients for earlier diagnosis
and more refined individualized anti-VEGF therapies with the aim of optimizing
functional outcomes.
Commercial Relationships: Alexander R. Cunea, Carl Zeiss Meditec,
Heidelberg Engineering (F); Johanna Meyer, Carl Zeiss Meditec, Heidelberg
Engineering (F); Pia Welker, Mivenion GmbH (E), WO 2011/095311 (P); Kai
Licha, Mivenion GmbH (E), WO 2011/095311 (P); Dagmar Sonntag-Bensch,
Carl Zeiss Meditec, Heidelberg Engineering (F); Steffen Schmitz-Valckenberg,
Carl Zeiss Meditec, Heidelberg Engineering, Optos Ltd., Topcon UK. (F),
Heidelberg Engineering (R); Frank Holz, Carl Zeiss Meditec, Heidelberg
Engineering, Optos Ltd. (F), Heidelberg Engineering (R), Heidelberg Engineering,
Carl-Zeiss Meditec AG (C)
Support: German Ministry of Education and Research, BMBF: FKZ 13N10285
Program Number: 2991 Poster Board Number: D840
Presentation Time: 8:30 AM - 10:15 AM
Lipid-derived Energy Shortage Precedes Subretinal Neovascularization In A
Mouse Model Of Mactel
Jean-Sebastien Joyal1, Jing Chen1, Aimee Juan1, Colman J. Hatton1, Dorothy Pei1,
Christian Hurst1, Molly R. Seaward1, Patrick Bherer2, Bruno Maranda2, Lois E.
Smith1. 1Ophthalmology, Harvard University, Childrens Hospital, Boston, MA;
Genetics, Université de Sherbrooke, Sherbrooke, QC, Canada.
Purpose: Macular telangiectasia (MacTel) is a blinding disease of unknown
etiology characterized by the development of pathological subretinal neovessels.
Very low density lipoprotein receptor mutant mice (Vldlr-/-) exhibit the abnormal
vascular phenotype of MacTel, resulting in photoreceptor degeneration. Vldlr is
expressed in high energy consuming organs such as the retina and contributes to the
uptake of fatty acids. Here we investigate the role of fatty acid β-oxidation during
retinal development and how energy shortage may lead to pathological subretinal
neovessels in Vldlr-/- mice.
Methods: Subretinal vascular lesions of Vldlr-/- mice were characterized by
confocal imaging of lectin-stained retinal flat mounts. Vldlr and Vegf expression
was evaluated by immunofluoressence (IF) and qRT-PCR of laser-capture
microdissected (LCM) retinal layers from Vldlr-/- and wild-type mice. Vascular
corrosion cast were used to assess the integrity of choroidal vessels. Total
acylcarnitine levels were measured by liquid chromatography followed by tandem
mass spectrometry (LC/MS/MS).
Results: Vldlr expression is localized (by LCM) to the outer nuclear layer (ONL)
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
and the retinal-pigmented epithelium (RPE) of the outer retina, as well as to
vessels. In Vldlr-/- mice, pathological neovessels originate from the deeper vascular
plexus and breach the outer plexiform layer (OPL) between postnatal day (P) 12
and 14, when photoreceptor energy requirements surge. The first vascular lesions
reach the retinal-pigmented epithelium (RPE) by P16. Choroidal vascular corrosion
casts of Vldlr-/- were comparable to wild-type mice, suggesting a normal delivery
of oxygen to the outer retina. Nonetheless, Vegf expression was increased in the
ONL and RPE adjacent to subretinal neovessels (LCM). Because of the important
role of VLDLR in lipid metabolism, we explored the energy derived through fatty
acid β-oxidation in the retinas of our mice. Free carnitine and total acylcarnitine
levels were significantly reduced in Vldlr-/- retinas (P12) compared to wild-type
mice, suggesting a lipid-derived energy shortage preceding the appearance of
vascular lesions.
Conclusions: As the energy requirements of photoreceptors increase during
development, an energy deficiency in lipid metabolism precedes the appearance of
pathological neovessels in Vldlr-/- mice. This may be pertinent to the pathology of
Commercial Relationships: Jean-Sebastien Joyal, None; Jing Chen,
None; Aimee Juan, None; Colman J. Hatton, None; Dorothy Pei,
None; Christian Hurst, None; Molly R. Seaward, None; Patrick Bherer,
None; Bruno Maranda, None; Lois E. Smith, None
Support: MacTel Foundation (LEHS), Research to Prevent Blindness Senior
Investigator Award (LEHS), Canadian Institute of Health Research Fellowship
(JSJ), Charles H. Hood Foundation Research Award (JC)
Program Number: 2992 Poster Board Number: D841
Presentation Time: 8:30 AM - 10:15 AM
Angioblasts Differentiate Into Endothelial Cells Or Pericytes In Vitro But
Favor A Pericyte Position When Injected Intravitreally
Malia M. Edwards1, D S. McLeod1, Imran A. Bhutto1, Carol Merges1, Takayuki
Baba2,1, Vikash Juriasinghani1, Gerard A. Lutty1. 1Wilmer Eye Institute, Johns
Hopkins Univ Sch of Med, Baltimore, MD; 2Ophthalmology & Visual Science,
Chiba Univ Grad School of Med, Chiba, Japan.
Purpose: Cultured neonatal canine retinal angioblasts (NCRA) differentiate into
endothelial cells (EC) when given basic fibroblast growth factor (bFGF) or
pericytes in the presence of platelet derived growth factor BB (PDGF-BB) [1]. The
present study investigated tube formation by these differentiated cells using 3-D
collagen gels. The influence of different growth factors on tube formation was
investigated as was the position of cells when co-cultured. The incorporation of
NCRA into developing vessels and neovascular tufts was also investigated using a
model for oxygen induced retinopathy.
Methods: NCRA were cultured and trained from neonatal canine retinas as
previously described [1] and labeled with PKH dyes (26 or 67) according to the
manufacturer’s instructions. For the collagen tube assay, labeled cells were mixed
with collagen solution and plated into a 96 well plate [2]. After 1 hr, media
containing different growth factors was added and cells were imaged every 24 hrs.
The incorporation of differentiated angioblasts into developing retinal vessels was
tested by injecting labeled cells intravitreally into post-natal day (P) 2 dogs.
Animals were sacrificed at P5, retinas dissected and stained with anti-von
Willebrands Factor (vWF). The localization of cells in relation to blood vessels was
observed. For the oxygen-induced retinopathy studies, pups were exposed to 100%
oxygen for 5 days prior to the injection of trained angioblasts at P8. At P21, pups
were sacrificed and the vitreous and retinas labeled with anti-vWF.
Results: NCRA trained with bFGF but not PDGF-BB formed tubes. Tube
formation by bFGF-trained cells was enhanced by VEGF and bFGF. When the two
cell types were cultured together, the bFGF-trained cells formed tubes and the
PDGF-BB-trained cells were observed almost exclusively on the outside walls of
these tubes.
Both types of NCRA injected into the developing canine vitreous were found in a
pericyte position at P5. Similarly, trained cells injected following exposure to
oxygen took a pericyte position on intravitreal neovascularization and retinal blood
Conclusions: The present study demonstrates that cultured canine angioblasts
trained to be either pericytes or endothelial cells function as these cell types during
in vitro tube formation but favored a pericytic position in vivo.
1. Lutty, G. et al., Exp Eye Res, 2006. 83: 183.
2. Liu H. et al Angiogenesis, 2008. 11:223.
Commercial Relationships: Malia M. Edwards, None; D. S. McLeod,
None; Imran A. Bhutto, None; Carol Merges, None; Takayuki Baba,
None; Vikash Juriasinghani, None; Gerard A. Lutty, None
Support: NIH grants EY016551 (GAL); EY01765 (Wilmer Core Grant), Research
to Prevent Blindness (Unrestricted funds to Wilmer)
Program Number: 2993 Poster Board Number: D842
Presentation Time: 8:30 AM - 10:15 AM
Non-Canonical VEGF Receptor Signaling Regulates Retinal
Jun Cai1A, Xiaoping Qi1A, Qing Ruan1A, Song Han1B, Zhijuan Chen1A, Alex
Podlaski1A, Maria B. Grant1C, Michael E. Boulton1A. AAnatomy and Cell Biology,
Surgery, CPharmacology and Therapeutics, 1University of Florida, Gainesville,
Purpose: VEGF binds to its cognate receptors VEGFR1 (Flt-1) and VEGFR2
(KDR/Flk-1) to regulate endothelial function and survival. Although classic
canonical kinase signaling pathways can determine angiogenic outcome it is now
realized that the intracellular trafficking and localization of VEGFRs make a
critical contribution to cellular function. The purpose of this study was to
investigate the role of the subcellular translocation of VEGFRs to the nucleus and
adheren junctions (AJ) and to assess if the ratio of VEGFR1:VEGFR2 at these
intracellular locations determines retinal neovascularization.
Methods: Retinal microvascular endothelial cells (RMECs) exposed to VEGF,
PEDF or VEGF+PEDF were subjected to subcellular fractionation followed by
Western blot analysis with antibodies against the C-terminal domain of VEGFR1
and VEGFR2. Immunocytochemistry was used to characterize the subcellular
translocation of VEGFRs. The reciprocal immunoprecipitation/Western blot
approach was used to detect: 1) the association of VEGFRs with candidate
transcription factors and 2) the association of VEGFRs with AJ proteins (e.g. VEcadherin and β-catenin). To confirm the nuclear localization of VEGFRs in vivo,
C57BL mice received an intravitreal injection of VEGF, PEDF or VEGF+PEDF
and nuclear expression of VEGFR1 and VEGFR2 in retinal endothelial cells was
determined by immunogold cytochemistry.
Results: VEGF induced nuclear translocation of both VEGFRs in RMECs and
decreased VEGFR1:VEGFR2 ratio compared to control. However, addition of
PEDF blocked the VEGF-induced effect and maintained a high VEGFR1:VEGFR2
ratio. Full length VEGFRs were differentially associated with three transcript
factors implicated in angiogenesis; CREB1, NFATc2 and ETS1. In vivo studies
confirmed our in vitro results and showed intravitreal VEGF significantly increased
VEGFR2 levels within the nucleus while VEGFR1 was reduced. This was blocked
if VEGF was co-injected with PEDF. We also observed a strong and dynamic
association between VEGFRs and AJ proteins. Under basal conditions, VEcadherin and β-catenin were strongly associated with VEGFR1 but after VEGF
treatment VEGFR1 association significantly decreased resulting in a reduced
VEGFR1:VEGFR2 ratio which eventually resulted in dissociation of the VEcadherin/β-catenin complex and increased permeability.
Conclusions: A dynamic subcellular translocation of VEGFRs occurs in
microvascular endothelial cells that is dependent on the balance of growth factors
in the local microenvironment. The ratio of VEGFR1 to VEGFR2 is a critical
determinant in retinal angiogenesis and vascular permeability.
Commercial Relationships: Jun Cai, None; Xiaoping Qi, None; Qing Ruan,
None; Song Han, None; Zhijuan Chen, None; Alex Podlaski, None; Maria B.
Grant, None; Michael E. Boulton, None
Support: NIH grant EY018358
Program Number: 2994 Poster Board Number: D843
Presentation Time: 8:30 AM - 10:15 AM
Fenretinide Prevents Retinal Neovascularization in Mouse Models of
Brian C. Oveson1, Takeshi Iwase1, Sean Hackett1, Christopher Seidel1, Nathan L.
Mata2, Peter A. Campochiaro1. 1Ophthalmology, Johns Hopkins University,
Baltimore, MD; 2Research, ReVision Therapeutics, La Jolla, CA.
Purpose: To assess the effects of fenretinide treatment in mouse models of
choroidal and retinal neovascularization (NV).
Methods: Four week-old C57Bl/6 mice received either fenretinide-supplemented
or control diet for four weeks before and 2 weeks after rupture of Bruch’s
membrane by laser photocoagulation and then the area of choroidal
neovascularization (CNV) at rupture sites was measured. Pregnant C57Bl/6 mice
were given fenretinide-supplemented or control diet starting 1 week before giving
birth. Oxygen-induced ischemic retinopathy was induced in the pups and the area
of retinal NV was measured at P17. Pregnant rho/VEGF mice were given
fenretinide-supplemented or control diet starting 1 week before giving birth. At
P28, the pups were perfused with fluorescein-labeled dextran and the area of NV
per retina was measured.
Results: Compared to controls, fenretinide-treated mice had a significant reduction
in the area of CNV at Bruch’s membrane rupture sites (0.003+0.0004 vs
0.006+0.0009 mm2; p<0.002). Fenretinide-treated mice also had a significant
reduction in ischemia-induced retinal NV (0.012+0.004 vs 0.032+0.003 mm2;
p<0.001). This was associated with a significant reduction in the number of
macrophages in ischemic retina. In rho/VEGF transgenic mice, fenretinide
treatment significantly reduced the area of subretinal NV per eye (0.004+0.0009 vs
0.018+0.003 mm2; p<0.001).
Conclusions: Fenretinide reduces macrophage influx into the retina and suppresses
retinal and choroidal NV.
Commercial Relationships: Brian C. Oveson, None; Takeshi Iwase,
None; Sean Hackett, None; Christopher Seidel, None; Nathan L. Mata,
None; Peter A. Campochiaro, None
Support: None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 2995 Poster Board Number: D844
Presentation Time: 8:30 AM - 10:15 AM
Leucine-rich Alpha-2 Glycoprotein 1 (Lrg1) Contributes To The Development
Of Ocular Neovascularisation
Xiaomeng Wang1, Jenny McKenzie1, Natasha R. Jeffs2, Sabu Abraham1, Matthew
Swire1, Clemens A. Lange3A, James W. Bainbridge4, Stephen E. Moss3B, John
Greenwood4A. 1Cell Biology, Institute of Ophthalmology, UCL, London, United
Kingdom; 2Cell Biology, UCL, London, United Kingdom; AGenetics, BCell
Biology, 3Institute of Ophthalmology, London, United Kingdom; ACell Biology,
UCL Institute of Ophthalmology, London, United Kingdom.
Purpose: Aberrant angiogenesis is the hallmark of many blinding eye diseases
including retinopathy of prematurity, proliferative diabetic retinopathy, age-related
wet macular degeneration (AMD) and macular telangiectasia. The formation of
new vessels is a complex and tightly regulated process involving multiple
intercellular pro- and anti-angiogenic molecules. We have previously demonstrated
that leucine rich α-2-glycoprotein-1 (Lrg1) is involved in the fine-tuning of
angiogenesis and drives this delicate process in a pro-angiogenic direction. The aim
of this study was to gain new insight into the molecular mechanisms of Lrg1
signalling and its interaction with other signalling pathways involved in
angiogenesis, as well as to evaluate its potential as a therapeutic target.
Methods: Co-immunoprecipitation and GST-pulldown assay were used to identify
Lrg1 binding proteins. The interaction between Lrg1 and the TGFβ signalling
pathway in angiogenesis was studied in vitro (proliferation assay and organotypic
angiogenesis assay), ex vivo (metatarsal assay and aortic ring assay) and in vivo.
The therapeutic potential of a Lrg1 neutralizing antibody was studied in vitro
(Matrigel assay) and in vivo (mouse model of wet AMD).
Results: We observed that Lrg1 interacts with TGFβ1 and its receptors in vitro.
Recombinant human Lrg1 (rhLrg1) promotes TGFβ1 stimulated Smad1/5
phosphorylation and subsequent endothelial cell (EC) proliferation. rhLrg1 and
TGFβ1 promote vessel outgrowth from metatarsal and aortic ring explants, which
could be attenuated by polyclonal antibody against full length human Lrg1 protein.
Lrg1 neutralizing antibody completely blocks HUVEC tube formation in Matrigel
and inhibits laser induced choroidal neovascularization.
Conclusions: This study demonstrates that Lrg1 plays an important role in
angiogenesis through regulating the TGFβ signalling pathway and is a potentially
promising therapeutic target.
Commercial Relationships: Xiaomeng Wang, None; Jenny McKenzie,
None; Natasha R. Jeffs, None; Sabu Abraham, None; Matthew Swire,
None; Clemens A. Lange, None; James W. Bainbridge, None; Stephen E. Moss,
None; John Greenwood, None
Support: Lowy Medical Research Institute Ltd, the MRC and a UCLB POC
Program Number: 2996 Poster Board Number: D845
Presentation Time: 8:30 AM - 10:15 AM
Molecular Imaging of mRNA in Choroidal Neovascularization
Ashwath Jayagopal1, Andrew Y. Gordon2A, Megan E. Capozzi2B. 1Ophthalmology
and Visual Sciences, Vanderbilt Eye Institute, Nashville, TN; ASchool of Medicine,
Vanderbilt Eye Institute, 2Vanderbilt University, Nashville, TN.
Purpose: Early detection of disease biomarkers in the retina in vivo would
facilitate earlier detection of disease and enhance effectiveness of therapy.
However, the development of protein-targeted agents for this purpose has been
limited. The goal of this study was to evaluate a new approach for in vivo imaging
of mRNA biomarkers in living ocular tissue for the purpose of detecting
neovascular AMD-associated biomarkers.
Methods: Infared dye-linked hairpin DNA-functionalized gold nanoparticles
(hAuNP) were engineered for targeting disease-relevant mRNA biomarkers
including HIF1 alpha, VCAM-1, and VEGFR2, as well as housekeeping transcripts
such as beta actin. Nanoparticles were evaluated in a mouse model of laser-induced
choroidal neovascularization as well as age-matched controls. hAuNP were injected
systemically or intraocularly, and imaged using optical imaging in vivo and ex vivo
in animal models to evaluate specificity and sensitivity.
Results: hAuNP facilitated imaging of neovascular AMD biomarkers within
neovascular lesions throughout the timecourse of laser induced choroidal
neovascularization, with signal to noise ratios exceeding 10:1. Nonspecific
controls, including scrambled hairpin DNA-functionalized nanoparticles, did not
emit appreciable signal in the retina. hAuNP administered by either injection route
were capable of binding to target mRNA within lesions. hAuNP were capable of
simultaenous imaging of multiple mRNA biomarkers within the same animal retina
in vivo.
Conclusions: hAuNP are promising nanoscale optical agents for imaging mRNA
biomarkers in ocular diseases such as neovascular AMD. This imaging approach
will facilitate construction of molecularly-targeted contrast agents capable of
imaging virtually any disease biomarker in neovascular disease, due to the ease of
design and synthesis and homing capability.
Commercial Relationships: Ashwath Jayagopal, None; Andrew Y. Gordon,
None; Megan E. Capozzi, None
Support: P30-EY008126 (Core Grant in Vision Research), International Retinal
Research Foundation, Unrestricted Grant from Research to Prevent Blindness
Program Number: 2997 Poster Board Number: D846
Presentation Time: 8:30 AM - 10:15 AM
The effects of pharmacologic manipulation of Peroxisome ProliferatorActivated Receptor β/δ on pathological angiogenesis
Sara R. Savage1A, Megan E. Capozzi1B, Gary W. McCollum1B, John S. Penn1B.
Pharmacology, BOphthalmology, 1Vanderbilt Univ Medical Center, Nashville,
Purpose: Peroxisome proliferator-activated receptors (PPARs) are members of the
nuclear receptor superfamily of transcription factors activated by a number of
ligands, including naturally occurring fatty acids, NSAIDS, and prostaglandins.
There are three PPAR subtypes: α, β/δ, and γ. Evidence suggests that PPAR β/δ
may regulate VEGF-induced pathological angiogenesis. Therefore, we investigated
the effect of pharmacologic activation and inhibition of PPAR β/δ on human retinal
microvascular endothelial cell (HRMEC) behavior and in a model of oxygeninduced retinopathy (OIR).
Methods: The angiogenic behavior of HRMEC was assessed by proliferation and
tubulogenesis assays in the presence of increasing doses of the PPAR β/δ agonist
GW0742 or antagonist GSK0660. An OIR rat model was used to assess the effect
of PPAR β/δ on development of neovascularization (NV). GW0742 and GSK0660
were administered locally by intravitreal injection and systemically by oral gavage
and intraperitoneal injection, respectively.
Results: Activation of PPAR β/δ by GW0742 had no effect on HRMEC
proliferation, but resulted in a 118% increase (p < 0.0001) of tube formation at the
highest concentration (1.0 µM). Inhibition by GSK0660 caused a dose-dependent
decrease in serum-induced proliferation (39%; p = 0.0034) and tube formation
(40%; p = 0.0039) at the highest concentration (1.0µM). Intravitreal injection of
GW0742 at the highest concentration (500nM) increased NV by 52% (p = 0.044)
and GSK0660 at the highest concentration (500nM) inhibited NV by 45% (p =
0.015). A similar but more pronounced effect was observed with systemic
injections; 10mg/kg GW0742 caused a 190% increase (p = 0.024) and 1mg/kg
GSK0660 caused a 67% reduction (p = 0.0003) of NV.
Conclusions: Activation of PPAR β/δ significantly increases pathological NV in
OIR, while antagonism significantly inhibits NV. This suggests that this
transcription factor may constitute a rational target for chemotherapeutic
intervention in ocular angiogenesis.
Commercial Relationships: Sara R. Savage, None; Megan E. Capozzi,
None; Gary W. McCollum, None; John S. Penn, None
Support: EY007533, Unrestricted Grant from Research to Prevent Blindness
Program Number: 2998 Poster Board Number: D847
Presentation Time: 8:30 AM - 10:15 AM
Retinal Expression Of Wnt-pathway Mediated Genes In Low-density
Lipoprotein Receptor-related Protein 5 (Lrp5) Knockout Mice
Christian G. Hurst1, Jing Chen1, Andreas Stahl1,2, Nathan M. Krah1, JeanSebastien Joyal1, Aimee M. Juan1, Colman J. Hatton1, Dorothy T. Pei1, Przemyslaw
Sapieha1,3, Lois E. Smith1. 1Dept. of Ophthalmology, Harvard Medical School,
Children's Hospital Boston, Boston, MA; 2University Eye Hospital Freiburg,
Freiburg, Germany; 3Dept. of Ophthalmology, Maisonneuve-Rosemont Hospital
Research Centre, University of Montreal, Montreal, QC, Canada.
Purpose: Mutations in low-density lipoprotein receptor-related protein 5 (Lrp5), a
Wnt co-receptor, impair retinal angiogenesis in patients with familial exudative
vitreoretinopathy (FEVR), a rare type of blinding vascular eye disease. FEVR is a
Wnt-related genetic disease in which abnormal retinal vascular development leads
to pathological neovascularization and vascular leakage. Similar to human patients,
mice lacking Lrp5 (Lrp5-/-) exhibit delayed and incomplete growth of retinal
vessels. In order to gain insight into the molecular mechanisms behind the
pathology of FEVR and related eye diseases also impacted by mutations in Wnt
signaling, we analyzed gene expression in the developing retinas of Lrp5-/- mice.
Methods: Lrp5-/- and wild type (WT) retinas from postnatal day (P) 8 mice were
isolated and either stained with lectin for quantification of vascular growth or
processed for RNA isolation (n=6 per group). Retinal and brain samples from both
groups were also sectioned and lectin-stained for observance of vasculature. Gene
expression levels in total RNA from WT and Lrp5-/- retinas isolated on P8 were
evaluated with an Illumina microarray analysis using the Mouse-WG6 expression
BeadChip (n=3 per group), followed by validation with RT-qPCR analysis.
Results: Retinas from P8 Lrp5-/- mice exhibited significantly delayed retinal
vascular development as compared to WT retinas (WT: 92.2±1.6% vs. Lrp5-/- :
69.3+2.7%; p≤0.0001). Further, the normal regression of hyaloid vessels and
formation of deep layer retinal capillary networks observed in WT mice is absent in
the Lrp5-/- mice. Lrp5-/- mice also exhibited lower vascular brain density at P8 and
developed dilated retinal vasculature with enlarged microaneurysm-like lesions
from P12 through adulthood. In the microarray analysis, we found a significant
downregulation of tight junction protein claudin5 and amino acid transporter
slc38a5 in Lrp5-/- retina, as well as an increase in expression of endothelial
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
permeability marker plvap. Additionally, we found certain Wnt ligands to be
significantly down regulated in Lrp5-/- retinas, particularly Wnt7b.
Conclusions: These data suggest that the downregulation of cell adhesion proteins
and increase in vessel permeability markers in Lrp5-/- mice may contribute to their
observed defects in retinal vasculature, as well as those found in patients with
FEVR. Thus, the Wnt signaling pathway presents as a potential future target for
prevention and treatment of vascular eye diseases.
Commercial Relationships: Christian G. Hurst, None; Jing Chen,
None; Andreas Stahl, None; Nathan M. Krah, None; Jean-Sebastien Joyal,
None; Aimee M. Juan, None; Colman J. Hatton, None; Dorothy T. Pei,
None; Przemyslaw Sapieha, None; Lois E. Smith, None
Support: CHB Manton Center, JDRF, Hood Foundation (JC), NIH (EY017017,
EY017017-04S1, EY017017-05), MacTel Foundation (LEHS), Research to Prevent
Blindness Senior Investigator Award (LEHS)
Program Number: 2999 Poster Board Number: D848
Presentation Time: 8:30 AM - 10:15 AM
Pathophysiological Role Of Adrenomedullin-ramp2 System In Retinal
Yasuhiro Iesato1A,1B, Takayuki Sakurai1B, Akiko Kamiyoshi1B, Yuka Shindo1A,1B,
Teruhide Koyama1B, Takahiro Yoshizawa1B, Akihiro Yamauchi1B, Takayuki
Shindo1B, Toshinori Murata1A. AOphthalmology, BOrgan Regeneration, 1Shinshu
Univ Grad Sch of Med, Matsumoto, Japan.
Purpose: Ischemic proliferative retinopathy, characterized by pathological retinal
neovascularization, is a major cause of blindness. Adrenomedullin (AM) is an
endogenous peptide first identified as a strong vasodilating molecule. We showed
AM knockout mice (AM-/-) are embryonic lethal with abnormal vascular
development and first proved AM also possesses angiogenic function. AMreceptor, CLR associates with one of the subtype of accessory proteins, RAMPs.
We also showed among knockout mice of RAMPs, only RAMP2-/- are lethal with
the similar phenotypes of AM-/-, suggesting AM-RAMP2 system is specifically
important for angiogenesis. AM is also expressed in retina and strongly induced
under ischemia. In this study, to clarify the pathophysiological roles of AMRAMP2 system in retina, we analyzed oxygen-induced retinopathy (OIR) model
using RAMP2 knockout mice.
Methods: Wild-type C57BL/6 mice (WT) and heterozygotic RAMP2 knockout
mice (RAMP2+/-) were exposed to 75% oxygen from postnatal day (P)7 to P12
and returned to room air. Both genotypes of mice, which were kept in room air,
were used for control. Eyes were collected at P17 and mRNA was extracted from
retinas to analyze the gene expression by quantitative real-time PCR. Retinal
neovascularization, avascular area, and hypoxic area were analyzed using flatmount specimens of retina stained with isolectin B4 and hypoxyprobe-1.
Neovascularization was also assessed in retinal sections by HE staining.
Results: Both in WT and RAMP2+/- retinas with OIR, AM expression was
upregulated than those in room air. In the OIR, neovascularization in RAMP2+/was significantly reduced than that in WT. On the other hand, there was no
significant difference in avascular area and hypoxic area between WT and
Conclusions: We first found that AM-RAMP2 system is crucially involved in
retinal pathological angiogenesis. Modulation of AM-RAMP2 signaling could be
applied for the management of retinal neovascularization in future.
Commercial Relationships: Yasuhiro Iesato, None; Takayuki Sakurai,
None; Akiko Kamiyoshi, None; Yuka Shindo, None; Teruhide Koyama,
None; Takahiro Yoshizawa, None; Akihiro Yamauchi, None; Takayuki Shindo,
None; Toshinori Murata, None
Support: None
Program Number: 3000 Poster Board Number: D849
Presentation Time: 8:30 AM - 10:15 AM
Shifting expression of membrane Vascular Endothelial Growth Factor
Receptor-2 (mVEGFR2) to soluble Vascular Endothelial Growth Factor
Receptor-2 (sVEGFR2) reduces CNV volume and tumor growth
Brian C. Stagg1, Hironori Uehara2, Taylor Bates2, Yang Kyung Cho2,3, Tadashi
Miya2, Balamurali K. Ambati2. 1University of Utah School of Medicine, Salt Lake
City, UT; 2John Moran Eye Center, Salt Lake City, UT; 3St.Vincent Hospital,
Catholic University of Korea, Seoul, Republic of Korea.
Purpose: Angiogenesis plays a key role in the pathophysiology of many ocular
conditions such as age-related macular degeneration (AMD). Angiogenesis is also
important in cancer growth in order to provide nutrients and metabolic support to
tumors. VEGFR2 is an important receptor involved in neovascular signaling.
Because of alternative splicing, it is present in both membrane-bound (mVEGFR2)
and soluble (sVEGFR2) forms. mVEGFR2 is pro-angiogenic, while sVEGFR2 is
anti-angiogenic. We have developed a morpholino-based gene therapy technique
that shifts expression from mVEGFR2 to sVEGFR2. Here we present the success
of this technique in treating a murine model of choroidal neovascularization
(CNV). In a follow-up experiment we show that this treatment is able to reduce
tumor growth in a xenograft tumor model.
Methods: CNV was induced in C57 mice using laser photocoagulation. On day 1
and day 4 after photocoagulation, either control morpholino, sVEGFR2-inducing
morpholino, or DBPS was injected intravitreously. On day 7, CNV volumes were
measured by confocal microscopy.
NMRI nu/nu mice received subcutaneous injections of HCT 116 colon cancer cells
for the xenograft tumor experiments. One week after the injection of cells,
treatment began with control morpholino, sVEGFR2-inducing morpholino, or
HBSS. Tumors were injected with treatment twice weekly for a period of three
weeks. Tumor size was measured twice weekly using calipers. A subset of tumors
was harvested after the first treatment injection. RNA from these tumors was
extracted, and RT-PCR was performed to evaluate levels of both mVEGFR2 and
Results: There was a 50% reduction of CNV in the mice treated with the
sVEGFR2-inducing morpholino compared to the mice treated with control
morpholino (n=11-17).
There was a 53% reduction in tumor volume in the tumors treated with the
sVEGFR2-inducing morpholino compared to the mice treated with control
morpholino (n=5). Additionally, the sVEGFR2-inducing morpholino showed a
reduced expression of mVEGFR2 and increased expression of sVEGFR2 as
confirmed by RT-PCR.
Conclusions: Anti-sense morpholinos targeting the exon13-intron13 junction of
VEGFR2 shift expression from mVEGFR2 to sVEGFR2. Here we have shown that
this results in a decrease in CNV volume in a laser CNV model. We have also
demonstrated that increased sVEGFR2 can reduce tumor growth in a xenograft
model. This is an interesting example of a therapy developed for ocular diseases
being used for cancer therapy. The splicing shift described in this study shows
promise as a therapeutic method for neovascular-related disorders.
Commercial Relationships: Brian C. Stagg, None; Hironori Uehara,
None; Taylor Bates, None; Yang Kyung Cho, None; Tadashi Miya,
None; Balamurali K. Ambati, None
Support: RPB Physician Scientist Award, VA Merit Award and NEI
Program Number: 3001 Poster Board Number: D850
Presentation Time: 8:30 AM - 10:15 AM
Regulation Of Angiogenesis By Apelin And Its Receptor Apj
Sabu Abraham, Stephen E. Moss, John Greenwood. Department of Cell Biology,
Institute of Ophthalmology, UCL, London, United Kingdom.
Purpose: Apelin together with its cognate G-protein coupled receptor, APJ has
been implicated in retinal angiogenesis and also in retinal vascular remodelling.
There have been recent reports of apelin signalling being important in the OIR
model of angiogenesis, whereas in the CNV and VLDLR-/- mouse models of
pathological angiogenesis, apelin has no discernable role. The aim of this study was
to investigate how apelin-APJ signalling regulates vascular remodelling and
angiogenesis in the context of other pro-angiogenic factors.
Methods: In order to assess the angiogenic response to apelin under different
conditions we have employed various in-vitromodels including the metatarsal exvivo angiogenesis assay and the organotypic co-culture
angiogenesis assay. The effect of treating these assays with apelin and the effect of
apelin depletion in the presence and absence of other pro-angiogenic factors such as
VEGF was determined. Endothelial tip cell formation, tube formation, vessel
bifurcation and calibre were determined and quantified using Imaris and angiosys
Results: Targeted deletion of Apl resulted in a significant reduction in angiogenesis
in the metatarsal angiogenesis assay. In an organotypic co-culture assay where
HUVEC were grown on human dermal fibroblasts, siRNA knockdown of apelin
and APJ led to a significant decrease in endothelial tubule formation. Exogenous
addition of apelin led to an increase in vessel branching in the co-culture assay. We
then used these angiogenesis models to dissect the effects of apelin on downstream
signalling from other pro-angiogenic molecules such as VEGF and Wnt. Addition
of VEGF significantly increased vessel branch
formation (4 fold) and total tubule length in the co-cultures in comparison to the
modest increase (1.4 fold) of these parameters upon apelin treatment. When VEGF
and apelin were applied together, we observed up to a 6 fold increase in vessel
branch formation. These results suggest that apelin and VEGF have a synergistic
effect on vessel branch formation.
Conclusions: Angiogenic factors that regulate vessel morphogenesis are tightly
regulated in their expression and co-operation in signalling. However, in diseases
such as diabetic retinopathy, age-related macular degeneration or macular
telangiectasia, such regulation is disrupted resulting in abnormal vessel growth.
The study here suggests that apelin, which we have previously shown to be
involved in vessel remodelling, can also modify the angiogenic outcome of VEGF
signalling and could play a role in disease pathology.
Commercial Relationships: Sabu Abraham, None; Stephen E. Moss,
None; John Greenwood, None
Support: This work was supported by a grant from the Wellcome Trust
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 3002 Poster Board Number: D851
Presentation Time: 8:30 AM - 10:15 AM
The Effect Of Epo On Human Retinal Endothelial Cell Migration,
Proliferation, And Apoptosis
Ana M. de Lucas-Cerrillo1, Jena J. Steinle2, Tonia Rex1. 1Ophthalmology,
University of Tennessee Health Science Center, Memphis, TN; 2Ophthalmology,
Univ of Tennessee Hlth Sci Ctr, Memphis, TN.
Purpose: To determine if EPO or EPO-R76E induce angiogenesis in primary
human retinal endothelial cells (RECs) in vitro.
Methods: Serum deprived HEK293 cells were transfected with plasmids
containing wild type EPO (EPO) or EPO-R76E. The medium was collected and
concentrated, and EPO or EPO-R76E was quantified by ELISA and used in the cell
death, proliferation and migration assays. REC death was induced by starving cells
overnight in the presence or absence of 0.5 to 5 U/ml EPO or EPO-R76E beginning
6 hours prior to serum deprivation. The Roche Cell Death Detection ELISA-Plus
kit was used according to manufactures protocol. REC proliferation was assessed
using the Millipore Cell Proliferation Assay Kit. RECs were plated in a 96-well
plate, starved overnight, and incubated with: no serum, 10% serum, or 0.5 to 5
U/ml EPO or EPO-R76E. Absorbance was measured at 24 and 48 hr following
drug treatments. Cell migration was assayed using the BD BioCoat Angiogenesis
System Endothelial Cell Invasion kit. RECs were plated in the top chambers and
the bottom chambers contained media with: no serum, 10% serum, or 0.5 to 5 U/ml
EPO or EPO-R76E. Plates were incubated 24 hr at 37°C and then labeled with
Calcein AM. Fluorescence was read at 485/528 nm.
Results: Serum deprivation induced a 5-fold increase in cell death as compared to
the positive control, this was unchanged by treatment with 0.5 to 5U/ml EPO or
0.5U/ml EPO-R76E. Treatment with 1.5 or 5U/ml EPO-R76E induced a 4-fold
increase in cell death compared to the positive control, however this decrease was
not statistically significant. Serum deprivation inhibited cell proliferation and
addition of EPO or EPO-R76E had no effect at any dose or time point. Serum
treatment induced migration of the RECs. No migration was detected in serumdeprived RECs with or without treatment of EPO or EPO-R76E at any dose tested.
Conclusions: Our results indicate that neither EPO nor EPO-R76E induce
angiogenesis in vitro in serum-deprived RECs. There was a trend toward a slight
anti-apoptotic effect in cells treated with EPO-R76E, but more experiments need to
be performed to determine if this was a real effect. Additional future studies will
assess the ability of EPO and EPO-R76E to induce angiogenesis in the presence of
1% serum.
Commercial Relationships: Ana M. de Lucas-Cerrillo, None; Jena J. Steinle,
None; Tonia Rex, 61433186, 61441512 (P)
Support: DoD W81XWH-10-1-0528; NIH 5P30EY13080; Research to Prevent
Blindness Unrestricted funds to Dr. Haik; Research to Prevent Blindness Career
Development Award
Program Number: 3003 Poster Board Number: D852
Presentation Time: 8:30 AM - 10:15 AM
Connective Tissue Growth Factor Regulates Neovessel Formation via
Targeting Structurally Conserved Cystine Knot Motifs in Multiple Angiogenic
Liya Pi1A, Jianwen Liu2, Anitha Shenoy1A, Paulette Robinson1A, Daniel Gibson1B,
Gregory Schultz1B, Edward Scott1A. AMolecular Genetics & Microbiology,
Department of Obstetrics and Gynecology, 1University of Florida, Gainesville,
FL; 2Bascom Palmer Eye Institute, University of Miami Miller School of Medicine,
Purpose: This study investigated the molecular mechanism of CTGF regulation of
retinal angiogenesis by characterizing CTGF binding proteins.
Methods: Binding of CTGF to angiogenic growth factors was analyzed using
immunoprecipitation assay and yeast two-hybrid cDNA analysis. Gene expression
was assessed using transgenic mice carrying CTGF promoter-driven-GFP,
immunohistochemistry, and confocol microscopy during retinal angiogenesis. Mice
at postnatal day 7 (P7) were treated by 75% oxygen for 5 days to induce retinal
retinopathy (OIR). A CTGF mutant carrying the first three modules was injected
into the vitreous at P12 and P14 and retinal neovascularization was analyzed at P17
by retinal flat-mounting using Griffonia simplicifolia Isolectin B4 to stain blood
Results: Key angiogenic regulators of the cystine knot superfamily including
vascular endothelial growth factor (VEGF)-A, Slit3, platelet derived growth factor
(PDGF)-B, and von Willebrand factor (vWF), bound with CTGF through its first
three modules. The immunofluorescent staining showed that CTGF and Slit3 were
co-localized during retinal angiogenesis in neonatal mice. CTGF functioned in
concert with Slit3 to promote endothelial tube formation and downstream
activation of Cdc42, whereas a truncation mutant that contained the first three
modules of CTGF and retained the full ability to bind all the tested cystine knot
motifs inhibited the angiogenic activities of CTGF and Slit3. Intravitreal injection
of this CTGF fragment significantly inhibited retinal neovascularization at P17 in
the OIR model.
Conclusions: These results demonstrate that CTGF plays key roles in regulating
retinal neovessel formation by binding other key angiogenic regulators with cystine
knot motifs. A CTGF fragment consisting of the three N-terminal modules that
binds key angiogenic growth factors with cystine knot motifs inhibits OIR in mice
and may provide a unique therapeutic strategy to reduce pathological angiogenesis
in eye diseases.
Commercial Relationships: Liya Pi, None; Jianwen Liu, None; Anitha Shenoy,
None; Paulette Robinson, None; Daniel Gibson, None; Gregory Schultz,
None; Edward Scott, None
Support: EY018158
Program Number: 3004 Poster Board Number: D853
Presentation Time: 8:30 AM - 10:15 AM
Differential Regulation of Retinal Neovascularization by the Modular
Domains of The Matricellular Protein Cysteine-Rich 61 (CCN1/Cyr61)
Brahim Chaqour1, Maria B. Grant2, Hemabindu Chintala1, Rahul Parmar1,
Moneka Kamalska1. 1Cell Biology, SUNY Downstate Medical Center, Brooklyn,
NY; 2Pharmacology and Therapeutics, University of Florida, Gainesville, FL.
Purpose: The CCN1 protein also known as cysteine-rich protein 61 (Cyr61) is a
dynamically expressed, matricellular protein required for proper angiogenesis and
vasculogenesis during development. We have previously demonstrated that
lentivirus-mediated expression of CCN1 in the mouse eye, promoted normalization
of the retinal vasculature in the mouse model of oxygen-induced retinopathy (OIR).
Structurally, the CCN1 protein is organized into 4 modular domains including
IGFBP, vWF, TSP and CT domain. This multimodular organization is a means of
generating through proteolytic cleavage, variants/bioactive domains that exhibit a
diverse range of activities. This study examined the angiogenic potential of CCN1
and CCN1-truncated forms in the mouse OIR model.
Methods: Lentiviral vectors expressing either the full-length or serially-truncated
forms of the CCN1 protein were prepared (i.e., lnv-CCN1: full-length; lnv-CCN113: CT truncated form; lnv-CCN11-2: CT and TSP-truncated form; lnv-CCN11: CT,
TSP and vWF truncated form). Each vector was intravitreally injected in the eyes
of mouse pups at postnatal day (P) 4. Mouse pups were then subjected to OIR and
the retinas were analyzed for vaso-obliteration at P12 and neovascularization at
Results: Truncation of the CT domain through injection lnv-CCN11-3 had no
effect on hyperoxia-induced vasoobliteration at P12, but the reduction of
neovascular tufts at P17 was greater than those induced by the full-length protein,
lnv-CCN1. Truncation of both the CT and TSP1 domains through injection lnvCCN11-2, reduced avascular areas at P12 but significantly increased neovascular
tuft formation at P17. Truncation of the CT, TSP-1 and vWF domains through
injection lnv-CCN11 had no effects on hyperoxia-induced vasoobliteration but
further reduced neovascular tuft formation in comparison with the full-length
Conclusions: The presence of IGFBP and TSP domains inherently inhibits
neovascular tuft formation while vWF and CT domains increase resistance to
hyperoxia-induced vaso-obliteration.
Commercial Relationships: Brahim Chaqour, None; Maria B. Grant,
None; Hemabindu Chintala, None; Rahul Parmar, None; Moneka Kamalska,
Support: EY022091-01
Program Number: 3005 Poster Board Number: D854
Presentation Time: 8:30 AM - 10:15 AM
PDCL3 Regulates Endothelial Cell Proliferation And Angiogenesis Through
The Generation Of Functional VEGFR-2
Manisha N. Mehta1, Srimathi Srinivasan2, Rosana D. Meyer2, Ricardo Lugo2, Vipul
Chitalia3, Nader Rahimi2. 1Pathology, Boston University, Newton, MA;
Ophthalmology and Pathology, Boston University, Boston University, MA;
Massachusetts Institute of Technology, Harvard-MIT Division of Health Science
and Technology, Harvard University, MA.
Purpose: Angiogenesis, a hallmark step in ocular neovascularization, is primarily
driven by the function of VEGF on its receptor, VEGFR-2. Central to the initiation
of angiogenesis by VEGF, the abundance of VEGFR-2 on the surface of
endothelial cells is essential for VEGF to recognize and activate VEGFR-2. We
have identified PDCL3 (phosducin like 3) as a novel protein involved in the
stabilization of VEGFR-2 by serving as a VEGFR-2 co-chaperone protein.
Methods: Various molecular and cell biology methods including Western blotting,
site direct mutagenesis, endothelial cell culture systems, knock-down experiments
with siRNA, invitro-angiogenesis assays and immunohistochemical studies on
mouse eyes exposed to hypoxia were used to study the role of PDCL3 in regulation
of VGFR2 in endometrial cells.
Results: PDCL3 expression is upregulated by hypoxia in endothelial cells and its
expression is responsible for hypoxia-induced expression of VEGFR-2. PDCL3 colocalizes with VEGFR-2 in cells and recognizes the JM domain of VEGFR-2.
Over-expression of PDCL3 increases, whereas depleting its expression by siRNA
in endothelial cells reduces the abundance of VEGFR-2 protein. Upregulation of
PDCL3 markedly increases ligand-mediated activation of VEGFR-2.
Conclusions: Our studies provide experimental evidence for the role of PDCL3 in
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
governing the angiogenic pathway by controlling VEGFR-2 destruction. PDCL3
elicits its activity by rendering VEGFR-2 to a more stable protein by preventing its
ubiquitylation and degradation. PDCL3 activity is required for endothelial cell
proliferation and tube formation of endothelial cells and angiogenesis.
Commercial Relationships: Manisha N. Mehta, None; Srimathi Srinivasan,
None; Rosana D. Meyer, None; Ricardo Lugo, None; Vipul Chitalia,
None; Nader Rahimi, None
Support: National Institute of Health (NIH/NEI) to NR and Pathology, Boston
University and Massachusetts Lions Foundation grant to Department of
Program Number: 3006 Poster Board Number: D855
Presentation Time: 8:30 AM - 10:15 AM
Nanotechnology Guided Molecular Imaging of Choroidal Neovascularization
Colin A. Bretz1, Ashwath Jayagopal2. 1Vanderbilt Eye Institute, Vanderbilt Univ
Med Center, Nashville, TN; 2Vanderbilt Eye Institute, Vanderbilt University
Medical Center, Nashville, TN.
Purpose: Strategies for the early detection of choroidal neovascularization are
needed to facilitate timely therapeutic intervention and to monitor disease
progression and treatment response. Ligand-coated nanoparticles were developed
for site-specific imaging of neovascular lesions in animal models of laser-induced
choroidal neovascularization.
Infrared dyes and fluorescent semiconducting nanocrystals (quantum dots) were
conjugated to peptides or antibodies directed against vascular cell adhesion
molecule 1 (VCAM-1), an early marker of choroidal neovascularization, using
covalent conjugation techniques. Negative controls were synthesized incorporating
scrambled peptide or nonspecific antibody conjugated imaging agents. Conjugates
were injected in mouse models of laser-induced choroidal neovascularization at
timepoints up to 31 days post laser injury, and age matched controls. Ocular tissue
was imaged ex vivo and in vivo throughout the time course of choroidal
neovascularization for evaluation of specificity and sensitivity.
VCAM-1 targeted imaging agents were capable of early detection of choroidal
neovascularization via binding to VCAM-1. VCAM-1 signal remained elevated
throughout the time course of pathology, up to 1 month post laser injury. Signal to
background ratios of VCAM-1 specific imaging agents ranged from 10-25:1
depending on the disease timepoint, and fluorescence emission was focal to CNV
lesions. Negligible binding of imaging agents was observed in control animals.
Successful detection of early choroidal neovascularization was possible in the
mouse model of laser induced choroidal neovascularization. Molecular imaging of
the eye is a promising strategy for facilitating early detection of disease, which may
improve clinical management of neovascular AMD.
Commercial Relationships: Colin A. Bretz, None; Ashwath Jayagopal, None
Support: P30-EY008126 (Core Grant in Vision Research), International Retinal
Research Foundation, Unrestricted Grant from Research to Prevent Blindness
Program Number: 3007 Poster Board Number: D856
Presentation Time: 8:30 AM - 10:15 AM
UCHL1 Promotes Proliferation of Human Retinal and Choroidal Endothelial
Cells In Vitro
Yuzhen Pan, Binoy Appukuttan, Kathleen Mohs, Justine R. Smith. Casey Eye
Institute, Oregon Health & Science University, Portland, OR.
Purpose: In a published gene expression microarray study (JR Smith et al, Invest
Ophthalmol Vis Sci, 2007), we identified UCHL1 expression by cultured human
choroidal and retinal endothelial cells. Ubiquitin carboxyl-terminal hydrolase
isozyme L1 (UCHL1) is a deubiquitinating enzyme that counteracts VHL genedriven ubiquitination of hypoxia-inducible factor (HIF)-1alpha, stabilizing HIF1alpha. These observations led us to investigate the potential role of UCHL1 in
human ocular angiogenesis.
Methods: 70% ethanol-fixed human retina and choroid from 3 human cadaver eyes
were indirectly immunostained with rabbit anti-human UCHL1 antibody (Cedar
Lanes, 1:5000 dilution) to examine in vivo expression of UCHL1. Endothelial
proliferation assays were performed using retinal and choroidal endothelial cells,
isolated from additional human cadaveric eyes by established methods and
immortalized by transduction with the murine recombinant amphotropic retrovirus,
LXSN16E6E7, which encodes HPV E6 and E7 oncogenes. When no less than 80%
confluent, cells were transfected with UCHL1-targeted or non-targeted small
interfering (si)RNA, designed in-house and synthesized by Invitrogen, using
Targefect (Targeting Systems), according to the manufacturer’s instructions.
Subsequently transfected endothelial cells were incubated at 37 °C and 3.5% CO2
in MCDB-131 medium with 5-10% FBS and endothelial growth factors, and
growth was evaluated by CyQUANT Cell Proliferation Assay (Invitrogen) 96
hours later.
Results: Immunohistochemistry confirmed expression of UCHL1 by vascular
endothelium in intact retina and choroid. siRNA treatment targeting UCHL1
resulted in significant reduction in endothelial proliferation when compared to
treatment with non-targeted siRNA for choroidal (p ≤ 0.008, n= 3 independent
donors) and retinal (p ≤ 0.004, n= 3 independent donors) endothelial cells. Results
were confirmed for both cell types on one additional donor using siRNA targeting a
different section of transcript sequence. Successful UCHL1 knock-down with
UCHL1-targeted siRNA was confirmed by Western blot for all experiments, using
protein harvested 48 hours after transfection.
Conclusions: UCHL1 protein is expressed by retinal and choroidal endothelium in
vivo. Targeted knock-down of UCHL1 has an anti-proliferative effect on retinal
and choroidal endothelial cells in vitro. Our results suggest that targeting UCHL1
may have therapeutic benefit for neovascular eye diseases, including diabetic
retinopathy and age-related macular degeneration.
Commercial Relationships: Yuzhen Pan, None; Binoy Appukuttan,
None; Kathleen Mohs, None; Justine R. Smith, None
Support: American Health Assistance Foundation, NIH R01 EY019875, Research
to Prevent Blindness (unrestricted grant to Casey Eye Institute)
Program Number: 3008 Poster Board Number: D857
Presentation Time: 8:30 AM - 10:15 AM
Safe and Effective Polymeric-Doxorubicin Conjugate Nanoparticles for
Prolonged Antiangiogenic Activity in the Eye
Takeshi Iwase1, Jie Fu2, Tsunehiko Yoshida1, Daisuke Muramatsu1, Brian Oveson1,
Bing Han2, Mingsheng Wu2, Gregg Semanza3, Justin Hanes2, Peter A.
Campochiaro1. 1Ophthalmology and Neuroscience, Johns Hopkins Wilmer Eye
Inst, Baltimore, MD; 2The Center for Nanomedicine, The Wilmer Eye Institute,
Johns Hopkins school of medicine, Baltimore, MD; 3Departments of Pediatrics,
Medicine, Oncology, Radiation Oncology, and Biological Chemistry, Johns
Hopkins University School of Medicine, Baltimore, MD.
Purpose: To investigate the effects of doxorubicin (DXR), daunorubicin (DNR),
and nanoparticles of a DXR-polymer conjugate in models of choroidal and retinal
neovascularization (NV).
Methods: The effect of DNR, DXR, and DXR- poly(sebacic acid)- poly(ethylene
glycol)3 nanoparticles (DXR-PSA-PEG3) were tested in mouse models of ocular
NV (choroidal NV due to laser-induced rupture of Bruch’s membrane, oxygeninduced ischemic retinopathy, and rho/VEGF transgenic mice with overexpression
of VEGF in photoreceptors). Retinal toxicity was assessed by performing
electroretinograms (ERGs) and by measuring outer nuclear layer thickness.
Results: Intraocular injections of 10 µg of DXR or DNR suppressed choroidal NV
and injections of 0.1 or 1 µg suppressed retinal NV; however, ERG b-wave
amplitudes were decreased after injection of 1 or 10 µg of DXR or DNR, indicating
significant toxicity. Intraocular injection of 1 or 10 µg of DXR-PSA- PEG3
nanoparticles strongly suppressed choroidal and retinal NV and did not cause
retinal toxicity. In rho/VEGF transgenic mice, intraocular injection of 10 µg of
DXR-PSA- PEG3 nanoparticles suppressed NV for at least 35 days demonstrating
that the nanoparticles provide sustained release of efficacious and safe levels of
Conclusions: These data demonstrate a novel DXR-polymer conjugate
nanoparticle that maximizes efficacy and minimizes toxicity providing a promising
new chemical entity for treatment of ocular NV.
Commercial Relationships: Takeshi Iwase, None; Jie Fu, None; Tsunehiko
Yoshida, None; Daisuke Muramatsu, None; Brian Oveson, None; Bing Han,
None; Mingsheng Wu, None; Gregg Semanza, None; Justin Hanes, None; Peter
A. Campochiaro, None
Support: None
Program Number: 3009 Poster Board Number: D858
Presentation Time: 8:30 AM - 10:15 AM
Inhibition Of Vascular Leakage And Choroidal Neovascularization Using
Vegf-inhibiting Darpins In Rodents
Andreas Stahl1, Michael T. Stumpp2, Anja Schlegel2, Savira Ekawardhani2,
Gottfried Martin1, Maya Gulotti-Georgieva2, Denis Villemagne2, Patrik Forrer2,
Hansjürgen T. Agostini1, Kaspar H. Binz1. 1University Eye Hospital Freiburg,
Freiburg, Germany; 2Molecular Partners AG, Zurich, Switzerland.
Purpose: Vascular endothelial growth factor (VEGF) is a key controller of
pathological angiogenesis and leakage in various retinal diseases. Clinically, antiVEGF agents have revolutionized treatment of exudative age related macular
degeneration (AMD), diabetic retinopathy and retinal vein occlusion. However, one
of the main limitations of current VEGF inhibitors remains the need for repeated
intravitreal injections. Second-generation VEGF inhibitors should thus aim at
providing more efficient VEGF-inhibition to reduce injection frequency.
Methods: In the current study, we use highly potent anti-VEGF DARPins
(designed ankyrin repeat proteins) to inhibit VEGF receptor phosphorylation and
endothelial cell sprouting in vitro, retinal vascular leakage in rabbits and choroidal
angiogenesis in the Laser CNV model in rats.
Results: Our results demonstrate the successful isolation and purification of several
DARPins with picomolar affinity to VEGF. In vitro, VEGF receptor
phosphorylation was strongly inhibited compared to controls. Functional
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
experiments using endothelial spheroid sprouting demonstrated potent inhibition of
VEGF-induced angiogenic tube formation. Upon intravitreal injection, DARPins
were found to penetrate into the retina and inhibit VEGF-induced retinal vascular
leakage (p=0.025 vs. controls). In addition, topical DARPin application was found
to suppress Laser-induced choroidal neovascularization in rats (p=0.025 vs.
controls). No ocular or systemic side effects were observed in both in vivo models.
Conclusions: DARPins are a new class of antibody mimetic molecules with
increased anti-VEGF potency and prolonged intraocular activity that are derived
from the abundant protein family of naturally occurring ankyrin repeat proteins.
Anti-VEGF DARPins are promising candidates for the treatment of neovascular
eye diseases in humans and currently under investigation in phase I/II clinical trials.
Commercial Relationships: Andreas Stahl, None; Michael T. Stumpp, Inventor
at Molecular Partners AG (P), Shareholder of Molecular Partners AG (I); Anja
Schlegel, Employee of Molecular Partners (E); Savira Ekawardhani, Employee at
Molecular Partners (E); Gottfried Martin, None; Maya Gulotti-Georgieva,
Employee at Molecular Partners (E); Denis Villemagne, Employee at Molecular
Partners (E); Patrik Forrer, Inventor at Molecular Partners AG (P), Shareholder of
Molecular Partners AG (I); Hansjürgen T. Agostini, None; Kaspar H. Binz,
Inventor at Molecular Partners AG (P), shareholder of Molecular Partners AG (I)
Support: None
Program Number: 3010 Poster Board Number: D859
Presentation Time: 8:30 AM - 10:15 AM
Increased Expression of Integrin α Chains and Their Ligands during LaserInduced Neovascularization of Rat Choroid
Takeshi Nakajima1, Thomas R. Shearer2, Mitsuyoshi Azuma3,2. 1Senju Laboratory
of Ocular Sciences, Senju Pharmaceutical Co Ltd, Kobe, Japan; 2Department of
Integrative Biosciences, Oregon Health & Science University, Portland, OR; 3Senju
Laboratory of Ocular Sciences, Senju Pharmaceutical Co Ltd, Beaverton, OR.
Purpose: Binding inhibitors of integrins αv and α5 were anti-angiogenic in
experimental choroidal neovascularization (CNV). However, a comprehensive
understanding of the expression of the integrin isoforms, ligands, and interassociations is limited in the CNV model. Thus, the purpose of present study was to
examine the expression and localization of integrin α chains and their extracellular
matrix (ECMs) ligands in retinal/choroidal tissue after laser injury.
Methods: CNV, observed by staining with FITC-labeled lectin, was produced in
Brown Norway rats with a 532 nm diode laser. Expression and localization of αv
and α5 integrins and their ligands was performed by immunohistochemistry in
consecutive cryosections. An in vitro cell adhesion assay using retinal endothelial
cells and specific antibodies confirmed binding specificity between integrin α
chains and ECMs.
Results: Angiogenesis was observed on day 7 after laser injury in choroidal
flatmounts and cryosections. Integrin α5 and αv expression massively increased at
day 3 and then gradually decreased, but were still elevated on day 14. Increased
ligands for α integrins localized closely to their integrins in the laser-injured region.
One day after photocoagulation, integrin ligands fibronectin (FN) and vitronectin
(VN) were already massively increased. Expression of FN remained stable, but VN
returned to normal by day 14. Expression of laminin was only minimally increased
at day 1 and then returned to normal by day 3. Cell adhesion assays confirmed
specific association of integrin α5 to FN, and αV to VN. Negative control IgG did
not stain.
Conclusions: Choroidal injury increased FN and VN ligands followed by timedependent increases in integrins α5 and αv. The interaction of FN and VN with
their specific integrin α chains may be part of the mechanism producing CNV.
Commercial Relationships: Takeshi Nakajima, Senju Pharmaceutical Co Ltd
(E); Thomas R. Shearer, Senju Pharmaceutical Co Ltd (C); Mitsuyoshi Azuma,
Senju Pharmaceutical Co Ltd (E)
Support: None
Program Number: 3011 Poster Board Number: D860
Presentation Time: 8:30 AM - 10:15 AM
Attenuation of the Proangiogenic Properties of Retinal Endothelial Cells by
Inflammatory Cytokines
Nader Sheibani1, Tammy L. Palenski1, Christine M. Sorenson2. 1Ophthalmology
and Visual Sciences, Univ of Wisconsin-Madison, Madison, WI; 2Pediatrics, Univ
of Wisconsin School of Med, Madison, WI.
Purpose: Diabetic retinopathy (DR) is a major complication of diabetes afflicting
roughly 40% of adults with the disease, with 5-10% developing severe visionthreatening complications. The basic mechanisms underlying the pathogenesis of
DR are poorly understood. Many studies have demonstrated that increased
production of inflammatory mediators is strongly associated with vascular changes
in DR. However, the source and targets of these inflammatory mediators remain
elusive. The objective of this study was to determine the impact of inflammatory
cytokines TNF-α, IL-1β and MCP-1 on retinal EC function using a variety of
assays including viability, apoptosis, proliferation, migration, and capillary
Methods: The expression of endothelial nitric oxide synthase and nitric oxide
levels, as well as the production of various extracellular (ECM) proteins were
determined. We also assessed retinal EC adhesion on various ECM proteins, their
junctional organization, VEGF expression, and oxidative stress state after
incubation with various inflammatory cytokines.
Results: Cell viability and proliferation of retinal EC incubated with TNF-α, IL-1β
or MCP-1 for 24 h was not affected. Incubation with TNF-α and IL-1β, but not
MCP-1, decreased retinal EC migration and their ability to undergo capillary
morphogenesis. Incubation with TNF-α and IL-1β, but not MCP-1, for 48 h
resulted in increased production of VEGF, increased oxidative stress, and abnormal
junctional localization of VE-cadherin at sites of the cell-cell contact consistent
with altered cellular permeability.
Conclusions: Together our results demonstrate that the inflammatory cytokines
have specific adverse effects on angiogenic properties of retinal EC which is
associated with increased oxidative stress and altered vascular permeability. Thus,
altered production of proinflammatory cytokines during diabetes has a significant
impact on retinal vascular function and pathogenesis of DR.
Commercial Relationships: Nader Sheibani, None; Tammy L. Palenski,
None; Christine M. Sorenson, None
Support: NIH Grants: EY016695 and EY021357 ; ADA Grant:: 1-10-BS-160
Program Number: 3012 Poster Board Number: D861
Presentation Time: 8:30 AM - 10:15 AM
Modified Anthrax Toxin Selectively Targets Activated Endothelial Cells: A
Novel Therapeutic Approach for the Treatment of Retinal Neovascular
Murilo W. Rodrigues, Jr.1A, Fabiana K. Kashiwabuchi1A, Xiaoban Xin1A, Daniel
Leonard2A, Stephen Leppla2B, Thomas Bugge2A, Akrit Sodhi1B. AOphthalmology,
Ophthalmology - Retina Division, 1Johns Hopkins Medical Institution, Baltimore,
MD; AProteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer
Branch, BLaboratory of Bacterial Diseases, National Institute of Allergy and
Infectious Diseases, 2National Institutes of Health, Bethesda, MD.
Purpose: The recent introduction of monoclonal antibodies against VEGF for the
treatment of retinal and choroidal neovascular disease demonstrates the importance
of VEGF in pathological angiogenesis in the eye. Nonetheless, patients with
“mature” retinal neovascular lesions are often remarkably insensitive to VEGF
inhibition, prompting the exploration of other genes that may serve as appropriate
therapeutic targets for these patients. In this regard, the extensive interplay between
endothelial cells, soluble factors, and the extracellular matrix is an emerging target
for anti-angiogenic therapies. In particular, extracellular proteolysis has been
implicated as one of the first and most sustained activities involved in pathological
angiogenesis. Our goal is to identify and target key extracellular proteases
upregulated by activated endothelial cells to develop new therapies for patients
with ischemic retinopathies.
Methods: Vascular endothelial cells (VECs) were exposed to hypoxia or
recombinant VEGF protein and the expression of extracellular proteases was
assessed. These results were corroborated in vivo using the oxygen-induced
retinopathy (OIR) model for ischemic retinopathy. Modified bacterial (anthrax)
toxins - engineered to be active only in the presence of specific proteases - were
then used to target activated VECs and their toxicity examined.
Results: Matrix metalloprotease-2 (MMP-2) RNA and protein are increased
several fold in VECs exposed to hypoxia or recombinant VEGF protein in vitro and
increase within 24 hours of ocular ischemia in vivo (p<0.05). Moreover, while
VEGF RNA levels drop precipitously after 24 hours in the OIR model after return
to normoxia, MMP-2 RNA levels remain elevated for at least 72 hours (p<0.05).
Using a modified anthrax toxin activated by MMPs, we further observed rapid cell
death of activated VECs treated with the toxin.
Conclusions: We demonstrate that MMP-2 is upregulated in activated VECs by
VEGF and can be used therapeutically to specifically target and kill these cells in
retinal neovascular lesions. This study provides the foundation for the assessment
of modified bacterial toxins as a novel therapeutic approach for patients with
ischemic retinopathies.
Commercial Relationships: Murilo W. Rodrigues, Jr., None; Fabiana K.
Kashiwabuchi, None; Xiaoban Xin, None; Daniel Leonard, None; Stephen
Leppla, 5,591,631 (P), 5,677,274 (P), 6,485,925 (P), 6,893,835 (P), 6,911,203: (P),
7,056,693 (P), 7,183,071 (P), 7,468,352 (P), 7,947,289 (P); Thomas Bugge,
5,591,631 (P), 5,677,274 (P), 6,485,925 (P), 6,893,835 (P), 6,911,203 (P),
7,056,693 (P), 7,183,071 (P), 7,468,352 (P), 7,947,289 (P); Akrit Sodhi, None
Support: None
Program Number: 3013 Poster Board Number: D862
Presentation Time: 8:30 AM - 10:15 AM
Laser Induced-choroidal Neovascularisation Enhances The Mrna Expression
Of Sphingosine-1-phophate Receptors In The Retina
Norbert Kociok, Sergej Skosyrski, Irina Semkova, Antonia M. Joussen.
Ophthalmology, Charite Universitaetsmedizin Berlin (CVK), Berlin, Germany.
Purpose: Sphingosine-1-phophate (S1P) is a multifunctional lipid molecule that
stimulates endothelial cell migration and proliferation via the S1P family of G
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
protein-coupled receptors (S1P(1-5)R). Activation of the sphingosine 1-phosphate
receptor 1 (S1P(1)R) protects against renal ischemia-reperfusion injury and
inflammation. Recently, the inhibition of S1P was considered as a therapeutic
treatment of AMD patients. Here we analyze the mRNA expression of S1P(1)R and
S1P(3)R in a mice model of choroidal neovascularisation.
Methods: Choroidal neovascularisation was induced in C57Bl/6-mice by
disrupting the Bruch’s membrane by laser treatment. 14 days after laser treatment
the retina was removed, the RNA was isolated and a Real Time RT-PCR analysis
for S1P(1)R and S1P(3)R was performed.
Results: A clear scare formation in the choroid was detected by angiography in
laser treated eyes. In the retinas of laser treated mice the mRNA expression of
S1P(1)R was enhanced between 26.6 ± 5.6 and 4.3 ± 1.4 when compared to not
treated control eyes. The expression of S1P(3)R in these retinas was enhanced
between 5.1± 0.9 and 20.5 ± 2.5.
Conclusions: The enhanced mRNA expression if these S1P receptors in the mouse
retina after laser treatment was shown. Choroidal neovascularisation has an effect
on the sphingosine-1-phosphate receptor gene expression in the retina. This
pathological effect on the retina may be attenuated by blocking the action of
sphingosine-1-phosphate or its receptors.
Commercial Relationships: Norbert Kociok, None; Sergej Skosyrski,
None; Irina Semkova, None; Antonia M. Joussen, None
Support: DFG JO324/10-1; Dr. Werner Jackstädt-Stiftung
Program Number: 3014 Poster Board Number: D863
Presentation Time: 8:30 AM - 10:15 AM
Inhibition Or Genetic Knockout Of More Than One Cathepsin Reduces
Angiogenic Sprout Formation In Vitro And Laser-CNV Formation In Mice
Anima D. Buehler1, Stefanie Berger1, Fee Werner2, Gottfried Martin1, Hansjuergen
Agostini1, Thomas Reinheckel2, Andreas Stahl1. 1Ophthalmology, University Eye
Hospital Freiburg, Freiburg, Germany; 2Institute for Molecular Medicine and Cell
Research, Faculty of Biology, Albert-Ludwigs-University Freiburg, Freiburg,
Purpose: Cathepsins are a family of lysosomal and secreted proteases that are
involved in intracellular protein turnover and matrix invasion, respectively.
Cathepsin B (Ctsb) and Z (Ctsz) are two cysteine cathepsin family members with
possible roles in angiogenesis. We therefore investigated the angiomodulatory
function of these cathepsins in vitro as well as in a mouse model of laser-induced
choroidal neovascularization (Laser-CNV).
Methods: Ctsb-/- mice, Ctsz-/- mice, Ctsb/Ctsz double knockouts (Ctsb/z DKO)
and C57BL/6 wildtype controls underwent argon laser treatment to induce
choroidal neovascularization (CNV). 10 days after laser treatment, animals were
perfused with FITC dextran, the eyes were collected and choroidal flatmounts were
prepared. The CNV area was quantified individually for each lesion. For in vitro
analysis, endothelial cell (EC) sprouting was analyzed using a spheroidal sprouting
assay in collagen matrix under VEGF stimulation. A pan-cysteine cathepsin
inhibitor was added to the matrix. VEGF 165 stimulation alone served as positive
Results: In the Laser CNV model, neither Ctsb-/- nor Ctsz-/- mice show a
significant difference in CNV area compared to wildtype controls (p=0.7 and p=0.1
respectively). Ctsb/z DKO, however display a significantly reduced area of CNV
formation compared to wild type controls (p=0.001). Similarly, VEGF-induced EC
spheroid sprouting in vitro was significantly suppressed using the cell-permeable
broad-spectrum cysteine cathepsin inhibitor E64d (p=0.0001).
Conclusions: Our results show that knockout of either Ctsb or Ctsz alone does not
significantly alter laser-induced CNV formation in mice. However, laser-CNV
formation is significantly reduced in Ctsb/z DKO mice suggesting a compensatory
mechanism between different members of the cathepsin family. In accordance, EC
sprout formation in vitro is significantly reduced with broad-spectrum cathepsin
inhibition. The EC spheroid assay also gives a strong hint that the anti-angiogenic
effect of cathepsin inhibition is mediated directly via an endothelial mechanism
without the involvement of other cell types.
Commercial Relationships: Anima D. Buehler, None; Stefanie Berger,
None; Fee Werner, None; Gottfried Martin, None; Hansjuergen Agostini,
None; Thomas Reinheckel, None; Andreas Stahl, None
Support: None
Program Number: 3015 Poster Board Number: D864
Presentation Time: 8:30 AM - 10:15 AM
Implementation of Spectral Domain Optical Coherence Tomography for Post
Laser Verification of Thermal Laser Induced Choroidal Neovascularization in
the Primate Model
Brittany A. Jackson, Richard L. Ornberg. Biological Sciences, Alcon Research,
Fort Worth, TX.
Purpose: Focal thermal laser ablation to the peri-macular retina is a common
method for creating choroidal neovascular (CNV) lesions for evaluating
therapeutics for age related macular degeneration (AMD). To date, a variety of
laser parameters and evaluation methods used have resulted in CNV development
in less than 50% of laser burns delivered. In this study, SDOCT was used to
objectively assess Bruch’s membrane rupture immediately post laser against a
range of commonly used thermal laser parameters. The SDOCT findings were then
correlated with the creation of leaky CNV lesions in a primate model of AMD
Methods: Naïve Cynomolgus monkeys (n=3, 2.4-5.8kg) were sedated and given
topical mydriatics and anesthetic to effect for the bilateral laser procedure. Using
the Novus Varia three mode laser system, thermal laser lesions were induced using
argon green (532nm, 0.1 sec, 50um) with power set at 350, 550, 750 or 900 mW) or
krypton red (657nm, 600mW, 0.1sec.) with spot size set 50 or 75um to examine a
single eye with 6 spots per treatment condition. SDOCT was acquired for each
lesion in a 6-burn pattern around the fovea immediately post laser. Fluorescein
angiography, color funduscopy and SDOCT were performed biweekly through 6
weeks post procedure to document CNV leakage and vascular network formation.
Results: The majority of argon green laser settings did not rupture Bruch’s
membrane when viewed with SDOCT, and indeed did not produce clinically
relevant CNV lesions. SDOCT verification of Bruch’s membrane rupture was
observed for 5 of 6 of the laser lesions made with 657nm at 75um spot size, with 6
of 6 CNV showing leak to 6 weeks. Only 3 of 6 laser lesions made with argon
green at750mW power appeared to have ruptured Bruch’s membrane. Of these, 6
of 6 lesions demonstrated leaked through 4 weeks but only the 3 the lesions with a
ruptured Bruch’s membrane were leaky at 6 weeks post laser
Conclusions: SDOCT examination of Bruch’s membrane rupture following laser
treatment is more reliable than a subjective assessment of thermal laser burns as a
predictor of CNV development. Lesions produced by krypton red excitation at
75um spot size produced a higher percentage of CNV compared to those produced
by the range of power settings for argon green
Commercial Relationships: Brittany A. Jackson, Alcon Research Ltd (E);
Richard L. Ornberg, Alcon Research Ltd (E)
Support: None
Program Number: 3016 Poster Board Number: D865
Presentation Time: 8:30 AM - 10:15 AM
Sorsby Fundus Dystrophy S156C-TIMP3 mutation promotes angiogenesis and
choroidal neovascularization via an MMP2-dependent mechanism
Jian H. Qi, Sr.1A, Heidi Stoehr2, Bela Anand-Apte1B. AOphthalmic Research-Cole
Eye Inst, BOphthalmology, 1Cleveland Clinic, Cleveland, OH; 2Human Genetics,
University of Regensburg, Regensburg, Germany.
Purpose: TIMP3 is a regulator of matrix metalloproteinases (MMPs) and a potent
angiogenesis inhibitor. Mutations in the TIMP3 gene cause Sorsby Fundus
Dystrophy (SFD), an inherited, early onset macular degenerative disorder. SFD
closely resembles age-related macular degeneration (AMD), including the
development of choroidal neovascularization (CNV). We have demonstrated
previously that expression of SFD-related S156C mutation in human retinal
pigment epithelial (RPE) cells reduces MMP inhibition and may promote
angiogenesis. The objective of this study was to detertmine if S156C-TIMP3
mutation promotes angiogenesis or CNV via an MMP dependent mechanism.
Methods: Methods: Human RPE cells expressing WT and S156C-TIMP3 or empty
vector as well as the RPE-choroid tissue of Timp3 S156C/S156C knock-in mice
and their corresponding WT littermates were analyzed by reverse zymography and
zymography to detect MMP inhibitory activity and gelatinase activity respectively.
ELISAs were used to measure MMP2, MMP9, bFGF or VEGF. An in vitro tube
formation assay was utilized to analyze angiogenic responses in endothelial cells .
In vivo CNV response in mice was quantitated following laser-induced
Results: S156C-TIMP3 reduced MMP inhibitory activity, increased total MMP2
(pro- and active forms) and bFGF but had no effect on MMP9 and VEGF levels in
the conditioned medium (CM) of RPE cells relative to vector controls. In contrast,
either WT-TIMP3 or a selective MMP2/MMP9 inhibitor (SB-3CT) inhibited bFGF
and VEGF release. Addition of CM from mutant RPE cells to either PAE cells
expressing FGF receptor-1 (PAE/FGFR-1) or VEGF receptor-2 (PAE/VEGFR-2)
induced increased tube formation in three-dimensional (3D) collagen gels with
bFGF and VEGF dependency. Treatment with SB-3CT inhibited mutant cells CMinduced morphogenic effects in PAE/FGFR-1 or PAE/VEGFR-2 cells. Consistent
with the results from in vitro experiments, Timp3 S156C/S156C mice showed
reduced MMP inhibitory activity and increased active form of MMP2 rather than
MMP9 in RPE-choroid tissue, especially after a laser burn. Furthermore, at 2 weeks
post-laser induction, the area of CNV at Bruch’s membrane (BM) rupture sites was
observed to be significantly larger in mutant mice compared to their WT controls.
Conclusions: S156C-TIMP3 up-regulates RPE cell-mediated angiogenesis by
increasing bFGF and VEGF bio-availability and activity via elevated MMP2, and
thereby promotes CNV development.
Commercial Relationships: Jian H. Qi, Sr., None; Heidi Stoehr, None; Bela
Anand-Apte, None
Support: National Institute of Health EY016490, EY015638, Unrestricted Grant
from Research to Prevent Blindness and RPB Lew Wasserman award to BA-A.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 3017 Poster Board Number: D866
Presentation Time: 8:30 AM - 10:15 AM
Tivozanib, A Highly Potent Inhibitor Of Vascular Endothelial Growth Factor
Receptor Tyrosine Kinase, Has Antiangiogenic Properties On Experimental
Choroidal Neovascularization
Seungbum Kang1, Young-Jung Roh2, In-Beom Kim3. 1Department of
Ophthalmology and Visual Science, Deajeon St. Mary's Hospital, College of
Medicine, Catholic University of Korea, Republic of Korea; 2Department of
Ophthalmology and Visual Science, Yeouido St. Mary's Hospital, College of
Medicine, Catholic University of Korea, Republic of Korea; 3Department of
Anatomy, College of Medicine, Catholic University of Korea, Seoul, Republic of
Purpose: To investigate the effects of tivozanib, an inhibitor of vascular
endothelial growth factor receptors, on experimental choroidal neovascularization
(CNV) model.
Methods: C57BL/6 mice were treated with tivozanib (1 mg/kg/day) or vehicle at
the onset (day 0) of study. Experimental CNV was induced by laser
photocoagulation the following day. In other groups of mice, tivozanib or vehicle
treatment was started 7 days after the laser application to determine the effect of the
drug on established CNV. After 14 days, the extent of CNV was assessed from
choroidal flat mounts perfused with fluorescein-labeled dextran.
Immunofluorescence staining with isolectin IB4 was also used to quantify the CNV
Results: Tivozanib suppressed the development of CNV lesions, reducing the area
by 80.7 % compared to vehicle-treatment (p < 0.001). Tivozanib caused a
significant regression of established CNV, reducing the area by 67.7 % compared
to vehicle-treatment (p < 0.001). Isolectin IB4-labeled area was smaller in the
tivozanib-treated mice compared to the vehicle-treatment (p < 0.001).
Conclusions: Tivozanib effectively inhibits the progression of CNV in mice CNV
model. These results suggest that tivozanib could constitute a therapeutic
alternative for the treatment of neovascular AMD.
Commercial Relationships: Seungbum Kang, None; Young-Jung Roh,
None; In-Beom Kim, None
Support: Clinical Research Institute Grant (CMCDJ-P-2011-2) funded by The
Catholic University of Korea
Program Number: 3018 Poster Board Number: D867
Presentation Time: 8:30 AM - 10:15 AM
Blocking Endoglin Reduces Endothelial Progenitor Cell Angiogenic Capacity
and Retinal Neovascularization
Joshua M. Barnett1, Gary W. McCollum1, John S. Penn2. 1Ophthalmology and
Visual Sciences, Vanderbilt Eye Institute, Nashville, TN; 2Vanderbilt Eye Institute,
Vanderbilt Univ Schl of Med, Nashville, TN.
Purpose: Angiogenesis and neovascularization (NV) are common pathological
processes in age-related macular degeneration and neovascular retinopathies.
Endoglin (CD105) expression has been reported as being upregulated in
proliferative tumor vasculature, and, as an auxiliary receptor in the TGFβ super
family, it is suggested to be involved in cell proliferation, migration and
differentiation. This study sought to examine the contribution of endoglin to
endothelial progenitor cell (EPC) angiogenic activity and retinal NV.
Methods: Sprauge-Dawley rat litters were exposed to alternating 50% and 10%
oxygen atmospheres from birth through P14. Upon removal from the exposure
chamber, some rats received an intraperitoneal injection of a CXCR4 antagonist
(inhibitor of EPC homing), AMD3100, at post-oxygen days 0 and 3. Other animals
received intravitreal injections of vehicle, anti-VEGF, or anti-CD105. These
animals were sacrificed at 6 days later, and their retinas were ADPase-stained and
evaluated for NV and avascular area. The AMD3100-treated animals were
sacrificed at 3 and 6 days post-oxygen exposure, and retinal CD105 levels were
measured by ELISA.
Results: Endoglin protein was found to be upregulated 4.8-fold in retinal tissue
from oxygen-exposed rats relative to control animals (p<0.01). This increase in
endoglin was reduced 56% in animals treated with a CXCR4 antagonist
(AMD3100), presumably by inhibition of EPC homing to neovascular areas
(p<0.02). Anti-CD105 treatment reduced in vivo NV by 46% at the highest
concentration (p<0.05) and worked additively with anti-VEGF treatment to reduce
NV by an additional 23% (p<0.05).
Conclusions: Anti-CD105 was found to be effective in reducing neovascular lesion
size in oxygen-exposed rats and worked additively with anti-VEGF treatments.
Endoglin plays an angiogenic role in retinal NV, and blocking endoglin function is
an effective method of reducing NV, partially through a reduced angiogenic
capacity of EPCs.
Commercial Relationships: Joshua M. Barnett, None; Gary W. McCollum,
None; John S. Penn, None
Support: EY07533, AG031036, EY08126 and an Unrestriicted Grant from
Research to Prevent Blindness, Inc.
Program Number: 3019 Poster Board Number: D868
Presentation Time: 8:30 AM - 10:15 AM
Epigenetic Regulation of Choroidal Neovascularization
Nymph Chan1, Shikun He2A, Stephen J. Ryan, Jr.2B, David R. Hinton3. 1PathologyDVRC 313, Univ of Southern California, Los Angeles, CA; AOphthalmology-USC,
Ophthalmology, 2Doheny Eye Institute, Los Angeles, CA; 3Pathology, Keck
School of Medicine USC, Los Angeles, CA.
Purpose: Choroidal neovascularization (CNV) is a blinding complication of agerelated macular degeneration. The pathogenesis of CNV is still under investigation,
and epigenetic regulation can be a new target for a therapeutic approach in treating
CNV. The aim was to examine the role of histone deacetylation inhibitors (HDACi)
in the suppression of angiogenesis in vitro, and its regulation of pro- and antiangiogenic genes in new blood vessel formation in CNV.
Methods: Cultured early passage human fetal retinal pigment epithelial (RPE) cells
and bovine choroidal endothelial cells (BCEC) were used in the study. The role of
HDACs in the regulation of expression of HIF-1α, PEDF, VEGF and VEGFR2 was
analyzed by ChIP assay. 5×105 of RPE cells were treated with or without 0.5 µM of
trichostatin A (TSA) for 24 h, and fragmented chromatin was immunoprecipitated
with normal mouse IgG, anti-RNA Polymerase II or anti-Acetyl-Histone H3
antibodies. Pulled down DNA was amplified by PCR for the promoter regions of
the genes of interest and the DNA products were resolved on a 1% agarose gel. The
effect of TSA on the formation of tube-like structures was tested by treating
BCECs with or without 0.7 µM of TSA for 24 h. 1 × 104 of BCECs per condition
were transferred onto 50 µL of reconstituted basement membrane matrix and
incubated with or without 25 ng/mL of VEGF at 37°C for 6 h. The effect of another
HDACi, MS-275, on the expression of the four genes was examined by treating
RPE cells with 0 - 0.3 µM of MS-275 for 18 to 24 h, with or without 150 µM of
CoCl2 for 6 h. Western blots were performed to analyze expression of the four
Results: Results from the ChIP assay showed that in TSA-treated RPE cells, less
RNA Polymerase II and Acetyl-Histone H3 were associated with the promoter
regions of HIF-1α, VEGF and VEGFR2, but more RNA Polymerase II and AcetylHistone H3 were associated with the PEDF promoter. This suggests that under the
effect of TSA, the PEDF gene was actively being transcribed, while there was less
transcriptional activity at the HIF-1α, VEGF and VEGFR2 promoters. Another
HDACi, MS-275, also reduced the expression of HIF-1α, VEGF and VEGFR2, and
up-regulated PEDF, indicating that the expression of these genes was likely
regulated by epigenetics. Further, TSA inhibited the formation of capillary-like
structures in BCECs, which showed that TSA could inhibit angiogenesis.
Conclusions: The down-regulation of HIF-1α, VEGF and VEGFR2, and upregulation of PEDF after TSA treatment is likely affected by epigenetics.
Moreover, TSA also inhibits tube formation in BCECs. It is conceivable that
HDACi inhibits angiogenesis in CNV by modulating genes involved in regulating
new blood vessel formation. These data illustrate that epigenetic agents can emerge
as a new type of treatment for CNV.
Commercial Relationships: Nymph Chan, None; Shikun He, None; Stephen J.
Ryan, Jr., None; David R. Hinton, None
Support: EY01545, EY03040, RPB, Arnold & Mabel Beckman Foundation
Program Number: 3020 Poster Board Number: D869
Presentation Time: 8:30 AM - 10:15 AM
Involvement Of Apelin/APJ System In Laser-induced Choroidal
Chikako Ueno1, Atsushi Kasai2, Fumi Gomi1, Tatsuya Satooka2, Kei Nakai1,
Yasuhiro Yoshioka2, Akiko Yamamuro2, Sadaaki Maeda2, Kohji Nishida1.
Ophthalmology, Graduate School of Medicine, Osaka University, Suita, Japan;
Pharmacotherapeutics, Faculty of Pharmaceutical Sciences, Setsunan University,
Hirakata, Japan.
Purpose: The apelin/apelin receptor (APJ) system has been reported to modulate
developmental angiogenesis during early embryogenesis and pathogenic
angiogenesis. The deficiency of apelin was reported to inhibit abnormal vessels in a
mouse model of oxygen-induced retinopathy. We investigated its potential role in
the formation of choroidal neovascularization (CNV) in a laser-induced CNV
mouse model.
Methods: CNV was induced in normal wild-type (WT) and apelin knockout (KO)
mice by laser photocoagulation. The gene expression of apelin and APJ at 2, 4, and
7 days after laser photocoagulation was determined by quantitative real-time
reverse transcription-polymerase chain reaction. The expression of APJ in laser
induced CNV of WT mice was examined by immunohistochemistry with rat
monoclonal anti- PECAM-1. The incidence and the size of CNV were compared in
between apelin KO mice and WT mice 7 days after laser administration using the
image of Alexa488-isolectin B4 labeled tissue in the choroidal flat mounts on a
fluorescence microscope.
Results: In WT mice, the expression of apelin significantly increased with a peak
at 2 days after laser application (n=6, 5.98 ± 0.31 fold at 2 days (p<0.0001), 2.31 ±
0.27 fold at 4 days (p=0.099) and 1.92 ± 0.27 fold at 7 days (p=0.307) versus
control). The APJ expression significantly increased at day 4 and day 7 (n=5, 2.94
± 0.35 fold at 2 days (p=0.272), 7.13 ± 0.23 fold at 4 days (p<0.0001) and 7.74 ±
0.25 fold at 7 days (p<0.0001) versus control). APJ was detected in PECAM-1
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
positive endothelial cells in CNV by immunohistochemistry. The incidence of
CNV visualized was similar, 75.0% (33 lesions / 44 shots) and 78.1% (50 lesions /
64 shots) in WT and apelin-KO mice, respectively. On the other hand, the size of
CNV lesions in the apelin KO mice (22800 ± 6200µm2, n=9) were significantly
smaller than those in the WT mice (36300 ± 9600µm2 , n=10)(p=0.047).
Conclusions: The endogenous expression of apelin and APJ increased thorough
the course of the development of CNV. The size of laser-induced CNV in apelinKO mice was reduced comparing with WT mice. Those results suggest the
involvement of apelin/APJ system in the formation of CNV.
Commercial Relationships: Chikako Ueno, None; Atsushi Kasai, None; Fumi
Gomi, None; Tatsuya Satooka, None; Kei Nakai, None; Yasuhiro Yoshioka,
None; Akiko Yamamuro, None; Sadaaki Maeda, None; Kohji Nishida, None
Support: Grant-in-Aid Scientific Research (C) 22591942
Program Number: 3021 Poster Board Number: D870
Presentation Time: 8:30 AM - 10:15 AM
Low Shear Stress Up-Regulates Pro-Inflammatory Gene Expression in
Human Retinal Microvascular Endothelial Cells
Akihiro Ishibazawa, Taiji Nagaoka, Harumasa Yokota, Youngseok Song, Akitoshi
Yoshida. Ophthalmology, Asahikawa Medical University, Asahikawa, Japan.
Purpose: The vascular endothelium responds to shear stress generated by blood
flow and changes functions to regulate blood flow and maintain tissue homeostasis.
Recently, we found that physiologic high shear stress leads to increased expression
of vasodilatory and antithrombotic genes in human retinal microvascular
endothelial cells (HRMECs) (Ishibazawa et al. Invest Ophthalmol Vis Sci, 2011),
but the manner in which low shear stress affects retinal endothelial function
remains to be elucidated. We studied the effect of low shear stress especially in
pro-inflammatory gene expression in HRMECs.
Methods: HRMECs cultured on glass plates were exposed to laminar shear stresses
of 0 (=static), 1.5 and 15 dyne/cm2 for 24 hours using parallel plate-type flowloading devises. The mRNA expression of adhesion molecules,
cytokines/chemokines, pro-coagulant factors, and cyclooxygenase (COX) were
evaluated using real-time reverse-transcriptase polymerase chain reaction.
Results: HRMECs exposed to 1.5 dyne/cm2 significantly up-regulated the mRNA
expression of intracellular adhesion molecule-1 (4.2-fold vs static), vascular
adhesion molecule-1 (2.5-fold vs static, 4.3-fold vs 15 dyne/cm2), and E-selectin
(3.7-fold vs statc, 2.4-fold vs 15 dyne/cm2). The 1.5 dyne/cm2 group also had
increased cytokine/chemokine mRNA expression (> double vs static and 15
dyne/cm2), such as interleukin 1A (IL1A), IL6, IL8, platelet-derived growth factorB, and monocyte chemotactic protein-1. Pro-coagulant factors, i.e., tissue factor
and the plasminogen activator inhibitor-1 mRNA were significantly increased in
the 1.5 dyne/cm2 group (2.1-fold vs static, 1.5-fold vs 15 dyne/cm2). COX2 mRNA
expression increased in a shear-magnitude-dependent manner.
Conclusions: Our results demonstrate that relatively low shear stress causes the upregulation of pro-inflammatory gene in HRMECs.
Commercial Relationships: Akihiro Ishibazawa, None; Taiji Nagaoka,
None; Harumasa Yokota, None; Youngseok Song, None; Akitoshi Yoshida,
Support: None
Program Number: 3022 Poster Board Number: D871
Presentation Time: 8:30 AM - 10:15 AM
Myeloid Cells are Associated with Subretinal Neovascular Angiomas in a
Murine Model of Macular Telangiectasia
Edith Aguilar, Toshihide Kurihara, Peter D. Westenskow, Stephen Bravo, Martin
Friedlander. Cell Biology, Scripps Research Institute, La Jolla, CA.
Purpose: The pathogenesis of macular telangiectasia (MacTel) is unclear and
currently no approved treatment is available. We have previously characterized the
retinal phenotypes from very low density lipoprotein receptor (VLDLR) mutant
mice. We demonstrated that these mice exhibit very similar phenotypes to MacTel
patients including subretinal neovascularization and photoreceptor degeneration
(Dorrell J et al. J Clin Invest. 2009). However, the mechanisms initiating and
directing normal blood vessels ectopically into the outer nuclear layer have not
been identified. Since pronounced myeloid cell recruitment is commonly observed
during angiogenesis, we hypothesized that myeloid cells may also be recruited to
regions of VLDLR mutant eyes in which pathological angiogenesis is occurring.
Methods: Knock-in transgenic mice expressing GFP under the control of a
CX3CR1 promoter (CX3CR1GFP/GFP), were crossed with VLDLR mutant mice.
These crosses yielded pups with GFP positive myeloid cells in mice with MacTellike phenoytpes (VLDLR-/-;CX3CR1GFP/+). Immunohistochemistry was performed
on retinal flat mounts and on thick vibratome sectioned sensory retinas. Confocal
microscopy was utilized to image the spatial orientation of GFP positive myeloid
cells in relation to immunofluorescently labeled vascular networks.
Results: Myeloid cells are observed in a much higher frequency in the subretinal
space of VLDLR-/-;CX3CR1GFP/+ mice than in controls (CX3CR1GFP/+). Myeloid
cells are also consistently observed in very close proximity to neovascular tufts in
the deep plexus vascular layer and seem to wrap long processes around the
angiomas as they are forming.
Conclusions: While highly correlative, the distribution of myeloid cells in close
proximity to the angiomas of VLDLR mutant mice may provide evidence that
myeloid cells provide support to or regulate pathological angiogenesis. The results
of this study may have applications for the development of novel therapeutic
strategies for treating MacTel phenotypes.
Commercial Relationships: Edith Aguilar, None; Toshihide Kurihara,
None; Peter D. Westenskow, None; Stephen Bravo, None; Martin Friedlander,
Support: MacTel Foundation
Program Number: 3023 Poster Board Number: D872
Presentation Time: 8:30 AM - 10:15 AM
Pattern Scan Laser Photocoagulation induces less VEGF expression than
conventional laser in the murine retina
Shuichiro Hirahara, Takeshi Mizutani, Aiko Ito, Miho Nozaki, Yuichiro Ogura.
Department of Ophthalmology, Nagoya City Univ Medical School, Nagoya, Japan.
Purpose: The pattern scan laser is a semiautomated photocoagulator that delivers a
pattern array of multiple burns in a rapid predetermined sequence. Histological
evaluation revealed that the pattern scan laser system might restore inner retina.
Recently, not only green wavelength (532 nm) but also yellow wavelength (577
nm) became available with the pattern scan laser system. Previously, we have
reported that the pattern scan laser with green wavelength induced less
inflammatory cytokine including VEGF in the sensory retina compare with
conventional laser treatment (Hirano Y, et al, ARVO, 2010 and Sakamoto M, et al,
ARVO, 2011). The purpose of this study is to compare the expression pattern of
VEGF in the murine retina between the patter scan laser with green or yellow
Methods: Retinal scatter laser photocoagulation was performed on C57BL/6J mice
using the pattern scan laser with green wavelength (532 nm), yellow wavelength
(577 nm) (PASCAL Streamline,Topcon, Tokyo) or a conventional laser (532nm)
(Novus Varia®,Lumenis, CA). The eyes were enucleated 1 and 3 days after laser
treatment. The levels of VEGF in the sensory retina and RPE-choroid were
quantified by ELISA. Immunohistochemistry was also performed for VEGF and
macrophage infiltration (F4/80) on day 3.
Results: VEGF was significantly elevated in the sensory retina after laser treatment
in conventional laser group on day 1 (P<0.05). In pattern scan laser group, both 532
nm and 577 nm induced VEGF expression, but there was no significant difference
compared with the level on day 0.
Conclusions: We have shown that pattern scan laser induced less VEGF in the
sensory retina compare with conventional laser treatment. And there was no
significant difference between green and yellow wavelength. Collectively, the
pattern scan laser system, short duration and high power settings, may affect less on
inner retina, and led to support safety of panretinal photocoagulation with one
Commercial Relationships: Shuichiro Hirahara, None; Takeshi Mizutani,
None; Aiko Ito, None; Miho Nozaki, None; Yuichiro Ogura, None
Support: None
Program Number: 3024 Poster Board Number: D873
Presentation Time: 8:30 AM - 10:15 AM
Uncovering the VEGF Secretory Pathway in Retinal Pigment Epithelium: the
Rab Proteins’ Role
Ines P. Rodrigues1, Cristiana F. Pires1, Miguel C. Seabra1,2, Jose S. Ramalho1.
CEDOC, Faculdade de Ciencias Medicas, UNL, Lisboa, Portugal; 2Molecular
Medicine, National Heart and Lung Institute, Imperial College London, London,
United Kingdom.
Purpose: In the healthy eye, VEGF secretion occurs from the basolateral side of
retinal pigment epithelium cells (RPE), which are in close contact with the
choriocapillaries. However, VEGF secretion is impaired with aging and in retinal
degenerative disorders. The Rab family of GTPases is known to control several
steps of intracellular vesicular transport, but their involvement in the regulated
secretion of RPE cells is not yet understood. We aim at identifying the molecular
regulators underlying VEGF secretion in RPE cells.
Methods: To overexpress Rab proteins, vectors encoding their GFP-tagged version
were prepared. Silencing miRNA sequences against Rab genes were designed and
cloned into adenoviral and lentiviral vectors. Mouse RPE primary cultures were
used as an in vitro model, and the integrity of the cultures was evaluated by
immunofluorescence using specific cell markers, and by transepithelial resistance.
VEGF levels in cell culture media were measured by ELISA.
Results: Expression of tight junction proteins was assessed by confocal
microscopy analysis. Transepithelial resistance of mouse RPE cells monolayers
was 30-60 Ω*cm2. Overexpression of secretory pathway Rab proteins in RPE cells,
such as Rab3a and Rab14 led to a two-fold increase of VEGF secretion. Screening
of Rab silencing vectors led to several hits leading to deregulated VEGF secretion.
Conclusions: Modulation of expression of some Rab GTPases affect VEGF
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
secretion, suggesting the involvement of these Rab-dependent secretory pathways
in sustaining a healthy retina.
Commercial Relationships: Ines P. Rodrigues, None; Cristiana F. Pires,
None; Miguel C. Seabra, None; Jose S. Ramalho, None
Support: FCT Project Grant PTDC/SAU-OSM/104668/2008 & Individual PhD
Fellowship SFRH/BD/44389/2008
Program Number: 3025 Poster Board Number: D874
Presentation Time: 8:30 AM - 10:15 AM
Preclinical Pharmacology and Safety of ESBA1008, a Single-chain Antibody
Fragment, Investigated as Potential Treatment for Age Related Macular
Jacques Gaudreault1, Tea Gunde2, Heather S. Floyd3, Joel Ellis3, Julia Tietz1,
Daniela Binggeli1, Barbara Keller1, Anne Schmidt1, Dominik Escher1. 1ESBATech,
Schlieren, Switzerland; 2Numab, Wädenswil, Switzerland; 3Alcon Labs, Fort
Worth, TX.
Purpose: ESBA1008 is a humanized monoclonal single-chain FV (scFv) antibody
fragment targeting VEGFA and is currently investigated in patients with Age
Related Macular Degeneration. The smaller size of scFv (26kDa), compared to full
length IgG (150 kDa) or Fab fragments (50kDa), might allow better ocular tissue
penetration and is well suited for ophthalmology. Prior to initiation of clinical
trials, ESBA1008 pharmacology and toxicology were characterized in in vitro and
in vivo studies.
Methods: ESBA1008 in vitro pharmacology was characterized by BiaCore, ELISA
and inhibition of human umbilical endothelial cells (HUVEC) proliferation.
ESBA1008 potential toxicities were determined in cynomolgus monkeys (N=30)
who received a total of 3 IVT injections of ESBA1008 or vehicle (50 uL) every 3
weeks, in doses ranging from 0 to 6000 ug/eye.
Results: In vitro ESBA1008 binds to all isoforms of VEGFA, including VEGF165,
with a KD of 28.4 pM. ESBA1008 neutralized binding of VEGF165 to VEGFR2
with an IC50 of 0.86 nM and inhibited VEGF induced HUVEC proliferation with
an IC50 of 0.19 nM. In cynomolgus monkeys, ESBA1008 was well tolerated with
no ocular or systemic toxicity and with minimal ocular inflammation which was
comparable in the vehicle and active-treated groups. Mean serum cmax was 0.0405
and 0.286 ug/mL for the 500 ug and the 6000 ug group.
Conclusions: ESBA1008 is a potent inhibitor of VEGF and can be administered
safely in animals at doses as high as 6000 ug/eye, and results in more than 4-fold
lower systemic exposure compared to other antiVEGF agents, as shown in primate
toxicology studies. Since high doses were well tolerated in animals and ESBA1008
shows high affinity for VEGF, ESBA1008 has the potential in humans to be
administered at intervals greater than monthly, hence reducing the treatment burden
in patients. In addition, based on the predicted low systemic exposure in patients,
the risk of systemic adverse events might be lower with ESBA1008.
Commercial Relationships: Jacques Gaudreault, ESBATech (E); Tea Gunde,
ESBATech (E); Heather S. Floyd, Alcon Labs (E); Joel Ellis, Alcon Labs (E);
Julia Tietz, Esbatech (E); Daniela Binggeli, Esbatech (E); Barbara Keller,
Esbatech (E); Anne Schmidt, Esbatech (E); Dominik Escher, Esbatech (E)
Support: None
345 Smoking, the Retina and the RPE
Tuesday, May 8, 2012, 1:45 PM - 3:30 PM
Grand H Paper Session
Program #/Board # Range: 3189-3195
Organizing Section: Retinal Cell Biology
Contributing Section(s): Biochemistry/Molecular Biology
Program Number: 3189
Presentation Time: 1:45 PM - 2:00 PM
The Role of Nitric Oxide in Dry and Wet Age-related Macular Degeneration
Sarah B. Sunshine1A, Joel C. Sunshine1B, Marisol Cano1A, James T. Handa2.
Ophthalmology, BBiomedical Engineering, 1Johns Hopkins School of Medicine,
Baltimore, MD; 2Johns Hopkins Wilmer Eye Inst, Baltimore, MD.
Purpose: Age-related Macular Degeneration (AMD), the most common cause of
blindness among the elderly in the US, has two forms. The “dry” form,
characterized by retinal pigment epithelial (RPE) cell apoptosis and the “wet” form,
characterized by pathologic angiogenesis. Nitric Oxide (NO) is a chief component
of cigarette smoke (CS), the strongest risk factor for AMD. NO activates the
cytoprotective transcription factor Nrf2. CS and NO induce RPE apoptosis,
vascular permeability, and angiogenesis unless neutralized. NO from CS or
neuronal nitric oxide synthase (nNOS), the predominant isoform in the RPE, could
injure the RPE by impairing the Nrf2 response. We hypothesize that acute NO
exposure induces the Nrf2 response whereas chronic NO causes Nrf2 failure,
leading to dry AMD, and through NO’s pro-angiogenic properties, activate wet
Methods: NO was measured (DAF2DA) in ARPE19 cells treated with CS, SNAP
(NO donor), or transfected with nNOS. The Nrf2 response was tested by measuring
Nrf2 responsive gene (GCLM, NQO1, HO1) expression by RT-qPCR. Control and
nNOS deficient mice were treated with NOS’s substrate, Arginine (70mg/ml), to
increase NO, or water for 1-6 months. Laser induced rupture of Bruch’s membrane
was used to generate choroidal neovascularization (CNV).
Results: CS and nNOS transfected ARPE19 cells showed increased NO (10 and 2
fold) and increased expression of Nrf2 (1.6-fold p=0.05), GCLM (1.39-fold
p=0.02), NQO1 (2.7-fold, p=0.005). Mice treated with arginine had increased
expression of GCLM (2.13-fold, p=0.01), NQO1-(1.85-fold, p=0.04), HO1 (2.25fold, p=0.03) at 6 months in the RPE, and no Nrf2 response in nNOS deficient
mice. The area of CNV was paradoxically decreased by 41% (p=0.004) in arginine
treated mice compared to controls.
Conclusions: Nrf2 protects the RPE from acute NO stress. NO from nNOS
production likely mediated the Nrf2 response in the RPE of mice chronically
exposed to arginine. A dramatic decrease in CNV size was observed in mice treated
with arginine possibly through activated arginase. Further investigation of arginase
is warranted as a potential new therapy for wet AMD.
Commercial Relationships: Sarah B. Sunshine, None; Joel C. Sunshine,
None; Marisol Cano, None; James T. Handa, None
Support: Thome Foundation Grant (JTH), NIH Grant EY019904 (JTH),
Unrestricted grant from RPB (JTH), HHMI-FFB Medical Research Fellow (SBS)
Program Number: 3190
Presentation Time: 2:00 PM - 2:15 PM
Acute Periocular Exposure of Mouse Eyes to Hydroquinone Induces RPE
Cytoskeletal Changes, F-actin Aggregates, and Early Deposit Formation
Larry Koreen1, Albert R. Wielgus1, Grazia Spiga1, Peter Saloupis1, Diego G.
Espinosa-Heidmann2, Melody Chan1, Sina Farsiu1, Scott W. Cousins1.
Ophthalmology, Duke University Eye Center, Durham, NC; 2Ophthalmology,
Georgia Health Sciences University, Augusta, GA.
Purpose: Acute sub-lethal oxidative injury in cultured RPE causes extrusion of
intracellular and membranous material (i.e., cell membrane blebbing), which is
hypothesized to contribute to drusen formation. Our previous in vitro studies
showed that RPE cell blebbing is mediated by cytoskeleton reorganization and Factin aggregate formation. We sought to test the hypothesis that similar cytoskeletal
changes occur in vivo when mouse eyes are exposed to sub-conjunctival sub-lethal
doses of the cigarette smoke-associated oxidant, hydroquinone (HQ).
Methods: BALB/c mice received sub-conjunctival injection of 75mM HQ or
vehicle. At various time points after injection, mouse eyes were harvested, retinas
were removed, and 4-quadrant RPE flat mounts were prepared. RPE flat mounts
were stained with phalloidin and F-actin aggregates were imaged with confocal
microscopy. Computer software was developed to quantify actin aggregate
formation. Transmission electron microscopy (TEM) images were obtained from
some eyes to evaluate sub-RPE deposit formation.
Results: Periocular HQ exposure caused depolymerization of RPE cell actin
filaments and formation of F-actin aggregates in vivo. As we have observed
previously in vitro after ARPE-19 cells were exposed to HQ, mouse eyes treated
with HQ demonstrated significantly more actin aggregates and cytoskeletal changes
compared with vehicle-treated and untreated animals. Aggregate formation began
as early as 1 hour after exposure and appeared to maximize after 18 hours.
Aggregates accumulated much more in the basal portion of mouse RPE cells than
in the apical portion. Preliminary TEM imaging suggested that bleb-like structures
could be observed in association with mild transient sub-RPE deposit formation in
some eyes.
Conclusions: Local ocular exposure of mouse eyes to HQ causes RPE cell
cytoskeleton rearrangement and actin aggregate formation, an early stage in the
process of cell membrane blebbing, which may lead to formation of sub-retinal
deposits and drusen. This system will be helpful in understanding the in vivo
correlation with cell culture data from sub-lethal RPE oxidant injury and blebbing.
Commercial Relationships: Larry Koreen, None; Albert R. Wielgus,
None; Grazia Spiga, None; Peter Saloupis, None; Diego G. EspinosaHeidmann, None; Melody Chan, None; Sina Farsiu, None; Scott W. Cousins,
Imagen Biotech (E)
Support: NIH P30 EY-005722
Program Number: 3191
Presentation Time: 2:15 PM - 2:30 PM
Retinal Metabolic Response to Cigarette Smoke
Alexandra D. Butler1, William D. Ferrell1, Alex Woodell2, Carl Atkinson2, Robert
E. Marc1, Baerbel Rohrer2, Bryan W. Jones1. 1Ophthalmology, Moran Eye Center,
Salt Lake City, UT; 2Ophthalmology, Med Univ of South Carolina, Charleston, SC.
Purpose: Smoking is the single largest risk factor for age-related macular
degeneration, aside from age. Several of the main genetic risk factors for AMD are
polymorphisms occurring in complement genes involved in the alternative,
classical and common terminal pathways. To better understand the metabolic
impact of smoking on the retina, we used computational molecular phenotyping
(CMP) and examined the effects of cigarette smoke on wild type (wt) retinas and
mice in which either the alternative pathway (complement factor B, CfB) or the
common terminal pathway (complement component 3, C3) was removed.
Methods: Mice were exposed to either cigarette smoke or filtered air. Cigarette
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
smoke (CS) was generated using an automated cigarette-smoking machine (Model
TE-10, Teague Enterprises, Davis, CA) by burning 3R4F reference cigarettes (2.45
mg nicotine per cigarette; purchased from the Tobacco Health Research Institute,
University of Kentucky, Lexington, KY). Mice were exposed to CS for 6
hours/day, 5 days/week for 6 months. Age matched room filtered air exposed mice
were used as controls. Eyes were enucleated immediately post-mortem, fixed in
1% paraformaldehyde, 2.5% glutaraldehyde, dehydrated in graded methanols,
embedded in eponates and histologically analyzed with CMP.
Results: Alterations in retinal small molecule signatures from mice exposed to
cigarette smoke were observed compared to retinas from non-smoked mice in wt,
CfB and C3 knockout mice. Signal changes with arginine, glutamine and
glutathione progressively increased in the retinas of smoked exposed wt, CfB and
C3 knockout mice, indicating increased response profiles to cell stress. Both Müller
cells and photoreceptors of wt smoked retinas demonstrated changes relative to
non-smoked retinas.
Conclusions: Arginine, glutamine and glutathione, amino acids known to be
involved in cellular stress responses, were increased in retinal neurons and glial
cells upon smoke exposure. Eliminating essential components of the complement
system, a cascade required for the maintenance of the immune privilege of the eye,
appears to exacerbate responses to cigarette smoke in oxidative damage response
related pathways. Understanding complement-dependent alterations in the eye will
aid in our understanding of AMD pathology and may open new avenues for novel
treatment strategies.
Commercial Relationships: Alexandra D. Butler, None; William D. Ferrell,
None; Alex Woodell, None; Carl Atkinson, None; Robert E. Marc, Signature
Immunologics (E); Baerbel Rohrer, None; Bryan W. Jones, None
Support: RPB CDA (BWJ), Thome AMD Grant (BWJ), NIH EY02576 (RM),
NIH EY015128 (RM), NSF 0941717 (RM), NIH EY014800 Vision Core (RM),
NIH EY019320 (BR), VA merit award RX000444 (BR), grant to MUSC from RPB
Program Number: 3192
Presentation Time: 2:30 PM - 2:45 PM
Dysregulated Unfolded Protein Response: a Novel Mechanism for Cigarette
Smoke-induced RPE Cell Injury
Sarah X. Zhang, Chen Chen, Yimin Zhong, Jingming Li, Michael Daneshfar,
Joshua J. Wang. Medicine and Endocrinology, Univ of Oklahoma Hlth Sci Ctr,
Oklahoma City, OK.
Purpose: Endoplasmic reticulum (ER) stress and oxidative stress have been shown
to be the major causes of apoptosis and cell death. In response to ER stress, the cell
initiates a complex set of signaling pathways, which are collectively known as the
unfolded protein response (UPR). The objective of this study is to examine the role
and mechanism of the UPR signaling in cigarette smoke-induced RPE cell injury
and its implication in age-related macular degeneration (AMD).
Methods: Human RPE (ARPE-19) cells were exposed to hydroquinone (HQ), a
potent oxidant and a major component in cigarette smoke, for up to 48 h.
Expression of ER stress markers and UPR components were determined by
Western blot analysis, RT-PCR, and immunocytochemistry. Apoptosis and
mitochondria-related apoptotic pathways were examined by TUNEL assay,
activation of caspases, and expression of Bcl-2 proteins. ER stress and the UPR
signaling were manipulated by chemical chaperone 4-phenylbutyrate (4-PBA),
tauroursodeoxycholic acid (TUDCA), and adenoviruses expressing key UPR
regulators including XBP1, GADD34, and dominant negative mutant of PERK
Results: Expression of GRP78, XBP1 splicing, phosphorylation of PERK and
eIF2α were induced rapidly after HQ treatment. Nuclear levels of pro-apoptotic
transcription factors ATF4 and CHOP were significantly increased, accompanied
by elevated mitochondrial cytochrome c release and caspase 3 activation. Inhibition
of ER stress by 4-PBA or TUDCA, or suppression of CHOP activation by AdGADD34 or Ad-PERKDN, markedly alleviated HQ-induced apoptosis of RPE
cells. Interestingly, despite increased mRNA splicing, the protein level of active
XBP1, a master regulator of the adaptive UPR that eliminates ER stress, was
drastically decreased in HQ-treated cells. Further, XBP1-deficient RPE cells
isolated from conditional RPE-XBP1 knockout mice expressed much lower level of
anti-apoptotic protein Bcl-2, and were sensitive to HQ-induced CHOP activation
and apoptosis. Conversely, over-expression of active XBP1 abolished HQ-induced
CHOP expression and apoptosis in ARPE-19 cells.
Conclusions: Our findings provide strong evidence that cigarette smoke is
sufficient to induce ER stress, and, further, dysregulates the UPR signaling,
resulting in down-regulation of the protective UPR genes and activation of proapoptotic cascades. Enhancing the adaptive UPR function may, therefore, represent
a novel therapeutic strategy to combat cigarette smoke-associated RPE injury in
Commercial Relationships: Sarah X. Zhang, None; Chen Chen, None; Yimin
Zhong, None; Jingming Li, None; Michael Daneshfar, None; Joshua J. Wang,
Support: NIH grant EY019949; ADA Research Award 7-11-BS-182; AHAF grant
M2010088; OCAST Research Grant HR10-060; Dr. William Talley Research
Program Number: 3193
Presentation Time: 2:45 PM - 3:00 PM
The Role Of Sirt1 In The Pathogenesis Of Dry Age-related Macular
Takeshi Yoshida1,2, Shikun He1,2, Christine Spee1,2, Stephen J. Ryan, Jr.1,2, David R.
Hinton1,2. 1Ophthalmology, Doheny Eye Institute, Los Angeles, CA;
Ophthalmology, Keck School of Medicine of the University of Southern
California, Los Angeles, CA.
Purpose: Sirtuin1 (sirt1) is a NAD+-dependent deacetylase that modifies many of
the pleiotropic pathways associated with oxidative metabolism. The aim of the
present study is to investigate the role of sirt1 in the pathogenesis of dry age-related
macular degeneration (AMD).
Methods: Cryostat retinal sections from dry AMD patients and controls were used
to analyze the expression of sirt1. Early passage cultured human fetal RPE cells
were used for in vitro experiments. Sirt1 gene expression was knocked down using
specific siRNA, and then the RPE cells were then exposed to tertbutylhydroquinone (tBH; 0-100uM) for 16 hours. Acetylated-p53, cleavedcaspase3 protein and NF-E2-related factor 2 (nrf2) protein nuclear translocation in
the RPE cells were quantified by Western blotting after the treatment of sirt1
siRNA and tBH. Furthermore, apoptosis induced by knocking down of sirt1 and
tBH exposure was quantified by TUNEL assay.
Results: The expression of sirt1 in RPE of dry AMD patients was lower than in
age-matched control eyes. In vitro, after sirt1 knock down by siRNA, the oxidative
stress-induced acetylated-p53 and cleaved-caspase3 expression were significantly
increased in fetal RPE cells. Inhibition of sirt1 expression by siRNA also reduced
oxidative stress-induced nrf2 nuclear translocation in the RPE cells. Furthermore,
apoptosis in fetal RPE cells following knockdown of sirt1 by siRNA was markedly
increased after the oxidative stress.
Conclusions: Our data show that sirt1 plays an important role in anti-oxidative
stress and in pathogenesis of dry AMD. These results suggest that sirt1 might be a
therapeutic approach for the treatment of dry AMD.
Commercial Relationships: Takeshi Yoshida, None; Shikun He,
None; Christine Spee, None; Stephen J. Ryan, Jr., None; David R. Hinton,
Support: NIH grants EY01545, EY03040 & grants from RPB & the Arnold &
Mabel Beckman foundation
Program Number: 3194
Presentation Time: 3:00 PM - 3:15 PM
A Multi-Pronged Gene Expression Approach Identifies ADAM15 (Metargidin)
As A Potential Therapeutic Target For AMD
George Inana1, Christopher Murat1, Margaret J. McLaren2. 1Ophthalmology,
Bascom Palmer Eye Institute, Miami, FL; 2Graymatter Research, Miami, FL.
Purpose: To discover therapeutic targets for age-related macular degeneration
(AMD) using a multi-pronged gene profiling strategy incorporating gene
expression related to AMD, retinal pigment epithelium (RPE) phagocytosis, and
Methods: Gene expression profiling, including both a custom profiling of genes
expressed in RPE called CHANGE and an exon-based whole-genome strategy, was
performed with RNA from AMD patients’ retina and eyecup (EC, containing
RPE/choroid), RPE undergoing rod outer segment (ROS) phagocytosis, and retina
and EC of mice exposed to tobacco smoke, with controls. Genes showing similar
differential expression in all 3 criteria were identified and further analyzed by
individual expression studies, employing northern hybridization and quantitative
reverse transcription-polymerase chain reaction (qRT-PCR) amplification.
Results: Among the AMD-phagocytosis (AMDP) genes fulfilling the 3 criteria was
ADAM15 (metargidin), a gene with dual functions of a metalloproteinase and a
disintegrin. ADAM15 showed an increase in expression at 5 and 32 hr in ROS-fed
RPE, indicating a relationship to RPE phagocytosis. In AMD samples, the
expression of this gene was increased in retina and EC, in approximate correlation
with the severity of the disease (grade 1-4). In a 14-day laser photocoagulation
protocol to induce chorioneovascularization (CNV) in mice, ADAM15 showed a
delayed increase (at 6-14 days) in the EC, and a down-and-up pattern of expression
in the retina. In mice chronically exposed to tobacco smoke, a firmly established
environmental factor for AMD, an increase in this gene was observed after 3-5
weeks of exposure in the retina and EC. In the normal rodent retina and EC,
ADAM15 showed a diurnal pattern of expression with peaks at 6 PM and 12 AM,
respectively. Significantly, exposure to tobacco smoke phase-shifted both of these
peaks by 9 hours.
Conclusions: Increases in expression of ADAM15 were observed in human AMD
eyes, and in mouse models of laser-induced CNV, and of cigarette smoking, with
smoke exposure also causing a phase-shift in the expression peaks of ADAM15.
Given its known functions, and its involvement in circadian RPE phagocytosis, we
hypothesize that this gene plays a causative role in AMD, making it a strong
therapeutic candidate for this condition.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: George Inana, iTherapeutics (I), US-20110016544-A1 (P); Christopher Murat, None; Margaret J. McLaren,
iTherapeutics (I, C), US-2011-0016544-A1 (P)
Support: Flight Attendant Medical Research Institute, NIH P30 EY014801, an
unrestricted grant to the University of Miami from Research to Prevent Blindness,
Program Number: 3195
Presentation Time: 3:15 PM - 3:30 PM
The Effect of Combined Oxidant Exposure and AP Complement Attack on
Oxidant Production and Gene Expression in Human RPE Cells
Brett A. Toimil1, Ping Yang1, Jacob E. Berchuck1, Peter Baciu2, Glenn J. Jaffe1.
Ophthalmology, Duke University Medical Center, Durham, NC; 2Biology,
Allergan, Inc, Irvine, CA.
Purpose: Human RPE (hRPE) cells are chronically exposed to blood-borne
complement proteins and oxidative stress, a combination that is thought to
contribute to diseases such as AMD. At physiological levels, oxidants such as
hydrogen peroxide (H202) and hydroquinone (HQ) alter RPE cell function, as does
complement attack through alternative pathway (AP) activation. It is crucial to
understand how oxidants interact with AP activation to influence RPE cell survival
and gene expression. In the current study, we determined the effect of H202 or HQ
combined with AP activation on hRPE cell survival factor, cytokine, and
antioxidant enzyme gene expression.
Methods: Cultured confluent hRPE cells were treated with varying H202 or HQ
doses for 90 min. The cells were then primed with a complement-fixing Ab for 30
minutes, and incubated in C1Q-depleted serum (C1Qd). To examine intracellular
reactive oxidative species (ROS), cells were loaded with fluorophore dye and
fluorescence was quantified with a plate reader following 30 minute incubation in
C1Qd. Cells were counted following fluorescence measurement to calculate
fluorescence per cell for each treatment. For gene expression analysis, cells were
incubated in C1Qd for 1 to 4 hrs. RNA was harvested, and mRNA expression was
determined by qRT-PCR.
Results: Following AP activation, intracellular ROS increased, and further
enhancement occurred relative to oxidants alone and AP alone when AP activation
was coupled with H202 or HQ. Anti-survival factor Bax was expressed more highly
than Bcl-xL when AP activation was combined with H202 or HQ. MCP-1 was upregulated following AP activation, and this up-regulation was attenuated when AP
was combined with H202 or HQ. HO-1 and GCLC were unaffected by AP
activation, but were up-regulated by H202 and HQ. This oxidant- mediated upregulation was attenuated when AP was combined with H202 or HQ.
Conclusions: When AP activation is combined with oxidative stress, the balance
between pro- and anti- survival factors is shifted toward greater expression of antisurvival factors. The combination of oxidative stress and AP attenuated the effect
of AP or oxidant mediated up-regulation of cytokine and antioxidant enzyme
genes, providing a possible mechanism of hRPE dysfunction.
Commercial Relationships: Brett A. Toimil, None; Ping Yang, None; Jacob E.
Berchuck, None; Peter Baciu, Allergan, Inc. (E); Glenn J. Jaffe, None
Support: NIH P30 EY-005722 (Core grant), Research to Prevent Blindness, Inc.
353 Retinal Ganglion Cells
Tuesday, May 8, 2012, 1:45 PM - 3:30 PM
Hall B/C Poster Session
Program #/Board # Range: 3473-3496/D777-D800
Organizing Section: Retinal Cell Biology
Program Number: 3473 Poster Board Number: D777
Presentation Time: 1:45 PM - 3:30 PM
Efficacy Of TUDCA In Preventing Retinal Ganglion Cell Death In The RGC-5
Cell Line And Following Intravitreal Injection Of NMDA
Nicolas Cuenca1A, Violeta Gomez-Vicente1A, Francisco Germain2, Laura
Fernandez-Sanchez1A, Miguel Angel Company1B, Pedro de la Villa2, Mercedes
Palmero1B. APhysiology, Genetics and Microbiology, BOptics, Pharmacology and
Anatomy, 1University of Alicante, Alicante, Spain; 2Physiology, University of
Alcala, Alcala de Henares, Spain.
Purpose: Several studies have previously shown the anti-apoptotic properties of
the bile constituent, tauroursodeoxycholic acid (TUDCA), in diverse models of
photoreceptor degeneration. The aim of this study is to extend these findings to
other retinal neuronal types and to test whether systemic administration of TUDCA
protects retinal ganglion cells from death following oxidative or excitotoxic insults.
Methods: Apoptosis was induced in the RGC-5 cell line (kindly provided by Dr.
N. Agarwal) after exposure to the nitric oxide donor sodium nitroprusside, in the
presence of increasing concentrations of TUDCA. Viability was assessed by XTT
and crystal violet assays, and loss of mitochondrial potential with the JC-1 probe.
In vivo experiments were carried out in Sprague-Dawley rats and C57BL/6mice.
Retinal ganglion cell death was induced by intravitreal injection of N-methyl-daspartate (NMDA) in TUDCA (500 mg/kg i.p.) or vehicle-treated animals.
Immunohistochemistry on whole-mount retinas was used to evaluate retinal
ganglion cell survival.
Results: When cultured in the presence of TUDCA, survival of sodium
nitroprusside-treated RGC-5 cells significantly increased in a dose-dependent
manner. Viability values reached up to 80-90% of control untreated cells, which
correlated with the preservation of mitochondrial potential. In vivo, TUDCA
delayed NMDA-induced apoptosis of retinal ganglion cells, as revealed by Brn3a
immunostaining of whole-mounts. However, for high concentrations of NMDA the
effect of TUDCA is less significant.
Conclusions: Our results sustain the efficacy of TUDCA in preventing retinal
ganglion cell death, thus, TUDCA would be a good candidate for the
pharmacological treatment of degenerative diseases coursing with retinal ganglion
cell loss.
Commercial Relationships: Nicolas Cuenca, None; Violeta Gomez-Vicente,
None; Francisco Germain, None; Laura Fernandez-Sanchez, None; Miguel
Angel Company, None; Pedro de la Villa, None; Mercedes Palmero, None
Support: MICINN BFU2009-07793/BFI; ISCIII RETICS RD07/0062/0012;
ONCE; Fundación Mutua Madrileña to N. Cuenca; ISCIII RETICS
RD07/0062/0008 to P. de la Villa; JCI-2009-05224; UA GRE10-19 to V. GomezVicente
Program Number: 3474 Poster Board Number: D778
Presentation Time: 1:45 PM - 3:30 PM
Naloxone as a Neuroprotectant in Glaucoma: Its Role in the TLR4 Pathway
and Innate Immunity
Edward L. Wagner1, Algis Grybauskas1, Robert A. Burdi1, Loyal Walker1, Beatrice
Yue1, John Samples1, Paul A. Knepper1,2. 1Ophthalmology and Visual Sciences,
University of Illinois at Chicago, Chicago, IL; 2Ophthalmology, Northwestern
University Medical School, Chicago, IL.
Purpose: Primary open-angle glaucoma (POAG) is a neurodegenerative disease
causing retinal ganglion cell death which is usually associated with increased intraocular pressure (IOP). Previous studies have implicated an inflammatory
component in causing both neurotoxicity and increased IOP, and recently this
laboratory discovered that low-molecular-weight hyaluronic acid (LMW-HA)
fragments can initiate this pathway, interacting with toll-like receptor 4 (TLR4) to
activate the innate immune system. Therefore, finding a viable inhibitor to the
TLR4 pathway is of critical importance for neuroprotection in POAG. This study
examines viability of trabecular meshwork (TM) and naïve PC-12 cells when
challenged with LMW-HA in the presence and absence of naloxone, a known
TLR4-pathway inhibitor in non-ocular cells.
Methods: Cells were grown in 10% FBS in Eagle's medium until confluent, plated
at a density of 40,000 cells per well in full media for 24 hours, and then were
placed in 0.1% FBS and challenged with 0.2, 2.0, and 20.0 uM concentrations of
purified LWM-HA (50 kD; Sigma) in the presence or absence of 10.0 uM naloxone
(naloxone hydrochloride dihydrate, Sigma). The cells were treated for 24 hours
(n=6), harvested, and stained with trypan blue. Cell viability was determined by
two researchers without knowledge of experimental conditions.
Results: Naloxone showed significant protective properties in both TM and PC-12
cells. TM cells in the naloxone+LMW-HA condition were significantly more viable
than those treated with only LMW-HA, with an average viability increase of 19%
(P<0.02). Naloxone exhibited its greatest protection at higher concentrations of
LMW-HA; the 20 uM LMW-HA condition showed a 38.6% increase in viability
(P<0.005). Protection of PC-12 cells was even more dramatic, with an average
viability increase of 64% (P<0.005) in the naloxone+LMW-HA conditions relative
to LMW-HA alone.
Conclusions: These findings demonstrate that naloxone prevents the cytotoxic
effects of LMW-HA in vitro, confirming that LMW-HA acts through TLR-4 to
induce these effects. These findings are significant in that they provide promising
leads for protection of TM and retinal ganglion cells, a critical step in the treatment
of POAG.
Commercial Relationships: Edward L. Wagner, None; Algis Grybauskas,
None; Robert A. Burdi, None; Loyal Walker, None; Beatrice Yue, None; John
Samples, None; Paul A. Knepper, None
Support: American Health Assistance Foundation
Program Number: 3475 Poster Board Number: D779
Presentation Time: 1:45 PM - 3:30 PM
Progesterone Treatment In Rodent Anterior Ischemic Optic Neuropathy
Rachael S. Allen1A,1B, Timothy W. Olsen1B, Heather A. Cale1A, Katherine C.
Morrison1A, Jeffrey H. Boatright1B, Machelle T. Pardue1B,2, Donald G. Stein1A.
Emergency Medicine, BOphthalmology, 1Emory University, Atlanta, GA; 2Rehab
R&D Center of Excellence, Atlanta VA Medical Center, Decatur, GA.
Purpose: Optic nerve stroke, or anterior ischemic optic neuropathy (AION), is the
leading cause of sudden vision loss related to optic nerve dysfunction in older
adults. No effective treatment currently exists. The neurosteroid progesterone has
protective effects in animal models of traumatic brain injury, stroke, and spinal
cord injury, including reduced inflammation, swelling, and neuronal death; and
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
improved behavioral and functional recovery. The substantial overlap of
mechanisms involved in optic nerve stroke and mechanisms modulated by
progesterone leads us to hypothesize that progesterone protects against retinal
ganglion cell death in optic nerve stroke.
Methods: The rodent AION model was used to induce monocular optic nerve
stroke, while the contralateral eye served as a control. Rats were treated
systemically with vehicle or progesterone at 1, 6, 24, 48, 72, 96, and 120 hours post
surgery. Electroretinograms and visual evoked potentials (VEPs) were performed at
3 days post surgery. At 14 days, rats were euthanized, eyes were enucleated, and
retinal flat mounts were immuno-labeled with the retinal ganglion cell specific
antibody, Brn3a. Retinal ganglion cells were counted using an imaging analysis
Results: As measured by VEPs, stroke eyes from vehicle treated animals (n = 8)
performed at 68% of the level of their contralateral control eyes. Meanwhile, stroke
eyes from progesterone treated animals (n = 7) performed at 95% of the level of
their contralateral controls (p = 0.08). These results indicate clinically relevant
improvement in visual function. Additionally, stroke retinas from progesterone
treated animals (n = 8) had more Brn3a stained retinal ganglion cells than their
vehicle treated counterparts (n = 8) (approximately 10,000 more or 15% on
Conclusions: Given the current observations along with the growing literature on
the neuroprotective effects of progesterone in cerebral stroke and TBI, progesterone
may prove to be an effective treatment in the rodent anterior ischemic optic
neuropathy model as well.
Commercial Relationships: Rachael S. Allen, None; Timothy W. Olsen,
None; Heather A. Cale, None; Katherine C. Morrison, None; Jeffrey H.
Boatright, None; Machelle T. Pardue, None; Donald G. Stein, None
Support: H. Allen & Co., The Abraham J. & Phyllis Katz Foundation, Foundation
Fighting Blindness, Research to Prevent Blindness, NIH NEI R01EY014026,
R01EY016470, R24EY017045, P30EY006360, and T32EY007092-25
Program Number: 3476 Poster Board Number: D780
Presentation Time: 1:45 PM - 3:30 PM
Role Of Focal Adhesion Kinase In RGC Death And Axon Regeneration After
Mark Magharious, Philippe D'Onofrio, Meghan Lysko, Paulo D. Koeberle.
Surgery, University of Toronto, Toronto, ON, Canada.
Purpose: Our recent data demonstrates that Caspase-6 (CASP6) inhibition
promotes Retinal Ganglion Cell (RGC) survival and axon regeneration; however,
the downstream targets of CASP6 remain unclear. In the present study, we
examined the role of a CASP6-specific substrate (Focal Adhesion Kinase- FAK) in
RGC axon regeneration.
Methods: Optic nerve transection or optic nerve crush was performed in adult rats.
FAK cleavage and phosphorylation after optic nerve transection was assayed by
Western immunoblots of retinal lysates at 4 days after axotomy. After optic nerve
crush, animals received intraocular injections of GM6001 (broad spectrum MMP
inhibitor), Necrostatin-1 (Nec-1; RIP phosphorylation inhibitor), Y-27632 (ROCK
inhibitor), or control (PBS) to determine if pathways that stimulate FAK activation
promote axon regeneration in adult RGCs. Intraocular injections were delivered at
3 and 10 days after optic nerve crush. FAK phosphorylation in RGC growth cones
(21 days after crush) was assessed by immunohistochemistry in longitudinal optic
nerve sections, where RGC axons had been anterogradely labeled by intraocular
injection of Cholera-Toxin B-FITC (CTB-FITC). RGC axon regeneration was
quantified in fixed longitudinal sections of optic nerve at three different bins from
the lesion site: 0-250 microns, 250-500 microns, and >500 microns.
Results: Western blots of axotomized retinas showed that FAK cleavage products
were increased at 4 days postaxotomy, relative to normal retinas. Furthermore,
FAK-phosphorylation (Y397) in the retina was reduced after axotomy, indicative of
decreased FAK activation. In the crushed optic nerve, phospho-FAK was localized
to regenerating RGC axons at 21 days after optic nerve crush, when Y-27632 was
used to promote regeneration. Intraocular injections of the broad spectrum MMP
inhibitor, GM6001, or the RIP phosphorylation inhibitor, Nec-1, significantly
increased RGC axon regeneration through the lesion site at 21 days postaxotomy
(p<0.01), resulting in a 3-fold increase in the mean number of growth cones in each
of the three bins. Axons >500 microns in length were only observed after GM6001
or Necrostatin-1 treatment and not in controls.
Conclusions: Optic nerve transection causes the dephosphorylation and
degradation of FAK. Activated phospho-FAK is an integral component of
regenerating RGC growth cones, and enhancing FAK activation promotes RGC
Commercial Relationships: Mark Magharious, None; Philippe D'Onofrio,
None; Meghan Lysko, None; Paulo D. Koeberle, None
Support: CIHR
Program Number: 3477 Poster Board Number: D781
Presentation Time: 1:45 PM - 3:30 PM
Exposure To An Enriched Environment As A Novel Strategy To Prevent
Acute Ischemic Damage Of The Visual Pathway In Adult Rats
Damian Dorfman, Diego C. Fernandez, Ruth E. Rosenstein. Human Biochem/Sch
of Med, University of Buenos Aires, Buenos Aires, Argentina.
Purpose: Ischemia is a key component of several retinal diseases that are leading
causes of irreversible blindness. At present, there are no effective strategies to
prevent or reverse retinal ischemic damage. Recent evidences indicate that the
exposure to an enriched environment (EE) affects the functioning of the visual
system. The EE constitutes a strategy that boosts exploratory, visual, and cognitive
activities as well as social interaction and voluntary physical exercise. In this
context, the aim of the present work was to analyze the effect of exposure to an EE
in an acute retinal ischemia model.
Methods: Adult male Wistar rats were exposed to a standard environment (SE) or
EE 3 weeks before and 2 weeks after retinal ischemia. EE consisted of big cages
housing 6 animals and containing several food hoppers, wheels and different
objects repositioned once/day and fully substituted once/week. Retinal ischemia
was induced by increasing intraocular pressure to 120 mm Hg for 40 min. Retinal
function (electroretinography, ERG), and histology were analyzed at 7 and 14 days
post-ischemia. Anterograde transport from the retina to the superior colliculus (SC)
was examined after an intravitreal injection of cholera toxin β-subunit, and retinal
and SC glial fibrillary acidic protein (GFAP) levels were assessed by
Results: In control animals, ischemia induced a significant decrease in ERG a- and
b- wave amplitude, whereas the exposure to EE reduced these alterations. In
animals exposed to SE, ischemia provoked retinal ganglion cell (RCG) loss which
was decreased by exposure to EE. Moreover, ischemia induced a significant
increase in GFAP levels in the retina and the SC, and a deficit in the anterograde
transport which were reduced in animals exposed to EE.
Conclusions: These results suggest that the exposure to an EE could become a new
strategy for retinal ischemia treatment.
Commercial Relationships: Damian Dorfman, None; Diego C. Fernandez,
None; Ruth E. Rosenstein, None
Support: Prestamo BID convocatoria 2006 PICT 1623
Program Number: 3478 Poster Board Number: D782
Presentation Time: 1:45 PM - 3:30 PM
Direct Neuroprotection by Exogenous and Endogenous VEGF-A in Rodent
Models of Glaucoma
Richard H. Foxton1, Arthur Finkelstein1, Sauparnika Rayapureddi1, Annegret
Dahlmann-Noor2, Peng Khaw2, David Shima1, Yin-Shan Ng1. 1Institute of
Ophthalmology, UCL, London, United Kingdom; 2Moorfields Eye Hospital,
London, United Kingdom.
Purpose: Evidence suggests that VEGF-A plays a significant role in CNS neuron
development and neuroprotection. We have previously demonstrated a direct
neuroprotective function for VEGF-A in the retina and isolated RGC cultures, and
found VEGFR2 signalling via PI3K/Akt was primarily involved in the survival
response. Using animal models of acute drug-induced retinal cell death and
experimental hypertensive glaucoma we sought to further evaluate the role of
VEGF-A, and the effect of VEGF-A blockade on neuronal survival.
Methods: In the acute model, mice were pre-treated for 24 hours with intravitreal
VEGF120 then injected with staurosporine for a further 24 hours to induce cell
death. The experimental model of hypertensive glaucoma was performed in rats by
anterior chamber injection of magnetic microspheres to increase IOP, followed by
two intravitreal injections of VEGF120 or soluble VEGFR2 to neutralize
endogenous VEGF-A over 2 weeks post-glaucoma induction. TUNEL staining was
done on whole mount retinas to quantify cell death in the ganglion cell layer
(GCL), and immunohistochemistry was used to investigate receptor expression and
signalling pathways.
Results: Staurosporine induced extensive cell death in the GCL, which was
reduced by 55% after VEGF120 pre-treatment. PI3K pathway inhibitors abolished
the protective effect of VEGF120 pre-treatment. Anterior chamber injection of
magnetic beads in the rat induced a robust rise in IOP and a significant increase in
GCL death, and VEGF120 treatment attenuated RGC apoptosis by 75%, without
affecting the IOP. In contrast, injection of soluble VEGFR2 to block endogenous
VEGF-A increased GCL death by approximately 4-fold.
Conclusions: These data extend and reinforce the concept that VEGF-A is
neuroprotective in the retina, using both acute drug-induced and ocular
hypertension-induced models of retinal ganglion cell death. Importantly, VEGF-A
blockade appeared to exacerbate neuronal injury in the GCL, suggesting a
neuroprotective role of endogenous VEGF. These findings highlight that further
studies are necessary to determine if retinal neurons will be adversely affected
following long-term treatment with VEGF-A antagonists for ocular neovascular
Commercial Relationships: Richard H. Foxton, None; Arthur Finkelstein,
None; Sauparnika Rayapureddi, None; Annegret Dahlmann-Noor, None; Peng
Khaw, None; David Shima, None; Yin-Shan Ng, None
Support: Medical Research Council
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 3479 Poster Board Number: D783
Presentation Time: 1:45 PM - 3:30 PM
The Role of Matrix Metalloproteinases and Focal Adhesion Kinase in Retinal
Ganglion Cell Viability after Axotomy
Philippe M. D'Onofrio1A, Meghan D. Lysko1A, Mark M. Magharious1A, Paulo D.
Koeberle1B. ADepartment of Rehabilitation Science, BSurgery, 1University of
Toronto, Toronto, ON, Canada.
Purpose: The adherence of cells to extracellular matrix (ECM) provides important
survival signals through the activation of integrin receptors and Focal Adhesion
Kinase (FAK) activation. Matrix Metalloproteinases (MMPs) are a group of
enzymes that degrade ECM, and induce apoptosis. We examined the role of MMPs
in RGC apoptosis after axotomy, and whether MMP inhibition could extend the
therapeutic window of neurotrophic factors such as GDNF and Neurturin.
Methods: Optic nerve transection was performed in adult rats. Animals received
intraocular injections of: MMP-2/9 inhibitor ((2R)-2-[(4Biphenylylsulfonyl)amino]-3-phenylpropionic acid), MMP-3 inhibitor (N-IsobutylN-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid), MMP-8 inhibitor ((3R)-(+)[2-(4-Methoxy-benzenesulfonyl)-1,2,3,4-tetrahydroisoquinoline-3-hydroxamate]),
GM6001 (broad spectrum MMP inhibitor), FAK inhibitors (PF-573228, FAK-I14), GDNF, or Neurturin, at 3 and 10 days postaxotomy. RGC survival was
quantified in fixed flat-mounted retinas at 7, 14 or 21 days postaxotomy. Western
blots, in situ fluorescent zymography, and immunohistochemistry were used to
evaluate MMP activation after axotomy. Constitutively-active or dominantnegative FAK encoding plasmids were injected into the transected optic nerve in
order to study the role of FAK in RGC apoptosis.
Results: We observed increased levels of MMP-2 and -9, coincident with increased
MMP activity in the inner retina after axotomy. MMP-3 was localized to Müller
glia, and MMP-9 and -12 were both localized to injured RGCs and cells in the
inner nuclear layer. Intraocular delivery of an MMP-2/9 inhibitor, MMP-3
inhibitor, MMP-8 inhibitor, or GM6001 significantly enhanced RGC survival by 2
to 3-fold at 14 days postaxotomy (p<0.001). Transfection of axotomized RGCs
with constitutively-active FAK significantly increased RGC survival while
dominant-negative FAK decreased viability. FAK inhibitors (PF-573228 or FAK-I14) reduced RGC survival at 7 days postaxotomy. Dominant-negative FAK
abolished RGC rescue after co-delivery with GM6001. GM6001 prolonged the
anti-apoptotic effect of GDNF or Neurturin treatment from 14 to 21 days
Conclusions: ECM degradation by MMPs plays an important role in the
degeneration of axotomized RGCs. MMP inhibition prolongs the therapeutic
window of GDNF and Neurturin suggesting that FAK activity, concurrent with
neurotrophic-dependent signals, is required for long-term RGC survival after
Commercial Relationships: Philippe M. D'Onofrio, None; Meghan D. Lysko,
None; Mark M. Magharious, None; Paulo D. Koeberle, None
Support: CIHR
Program Number: 3480 Poster Board Number: D784
Presentation Time: 1:45 PM - 3:30 PM
The Neuronal EGF-related Gene Nell2 Supports Survival Of Retinal Ganglion
Cells After Optic Nerve Axotomy
Natik Piri1A, Jacky Man Kwong Kwong1A, Haksu Kyung1A, Joseph Caprioli1B,
Yasunari Munemasa2. AOphthalmology, BGlaucoma, 1Jules Stein Eye Institute,
UCLA, Los Angeles, CA; 2Ophthalmology, St Marianna University, Kawasaki,
Purpose: Nell2, a neuron-specific protein containing six epidermal growth factorlike domains, has been identified as a retinal ganglion cell (RGC)-expressed gene
by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of
this study was to evaluate the role of Nell2 in RGCs.
Methods: Proteome analysis was performed by matrix-assisted laser
desorption/ionisation-time of flight mass spectrometry (MS). EGFP-fused Nell2
expression construct was delivered to RGCs by electroporation-mediated cell
transfection. The optic nerve transection (ONT) model was generated in adult male
Wistar rats. Retrograde labeling to estimate the number of RGCs was performed by
application of the Fluorogold to the proximal cut surface of the optic nerve at the
time of optic nerve axotomy.
Results: Microtubule-actin crosslinking factor 1 (Macf1) was identified by MS as
Nell2 interacting protein in the retina in three consecutive experiments. The results
obtained with MS were confirmed by reverse immunoprecipitation with a Macf1
antibody and immunoblot with Nell2 antibody. The expression pattern of Macf1 in
the retina was determined by immunohistochemistry. Strong Macf1 expression in
the GCL and inner plexiform layer was observed. Macf1 expression in RGCs was
co-localized with Thy-1 staining, a commonly used marker for RGCs. The effect of
Nell2 overexpression on RGC survival was evaluated 2 weeks after ONT. RGCs in
the upper nasal retina were consistently more efficiently transfected than in other
areas. Approximately 49% of RGCs in the upper nasal area were transfected
whereas the transfection efficiency for the entire retina was only ~13% (n=5,
p<0.05). In non-transfected ONT retinas the loss of RGCs was approximately 95%
compared to the untreated control. In the upper nasal region, Nell2 transfection led
to the preservation of approximately 58% more cells damaged by axotomy
compared to untransfected (n=5, p<0.01) or pEGFP-transfected controls (n=5,
Conclusions: The results of MS that were further corroborated with
immunoprecipitation/immunoblot and immunohistochemistry data provide strong
evidence for the Nell2 interaction with Macf1 in RGCs. Furthermore, we evaluated
the cell-protective effect of Nell2 after ONT and demonstrated an increase in RGC
survival by approximately 58% compared to controls.
Commercial Relationships: Natik Piri, None; Jacky Man Kwong Kwong,
None; Haksu Kyung, None; Joseph Caprioli, None; Yasunari Munemasa, None
Support: NIH/NEI EY018644
Program Number: 3481 Poster Board Number: D785
Presentation Time: 1:45 PM - 3:30 PM
Effect Of Low Dose Advanced Glycation End-products On Neurite
Regeneration In Adult Rat Retinas And Regenerative Effects Of Nt-4 In
Advanced Glycation End-products Exposed Retinas
Guzel Bikbova, Toshiyuki Oshitari, Shuichi Yamamoto. Ophthalmology & Visual
Science, Chiba Univ Grad School of Medicine, Chuo-ku, Chiba, Japan.
Purpose: To determine the effect of low dose advanced glycation end-products
(AGE) on neurite regeneration in isolated rat retinas and the regenerative effect of
neurotrophin-4 (NT-4) in AGE exposed retinas.
Methods: All of the procedures were performed in accordance with the ARVO
Statement for the Use of Animals in Ophthalmic and Vision Research. Retinal
explants of 4 adult SD rats were studied. The explants were three-dimensionally
cultured in collagen gel and were incubated either in; 1) serum free control culture
media, 2) 10 µg/ml glucose-AGE-BSA media, 3) 10 µg/ml glycolaldehyde-AGEBSA media, 4) 10 µg/ml glyceraldehyde-AGE-BSA media, 5) 10 µg/ml glucoseAGE-BSA+100ng/ml NT-4 media, 6) 10 µg/ml glycolaldehyde-AGEBSA+100ng/ml NT-4 media, or 7) 10 µg/ml glyceraldehyde-AGE-BSA+100 ng/ml
NT-4 supplemented culture media. After 7 days, the number of regenerating
neurites from the explants was counted under a phase-contrast microscope.
ANOVA with Scheffe’s F test was performed to determine whether the number of
neuritis were significantly different among the different culture conditions.
Results: In retinas incubated with glucose-AGE-BSA, glycolaldehyde-AGE-BSA,
and glyceraldehyde-AGE-BSA, the number of regenerating neurites was
significantly fewer than in retinas without AGE (68.56±4.68/mm2 vs.
30.81±4.50/mm2; P=0.0033, 68.56±4.68/mm2 vs. 31.25±5.71/mm2; P=0.0044,
68.56±4.68/mm2 vs. 31.75±3.68/mm2; P=0.0238). In AGE exposed retinas
supplemented with NT-4, the numbers of regenerating neuritis were significantly
higher than in glucose-AGE-BSA without NT-4 (210.00±26.25/mm2 vs.
30.81±4.50/mm2; P<0.0001), in glycolaldehyde-AGE-BSA without NT-4
(193.75±24.06/mm2 vs. 31.25±5.71/mm2; P<0.0001), and in glyceraldehyde-AGEBSA without NT-4 (168.75±10.56/mm2 vs. 31.75±3.68/mm2; P<0.0001).
Conclusions: Low dose of AGE impedes neurite regeneration in adult rat retinas.
This is important in relation to the pathogenesis of several retinal diseases that form
and accumulate AGE such as diabetic retinopathy and other age-related neuronal
diseases. NT-4 significantly enhances neurite regeneration in retinas exposed to
Commercial Relationships: Guzel Bikbova, None; Toshiyuki Oshitari,
None; Shuichi Yamamoto, None
Support: None
Program Number: 3482 Poster Board Number: D786
Presentation Time: 1:45 PM - 3:30 PM
β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal
Ganglion Cells Survival
M. Livia Bajenaru1, Andrea Rachelle C. Santos1, Betty A. Obeso1, Ephraim
Trakhtenberg1, Dimitry Ivanov1, Jamie Ponmattam1, Sebastian Gutierrez1, Eleut
Hernandez1, M Elizabeth Fini2, Jeffrey L. Goldberg1. 1Bascom Palmer Eye
Institute, Miami, FL; 2USC Institute for Genetic Medicine, Keck School of
Medicine of USC, Los Angeles, CA.
Purpose: Retinal ganglion cells (RGCs) survival and neurite outgrowth are
promoted by neurotrophins and extracellular matrix (ECM) molecules, such as
laminin. Integrins are the main receptors for ECM. Previously, we showed that rat
retinal ischemic injury (RIRI) results in down-regulation of the integrin survival
pathway in RGC as evidenced by decreased expression of β1 integrin, FAK, and
Akt dephosphorylation, and a reduction in anti-apoptotic protein bcl-xL. In this
study, we further investigate the role of β1 integrin, and FAK in RGC survival, and
neurite growth.
Methods: β1 integrin activating (HUTS-21), or isotype control antibodies were
added to purified RGCs, 12 hours after plating. In separate experiments, RGCs
were preincubated with PP2, before HUTS-21 addition. RGCs viability was
assessed 48, and 72 hours later by live cell imaging. To inhibit FAK expression,
RGCs were electroporated with custom-made FAK, or control siRNA (Dharmacon,
Inc) using Nucleofactor II (Amaxa). Knockdown of FAK in RGCs was tested 2472 hours later by RT-PCR and immunohistochemistry. RGC survival and neurite
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
outgrowth were evaluated by Cellomics (Thermo Scientific). RIRI was induced in
Sprague-Dawley rats by unilateral elevation of intraocular pressure to 110 mm Hg
for 1 hour (n=16). The animals were divided in 4 groups and HUTS-21, isotype
control antibody, and PBS control were administered intravitreally into the rat eye
30 minutes, and 2 days post-RIRI. Animals were euthanized 5 days post-RIRI and
fluorogold (FG) retrogradely labeled RGCs were counted in flat mounted retinas
using AxioVision 4.7.
Results: The activating antibody HUTS-21 mimicked the effect of laminin, and
resulted in 70% RGC survival in vitro, compared to 50% by PDL. The FAK
inhibitor PP2 abolished both laminin and HUTS-21 survival effect, suggesting that
FAK is downstream of β1 integrin. siRNA treatment knocked-down FAK
expression by 92% after 72 hrs, and resulted in 2-fold reduced survival, and 1.7fold reduced neurite growth in RGC. RIRI resulted in 55% RGC death after 5 days,
but intravitreal HUTS-21 administration in vivo significantly reduced RGC death
to 20%, while the control antibody did not change RGC survival.
Conclusions: We demonstrated that β1 integrin and its downstream effector FAK
are key regulators of the integrin survival pathway in RGCs. Intravitreal
administration of the HUTS-21 antibody in the RIRI model has neuroprotective
effect, and may be exploited to prevent RGCs death in ischemic optic neuropathy,
and glaucoma.
Commercial Relationships: M. Livia Bajenaru, None; Andrea Rachelle C.
Santos, None; Betty A. Obeso, None; Ephraim Trakhtenberg, None; Dimitry
Ivanov, None; Jamie Ponmattam, None; Sebastian Gutierrez, None; Eleut
Hernandez, None; M Elizabeth Fini, None; Jeffrey L. Goldberg, None
Support: American Heart Association SDG 09SDG2280555 (MLB); NIH Core
Grant NIHP301430 (BPEI); Unrestricted grant from Research to Prevent
Blindness (BPEI).
Program Number: 3483 Poster Board Number: D787
Presentation Time: 1:45 PM - 3:30 PM
The role of Receptor Interacting Proteins in Retinal Ganglion Cell apoptosis
following axotomy
Meghan D. Lysko1A, Philippe M. D'Onofrio1A, Mark M. Magharious1A, Paulo D.
Koeberle1B. AGraduate Department of Rehabilitation Science, BSurgery, 1University
of Toronto, Toronto, ON, Canada.
Purpose: We have recently shown that caspase-8 (CASP8) promotes Retinal
Ganglion Cell (RGC) degeneration and contributes to regenerative failure after
optic nerve axotomy or crush. Nevertheless, the activators and targets of CASP8
remain unclear. CASP8 activation is associated with Tumor Necrosis FactorReceptor 1 (TNF-R1) and TRAIL receptor signaling. We examined the role
Receptor Interacting Proteins (RIPs: molecular switches in the TNF-R1 cell death
pathway) in RGC apoptosis after axotomy. We also evaluated whether TRAIL
plays a role in RGC death.
Methods: Optic nerve transection was performed in adult rats. Animals received
intraocular injections (3 days postaxotomy; 4 microliters) of either Necrostatin-1
(Nec-1: a RIP1 phosphorylation inhibitor), Pre-084 (a Sigma-1 receptor agonist), or
recombinant TRAIL protein. RIP3 and RIP5 were identified and quantified in an
iTRAQ proteomic screen of axotomized retinas. In order to examine the role of
RIPs in RGC apoptosis following axotomy, short interfering RNAs (siRNAs)
directed against RIP1, RIP3, or RIP5 were injected into the transected optic nerve
to knockdown these proteins in RGCs. RGC survival was quantified in fixed flatmounted retinas at 7 or 14 days postaxotomy.
Results: Intraocular delivery of Nec-1 at 3 days postaxotomy increased RGC
survival by threefold (p<0.001) at 14 days, showing that RIP1 activation promotes
RGC death. Furthermore, in vivo transfection of axotomized RGCs with RIP1
siRNAs enhanced RGC survival by threefold (p<0.001). The levels of RIP3
increased between 3 and 7 days postaxotomy and then steadily declined. In
contrast, the expression of RIP5 remained normal until 3 days postaxotomy and
then decreased continuously until 14 days. By 21 days postaxotomy, the levels of
both RIP3 and RIP5 were 50% or less relative to normal retinas. siRNAs directed
against RIP3 or RIP5 increased RGC survival by threefold (p<0.001) at 14 days
postaxotomy. Intraocular injection of recombinant TRAIL protein did not induce
RGC apoptosis in uninjured or axotomized retinas. Similarly, Pre-084, which has
been shown to antagonize TRAIL activation, had no effect on RGC survival.
Conclusions: Our results show that RIP1 activation plays an important role in
RGC apoptosis following axotomy. Furthermore, RIP3 and RIP5 also promote
RGC degeneration. However, TRAIL does not affect RGC survival.
Commercial Relationships: Meghan D. Lysko, None; Philippe M. D'Onofrio,
None; Mark M. Magharious, None; Paulo D. Koeberle, None
Support: CIHR
Program Number: 3484 Poster Board Number: D788
Presentation Time: 1:45 PM - 3:30 PM
High-throughput siRNA Screening in Primary Retinal Ganglion Cells
Derek S. Welsbie1, Scott E. Martin2, Katy L. Mitchell1, Yan Ge1, Zhiyong Yang1,
Donald J. Zack1. 1Glaucoma, Wilmer Ophthalmological Institute, Baltimore, MD;
RNAi Screening Facility, NIH Chemical Genomics Center, Bethesda, MD.
Purpose: Glaucoma is defined by the death of retinal ganglion cells (RGCs), the
neurons that transmit vision from the retina to the brain. Current therapies all act by
lowering the intraocular pressure (IOP). Unfortunately, lowering IOP can produce
undesirable side effects and/or be difficult to achieve. Lacking are neuroprotective
agents that directly interfere with the cell death process in RGCs. To identify such
agents, we created a high-throughput drug target screening platform using RNA
interference in primary mouse RGCs. We hypothesized that gene targets whose
knockdown by small interfering RNAs (siRNAs) promotes survival would
represent potential drug targets.
Methods: Primary mouse RGCs were isolated by immunopanning dissociated
retinal cells from P0-4 pups using an antibody against the RGC surface antigen
Thy1.1. Cells were transfected with siRNA using the NeuroMag reagent (Oz
Biosciences). After 72 hours of culture in neutrophin-depleted media, viability was
measured with a plate reader using CellTiter-Glo luminescence or with automated
fluorescent imaging and calcein AM-staining.
Results: A limited subset of the Ambion Silencer druggable library (2112 siRNAs
covering 704 genes), including mostly kinases and G-protein coupled receptors,
was screened in primary RGCs. Since no survival-promoting siRNAs were
previously known, the Qiagen All-Stars #3 proprietary toxic siRNA mix served as
the positive control for siRNA-mediated survival change. With this, screening
achieved a Z-factor of 0.65. Moreover, duplicate screens using the same library had
a correlation of determination coefficient (R2) of 0.63. Amongst the top survivalpromoting hits was the endothelin receptor type B, previously shown to be
important in RGC death. As a complementary approach, the same data set was
probed for receptors whose knockdown promoted cell death (i.e. hypothesizing that
the ligands may be neuroprotective). Amongst the most toxic receptor siRNAs
were estrogen receptor alpha, melanocortin 2 receptor and cholecystokinin B
receptor. Satisfyingly, estrogen receptor signaling is known to be neuroprotective
to RGCs.
Conclusions: High-throughput siRNA screening in primary mouse RGCs can be
used to identify potential neuroprotective agents. Ongoing efforts include the
confirmation of our current hits using mouse models of optic neuropathy and an
expanded, whole-genome siRNA screen.
Commercial Relationships: Derek S. Welsbie, None; Scott E. Martin,
None; Katy L. Mitchell, None; Yan Ge, None; Zhiyong Yang, None; Donald J.
Zack, None
Support: 5K12EY015025-07
Program Number: 3485 Poster Board Number: D789
Presentation Time: 1:45 PM - 3:30 PM
Blocking Gap Junction Hemichannels Reduces Vascular Leak and Protects
Ganglion Cells Following Retinal Ischemia
Colin R. Green, Nathan M. Kerr, Jie Zhang, Elizabeth K. Eady, Simon J.
O'Carroll, Louise F. Nicholson, Cameron S. Johnson, Helen V. Danesh-Meyer.
Ophthalmology, Univ of Auckland, Auckland, New Zealand.
Purpose: In response to central nervous system injury connexin43 (Cx43) gap
junction channels are upregulated in the vascular bed and the blood-brain barrier is
compromised, contributing to lesion spread and neuron loss. We used systemically
delivered Cx43 mimetic peptides to block gap junction hemichannel opening
following rat retinal ischemia-reperfusion in order to investigate the possibility of
reducing leak and inflammation, and so protect retinal ganglion cells (RGCs).
Methods: Raised intraocular pressure was used to induce one hour of hypoxia in a
rat retina ischemia-reperfusion model. Vessel leak was determined using Evans
Blue dye injection. Cx43 changes, vascular integrity (Isolectin-B4 label),
astrocytosis (GFAP levels) and RGC loss (Brn3A label) was analysed in
wholemounts at 4, 8 and 24 hours, or 7 and 21 days post-ischemia. Cx43 mimetic
peptide (or scrambled control peptide) was delivered by a single intraperitoneal
injection immediately following reperfusion and effects on all parameters
Results: Cx43 was significantly upregulated in endothelial cells after reperfusion,
peaking within 4 hours and remaining high until 24 hours (p<0.05). Vessel leak
resulting from hemichannel mediated endothelial cell rupture peaked at 4 hours
post reperfusion, but remained significant at 8 and 24 hours (p<0.05). Astrocytosis
was not initially universal, but correlated with regions of vascular leak. Within 21
days of ischemia 35% of RGCs were lost. A single injection of Cx43 mimetic
peptide reduced vascular leak and associated astrocytosis. Leak at 4 hours was 5%
of that in the control (then at its highest level), and peaked at 8 hours post perfusion
when it reached 20% of hypoxia only controls at that time point. The mimetic
peptide treatment significantly reduced RGC loss by two thirds to 9% - 14 %
Conclusions: Cx43 upregulation and hemichannel opening mediates vascular leak
which is associated with astrocytosis and subsequent RGC loss following retinal
ischemia. Reduction in vascular leak was brought about by systemic delivery of
Cx43 mimetic peptide at a level expected to block hemichannels, but not to
uncouple gap junctions. Modulation of Cx43 after retinal ischemia-reperfusion
injury alleviates vascular leakage, reduces inflammation and provides significant
neuron sparing.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: Colin R. Green, CoDa Therapeutics, Inc. (C,
P); Nathan M. Kerr, None; Jie Zhang, None; Elizabeth K. Eady, None; Simon
J. O'Carroll, None; Louise F. Nicholson, None; Cameron S. Johnson,
None; Helen V. Danesh-Meyer, None
Support: None
Program Number: 3486 Poster Board Number: D790
Presentation Time: 1:45 PM - 3:30 PM
Retinal Ganglion Cell Survival And Axon Regeneration In Wlds Transgenic
Rats After Optic Nerve Crush And Lens Injury
Barbara Lorber1, Alessia Tassoni1, Natalie D. Bull1, Marilita M. Moschos2, Keith
R. Martin1. 1Centre for Brain Repair, University of Cambridge, Cambridge, United
Kingdom; 21st Department of Ophthalmology, University of Athens, Athens,
Purpose: We have previously shown that the slow Wallerian degeneration
mutation (WldS) delays axonal degeneration after optic nerve crush (ONC) but
does not protect retinal ganglion cell (RGC) bodies in adult rats. To test whether a
combination approach protecting both axons and cell bodies would enhance the
therapeutic potential of the Wallerian degeneration mutation we performed ONC
and lens injury (LI), which results in both enhanced RGC survival as well as axon
regeneration past the lesion site in wildtype animals.
Methods: Adult Sprague Dawley (SD) or WldS rats received (a) ONC (SD: n=8;
WldS: n=9), (b) ONC + LI (SD: n=11; WldS: n=13), or were left untreated (SD:
n=8; WldS: n=9). 14 days after treatment, RGC survival (number of Islet+ RGC)
and retinal glia activation (intensity of GFAP labeling) were quantified in retinal
sections. In addition, the number of GAP43+ RGC axons regenerating past the
optic nerve lesion site was assessed.
Results: As previously reported, we found that the WldS mutation does not protect
RGC bodies after ONC alone (SD: 36.6 ± 2.6%; WldS: 36.1 ± 1.6% RGC survival
versus untreated). Surprisingly, we found that WldS transgenic rats did not exhibit
the enhanced RGC survival response after ONC + LI that was observed in
wildtypes (SD: 47.5 ± 2%; P<0.01 versus SD ONC; WldS: 35.2 ± 1.5%). After
ONC, only few GAP43+ axons were able to grow past the lesion site in both
wildtype and WldS rats (SD: 14.7 ± 5.2; WldS: 18.1 ± 10.8). ONC + LI led to a
significant increase in RGC axon regeneration past the lesion site in both wildtype
(140.4 ± 29.6; P<0.01 versus ONC) and WldS rats (95.1 ± 35.5; P<0.05 versus
ONC), although the difference between wildtype and WldS rats was not
statistically significant. Furthermore, activation of retinal glia, previously shown to
be associated with enhanced RGC survival and axon regeneration after ONC + LI,
was unaffected in WldS transgenic rats (SD: 2.9 ± 0.2; WldS: 2.7 ± 0.2 fold
increase over untreated).
Conclusions: The RGC axon regeneration potential is similar between WldS
transgenic and wildtype rats, but WldS transgenic rats do not exhibit enhanced
RGC survival after combined ONC + LI suggesting that the neuroprotective effects
of LI on RGC survival may be limited by the WldS protein.
Commercial Relationships: Barbara Lorber, None; Alessia Tassoni,
None; Natalie D. Bull, None; Marilita M. Moschos, None; Keith R. Martin,
Support: Fight for Sight
Program Number: 3487 Poster Board Number: D791
Presentation Time: 1:45 PM - 3:30 PM
Sectorial Loss Of Retinal Ganglion Cells In The RCS Rat
Diego Garcia-Ayuso1, Manuel Salinas-Navarro1, Marta Agudo-Barriuso2,
Francisco Manuel Nadal-Nicolás1, Manuel Jiménez-López1, Caridad GalindoRomero1, Manuel Vidal-Sanz1, María Paz Villegas-Pérez1. 1Oftalmologia,
Universidad de Murcia, Espinardo (Murcia), Spain; 2HUVA, Servicio Murciano de
Salud, FFIS, Murcia, Spain.
Purpose: To investigate whether the sectorial loss of retinal ganglion cells (RGC)
observed in previous studies of our laboratory in the dystrophic Royal College of
Surgeons (RCS) rat strain (Villegas-Pérez et al., J Comp Neurol 1998;392: 58-77)
is due to an axonal transport deficit problem or to retinal ganglion cell death.
Methods: Dystrophic (rdy−/p+) and non-dystrophic (rdy+/p+) pigmented RCS rats
with ages ranging from 12 to 20 months were used for this study. RGCs were
identified using Fluoro-Gold (FG) tracing from the Superior Collicullus (SC), to
label RGCs with a competent axonal transport and Brn3a immnunodetection, to
detect all RGCs. Retinas were processed as whole mounts and examined by
fluorescence microscopy. Reconstructions of the whole mounts were made using
Image-Pro Plus 5.0 for Windows®. FG-labelled and Brn3a positive RGCs were
automatically identified and counted in each retina using previously described
methods (Salinas-Navarro et al., Vision Res. 2009;49: 115-126, Nadal-Nicolás et
al., IOVS 2009;50: 3860-3868).
Results: Dystrophic retinas showed with age wedge-shaped sectors devoid of both
FG labelling and Brn3a detection. These sectors were observed first in the ventral
retina and later spread dorsally. The number of both FG-labelled and Brn3a
positive RGCs declined with age. However, at 20 months of age there were
significantly higher numbers of Brn3a positive than of FG-labelled RGCs. Taken
together, these data indicate that the sectors are due to RGC death, which is
preceded by an axonal transport deficit.
Conclusions: Sectorial RGC loss in the dystrophic RCS rat retina is due to an
axonal transport deficit that ultimately causes RGC death.
Commercial Relationships: Diego Garcia-Ayuso, None; Manuel SalinasNavarro, None; Marta Agudo-Barriuso, None; Francisco Manuel NadalNicolás, None; Manuel Jiménez-López, None; Caridad Galindo-Romero,
None; Manuel Vidal-Sanz, None; María Paz Villegas-Pérez, None
Support: Fundación Séneca 04446/GERM/07; Spanish Ministry of Education and
Science SAF-22010-10385; Spanish Ministry of Science and Innovation and
ISCIII-FEDER: PI10/00187, PI006/0780 and RETICS RD07/0062/0001
Program Number: 3488 Poster Board Number: D792
Presentation Time: 1:45 PM - 3:30 PM
Quantitative And Qualitative Analyses Of The Nerve Fiber Layer
Degeneration After Optic Nerve Axotomy In Adult Mice.
Maria-Cielo Sanchez-Migallon1, Manuel Salinas-Navarro1, Manuel JimenezLopez1, Francisco Nadal-Nicolas1,2, Leticia Nieto-Lopez1, F. Javier ValienteSoriano1, Paloma Sobrado-Calvo1, Maria-Paz Villegas-Perez1, Manuel VidalSanz1, Marta Agudo-Barriuso1,2. 1Oftalmologia. Facultad de Medicina, Universidad
de Murcia, Murcia, Spain; 2Unidad de investigacion, Hospital Universitario Virgen
de la Arrixaca, Murcia, Spain.
Purpose: To analyze the degenerative events taking place in the nerve fiber layer
of the mouse retina after optic nerve transection or optic nerve crush.
Methods: Adult albino Swiss male mice were used for this study. The left optic
nerve was intraorbitally transected (IONT) or crushed (IONC). The right eyes were
used as controls. Three, seven, fourteen and ninety days post-lesion (dpl), retinas
were dissected as whole flat mounts (n=6-8 per time point and lesion). Retinal
ganglion cells (RGCs) axons, i.e. the nerve fiber layer, were detected by
immunodetection of the highly phosphorylated heavy subunit of the neurofilament
triplet (pNFH). Retinas were photographed under an epifluorescence microscopy.
The aberrant expression of pNFH after axotomy was qualitatively studied, and the
number and distribution of RGCs expressing high or low levels of pNFH in their
somas assessed (pNFH+RGCs).
Results: In control retinas, pNFH is only expressed in the central and medial retina,
in fascicles of RGC axons converging towards the optic disc. These axons are
rectilinear and uniformly labeled. After both lesions, this expression pattern
changes dramatically and thus, 3 days later, pNFH signal is already observed in the
retinal periphery and in the RGCs somas. As the time post-lesion increases, pNFH
intra-axonal deposits are observed, reaching their maximum at day 14 post-lesion.
By day 90 post-lesion, only few axons remain in the retina. In both lesions the
number of pNFH+RGCs reaches its highest at 3 dpl.
Conclusions: Axonal damage to mice RGCs induces a change in the expression
pattern of pNFH which is similar in appearance to the one reported for rat (ParrillaReverter et al., Vis.Res, 49 (2009): 2808-2825). However, in mice appearance of
pNFH+RGCs occurs earlier. In addition, while in rat the course of the ganglion cell
layer degeneration was qualitatively and quantitatively different for IONT and
IONC, in mice both lesions trigger a similar response.
Commercial Relationships: Maria-Cielo Sanchez-Migallon, None; Manuel
Salinas-Navarro, None; Manuel Jimenez-Lopez, None; Francisco NadalNicolas, None; Leticia Nieto-Lopez, None; F. Javier Valiente-Soriano,
None; Paloma Sobrado-Calvo, None; Maria-Paz Villegas-Perez, None; Manuel
Vidal-Sanz, None; Marta Agudo-Barriuso, None
Support: Fundación Séneca 04446/GERM/07; Spanish Ministry of Education and
Science SAF-22010-10385; Spanish Ministry of Science and Innovation and
ISCIII-FEDER: PI10/00187, PI006/0780 and RETICS RD07/0062/0001
Program Number: 3489 Poster Board Number: D793
Presentation Time: 1:45 PM - 3:30 PM
Involvement of Calpain in Retinal Ganglion Cell Death in a Rat Model of
Anterior Ischemic Optic Neuropathy
Takayuki Oka1, Rie Suzuki1, Yoshiyuki Tamada1, Thomas R. Shearer2, Mitsuyoshi
Azuma1,2. 1Senju Pharmaceutical Co Ltd, Kobe, Japan; 2Integrative BiosciencesDentistry, Oregon Health Sciences University, Portland, OR.
Purpose: Anterior ischemic optic neuropathy (AION) is a serious, sudden loss of
vision caused by transient ischemia. AION is associated with common
cardiovascular risk factors, and no clinically effective treatment exists. Animal
studies show that activation of endogenous calpains, a family of Ca2+-activated,
neutral, cysteine proteases cause retinal degeneration and dysfunction induced by
ischemia/reperfusion, hypoxia, or acute ocular hypertension. The purpose of this
study was to determine if calpain-induced proteolysis likewise occurs in a rat
model of AION.
Methods: Rat AION was induced by photoactivation of intravenously injected
Rose Bengal by green laser irradiation of the optic nerve head, causing thrombosis
of anterior optic nerve capillaries. Degeneration of the retina and optic nerve was
histologically evaluated by H&E and Luxol fast blue staining. Retrograde labeling
with Fluorogold was performed to determine the number of surviving retinal
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
ganglion cells (RGCs), and apoptosis was evaluated by TUNEL staining of wholemounted retinas. Proteolysis of calpain substrates was detected by immunoblotting.
To activate endogenous calpains, soluble proteins from rat optic nerves were
incubated with calcium, with or without calpain inhibitors.
Results: After laser treatment, edema was observed in optic nerve head followed
by demyelination. The number of RGCs decreased in a time dependent manner.
TUNEL-positive cells were observed. Calpain substrates α-spectrin and myelinassociated glycoprotein MAG (also a marker protein for oligodendrocytes) were
proteolyzed. Incubation of optic nerve proteins with calcium led to similar
proteolysis of these substrates, and calpain inhibitors prevented the proteolysis.
Conclusions: Oligodendrocytes are essential for survival of the neuronal cell body
and axons, and they are needed for myelin assembly. Our results in the rat AION
model suggested that calpain proteolyzed critical proteins in the oligodendrocytes,
causing their degeneration and contributing to RGC death. Calpain inhibitor drugs
may be useful in AION patients by suppressing oligodendrocyte degeneration and
ameliorating RGC degeneration/dysfunction.
Commercial Relationships: Takayuki Oka, Senju Pharmaceuticals (E); Rie
Suzuki, Senju Pharmaceuticals (E); Yoshiyuki Tamada, Senju Pharmaceuticals
(E); Thomas R. Shearer, Senju Pharmaceuticals (C); Mitsuyoshi Azuma, Senju
Pharmaceuticals (E)
Support: None
Program Number: 3490 Poster Board Number: D794
Presentation Time: 1:45 PM - 3:30 PM
Stratification Stereotypy Of Genetically Defined Retinal Ganglion Cell Types
Uygar Sumbul1,2, Sen Song3, Kyle McCulloch4, Michael Becker4, Richard
Masland4, Sebastian Seung1,2. 1Brain and Cognitive Sciences, Massachusetts
Institute of Technology, Cambridge, MA; 2Howard Hughes Medical Institute,
Cambridge, MA; 3Biomedical Engineering, Tsinghua University, Beijing, China;
Department of Ophthalmology, Mass. Eye and Ear Infirmary, Harvard Medical
School, Boston, MA.
Purpose: The retinal ganglion cell(RGC) population in mice is thought to consist
of 20-30 cell types. Identifying these circuit elements is crucial towards mastery of
retinal circuitry. Genetically defined subsets of the RGCs can be imaged using
genetic engineering tools. This paves the way to test the limits of stratification
specificity in the retina. We have quantified the morphological stereotypy of
genetically defined RGC types, focusing on the depth at which dendrites of each
type stratify in the inner plexiform layer.
Methods: Confocal image stacks of genetically defined RGCs are enhanced using
a supervised machine learning algorithm to suppress the background and alleviate
the differences in brightness levels on the dendritic arbor. These enhanced images
are traced using the Simple Neurite Tracer in FIJI software package.
Image stacks need to be warped and registered for a depth analysis, to account for
the deformations occuring during preparation and imaging. We used the ChAT
bands as landmarks in the retina and warped the stacks so that the bands form two
parallel planes.
Depth profiles are obtained from the trace points, which are low-pass filtered to
suppress noise. Finally, they are normalized so that each neuron's profile has the
same energy.
Results: We show the depth profiles of 118 cells, including JAM-B, CB2, W3,
W7, and KIAA-CRE cells, as well as neurons from GFP-M animals. The depth
profiles are remarkably consistent. The alignment reveals that the peak
stratification plane can be determined with submicron accuracy. For instance, the
standard deviation of peak depth is 0.04 times the distance between ChAT bands
for CB2 cells, which corresponds to ~0.5um. The KIAA-CRE cells are
accompanied by a second, less strongly GFP-expressing, population. These are
easily distinguished on the basis of stratification.
Conclusions: RGCs target their synaptic partners in narrow through-plane regions.
For homogeneous, genetically defined cell types they show submicron specificity.
This precision will be useful for further studies classifying the
Commercial Relationships: Uygar Sumbul, None; Sen Song, None; Kyle
McCulloch, None; Michael Becker, None; Richard Masland, None; Sebastian
Seung, None
Support: None
Program Number: 3491 Poster Board Number: D795
Presentation Time: 1:45 PM - 3:30 PM
Enrichment of Retinal Ganglion Cells in Retinal Lysate by Excimer Laser
Ablation of the Outer Retina
Christian van Oterendorp, Wolf A. Lagreze, Philip Maier, Julia Biermann.
University Eye Hospital Freiburg, Freiburg, Germany.
Purpose: Retinal ganglion cells are a relatively small cell population in the retina.
This leads to an unfavorable signal to noise ratio when analysing RGC proteins in
whole retina lysate. We present a novel technique to obtain RGC enriched rat
retinal lysate by removing the outer retinal layers with an excimer laser prior to
Methods: Flatmounted retinae from adult albino rats were fixated with methanol
(n=4). The outer retinal layers were ablated with an excimer laser for clinical use
(Schwind Amaris, Germany) over a diameter of 7 mm. The non-treated peripheral
retina was removed and the remaining inner retina was either processed for
microscopy or lysed for western blotting. Microscopic images with DAPI stained
nuclei were used to measure the size of the remaining inner nuclear layer islets.
Western blot for layer specific markers (rhodopsin for photoreceptors; Thy1 for
RGCs) was used to quantify the effect of the ablation.
Results: Morphological analysis of four treated retinae showed that the ganglion
cell layer remained intact in 74 +/- 2.4 % (mean and SEM) of the total treated area.
The remaining islets of the inner nuclear layer comprised 11 +/- 3.5 % of the GCL
area. In western blots no rhodopsin was detected in any ablated retina while Thy1,
normalised with Actin, was increased.
Conclusions: As a proof of principle we demonstrate that excimer laser ablation of
outer retinal layers is feasible. The photoreceptors can be completely removed. The
inner nuclear layer can at least be reduced in thickness and the retinal lysate can be
used for western blotting.
Commercial Relationships: Christian van Oterendorp, None; Wolf A.
Lagreze, None; Philip Maier, None; Julia Biermann, None
Support: None
Program Number: 3492 Poster Board Number: D796
Presentation Time: 1:45 PM - 3:30 PM
A Comparative Study Of The Photoreceptor And Ganglion Cell Topography
In Arboreal And Terrestrial Snakes
Einat Hauzman1, Daniela Maria O. Bonci1, Andre Mauricio P. Liber1, Sonia L.
Martins1, Sonia R. Grotzner2, Maritana Mela2, Dora F. Ventura1. 1Experimental
Psychology, University of Sao Paulo, Sao Paulo, Brazil; 2Cell Biology, Federal
University of Parana, Curitiba, Brazil.
Purpose: To compare photoreceptor and ganglion cell densities and topography of
two species of Dipsadidae Snakes that live in different habitats - the arboreal
Philodryas olfersii and the terrestrial P. patagoniensis.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Methods: Photoreceptor types were labeled with antibodies JH455 and JH492
(against human S and L/M cones, respectively) in sections and wholemount retinas.
Ganglion cells were labeled with Nissl stain in wholemount retinas. Mean cell
density was calculated from counts made in photographs of the retinas and used to
obtain isodensity maps. Visual acuity was estimated from the ganglion cell peak
density. Morphology of photoreceptor types was viewed with Scanning EM (JEOL
JSM 6360LV).
Results: EM scans revealed four distinct photoreceptor subtypes, three of which
were labeled. Radial sections showed large single and double cones labeled by the
antibody JH492, and small single cones labeled by the antibody JH455. Densities
of the total photoreceptor population were obtained in 10 retinas of P. olfersii
(11,183 ± 1,107 cells/mm2) and 13 retinas of P. patagoniensis (11,531 ± 1,237
cells/mm2). Small cones comprised 3% of the photoreceptor population in P.
olfersii and 5% in P. patagoniensis. Large single and double cones represented
83% in P. olfersii and 85% in P. patagoniensis. The mean density of presumed
ganglion cells was obtained in 5 retinas of P. olfersii (10,118 ± 1,026 cells/mm2)
and 7 retinas of P. patagoniensis (9,835 ± 2,772 cells/mm2). The maximal spatial
resolution was 3 cpd in both species. P. olfersii ganglion cells and photoreceptors
isodensity maps revealed a horizontal streak and two areae of higher density, one
central and the other caudal. P. patagoniensis retinas presented an area centralis
located in the ventro-rostral region.
Conclusions: The two species have L/M and S (or UV) type photoreceptors plus a
non identified type, suggesting a potential for trichromacy. The different
specialization retinal areas might be related to specific habitat and life style. The
visual streak and the two areae in P. olfersii retinas may be relevant for locomotion
and hunting in the arboreal layer, while the area in the rostro-ventral quadrant in P.
patagoniensis retinas provides better spatial resolution in the superior visual field,
important for the survival of terrestrial snakes.
Commercial Relationships: Einat Hauzman, None; Daniela Maria O. Bonci,
None; Andre Mauricio P. Liber, None; Sonia L. Martins, None; Sonia R.
Grotzner, None; Maritana Mela, None; Dora F. Ventura, None
Support: None
Program Number: 3493 Poster Board Number: D797
Presentation Time: 1:45 PM - 3:30 PM
<b>Using the RGC-5 Cell Model for the Study of Retinal Ganglion Cell
Development and Axon Regeneration</b>
Justin Chew1, Chenying Guo1, Maximilian Staudt2, Dongfeng Chen1,3. 1Schepens
Eye Research Institute, Boston, MA; 2Institute of Medical and Human Genetics,
Charité – Universitätsmedizin Berlin, Berlin, Germany; 3Center for Innovative
Visual Rehabilitation Center, Boston, MA.
Purpose: To characterize and optimize the culture conditions for the RGC-5 cell
line as a model for the study of retinal ganglion cell (RGC) development and axon
Methods: RGC-5 cells were cultured in High Glucose DMEM at a cell density of 4
x 104 cells/mL. Cells were induced to undergo terminal differentiation under four
different conditions: 10 nM staurosporine + human non-pigmented epithelium
(HNPE)-conditioned medium, 50 nM staurosporine + HNPE-conditioned medium,
50 nM staurosporine, and 316 nM staurosporine. Optimal cell differentiation
conditions were assessed by gene expression profiles and quantification of RGC
neurite outgrowth. Genes examined included mature RGC markers, such as beta3tubulin, Map2ab, Thy 1.2, Tau, and Brn3b. Additionally, expression levels of
insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R were examined. Gene
expression was quantified using qRT-PCR and western blot analysis, and induction
of expression of RGC markers was observed through immunohistochemical
Results: The optimal differentiation condition for RGC-5 was found to be 316 nM
staurosporine, which caused cells to adopt a cell morphology and gene expression
profile most similar to primary RGCs. Differentiation events were found to occur
largely in the first 12-24 hours after addition of staurosporine, with neurite growth
occurring in conjunction with an observed upregulation of mature RGC markers.
All measured RGC markers were upregulated 3.5 to 4-fold within two hours after
addition of staurosporine, with the exception of Map2ab, which was not
significantly upregulated. IGF-1 was found to be drastically upregulated over 20fold during the first two hours after addition of staurosporine.
Conclusions: RGC-5 may serve as a valid RGC model through induction of
differentiation with staurosporine. Upregulation of RGC markers suggests that
differentiated RGC-5 resembles primary RGCs genetically. Furthermore, the
drastic upregulation of IGF-1 in tandem with neurite outgrowth agrees with
previous literature describing it as an important axonal growth factor.
Commercial Relationships: Justin Chew, None; Chenying Guo,
None; Maximilian Staudt, None; Dongfeng Chen, None
Support: NIH/NEI (R01EY017641), NIDA (R21DA024803), Department of
Veterans Affairs (1I01RX000110), Department of Defense (W23RYX-9104-N603)
to D.F.C.
Program Number: 3494 Poster Board Number: D798
Presentation Time: 1:45 PM - 3:30 PM
Analysis Of PKCα-IR Bipolar Cells And Ganglion Cells Of The Retina Of The
Tropical Fish Hoplias Malabaricus Intoxicated With Low Acute Doses Of
Andre M. Liber, Sr.1, Sonia R. Grötzner2, Izabela P. de Jesus2, Daniela M. Bonci1,
Ciro A. Oliveira Ribeiro2, Dora F. Ventura1. 1Psicologia Experimental,
Universidade de Sao Paulo, Sao Paulo, Brazil; 2Departamento de Biologia Celular,
Universidade Federal do Paraná, Curitiba, Brazil.
Purpose: This study aims to examine the morphological effects of low acute doses
of methylmercury (MeHg) on the retina of the tropical fish Hoplias malabaricus
Methods: Four levels of MeHg intoxication were induced by intraperitoneal
injection of doses of either 0.01, 0.05, 0.1 or 1.0 µg MeHg/g of body weight,
followed by a fifteen day period of depuration. The retinas were dissected and fixed
in 4% paraformaldehyde for 3 h. ON bipolar cells in the b sublamina of the inner
plexiform layer (IPL) immunoreactive to protein Kinase Cα (irPKCα) were labeled
and GCL cells were Nissl stained. Cell counts were performed in retina whole
mounts for quantitative analysis of ON bipolar and ganglion cell layer (GCL) cell
densities. These analyses were carried out in 3 or 4 retinas for each dose tested and
in the control retinas. Sample retinal fields were photographed throughout the
retina at intervals of 1 mm, with a digital camera attached to a microscope using
Axio Vision software coupled to a computer. ON bipolar and GCL cells within the
fields were counted with the help of the NIH Scion Image 2.0 software. The
average density (mm2) of both types of cells was estimated for each retina and the
data from each of the four MeHg-intoxicated groups were compared with the
control group values (Student t-test).
Results: Thraira irPKCα ON bipolar cells showed the characteristic morphological
aspect of this cell type both in retinas of control and exposed fish. The total
estimated average cell densities in control fish were 1780,672 ± 608,230 for ON
bipolar cells and 5247,214 ± 2539,142 for GCL cells. The density of ON bipolar
cells did not show a significant difference in the acute low doses tested. Nissl
stained cells (nuclei) of the GCL of exposed fish also showed no significant cell
loss in estimated average density. However, there is a tendency in the decrease of
the number of cells in both cells types analyzed, which can be seen in the
isodensity maps.
Conclusions: low acute doses of MeHg/g do not decrease cell densities of either
ON bipolar cells or cells in the GCL, compared to controls. There were no
significant decreases in cell density (counts) for either cell type in any of the retinal
regions, for any of the MeHg doses tested. This work supports previous findings
(Tanan et al. 2006) that cell responses were still present, although decreased, at the
same dose range but they were completely absent at higher doses.
Commercial Relationships: Andre M. Liber, Sr., None; Sonia R. Grötzner,
None; Izabela P. de Jesus, None; Daniela M. Bonci, None; Ciro A. Oliveira
Ribeiro, None; Dora F. Ventura, None
Support: None
Program Number: 3495 Poster Board Number: D799
Presentation Time: 1:45 PM - 3:30 PM
Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes
Ruth E. Rosenstein1A, Laura A. Pasquini1B, Damian Dorfman1A, Hernan J. Aldana
Marcos2, Diego C. Fernandez1A. ADept Human Biochem-Sch Med, BDepartment of
Biological Chemistry and IQUIFIB, School of Pharmacy and Biochemistry,
University of Buenos Aires, Buenos Aires, Argentina; 2Laboratory of Histology,
School of Science, University of Belgrano, Buenos Aires, Argentina.
Purpose: Diabetic retinopathy is a leading cause of acquired blindness. Visual
function disorders have been observed in diabetic patients with very early
retinopathy or even before the onset of retinopathy. The aim of the present work
was to analyze the visual pathway in an early stage of experimental diabetes.
Methods: Diabetes was induced in Wistar rats by an i.p. injection of
streptozotocin. Anterograde transport was examined after an intravitreal injection
of cholera toxin β-subunit. Apoptosis of ganglion cells was examined by TUNEL
analysis, and the optic nerve levels of glial fibrillary acidic protein, platelet-derived
growth factor receptor-α, and myelin basic protein were analyzed by
immunohistochemistry. The optic nerve structure was examined by electron
Results: A deficit in anterograde transport from the retina to the superior colliculus
was observed 6 weeks after streptozotocin injection. At this time point,
morphologic studies did not reveal retinal ganglion cell loss or substantial
alterations in the superior colliculus. The optic nerve was morphometrically
evaluated at intraorbital (unmyelinated and myelinated) and intracranial sections. In
animals that had been diabetic for 6 weeks, a large increase in astrocyte reactivity
occurred in the distal (but not the intraorbital) portion, which coincided with
significant axon loss. Moreover, profound myelin alterations and altered
morphologic features of oligodendrocyte lineage were observed at the distal (but
not the proximal) optic nerve portion.
Conclusions: The present results suggest that axoglial alterations at the distal
portion of the optic nerve could be the first structural change in the diabetic visual
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: Ruth E. Rosenstein, None; Laura A. Pasquini,
None; Damian Dorfman, None; Hernan J. Aldana Marcos, None; Diego C.
Fernandez, None
Program Number: 3496 Poster Board Number: D800
Presentation Time: 1:45 PM - 3:30 PM
Enhanced Survival Of Injured Retinal Ganglion Cells By Depletion Of
Dendritic Cells
Dale S. Gregerson, Thien N. Sam, Neal D. Heuss, Scott W. McPherson.
Ophthalmology, University of Minnesota, Minneapolis, MN.
Purpose: We previously reported that injury of retinal ganglion cells (RGC) by an
optic nerve crush (ONC) led to the close association of dendritic cells (DC) with
RGC axons in the nerve fiber layer. The DC dominated this response, raising the
question of whether their response to RGC injury was protective, harmful, or
unrelated to the survival of RGC. The role of the DC, and the potential
contributions of other immune system activities, was examined.
Methods: Unilateral ONCs were done in several strains of B6 transgenic mice to
examine different activities of the immune system for effects on RGC survival
post-injury. These strains included CD11c-DTR/GFP mice whose DC express the
diphtheria toxin receptor (DTR) and GFP. The DC of this strain can be locally
depleted with diphtheria toxin (DTx). Other strains included mice deficient in
MHC Class I, MHC Class II, or Fas. Wild type mice were used as controls. All
mice were compared to uninjured mice, as well as the contralateral retinas. An
ONC protocol limited in severity was used in all mice to increase the baseline
survival of RGC. RGC were identified by their uptake of Fluorogold injected into
the superior colliculus at 4 wks post-ONC. Retinal wholemounts were made 1 wk
after Fluorogold injection, using Vectashield with propidium iodide to stain nuclei.
Injection of DTx, or saline, was done into the anterior chamber in 1 microL
containing 5 ng DTx.
Results: Normal, unmanipulated retinas from all mice, wt and transgenic,
contained similar numbers of RGC. Ipsilateral retinas from ONC-crushed eyes of
wt mice showed that the survival of RGC was 40.4%. The RGC counts in the
contralateral retinas were unchanged. MHC class I-deficient mice showed a
reduced survival of 25.9% of RGC post-ONC (Pvalue <0.05). The CD11cDTR/GFP mice, when treated with saline in the ipsilateral eye after the ONC, were
not different from controls (35.3%). However, when treated with DTx, the RGC
survived at a higher rate (54.8% if DTx-treated versus 35.3% if saline treated;
Pvalue <0.05).
Conclusions: Activation of innate immunity by an optic nerve crush leads to an
influx of myeloid cells into the retina and optic nerve including DC, macrophages
and granulocytes. Depletion of a portion of these cells, the DC in CD11c-DTR/GFP
mice, gave a higher RGC survival at 4 wks post-ONC. Use of a limited ONC to
increase RGC survival in control mice (approximately 40%) may have provided a
larger window of opportunity to detect differences in survival due to activities of
the immune system.
Commercial Relationships: Dale S. Gregerson, None; Thien N. Sam,
None; Neal D. Heuss, None; Scott W. McPherson, None
Support: NIH grant EY021003; Research to Prevent Blindness; and the Minnesota
367 RPE Metabolism and Cell Biology
Tuesday, May 8, 2012, 3:45 PM - 5:30 PM
Grand H Paper Session
Program #/Board # Range: 3698-3704
Organizing Section: Retinal Cell Biology
Program Number: 3698
Presentation Time: 3:45 PM - 4:00 PM
Regulation Of Constitutive Vascular Endothelial Growth Factor (VEGF) In
RPE/Choroid Ocular Tissue And RPE Cell Culture
Alexa K. Klettner, Jens Lassen, Daniel Westhues, Johann Roider. Ophthalmology,
Univ of Kiel, Kiel, University Medical Center, Kiel, Germany.
Purpose: The retinal pigment epithelium (RPE) is a major source of Vascular
Endothelial Growth Factor (VEGF) in the eye. Despite the role of VEGF in ocular
pathology, VEGF is an important factor in the maintenance of the choroid and of
the retinal pigment epithelium (RPE). VEGF expression is regulated by a plethora
of factors which are differently activated by different stimuli. The purpose of this
study was to investigate the regulation of constitutive VEGF expression in the RPE.
Methods: In order to investigate VEGF regulation, perfusion organ cultures and
primary RPE cells of porcine origin were used. VEGF content was evaluated with
ELISA and Western Blot. The influence of several molecular factors was assessed
by commercially available inhibitors. Toxicity of inhibitors was evaluated in MTT
Results: VEGF secretion as measured in RPE/choroid organ culture was
diminished after long term (48 h) inhibition of VEGFR-2 by VEGFR-2-antagonist
SU1498. It was also diminished after inhibition of phosphatidylinositol 3 kinase
(PI3K) by LY294002 for 48 h. Co-application of both substances did not show an
additive effect, suggesting that both use the same pathway in an autokrine positive
VEGF regulation loop. Inhibition of protein kinase C (PKC) by
Bisindolylmaleimide, on the other hand, did not influence VEGF secretion. VEGF
expression in primary RPE cell culture displayed a similar pattern, as the inhibition
of VEGFR-2 and as well as of PI3K diminished VEGF expression after 48 h.
However, in RPE cell culture, an additional effect of PKC inhibition could be
found. Inhibition of the transcription factor NFkB diminished VEGF secretion after
6 h (earliest measured time point) and remained diminished during all measured
time points (6 h, 24 h, 48 h), suggesting a vital role of NFkB in constitutive VEGF
regulation. Inhibition of the transcription factor SP-1 by Mithramycin also
displayed effects after 24 h and 48 h. Neither the inhibition of HIF-1 nor Stat3
displayed any influence on constitutive VEGF secretion.
Conclusions: Constitutive VEGF in the RPE is regulated by the transcription factor
NFkB and may be regulated to lesser extend by the transcription factor SP-1, while
Stat3 and HIF-1 do not seem to be involved. Additionally, VEGF secretion is
regulated by an autokrine positive loop via VEGFR-2 and PI3K.
Commercial Relationships: Alexa K. Klettner, None; Jens Lassen,
None; Daniel Westhues, None; Johann Roider, None
Support: CAU Intramural research grant
Program Number: 3699
Presentation Time: 4:00 PM - 4:15 PM
Effect Of Tnf-alpha On Barrier Function In Polarized Rpe Cells
makoto shirasawa, hiroto terasaki, noboru arimura, shozo sonoda, taiji sakamoto.
ophthalmology, kagoshima university, kagoshima city, Japan.
Purpose: Retinal pigment epithelial (RPE) cells form a blood-ocular barrier and its
polarized-nature is crucial for maintaining barrier functions. Nonetheless, highly
polarized RPE cells have not been used for in vitro studies. Considering the
frequent presence of tumor necrosis factor-a (TNF-a) in retinal disorders, it should
be studied using polarized RPE cells to elucidate the real mechanism of retinal
disorders. This study was conducted to examine the effects of TNF-a on barrier
integrity of polarized RPE cells.
Methods: Porcine RPE cells (1×10^5 cells) were seeded on fibronectin-coated
TranswellTM. The high polarization of RPE layer was confirmed both by its high
trans-epithelial resistance (TER >150 ohm/cm2) and polarized secretion of vascular
endothelial growth factor (ratio of lower chamber/upper chamber > 2.5 times),
which were used for the following experiments. After starvation, RPE cells were
incubated with/without 10 ng/ml TNF-a for 24 hour. Then TER was measured
chronologically. The effect of TNF-a on TER or intracellular signal pathway
proteins such as p38 MAPK, NF-kappa B, JNK, caspase was evaluated using
specific inhibitors as follows: SB203580 (p38 MAPK inhibitor), CAPE (NF-kB
inhibitor), SP600125 (JNK inhibitor), Z-VAD-fmk (caspase inhibitor). The
localization of ZO-1 was evaluated by immunohistochemistry. Morphological
evaluation was also performed by electron microscopy.
Results: After incubation with TNF-a, TER decreased over time and reached 13.1
+/- 3.4 % (average +/- SD; p<0.01) of reduction in comparison to control at 24
hours, which was abolished by anti-TNF-a antibody (3.1 +/- 4.4 %). This reduction
was inhibited by SB203580 inhibitor, but not by any other inhibitors. In control,
ZO-1 was located clearly on a margin of RPE cells, but this localization became
dispersed after co-incubation with TNF-a. Microvilli of RPE was flattened after
TNF-a stimulation by electron-microscopy.
Conclusions: The previous reports showed that TNF-a alone does not impair
barrier function of non-polarized RPE cells in vitro. While, in this study, RPE
barrier function was significantly damaged by TNF-a alone in highly polarized
RPE cells. This result indicates that even TNF-a alone may play an important role
even in pathogenesis of mild inflammatory retinal disorders such as chronic
macular degeneration.
Commercial Relationships: makoto shirasawa, None; hiroto terasaki,
None; noboru arimura, None; shozo sonoda, None; taiji sakamoto, None
Support: None
Program Number: 3700
Presentation Time: 4:15 PM - 4:30 PM
Role of DJ-1 in oxidative stress response in the Retinal Pigment Epithelium
Vera L. Bonilha1, Mary E. Rayborn2, Brent A. Bell3, Chengsong Xie4, Huaibin Cai5.
Ophthalmology, Cole Eye Inst/Cleveland Clin Lerner Ctr, Cleveland, OH;
Ophthalmic Res, Cole Eye Institute, Cleveland, OH; 3Department of
Ophthalmology, The Cleveland Clinic, Cleveland, OH; 4Neuroscience, National
Institutes of Health/ NIA, Cleveland, OH; 5Neuroscience, National Institutes of
Health/ NIA, Bethesda, MD.
Purpose: DJ-1 is a protein ubiquitously expressed in many tissues including the
brain where it has been shown to function as antioxidant, redox-sensitive molecular
chaperone and transcription regulator, which robustly protect cells from oxidative
stress. DJ-1 peptides were detected in rat retinal pigment epithelium (RPE) cell
fractions subjected to proteomic analysis. The present study was conducted to
define the precise molecular mechanisms regulating the expression and function of
DJ-1 in oxidative stress response in RPE and retina.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Methods: To decipher the DJ-1 function, RPE cultures were treated with H2O2
(100 to 800uM) and 4-HNE (5 to 100uM) for various times followed by
biochemical and immunohistological analysis. In addition, cells were infected with
a replication-deficient adenovirus carrying the full-length human DJ-1 cDNA and a
mutant construct, which has the cysteine (C) residues at amino acid 46, 53 and 106
mutated to serine (S)(CtoS construct) prior to stress experiments. Results were
analyzed by immunofluorescence, and Westerns. In addition, the effects of DJ-1
deletion were examined in knockout (KO) mice through non-invasive, fundus
imaging in vivo using SLO and OCT, histological and immunohistological
evaluation of the retinas of young adult and aged DJ-1 KO and control mice.
Results: In RPE cells under baseline conditions, DJ-1 displays a diffuse
cytoplasmic and nuclear staining. Upon oxidative injury, a large portion of DJ-1
redistributed to mitochondria. Increase in DJ-1 expression was observed when cells
were exposed to oxidative stress as analyzed by immunocytochemical and
biochemical assays. In addition, cells exposed to oxidative stress generated
oxidized DJ-1. Overexpression of full-length DJ-1 prior to exposure to oxidative
stress led to significant decrease in the generation of reactive oxygen species (ROS)
stress species and oxidative stress-related cellular damage. In contrast, the
overexpression of CtoS DJ-1 prior to oxidative stress experiments did not affect
ROS generation by cells subjected to oxidative stress. Overexpressed CtoS DJ-1
also failed to change its cytoplasmic distribution to mitochondria in response to
oxidative stress. SLO and OCT imaging did not reveal any obvious, qualitative
differences between the retinal morphology of control and DJ-1 KO mice.
However, histology revealed altered inner and outer nuclei layer and RPE thinning
in young adult mice.
Conclusions: DJ-1 expression is increased upon exposure to oxidative stress. DJ-1
overexpression promotes protection of RPE cells to oxidative stress. The three DJ-1
cysteine residues are involved in the oxidative stress function of DJ-1 in RPE cells.
DJ-1 KO mice display early signs of retinal degeneration.
Commercial Relationships: Vera L. Bonilha, None; Mary E. Rayborn,
None; Brent A. Bell, None; Chengsong Xie, None; Huaibin Cai, None
Support: The Foundation Fighting Blindness, Research to Prevent Blindness, Wolf
Family Foundation and National Eye Institute (EY017153).
Program Number: 3701
Presentation Time: 4:30 PM - 4:45 PM
Phagosome Maturation And Interactions With The Endocytic Pathway In
Retinal Pigment Epithelial Cells
Clare Futter1, Ingrid P. Meschede1, Silene T. Wavre1, Mafalda Lopes Da Silva2,
Tanya Tolmachova3A, Miguel C. Seabra3B. 1Institute of Ophthalmology, University
College London, London, United Kingdom; 2National Heart and Lung Institute,
Imperial College, London, London, United Kingdom; AMolecular Medicine, NHLI,
Molecular Medicine, 3Imperial College London, London, United Kingdom.
Purpose: Phagosome maturation in macrophages involves sequential interactions
with the endocytic pathway and the acquisition of Rab GTPases before fusion with
the lysosome. Despite the huge phagocytic load of retinal pigment epithelial (RPE)
cells, which phagocytose shed photoreceptor outer segments everyday, the
regulation of phagosome maturation and degradation in the RPE is not well
characterised. We aimed to develop assays to monitor phagosome maturation in
RPE cells and determine whether the maturing phagosome interacts with the
endocytic pathway before lysosomal fusion and degradation.
Methods: To identify sequential phagocytic compartments in mouse retinal
sections rhodopsin processing was monitored, in conjunction with cathepsin D
staining, by cryo-immunoEM. To identify endocytic compartments, the endocytosis
of fluid phase probes and transferrin from apical and basal surfaces of cultured
primary porcine RPE was followed. To monitor interactions between phagocytic
and endocytic pathways RPE cells were incubated with gold-labelled endocytic
probes from the basal surface, and photoreceptor outer segments from the apical
surface, and the appearance of gold probes in the maturing phagosome was
identified by electron microscopy.
Results: Antibodies to different rhodopsin epitopes allowed the identification of
sequential stages of phagosome maturation. Cultured RPE cells engulf and process
photoreceptor outer segments and endocytose probes from both apical and
basolateral surfaces. Probes endocytosed from the basolateral surface meet apically
endocytosed probes in a pre-lysosomal compartment that contains transferrin
receptor. When the endocytic pathway is loaded with gold particles from the basal
surface gold particles appear in the maturing phagosome before lysosomal delivery.
Conclusions: Endosomes in RPE cells can be accessed from both apical and
basolateral surfaces via clathrin coated pits. Sequential stages of phagosome
maturation can be identified by monitoring rhodopsin processing. As phagosomes
mature they interact with the endocytic compartment before fusing with the
lysosome. The assays that we have developed will allow the determination of the
effects of retinal disease-causing mutations on phagosome maturation and
interactions with the endocytic pathway.
Commercial Relationships: Clare Futter, None; Ingrid P. Meschede,
None; Silene T. Wavre, None; Mafalda Lopes Da Silva, None; Tanya
Tolmachova, None; Miguel C. Seabra, None
Support: BBSRC, Fight for Sight and the Wellcome Trust
Program Number: 3702
Presentation Time: 4:45 PM - 5:00 PM
Systematic Mapping of RPE Phagocytosis Pathways
Nora B. Caberoy, Yixiong Zhou, Gabriela Alvarado, Wei Li. Ophthalmology,
Bascom Palmer Eye Inst Univ of Miami, Miami, FL.
Purpose: RPE phagocytosis plays an important role in maintaining retinal
homeostasis. Defects in RPE phagocytosis lead to retinal degeneration, and
dysfunction in RPE phagocytosis may contribute to age-related macular
degeneration. However, daunting challenge to define unknown phagocytosis
pathways has hindered our understanding of the physiological and pathological
roles of RPE phagocytosis. This study is to systematically identify unknown RPE
phagocytosis ligands and receptor-specific phagocytosis ligands.
Methods: An innovative strategy of phagocytosis-based functional cloning was
developed to identify RPE phagocytosis ligands in the absence of receptor
information. Furthermore, a novel dual functional cloning technique was developed
by combining phagocytosis-based functional cloning with receptor-based affinity
cloning to identify receptor-specific RPE phagocytosis ligands. Identified ligands
were expressed as recombinant proteins, purified and independently verified by
assays for RPE phagocytosis, receptor binding and receptor activation with
intracellular signal cascades.
Results: Phagocytosis-based functional cloning identified tubby and Tulp1 as
novel phagocytosis ligands for RPE cells. Both proteins were characterized as
bridging molecules for MerTK, a well-known phagocytic receptor. Phagocytic
receptor-binding domain (PRBD) of tubby and Tulp1 was mapped to their Nterminal K/R(X)1-2KKK motif(s). PRBP was essential for MerTK binding and
receptor phosphorylation induced by tubby and Tulp1. Phagocytosis prey-binding
domain (PPBD) of tubby and Tulp1 was mapped to their highly conserved Cterminal 44 amino acids, deletion of which cause retinal degeneration. Removal of
either PRBD or PPBD disrupted the bridging function of tubby and Tulp1,
abolishing their stimulation of RPE phagocytosis. Dual functional cloning
identified galectin-3 (Gal-3) as new MerTK-specific phagocytosis ligand. Similar
to tubby and Tulp1, Gal-3 was also characterized as a new bridging molecule for
MerTK to facilitate RPE phagocytosis.
Conclusions: These results identified three new phagocytosis ligands for MerTK.
These studies demonstrated that phagocytosis-based functional cloning is a valid
technology for unbiased identification of RPE phagocytosis ligands in the absence
of receptor information and that dual functional cloning is valid for systematic
identification of receptor-specific RPE phagocytosis ligands. These technologies
will improve our capability to systematically map RPE phagocytosis ligands,
receptors and signaling pathways, and open a new chapter for RPE molecular
phagocyte biology and exploration of their therapeutic potentials.
Commercial Relationships: Nora B. Caberoy, None; Yixiong Zhou,
None; Gabriela Alvarado, None; Wei Li, None
Support: This work is partially supported by NIH R01GM094449-01A1,
R01EY016211-05S1, 1K99EY020865-01A1, and an institutional grant from
Research to Prevent Blindness
Program Number: 3703
Presentation Time: 5:00 PM - 5:15 PM
Autophagy and Cytokine Release from RPE Cells
Claire H. Mitchell1A,1B, Sonia Guha1A, Jason C. Lim1A, Alan M. Laties1C. AAnatomy
and Cell Biology, BPhysiology, COphthalmology, 1University of Pennsylvania,
Philadelphia, PA.
Purpose: Cytokines can be released through a variety of mechanisms. In addition
to the classical release pathways, recent evidence suggests a role for autophagy in
cytokine release. As autophagy may be perturbed in aging RPE cells, this could
alter the signaling dependent release of cytokines. Release of IL-6 may be
particularly important in this regard as it may impact macrophage activity. This
study probes the relationship between autophagy, lysosomal pH and IL-6 release
from RPE cells.
Methods: IL-6 release from fresh mouse RPE and ARPE-19 cells was measured
with Elisa assays. Ca2+ was determined using Fura-2 and lysosomal pH using
Lysosensor Yellow/Blue.
Results: Fresh and cultured RPE cells released baseline levels of the cytokine IL-6.
The vHATPase inhibitor bafilomycin reduced baseline secretion of IL-6 from RPE
cells. Tamoxifen, which like bafilomycin can raise lysosomal pH, also reduced
secretion of IL-6, suggesting that lysosomal alkalinization can decrease cytokine
release. The P2X7 receptor agonist BzATP also raised lysosomal pH and increased
LC3BII levels, consistent with impaired autophagy. In contrast, however, it
triggered an increased release of IL-6 release from cultured and fresh mouse RPE.
This BzATP-stimulated release was even greater for ABCA4-/-mice than control.
BzATP also raised intracellular Ca2+; the ability to simultaneously raise Ca2+ may
be critical in triggering the cytokine release as the release of IL-6 by BzATP was
prevented by the removal of extracellular Ca2+.
Conclusions: The interplay between autophagy and cytokine release is complex.
While decreased autophagy levels and/or elevated lysosomal pH can decrease
secretion of IL-6, simultaneously raising intracellular Ca2+ leads to a net rise in IL-
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
6 release. The increased release of IL-6 in ABCA4-/- mice suggests the process may
contribute to the pathology in AMD.
Commercial Relationships: Claire H. Mitchell, 61/480,055 (P); Sonia Guha,
None; Jason C. Lim, None; Alan M. Laties, 61/480,055 (P)
Support: EY013434
Program Number: 3704
Presentation Time: 5:15 PM - 5:30 PM
Endogenously Expressed Bestrophin-1 Influences Store-operated Ca2+ Entry
Mediated By Orai-1 In The RPE
Olaf Strauss, Nestor Mas Gomez. Experimental Ophthalmology, Klinikum der
Univ Regensburg, Regensburg, Germany.
Purpose: : Recent publications suggest that endogenously expressed bestrophin-1
might function as an intracellular Cl channel localized to cytosolic Ca2+ stores.
With this localization bestrophin-1 was found to interfere with intracellular Ca2+
signalling . In order to clarify the intracellular function of bestrophin-1 we
investigated store-operated Ca2+ entry (SOCE) in primary porcine retinal pigment
epithelial (RPE) cells.
Methods: Ca2+ signalling was investigated by means Ca2+ imaging using fura-2 as
Ca2+sensitive fluorescence dye. Expression of Orai-1 and bestrophin-1 was downregulated by siRNA transfection. Expression of Orai-1 Stim-1 and bestrophin-1
was investigated by means of western-blot analysis and immunohistochemistry.
Results: Short-time cultured primary porcine RPE cells robustly expressed
bestrophin-1 and the proteins of SOCE: Orai-1 and stromal-interacting molecule-1
(Stim-1). SOCE was elicited by application of sarcoplasmic Ca2+-ATPase
(SERCA) inhibitor thapsigargin (1 µM) under extracellular Ca2+-free conditions
followed by re-addition of extracellular Ca2+. Amplitude of SOCE was increased by
5 µM 2-APB, reduced by 75 µM 2-APB and not influenced by 50 µM SKF96563.
RNAi knock-down of Orai-1 strongly reduced SOCE amplitude by 70%. RNAi
knock-down of bestrophin-1 also reduced the SOCE amplitude by 80 %.
Quantification of the amount of Ca2+ released from Ca2+ stores by thapsigargin
application showed that less Ca2+ is stored in cells which have treated with RNAi
against bestrophin-1 but not in cells treated with RNAi against Orai-1. Colocalization analysis by immunohistochemistry showed stronger co-localization of
bestrophin-1 and Stim-1 than bestrophin-1 with beta-catenin. Immunoprecipitation
experiments revealed that Stim-1 does not interact with bestrophin-1.
Conclusions: Porcine RPE cells exhibit SOCE mediated by activation of Orai-1
Ca2+ channels in response to release of Ca2+ from cytosolic stores. Bestrophin-1
function as Cl channel which conducts the counter-ion for Ca2+ uptake into
cytosolic stores by SERCA activity. A loss of bestrophin-1 function would change
intracellular Ca2+ signalling which involves release of Ca2+ from cytosolic stores.
Commercial Relationships: Olaf Strauss, None; Nestor Mas Gomez, None
Support: DFG STR480/10-1, STR480/10-2
374 Development: RPE, Photoreceptors, Retina
Tuesday, May 8, 2012, 3:45 PM - 5:30 PM
Hall B/C Poster Session
Program #/Board # Range: 3937-3972/D801-D836
Organizing Section: Retinal Cell Biology
Contributing Section(s): Visual Psychophysics/Physiological Optics
Program Number: 3937 Poster Board Number: D801
Presentation Time: 3:45 PM - 5:30 PM
Reprogramming Retinal Progenitor Cells by Exchanging Neurod1 for
Chai-An Mao1, Jang-Hyeon Cho1, Jing Wang2, Zhiguang Gao1, Ping Pan1, Diana
Lozano2, Laura J. Frishman2, William H. Klein1. 1Biochemistry and Molecular
Biology, The Univ of TX MD Anderson Cancer Ctr, Houston, TX; 2College of
Optometry, University of Houston, Houston, TX.
Purpose: In response to changes in the local environment, vertebrate retina
progenitor cells (RPCs) continuously adjust their intrinsic programs to acquire
distinct competency states. Atoh7/Math5, a basic helix-loop-helix (bHLH)
proneural transcription factor, is first expressed in a subset of RPCs and sets their
competency for a retinal ganglion cell (RGC) fate. Soon afterward, RGC
differentiation begins in a subpopulation of Atoh7-expressing RPCs. At the same
time, several other bHLH factors required for establishing non-RGC fate are
expressed in distinct subpopulations of RPCs. The mosaic spatiotemporal pattern of
the bHLH factors mirrors the competency state in the RPCs. The process by which
an individual RPC destine for one fate yet surrounded by RPCs destine for other
fates is exceedingly complex and is not well understood. In this study, we
determine whether RPC fate can be reprogrammed by replacing one bHLH factor
with another.
Methods: Previously, we replaced Atoh7 with Neurod1, a bHLH factor required
for amacrine cell fate, and showed that Neurod1 could replace Atoh7’s function in
specifying RGC fate, suggesting that Atoh7+ RPCs are largely programmed by
intrinsic mechanisms that are not solely dependent on a particular bHLH gene.
Here, we performed the converse experiment by replacing Neurod1 with Atoh7 by
gene targeting.
Results: Retinas from Neurod1Atoh7 mice produced approximately twenty percent
more RGCs than retinas from wild type mice. In the absence of endogenous Atoh7,
Neurod1Atoh7 activated RGC genes, and partially restored the optic nerve. The
Neurod1Atoh7-induced RGC axons projected to the lateral geniculate nucleus (LGN)
and superior colliculus (SC). Full field scotopic (dark-adapted) electroretinograms
(ERG) analysis showed that RGCs in Neurod1Atoh7 mice partially rescue the ERG
defects found in Atoh7-/- retinas. Moreover, expression of Atoh7 from the
NeuroD1Atoh7/Atoh7 allele did not rescue any of the phenotypic defects associated
with Neurod1-/- retinas.
Conclusions: We conclude that Atoh7 alone is sufficient to redirect an RPC destine
for an anacrine cell fate to an RGC fate, and that early arising RPCs are inherently
programmed to produce RGCs, although they require Atoh7 to do so. Together with
our previous results, we propose that in early retinogenesis, most RPCs have the
developmental potential to differentiate into RGCs. Although the extrinsic
environment plays a role in this process, subtle intrinsic differences must exist
among different RPC subpopulations which can be overcome by the expression of
Commercial Relationships: Chai-An Mao, None; Jang-Hyeon Cho, None; Jing
Wang, None; Zhiguang Gao, None; Ping Pan, None; Diana Lozano,
None; Laura J. Frishman, None; William H. Klein, None
Support: NEI (EY011930, EY010608-139005), Robert A. Welch Foundation (G0010) to W.H.K. NEI (EY06671) to L.J.F.
Program Number: 3938 Poster Board Number: D802
Presentation Time: 3:45 PM - 5:30 PM
Characterization Of Isolated Retinal Progenitor Cells In Polymeric Scaffolds
Miguel Flores-Bellver, Sr.1, Violeta Sánchez-Vallejo1, Raquel Álvarez-Nölting1,
Manuel Monleón Pradas2, Jose Miguel Soria1, María Miranda1, Francisco Javier
Romero3,4. 1Ciencias Biomédicas, Universidad Ceu-Cardenal Herrera, Moncada,
Spain; 2Center for Biomaterials and Tissue Engineering, Universitat Politecnica de
Valencia, Valencia, Spain; 3Fundación Oftalmológica del Mediterráneo (FOM),
Valencia, Spain; 4Facultad de Medicina, Universidad Católica de Valencia ‘San
Vicente Mártir’, Valencia, Spain.
Purpose: Regenerative or cellular therapy has emerged in recent years as one of
the most promising alternatives in the treatment of many diseases, including retinal
degenerations such as retinitis pigmentosa or age related macular degeneration. A
strategy to restore visual function in these patients is to replace the cells affected by
retinal progenitor cells (RPC) that are predisposed to differentiate into neurons in
the retina. On the other hand, obtaining and characterizing neurospheres from
progenitor cells of different animal models of retinal disease can also be a way to
study the mechanisms involved in these diseases. With this background, the
objective of this work was to characterize and differentiate neurospheres derived
from retina of mice into scaffolds or biomaterial thereby studying their integration
and survival.
Methods: GFP+ Mice (C57BL/6J genetic background) were used in this study
(n=5). The animal care and protocols were approved by the Ethics Committee of
the institution and adjusted to the Spanish law concerning animal experiments.
Progenitor cells were isolated from retina at postnatal day 1. We proceeded to
culture these cells until day 7 and then seeded them on a biomaterial or scaffold.
Biomaterials based on the hydrophobic polymer ethylacrylate, EA, and
hydroxyethyl acrylate, p (EA-co-HEA 90-10) were prepared as polymeric
Cells grown in the scaffold were characterized with the following markers: nestin,
Ki-67, GFAP, MAP2, recoverin, TUJ-1 and NF200. We also proceeded to the
differentiation of neurospheres to day 14 culturing them in the absence of growth
factors to further characterize them with the same markers.
Results: Neurospheres from progenitor cells express markers of retinal
neurofilament and proliferation, like GFAP, TUJ1, Ki-67 and nestin. Differentiated
cells express neuronal markers like TUJ-1 and photoreceptors markers (i.e.
recoverin, rhodopsin).
Conclusions: Cells obtained from retinas have progenitor properties and
neurogenic potential, providing potential sources of cells for transplantation
through scaffolds.
Commercial Relationships: Miguel Flores-Bellver, Sr., None; Violeta SánchezVallejo, None; Raquel Álvarez-Nölting, None; Manuel Monleón Pradas,
None; Jose Miguel Soria, None; María Miranda, None; Francisco Javier
Romero, None
Support: None
Program Number: 3939 Poster Board Number: D803
Presentation Time: 3:45 PM - 5:30 PM
Identification of an Early-Specified Population of Cone Progenitors Marked
by Islet1 and Recoverin in the Developing Swine Retina
Wei Wang1A, Liang Zhou2, Sang-Joon Lee1A,3, Henry J. Kaplan1A, Douglas C.
Dean1A,1B. AOphthalmology & Visual Sciences, BJames Graham Brown Cancer
Center, 1University of Louisville, Louisville, KY; 2Ophthalmology, The second
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Xiangya Hospital,Central South University, Changsha, China; 3Ophthalmolgy,
Kosin University, College of Medicine, Busan, Republic of Korea.
Purpose: We examined the generation of rod and cone photoreceptors from retinal
progenitors in the developing outer nuclear layer (ONL) of the pig retina.
Methods: Retinas were removed from pigs at various development ages, and
retinal development was followed by H&E staining of retinal sections. Retinal
progenitors and rod and cone photoreceptors progenitors were followed by
immunostaining of retina sections.
Results: We identify a population of cone progenitors which are specified and in
place in the outer two rows of the developing swine retina before mid-gestation.
These cells are marked by Islet1 and recoverin, and they appear before NRL+ rods
are specified from PAX6+ retinal progenitor cells. Further, we demonstrate that
L/M opsin cones are generated from this population before S opsin cones.
Conclusions: These results provide the first evidence of a distinct population of
cone progenitors, which are specified before mid-gestation. These cone progenitors
do not differentiate and express opsins for 35 days, and they are distinct from the
later-appearing rod progenitors. As they differentiate, the cone progenitors give rise
sequentially to L/M opsin cones and the S opsin cones. The positioning of the cone
progenitors in the outer retina leads to complete segregation of cones and rods in
the adult with cones comprising the outer two rows of the ONL and rods all
confined to the interior rows. Taken together, the results suggest differences in
pathways leading to rod and cone specification in the mouse and pig.
Commercial Relationships: Wei Wang, None; Liang Zhou, None; Sang-Joon
Lee, None; Henry J. Kaplan, None; Douglas C. Dean, None
Support: Research to Prevent Blindness, American Health Assistance Foundation,
NIH Grants (P20 RR018733 and EY015636) and The Commonwealth of Kentucky
Research Challenge.
Program Number: 3940 Poster Board Number: D804
Presentation Time: 3:45 PM - 5:30 PM
Partial Reprogramming of Adult Mouse Rods Into Cones Rescues Retinal
Cynthia L. Montana, Joseph C. Corbo. Pathology and Immunology, Washington
University School of Medicine, Saint Louis, MO.
Purpose: Recent studies have demonstrated a remarkable degree of plasticity
within adult mammalian cell types: fibroblasts can be converted into iPS cells and
some differentiated cell types can be directly transformed into others. We wished to
determine whether adult rod photoreceptors could be directly converted into cones.
If this were possible, it might serve as a gene-independent approach to therapy for
retinitis pigmentosa, since converting rods into cones might make the cells resistant
to the effects of mutations in rod-specific genes. To test this idea, we took
advantage of the fact that the photoreceptor transcription factor Nrl acts as a cell
fate switch during retinal development: photoreceptor precursors that turn on Nrl
become rods while those that do not become cones.
Methods: We created a mouse line containing a floxed Nrl allele and crossed it to a
drug-inducible Cre recombinase-expressing line. Nrl was acutely deleted in these
mice at an adult stage and retinas were evaluated several weeks later for evidence
of rod-to-cone conversion.
Results: Extensive experimental characterization of these mice showed evidence of
partial reprogramming of rods into cones: many rod genes were markedly
downregulated and some cone gene were derepressed. However, reprogramming
was incomplete in that many cone genes, including the cone opsins, failed to be
expressed in the reprogrammed rods. Next, we tested whether partial
reprogramming of rods into cones might make the cells immune to the effects of a
rod-specific mutation. Remarkably, we found that acute elimination of Nrl from
adult rods can effect significant cellular and functional rescue in the Rho-/- mutant
Conclusions: Acute Nrl knockdown in the adult retina can rescue retinal
degeneration in the Rho-/- mouse. Studies in other retinal degeneration mutants are
ongoing. In addition, efforts are underway to overcome the genetic and epigenetic
barriers to complete reprogramming of rods into cones.
Commercial Relationships: Cynthia L. Montana, None; Joseph C. Corbo,
Support: NIH Grants EY018826, 5-T32-EY13360-08
Program Number: 3941 Poster Board Number: D805
Presentation Time: 3:45 PM - 5:30 PM
Misplaced Photoreceptors in the Retina of Developing Rodents
Klaudia Szabo1A, Arnold Szabo1A, Anna Enzsoly1B, Pál Röhlich1A, Agoston Szel1A,
Akos Lukats1A. ADept. of Human Morphology and Developmental Biology, BDept.
of Ophthalmology, 1Semmelweis University, Budapest, Hungary.
Purpose: In the retina of the primarily nocturnal animals, such as rodents, rod
photoreceptors are the dominant components in signal perception. Their
photosensitive pigment -rhodopsin- can be detected with immunocytochemistry
from birth. Previous studies showed, that besides the extensive staining of the
photoreceptors, a less numerous population of cells was also labeled in the inner
nuclear, and ganglion cell layers during postnatal development. The aim of the
present study was to describe and compare the morphology, the number and the
staining characteristics of this peculiar population in various rodent species.
Methods: Retinas of Spague-Dawley rats, Siberian hamster, Syrian golden hamster
and mouse of different ages (P0-P21) were studied. Immuncytochemical labeling of
different rhodopsin specific antibodies were used on whole mounted retinas and
cryosections. Double labeling with several retinal cell type specific antibodies was
used to further characterize the rhodopsin positive cell populations located in the
inner nuclear and ganglion cell layers.
Results: We observed rhodopsin expressing cells in the inner nuclear and ganglion
cell layers of the retina in all four species studied. They comprised approximately
1% of all labeled cells, often appeared in smaller clusters and morphologically
resembled bipolar, amacrine or ganglion cells. They could be reliably detected
using several different sets of antibodies, raised against the C-, and the N-terminal
of rhodopsin, respectively. These cells first appeared at the 4th postnatal day (P4),
reached their maximum density at P14 and disappeared entirely by the end of the
3rd week. Double labeling could not confirm the presence of any other type of
photopigment in their cytoplasm. Staining with different inner retinal cell type
specific antibodies showed colocalization only in case of recoverin, that is known
to label photoreceptors besides bipolar and amacrine cells.
Conclusions: Our results showed that approximately 1% of all rhodopsin
expressing cells are not located amongst other photoreceptors during development,
but are displaced to the inner retinal layers. Although they lose almost all
morphological resemblance to photoreceptors, colabeling indicates that they are
most probably misplaced rods that fail to integrate in the retinal mosaic.
Commercial Relationships: Klaudia Szabo, None; Arnold Szabo, None; Anna
Enzsoly, None; Pál Röhlich, None; Agoston Szel, None; Akos Lukats, None
Support: OTKA 73000, TÁMOP-4.2.1.B-09/1KMR-2010-0001, TÁMOP-4.2.2/B10/1-2010-0013, OTKAF-61717
Program Number: 3942 Poster Board Number: D806
Presentation Time: 3:45 PM - 5:30 PM
Thyroid Hormone Dependent Differentiation of M/L-cones in Organotypic
Cultures of the Rat and Syrian Hamster Retina
Akos Lukats1, Arnold Szabo1, Viktoria Doma1, Gergely Halasz1, Attila Magyar1,
Gyorgy Vegvari2, Agoston Szel1. 1Human Morphology & Dev Biol, Semmelweis
University, Budapest, Hungary; 2Department of Fruit Science, Corvinus University,
Budapest, Hungary.
Purpose: Literature data indicate that thyroid hormones play an important role in
M/L-cone differentiation of the mouse. Little data exist however about cone
development in other species of mammals, mainly due to the lack of proper
methods. In a previous work we demonstrated the expression of TRβ2 thyroid
hormone receptor in the photoreceptors of the rat and the Syrian hamster. The aim
of the present study was to examine if the hormone indeed controls M/L-cone
development in these rodent species.
Methods: Retinas of early postnatal (P0-P4) Sprague-Dawley rats and Syrian
golden hamsters were explanted onto semiporous membranes, and kept in culture
for 14-28 days. Culturing media contained a 1:1 mixture of DMEM and F12,
supplemented with vitamins, amino acids and hormones, with or without serum
(FCS, 10%) added. Thyroid hormone (T3) was added to, or omitted from the
cultures. After fixation, general retinal morphology was compared on radial
sections and opsin expression patterns were analyzed using immunocytochemistry
and PCR.
Results: The retina of both species exhibited a near normal differentiation pattern
in control cultures, with M/L-cone densities and morphology comparable to that
observed in vivo, even without serum added to the medium. The structure was also
maintained under prolonged culturing conditions. Withdrawal of T3 from the media
affected only the cultures lacking serum substitution, indicating that serum alone
contains enough thyroid hormone to allow full differentiation. The lack of hormone
under serum free conditions significantly altered the staining pattern and number of
M/L-opsin expressing cells. Even after 15 days in vitro, labeling with monoclonal
probe completely disappeared, and the number of elements stained by polyclonal
antiserum decreased significantly. The change was more prominent after 3 or 4
weeks in culture, but M/L-opsin expression could still be detected in all cultures
using PCR reactions.
Conclusions: Our results show that thyroid hormone plays an important role in
M/L-cone differentiation also in the two rodent species reported, and indicate that it
may be a universal regulator in mammals. The organotypic culturing technique
enables us to study potential factors influencing photoreceptor development, and to
point out similarities and differences in retinal maturation between species.
Commercial Relationships: Akos Lukats, None; Arnold Szabo, None; Viktoria
Doma, None; Gergely Halasz, None; Attila Magyar, None; Gyorgy Vegvari,
None; Agoston Szel, None
Support: OTKA 73000, OTKA F61717, TAMOP-4.2.1.B-09/1KMR-2010-0001
Program Number: 3943 Poster Board Number: D807
Presentation Time: 3:45 PM - 5:30 PM
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
NRL Localizes to Discrete Regions of Euchromatin in Rod Photoreceptors of
Developing and Adult Mouse Retina
Awais Zia, Jacob Nellissery, Jerome Roger, Anand Swaroop, Tiansen Li. NNRL,
National Eye Institute, NIH, Bethesda, MD.
Purpose: NRL (neural retina leucine zipper) is a key transcription factor that
regulates rod photoreceptor cell fate and homeostasis. Mutations in NRL are
associated with retinal degeneration and enhanced S cone syndrome. Previous
studies reported NRL localization in human rod photoreceptors, but investigations
in mice have been lacking due to technical difficulties and appropriate antibodies
for use in mouse immunohistochemistry (IHC). The goal of these studies is to
generate and evaluate anti-NRL antibodies and perform subcellular localization in
developing and mature mouse retina.
Methods: Antibodies were raised against recombinant mouse NRL (aa23-130).
IHC was performed using different processing conditions. We used wild-type
(WT), Nrl-knockout and two mouse transgenic lines - one carrying a GFP reporter
(Nrl-GFP) and the other carrying mNrl (Nrl-mNrl) under the control of 2.5 kb
mouse Nrl promoter.
Results: Immunoblot analysis identified a 28 kDa NRL protein in WT but not in
Nrl-knockout retina. In adult WT retina, Nrl localizes to peripheral region of rod
nuclei. Co-labeling of Nrl with euchromatin marker H3K4Me3 shows that Nrl
associates exclusively with euchromatin. Nrl immunostaining appears as discrete
puncta dispersed along the euchromatin. Double staining for Nrl with anti-RNA
Polymerase II reveals significant colocalization, suggesting that Nrl resides in
active transcription regions. Other photoreceptor transcription factors, including
CRX, OTX2, NR2E3, and ROR-beta, also colocalize with Nrl in these actively
transcribed regions. Co-labeling of Nrl with BrdU pulse in P0 retina shows that Nrl
is expressed in post-mitotic cells at least 4 hrs after the BrdU injection. In the NrlmNrl transgenic; Nrl-knockout retina, which express Nrl only in a small percentage
of cells and at lower than physiological levels, co-localization of Nrl with
rhodopsin but not S-opsin, and cone arrestin suggests that Nrl-positive cells commit
to rod phenotype and do not reside in a hybrid state. We also show that Nrl staining
pattern largely overlaps with GFP-positive cells in Nrl-GFP retina.
Conclusions: NRL associates exclusively with euchromatin of rod nuclei and
appears to reside in actively transcribed regions. We confirm that Nrl is expressed
in post-mitotic rods and that GFP expression in Nrl-GFP mice broadly reflects
native Nrl expression. We also demonstrate that Nrl expression in Nrl-knockout
cone photoreceptors can re-transform these cells to acquire rod phenotype.
Commercial Relationships: Awais Zia, None; Jacob Nellissery, None; Jerome
Roger, None; Anand Swaroop, None; Tiansen Li, None
Support: None
Immunocytochemical and Functional Analysis of TrkB Expression in the
Developing Cones of the Rat Retina
Arnold Szabo1A, Akos Lukats1A, Klaudia Szabo1A, Anna Enzsoly1B, Agoston Szel1A.
Dept. of Human Morphology and Developmental Biology, BDept. of
Ophthalmology, 1Semmelweis University, Budapest, Hungary.
Purpose: There is only controversial data in literature if photoreceptors express the
TrkB neurotrophin receptor. Some results indicate that the receptor is present in Mcones of the adult rat retina. The aim of the study was to examine whether cones
express TrkB receptor during development and to test if BDNF and NT-4 are
involved in cone differentiation.
Methods: Retinas of Sprague-Dawley rats were collected in different
developmental ages and analyzed by immunocytochemistry using cone- and TrkBspecific antibodies. For the functional studies, organotypic retina cultures allowing
M-cone development were prepared from P2 rats. In order to neutralize the
endogenous neurotrophins acting via the TrkB receptor, cultures were treated with
BDNF- or NT-4-specific antibodies continuously for 19 days. The cultures were
then processed histologically, and the M-cone densities of treated and non-treated
control retinas have been compared.
Results: The first M-opsin positive signals were detected at P10 in dual cones
expressing both the S- and M-opsin. The expression of TrkB receptor started at
P10, at the same time when the M-opsin appeared. In contrast to the adult, in which
only the mature M-cones expressed the receptor, in the developing retinas TrkB
receptor expression was detected in every cone that expressed M-opsin. Although
the majority of S-opsin positive cells were colocalized with the TrkB receptor
during development, some cones showed single S-opsin expression. The overall
retinal architecture was well preserved both in control and anti-NT-4 treated
cultures. Every retinal layer was well developed and no major differences were
found. Although the photoreceptor layer was comparable to that of the control and
anti-NT-4 treated retinas, anti-BDNF treatment resulted in a remarkable thinning of
the inner retina. The average M-cone density in the control cultures was ~4450
±870 cell/mm3. Neither the anti-BDNF nor the anti-NT-4 treatment effected Mcone density; similar values with no significant differences were calculated in both
treated groups.
Conclusions: The TrkB receptor is expressed in the developing, and later, in the
mature M-cones as well. Our results indicate that neurotrophins may be involved in
M-cone development and function, however, their role in the regulation of M-opsin
expression is unlikely.
Commercial Relationships: Arnold Szabo, None; Akos Lukats, None; Klaudia
Szabo, None; Anna Enzsoly, None; Agoston Szel, None
Support: OTKA-73000, OTKA F-61717, TAMOP-4.2.1.B-09/1KMR-2010-0001
Program Number: 3944 Poster Board Number: D808
Presentation Time: 3:45 PM - 5:30 PM
Synaptic Identity Of Photoreceptors And Their Connections With Bipolar
Neurons In Developing Retina Of Cone-only Nrl-/- and Rod-only Crxp-Nrl
Soo-Young Kim, Marie-Audrey Kautzmann, Robert Fariss, Wei Li, Tiziana
Cogliati, Anand Swaroop. Neurobiol-Neurodegnrtn & Repair, NEI, Bethesda, MD.
Purpose: Rod spherules and cone pedicles contain different number and size of
ribbons, and their synapses connect to distinct sets of bipolar neurons in the
discrete layers of the outer plexiform layer (OPL) in mammalian retina. With a goal
to understand the mechanism of circuit formation in OPL, we examined the
photoreceptor synapses, the connections with bipolar neurons and proper layering
in genetically modified cone-only (Nrl-/-) or rod-only (Crxp-Nrl) retinas.
Methods: In vivo electroporation was performed using Nrl promoter-driven EGFP
and S-opsin promoter-driven Td-Tomato plasmids to label rods and cones,
respectively. Whole mount retinas or vibratome-vertical sections were used for
immunohistochemistry with multiple synapse-specific antibodies.
Results: The size of Nrl-/- photoreceptor synaptic terminals increased during
postnatal development from P14 to P21, suggesting a progressive switch from rod
to cone terminal identity. Synaptic connections between Nrl-/- cones and rod
bipolar cells were less in number compared to wild type retina. Furthermore, the
lamination pattern in both Nrl-/- and Crxp-Nrl retinas was disrupted, with cone
bipolar dendritic arbors extending into the layer of rod bipolar dendritic arbors. The
analysis of rod-only retina also shows extensive changes and further quantification
is in progress.
Conclusions: Our findings support the hypothesis that intrinsic molecules in rods
and cones drive circuit-specificity between photoreceptors and second-order
Commercial Relationships: Soo-Young Kim, None; Marie-Audrey
Kautzmann, None; Robert Fariss, None; Wei Li, None; Tiziana Cogliati,
None; Anand Swaroop, None
Support: NEI intramural program No conflict of interest
Program Number: 3946 Poster Board Number: D810
Presentation Time: 3:45 PM - 5:30 PM
Stage and Gene Specific Signatures of H3K4me2 and H3K27me3 Accompany
Mammalian Retina Maturation in vivo
Evgenya Popova1A, Samuel Shao-Min Zhang1A, Xuming Xu2, Andrew DeWan2,
Josephine Hoh2, Colin J. Barnstable1B. ANeural & Behavioral Sciences, BNeural &
Behavioral Sciences, Hershey Eye Center, 1College of Medicine, Penn State Univ,
Hershey, PA; 2Department of Epidemiology and Public Health, Yale University,
New Haven, CT.
Purpose: Histone modifications are sensitive indicators, and possibly predictors, of
gene expression in developing tissues, but the complexity of the epigenetic code is
far from understood. In this study we have followed the distribution of H3K4me2
and H3K27me3 over the whole genome during mouse retina development and have
related modification patterns to specific classes of genes.
Methods: Animal use was in accordance with ARVO/IACUC guidelines.
Immunocytochemistry and biochemical studies were conducted with eyes collected
from C57BL/6j mice (Jackson Laboratory) at 4 developmental stages from E17 to
PN15. ChIP-Seq analysis of retinal chromatin was performed with antibodies
against H3K4me2 and H3K27me3. ChIP DNA was sequenced on the Illumina
Genome Analyzer. Data were analyzed with in-house program and NexGENe
Results: When visualized by immunocytochemistry H3K4me2 and H3K27me3
each showed changing cellular patterns of distribution during retina development
and a markedly different localization in the nuclei of rod photoreceptor cells. ChIPSeq analysis of H3K4me2 and H3K27me3 accumulation around the Transcription
Start Site (TSS) of genes uniquely expressed in rod photoreceptors showed a
dramatic de-novo increase of H3K4me2 around TSS after PN7 but complete
absence of H3K27me3 on the same genes throughout development. Other genes
upregulated in retina during development show a similar increase of H3K4me2
developmental accumulation at their TSS but a dramatic decrease in H3K27me3
during terminal differentiation. In addition to the specific patterns around the TSS,
H3K4me2 and H3K27me3 have gene specific distribution over the whole gene
body of tissue specific genes. Rod specific genes accumulate H3K4me2, but not
H3K27me3 over the whole gene whereas genes expressed in the retina non-rod
cells show extensive H3K27me3 accumulation over the gene in the mature retina.
Conclusions: We have identified unique epigenetic developmental signatures of
H3K4me2 and H3K27me3 around the TSS and on gene bodies that can distinguish
Program Number: 3945 Poster Board Number: D809
Presentation Time: 3:45 PM - 5:30 PM
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
cell type specific genes from genes expressed more widely. By comparing these
signatures for different types of genes with measured levels of expression, we
propose that different strategies are used to regulate chromatin structure around
different types of genes during development.
Commercial Relationships: Evgenya Popova, None; Samuel Shao-Min Zhang,
None; Xuming Xu, None; Andrew DeWan, None; Josephine Hoh, None; Colin
J. Barnstable, None
Support: NIH EY013865 and the Macula Vision Research Foundation
Program Number: 3947 Poster Board Number: D811
Presentation Time: 3:45 PM - 5:30 PM
Wnt Mediated Signaling From Surface Ectoderm Is Involved In RPE
Specification During Chick Eye Development
Joerg Steinfeld1, Ichie Ushijima2, Paul G. Layer1, Masasuke Araki2, Astrid VogelHoepker1. 1Developmental Biology and Neurogenetics, TU Darmstadt, Darmstadt,
Germany; 2Department of Biological Sciences, Nara Women`s University, Nara,
Purpose: The vertebrate eye forms through evagination of the optic vesicle from
the neuroepithelium of the prosencephalon. Extrinsic signals released from
surrounding tissues are involved in patterning the optic vesicle into a NR and RPE
domain. The current model of optic vesicle patterning assumes, that distally
Fibroblast Growth Factors (FGFs) released from the overlying surface ectoderm are
involved in NR specification, while proximally signals released from the ocular
mesenchyme are involved in RPE specification. However, our previous studies
suggested, that the chick optic vesicle is initially patterned into a ventral NR and
dorsal RPE domain. Bone morphogenetic proteins (BMPs) and FGFs are expressed
in the surface ectoderm and are involved in RPE and NR specification respectively.
These signals are initially expressed throughout the surface ectoderm, suggesting
that another signal might be involved in patterning the optic vesicle initially into a
dorsal RPE and ventral NR domain. Wnt signaling is required for dorsal patterning
of the neural tube, brain and limb. We therefore asked, whether WNTs might be
involved in patterning the dorsal optic vesicle and hence RPE development.
Methods: Gain- and Loss-of-function studies were carried out by implanting
protein soaked beads into the chick optic vesicle at stages 8 to 11. Effects were
analysed on a molecular level by immunohistochemistry using Mitf-Antibodies and
in situ hybridization for marker genes that distinguish the RPE and NR.
Results: During optic vesicle stages WNT-family members are expressed in the
dorsal ectoderm and in the presumptive RPE. Implantation of Wnt3a-soaked beads
into the optic vesicle converts the entire neuroepithelium of the optic vesicle,
including the presumptive optic stalk and neural retina region, into a single-layered
and strongly pigmented RPE. Moreover, these cells express the two key
transcription factors involved in RPE specification and differentiation, Mitf and
Otx2. In contrast, NR-specific genes are downregulated following WNT exposure.
On the other hand, interferring with WNT signaling results in the downregulation
of Mitf expression.
Conclusions: We propose a new model, where the optic vesicle is initially
patterned into a dorsal RPE domain by both BMP and WNT signals released from
the overlying ectoderm, while ventrally BMPs and FGFs are involved in NR
Commercial Relationships: Joerg Steinfeld, None; Ichie Ushijima, None; Paul
G. Layer, None; Masasuke Araki, None; Astrid Vogel-Hoepker, None
Support: DFG-AOBJ573797
Program Number: 3948 Poster Board Number: D812
Presentation Time: 3:45 PM - 5:30 PM
Retinal Ganglion Cell-specific Transcription Factors POU4F2 and ISL2 are
Positive Regulators of the Axonal-guidance Gene EphA5
Krista M. Beach1, Kuichun C. Zhu2, Steven W. Wang3, Takae Kiyama3, Deborah C.
Otteson1. 1Optometry, University of Houston, Houston, TX; 2Biologics Discovery,
HD Biosciences, Shanghai, China; 3Ophthalmology & Visual Science, University
of Texas Medical School, Houston, TX.
Purpose: EPHA5 is an ephrin receptor that patterns the retinotopic maps of retinal
ganglion cell (RGC) axons in the visual system. In the mouse retina, EphA5 mRNA
is expressed in a temporal>nasal gradient in RGCs. However, the mechanisms
regulating EphA5 expression are not well understood. This study addresses the role
of ganglion cell-specific transcription factors in regulating EphA5 expression in the
Methods: Transcription factor binding sites were identified by bioinformatics
using TESS and Genomatix software. Effects of overexpressing Pou4f2, Isl2, or
Zic2 on the mouse EphA5 promoter activity were tested using dual luciferase
assays. Expression of EPHA5 protein was assessed by immunofluorescence in
histological sections from C57BL/6, B6(Cg)-Tyrc-2J/J, and Pou4f2 null mice (P0.5).
Results: Potential binding sites for POU4F, ISL and ZIC transcription factors were
identified in the mouse EphA5 proximal promoter. In luciferase assays, deletion of
regions containing POU4F and/or ISL binding sites decreased EphA5 promoter
activity. In contrast, deletion of regions containing ZIC binding sites increased
EphA5 promoter activity. Co-transfection of Pou4f2 or Isl2 activated and Zic2
repressed EphA5 promoter activity. In C57BL/6 (pigmented) and B6(Cg)-Tyrc-2J/J
(unpigmented) mice at P0.5, EPHA5 protein was highly expressed in the ganglion
cell layer (GCL) with only subtle differences between nasal and temporal retina. In
Pou4F2 null mice on a B6(Cg)-Tyrc-2J/J background, EPHA5 immunoreactivity in
the nasal retina was greatly reduced despite a large number of cells remaining in the
GCL. In contrast, EPHA5 immunoreactivity in the temporal retina was robust,
resulting in a clear temporal to nasal gradient.
Conclusions: POU4F2 and ISL2 are transcriptional activators, and ZIC2 is a
repressor, of the EphA5 promoter in vitro. Persistence of EPHA5 expression in
Pou4F2 null retinas indicates that additional factors compensate for loss of
POU4F2. The nasal/temporal differences in EPHA5 expression in Pou4F2 null
mice could reflect different requirements for POU4F2 in nasal vs. temporal retina
or a previously unappreciated topographic difference in RGC loss in these mice.
Commercial Relationships: Krista M. Beach, None; Kuichun C. Zhu,
None; Steven W. Wang, None; Takae Kiyama, None; Deborah C. Otteson,
Support: Thomas R. Lee Award - National Glaucoma Research from AHAF, NIH
R01EY021792 (DCO); UH sVGR Grant (KMB); NIH EY018352, E. Matilda
Ziegler Foundation for the Blind (SWW); NIH P30EY07551 (core, UHCO)
Program Number: 3949 Poster Board Number: D813
Presentation Time: 3:45 PM - 5:30 PM
Development Of Retinal Pigment Epithelium From Human Parthenogenetic
Embryonic Stem Cells And Microrna Signature
Xiaorong Li1A, Wenbo Li1A, Zhenyu Lu2,3, Yunshan Zhang2, Lijie Dong1, Fei E.
Wang1, Rong Dong2. ARetina, 1Tianjin Medical Univ Eye Center, Tianjin, China;
Center for Reproductive Medicine, Tianjin Central Hospital for Obstetrics and
Gynecology, Tianjin, China; 3Union Stem Cell &Gene Engineering Co., Ltd.,
Tianjin, China.
Purpose: To investigate the potential of human parthenogenetic embryonic stem
cells (hPESCs) to differentiate into retinal pigment epithelium (RPE) cells, and
identify development-regulating microRNAs (miRNAs).
Methods: RPE cells were drived from hPESCs. The expression of markers,
miRNA expression profiles and positional information of RPE ‘Signature’ miRNAs
during differentiation were studied by using real-time RT-PCR, western blot and
miRNA expression array at three time points. Human fetal RPE (hfRPE) cells were
also analyzed. Then target genes of candidate miRNAs were validated.
Results: RPE cells can be derived from hPESCs with an efficiency similar to
human embryonic stem cells (hESCs). hPESCs-derived RPE cells exhibited similar
morphology and pigmentation to hfRPE cells. However, the expression of markers
(Oct4, Pax6, ZO-1, RPE65, MERTK) during retinal differentiation indicated that
hPESCs-derived RPE cells were in an immature state. Most specific miRNAs
played a role at some point in differentiation and maturation of RPE from hPESCs,
with the exception of only two miRNAs (miR-204 and the miR-302s family). miR204 showed an up-regulation, and miR-302 showed a down-regulation throughout
the process. CTNNBIP1 and TGFBR2 were confirmed to be the target genes of
miR-204 and miR-302 respectively, involved in mediating the activation of the
Meis2/Pax6, Wnt/beta-catenin and TGFβ/Nodal pathways.
Conclusions: hPESCs can develop into RPE-like cells, which provides an
additional promising source for RPE cells in cell therapy. miR-204, 302s and their
targets are involved in regulating directed differentiation during the full course,
which contribute to the search for a new method of improving the differentiation
efficiency using miRNAs.
Commercial Relationships: Xiaorong Li, None; Wenbo Li, None; Zhenyu Lu,
None; Yunshan Zhang, None; Lijie Dong, None; Fei E. Wang, None; Rong
Dong, None
Support: National Natural Science Foundation of China (No. 30973255)
Program Number: 3950 Poster Board Number: D814
Presentation Time: 3:45 PM - 5:30 PM
Roles For b2 And y3 Laminins In The Development Of Retinal Ganglion Cells
Lyl G. Tomlinson1, Shweta Varshney1, Elizabeth Chu1, William J. Brunken1,2.
Ophthalmology and Cell Biology, SUNY Downstate Medical Center, Brooklyn,
NY; 2SUNY Eye Institute, Brooklyn, NY.
Purpose: The β2 and y3 chains of the heterotrimeric molecule, laminin, are major
constituents of the retinal inner limiting membrane (ILM). The objective of this
study is to evaluate the loss of Lamb2 and Lamc3 genes on developing retinal
ganglion cells (GCs).
Methods: Axonal trajectories and mosaics of GCs in wholemount retinas (WM)
were assayed in wild type (WT) and Lamb2, Lamc3 and Lamb2;c3 (DKO)
knockout animals using immunohistochemistry (IHC). GC dendrite length, soma
size and surface area were studied in retinas from Thy1.1-EYFP mice.
Results: To study the effect of laminin chains on GC development, the morphology
of GCs was assayed in WT, Lamb2, Lamc3 and DKO mice. Axonal trajectories to
the optic nerve were assessed in P5 and P10 WM using a neurotubulin antibody. At
both time points, Lamb2 and DKO WM exhibit axon bundles with eccentric
curvature; this contrasts with the linear projections in WT and Lamc3 retinas; P0
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
animals are under study. Mice expressing EYFP controlled by the Thy1.1 promoter
were used to study a subset of GCs. P15 WT and DKO WM were assessed for
dendrite length, soma size and surface area. There was a shift to smaller average
dendrite length distributions in the DKO relative to WT. Additionally, peak GC
dendrite length in the DKO was shorter: 110µm (DKO) vs 140µm (WT). Soma size
and distribution also differed between the two: WT somas were between 15-30µm
and evenly distributed, whereas in DKO, somas were smaller and unevenly spread.
In addition, the area of GC dendritic arbors were assayed. WT exhibited a normal
Gaussian distribution of 5,000-40,0000 µm2; GCs in DKO had a tighter
distribution in the range of 5,000-10,000 µm2. Next we examined a single type of
GC; melanopsin IHC was used to study the mosaic of the intrinsically
photosensitive GCs (ipGCs). Two types of ipGCs, M1 and M2, were seen with
arborizations in the outer and inner IPL, respectively. The distribution of the
distances of these cells to their nearest homotypic neighbor were measured in P21
WT and Lamb2 groups. M1 cells in WT exhibited a normal Gaussian distribution
of cell distances with a peak spacing of 120-160µm; peak spacing of M1 cells in
Lamb2 was 80-120µm. M2 cells spacing in the WT was 40-80µm and 0-40µm in
Lamb2 retinas.
Conclusions: These results show that absence of laminin β2 and y3 adversely
affect the morphology and spacing of GCs.
Commercial Relationships: Lyl G. Tomlinson, None; Shweta Varshney,
None; Elizabeth Chu, None; William J. Brunken, None
Support: NIH Grant EY12676
Program Number: 3951 Poster Board Number: D815
Presentation Time: 3:45 PM - 5:30 PM
Linc Complexes Mediate The Apical Positioning Of Mouse Cone
Photoreceptor Nuclei
Didier M. Hodzic, David Razafsky. Ophthalmology, Washington University School
of Medicine, St Louis, MO.
Purpose: Linkers of the Nucleoskeleton to the Cytoskeleton (LINC complexes)
designate evolutionary-conserved macromolecular complexes that span the nuclear
envelope and physically connect the nuclear interior to cytoskeletal networks and
molecular motors. They form through direct interactions between inner nuclear
membrane Sun proteins (Sun1 and Sun2) and outer nuclear membrane Nesprins
(Nesprin 1, 2, 3 and 4) within the perinuclear space. In C.elegans and D.
melanogaster, mutation of Sun and Nesprin orthologs prevent nuclear anchorage
and/or migration within cells or syncitia. Mouse models deficient for both Sun1 and
Sun2 expression display abnormal migration of cortical neurons. Our goal is to
identify the role of LINC complexes during mammalian CNS tissue development
and homeostasis. To this end, the mouse retina offers several advantages because 1)
different types of nucleokinetic events such as interkinetic nuclear migration and
postmitotic neuronal migration can be targeted in vivo without affecting other CNS
tissues, 2) the retina consists of a laminated tissue where nuclei of various cell
types are anchored at specific spatial positions and 3) retinal neurons are amenable
to functional testing thereby allowing for functional analyses in vivo.
Methods: Using immunofluorescence microscopy, we characterized the expression
of LINC complex components during mouse retinal development. We further
developed a new versatile transgenic mouse model allowing for the conditional
disruption of LINC complexes in retinal neurons.
Results: We report the ectopic positioning of cone photoreceptor nuclei upon
LINC complex disruption. These results demonstrate the possibility to target the
disruption of LINC complexes in vivo in a cell type-specific manner and further
emphasize the relevance of Sun proteins and Nesprins in the positioning of cone
photoreceptor nuclei.
Conclusions: Our mouse model will allow the analysis of the contribution of LINC
complexes to nuclear positioning within additional retinal neurons and as well as in
the molecular mechanisms that govern the development of the laminar organization
of the mammalian retina.
Commercial Relationships: Didier M. Hodzic, None; David Razafsky, None
Support: NIH Training Grant (T32 EY013360 to DR), Research to Prevent
Blindness Inc. Unrestricted grant, NIH Vision Core Grant P30 EY002687
Program Number: 3952 Poster Board Number: D816
Presentation Time: 3:45 PM - 5:30 PM
RAX Homeoprotein and NOTCH-HES Signaling Regulate Otx2 Expression in
Embryonic Retinal Photoreceptor Cell Fate Determination
Takahisa Furukawa1, Yuki Muranishi1, Koji Terada1, Tatsuya Inoue1,2, Kimiko
Katoh1, Toshinori Tsujii1, Rikako Sanuki1, Yasuhiro Tamaki2. 1Developmental
Biology, Osaka Bioscience Institute & JST, CREST, Suita, Osaka, Japan;
Ophthalmology, Tokyo University School of Medicine, Tokyo, Japan.
Purpose: The molecular mechanisms underlying cell fate determination from
common progenitors in the vertebrate central nervous system remain elusive. We
previously reported that the OTX2 homeoprotein regulates retinal photoreceptor
cell fate determination. While Otx2 transactivation is a pivotal process for
photoreceptor cell fate determination, its transactivation mechanism in the retina is
unknown. We investigated the transactivation mechanism of Otx2 in retinal
photoreceptor precursors.
Methods: To identify the mouse Otx2 regulatory locus directing expression in the
embryonic photoreceptor precursors, we first analyzed two transgenic mouse lines
harboring bacterial artificial chromosomes (BAC), containing the mouse Otx2 gene
inserted with a Beta-galactosidase (LacZ) reporter. We examined LacZ expression
which was detected by X-gal staining, in the developing retina of transgenic lines at
embryonic day 13.5 (EI3.5), when mouse Otx2 expression becomes obvious in the
developing photoreceptors. We then narrowed down the critical region for the Otx2
regulatory activity by dividing the regulatory region identified in BAC transgenic
mouse analysis.
Results: We first found that OTX2 expression begins mainly in the final cell cycle
of RPCs. We analyzed the regulatory region of the Otx2 gene during embryonic
stages when Otx2 transcripts are distinctly expressed in the presumptive
photoreceptor layer in contrast to postnatal stages when Otx2 expression shifts to
the bipolar cell layer. We then identified an approximately 500 bp cis-regulatory
region we called embryonic enhancer locus for photoreceptor Otx2 transcription
(EELPOT) that can recapitulate initial Otx2 transcription in early developing
photoreceptors. We found that the RAX homeoprotein interacts with EELPOT to
transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional
inactivation of Rax results in down-regulation of Otx2 expression in vivo. We also
found that EELPOT is negatively regulated by the HES family of molecules, which
are bHLH transcription repressors.
Conclusions: Our results suggest that the integrated activity of cell intrinsic and
extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell
fate determination in the embryonic retina.
Commercial Relationships: Takahisa Furukawa, None; Yuki Muranishi,
None; Koji Terada, None; Tatsuya Inoue, None; Kimiko Katoh,
None; Toshinori Tsujii, None; Rikako Sanuki, None; Yasuhiro Tamaki, None
Support: CREST and PRESTO from Japan Science and Technology Agency,
Grant-in-Aid for Scientific Research
Program Number: 3953 Poster Board Number: D817
Presentation Time: 3:45 PM - 5:30 PM
Overexpression Of The Cilia Protein RPGRIPL1 Rescues Rhodopsin
Expression In cux1b Morphant Zebrafish
Pamela R. Pretorius, Julia M. Hatler, Elizabeth Speltz, Ashley M. Spahn, Stephanie
L. Lerach, Lisa A. Schimmenti. Department of Pediatrics, University of Minnesota,
Minneapolis, MN.
Purpose: During embryogenesis, proper eye development is critical for vision
function. Expression of the gene PAX2, is critical for normal ocular morphogenesis
in animal models and dominant mutations in PAX2 result in renal coloboma
syndrome. Previous studies in Drosophilia identified sparkling (spa) as a pax2
ortholog, spa mutants have abnormal eye development and reduced expression of
the DNA binding protein cut. This observation led us to hypothesize that cux1b acts
downstream of pax2 during early eye development, making it a good candidate to
investigate for involvement in vertebrate retinal development.
Methods: The genetic and functional similarities to the mammalian eye make
zebrafish (Danio rerio) an ideal model to study early vertebrate eye development.
To evaluate the functional role of cux1b in retinal development, a Morpholino
antisense oligonucleotide was utilized to knockdown expression in the zebrafish.
Immunohistochemistry was used to assess cux1b morphant and rescued
Results: Microinjection of a morpholino against cux1b into zebrafish embryos
results in a reduction of the overall eye size at 48hpf, with an absence of growth
between 48hpf and 72hpf. Additionally, an increase in proliferation is observed in
the 72hpf retina of cux1b morphant embryos. Although the overall eye size is
smaller, the retina is fully laminated and the optic chiasm forms. While
photoreceptors do form, the outer segments fail to fully develop and have reduced
rhodopsin expression. Overexpression of the ciliary protein RPGRIPl1 in cux1b
knockdown embryos normalizes eye size and partially restores rhodopsin
expression throughout the developing retina. Moreover, in cux1b morphants, green
opsin expression was not restricted to the photoreceptor outer segment, but was
also found in the cell body.
Conclusions: These data provide strong evidence that cux1b is required for proper
retinal development. Given the rescue of rhodopsin localization following
RPGRIPL1 overexpression, it seems likely that cux1b functions in cilia
development and/or maintenance in the retina.
Commercial Relationships: Pamela R. Pretorius, None; Julia M. Hatler,
None; Elizabeth Speltz, None; Ashley M. Spahn, None; Stephanie L. Lerach,
None; Lisa A. Schimmenti, None
Support: NIH NEI/5R01EY019267-02
Program Number: 3954 Poster Board Number: D818
Presentation Time: 3:45 PM - 5:30 PM
Neuronal Programmed Cell Death-1 Ligand Expression Regulates Retinal
Ganglion Cell Number in Neonatal and Adult Mice
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Caroline W. Sham1A,2, Ann M. Chan1A, Jacky Man Kwong Kwong1A, Joseph
Caprioli1B,1A, Steven Nusinowitz1A, Ling Chen3, Jonathan Braun2, Lynn K.
Gordon1A,4. AOphthalmology, BGlaucoma, 1Jules Stein Eye Institute, UCLA, Los
Angeles, CA; 2Pathology, David Geffen School of Medicine, UCLA, Los Angeles,
CA; 3Ophthalmology, Eye and ENT Hospital, Shanghai Medical School, Shanghai,
China; 4Ophthalmology Section, Greater Los Angeles Veterans Affairs Healthcare
System, Los Angeles, CA.
Purpose: During mouse retina maturation the final number of retinal ganglion cells
(RGCs) is determined by highly regulated programmed cell death. Previous studies
demonstrated that the immunoregulatory receptor programmed cell death-1 (PD-1)
promotes developmental RGC death. To identify the functional signaling partner(s)
for PD-1, we identified retinal expression of PD-1 ligands and examined the effect
of PD-1 ligand expression on RGC number. We also explored the hypothesis that
PD-1 signaling promotes development of functional visual circuitry.
Methods: Characterization of retinal and brain PD-L1 expression were examined
by immunofluorescence on tissue sections. The contribution of PD-ligands to RGC
number was examined in PD-ligand knockout mice. Retinal architecture was
assessed by spectral domain optical coherence tomography and retinal function was
analyzed by electroretinography in WT and PD-L1/L2 double deficient mice.
Results: PD-L1 expression is found throughout the neonatal retina and persists in
adult RGCs, bipolar interneurons, and Müller glia. In the absence of both PDligands, there is a significant numerical increase in RGCs (34% at P2, 18% in
adult), as compared to wild type, and PD-ligands have redundant function in this
process. Despite the increased RGC number, adult PD-L1/L2 double knockout
mice have normal retinal architecture and outer retina function.
Conclusions: This work demonstrates that PD-L1 and PD-L2 together impact the
final number of RGCs in adult mice and supports a novel role for active promotion
of neuronal cell death through PD-1 receptor-ligand engagement.
Commercial Relationships: Caroline W. Sham, None; Ann M. Chan,
None; Jacky Man Kwong Kwong, None; Joseph Caprioli, None; Steven
Nusinowitz, None; Ling Chen, None; Jonathan Braun, None; Lynn K. Gordon,
Support: NIH T32 GM08042; NIH/NIAID R01 AI021256; NIH/NEI T32
Program Number: 3955 Poster Board Number: D819
Presentation Time: 3:45 PM - 5:30 PM
Disrupted Circuitry In The Outer Retina Following Genetic Removal Of
Horizontal Cells
Patrick W. Keeley1A,1B, Kimberly A. Skyles1A, Mary A. Raven1A,1B, Ross A. Poche2,
Benjamin E. Reese1A,1C. ANeuroscience Research Institute, BMolecular, Cellular,
and Developmental Biology, CPsychological and Brain Sciences, 1University of
California, Santa Barbara, CA; 2Molecular Physiology and Biophysics, Baylor
College of Medicine, Houston, TX.
Purpose: To determine the role of horizontal cells upon the development of outer
retinal circuitry, we have analyzed the Lim1 conditional knockout (CKO) mouse
retina. Lim1 is expressed specifically in horizontal cells, and those lacking Lim1
fail to migrate to their correct stratum or to differentiate processes in the outer
retina, becoming ectopically positioned in the inner retina. The effect of this partial
depletion of horizontal cells upon the organization of the outer plexiform layer
(OPL) was examined.
Methods: Floxed Lim1 mice were crossed with Rx-Cre mice to create Lim1 retinaspecific CKO mice. Immunofluorescence and confocal microscopy were used to
examine the OPL in mature and developing mice, with antibodies to synaptic
markers, neurofilaments, calbindin, and cone arrestin as well as fluorescently
tagged PNA. Lim1 CKO mice were also crossed with Gustducin-GFP mice in order
to assess the dendritic morphology of single bipolar cells labeled with DiI.
Results: Lim1 CKO retinas showed a conspicuous decrease in the density of
horizontal cell processes and a corresponding collapse of the OPL. Despite a
conserved thickness of the outer nuclear layer (ONL), the OPL showed a large
reduction in the density of synaptic ribbons. This decrease coincided with sprouting
by rod bipolar cell dendrites into the ONL, where ectopic ribbon markers were
frequently detected. Cone pedicles displayed proper stratification in the OPL and
contained synaptic ribbons, but were less regularly distributed; additionally, the
area of their active zones was on average smaller, though more variable. Type 7
cone bipolar cells correctly targeted these pedicles but formed smaller dendritic
arbors with fewer terminal endings. Developmental analysis confirmed that
horizontal cells are never present in the OPL of the CKO retina; rod spherules and
rod bipolar dendrites, however, were properly stratified early in postnatal
development, and a normal complement of ribbon synapses was detected. Cone
pedicle distribution within the OPL appeared disrupted as early as postnatal day 9.
Conclusions: Horizontal cells are not required for the initial targeting and
synaptogenesis of the remaining OPL components; they are critical, however, for
the maintenance of normal photoreceptor-bipolar cell connectivity, although
different aspects of each circuit are disrupted in their absence.
Commercial Relationships: Patrick W. Keeley, None; Kimberly A. Skyles,
None; Mary A. Raven, None; Ross A. Poche, None; Benjamin E. Reese, None
Support: NIH Grant EY019968
Program Number: 3956 Poster Board Number: D820
Presentation Time: 3:45 PM - 5:30 PM
PTP-Meg2 - A New Functional Player During Early Retinogenesis?
Jacqueline Reinhard1, Andrea Horvat-Bröcker1, Tina Paech2, Yingchun Wang3,
Bernd Denecke4, Pjotr Knyazev5, Axel Ullrich5, Gregory Downey3, Andreas
Faissner1. 1Department of Cell Morphology and Molecular Neurobiology, RuhrUniversity Bochum, Bochum, Germany; 2Department of Neuroanatomy, IZN,
University of Heidelberg, Heidelberg, Germany; 3Department of Medicine,
University of Toronto, Toronto, ON, Canada; 4Interdisciplinary Centre Clinical
Research (IZKF) Aachen, RWTH Aachen University, Aachen, Germany;
Department of Molecular Biology, Max-Planck-Institute of Biochemistry,
Martinsried, Germany.
Purpose: The developing retina serves as one of the best characterized model
systems to investigate neuronal development. In this context, the phosphorylation
and dephosphorylation of proteins on tyrosine residues represents a key mechanism
for the control of numerous processes, including cell proliferation, differentiation,
migration and maturation. In order to investigate the potential functional role of the
protein tyrosine phosphatase (PTP) Meg2 during retinal development, we
performed studies in PTP-Meg2 knockout (KO) mice (Wang et al., 2005).
Methods: Histological studies, including immunohistochemistry and in situhybridization, as well as molecular biological techniques such as RT-PCR and
microarray analyses were performed to investigate retinal development of PTPMeg2 KO animals.
Results: In order to elucidate the functional role of PTP-Meg2 during
retinogenesis, we analyzed PTP-Meg2 deficient animals. We demonstrate that PTPMeg2 deficient mice exhibit a “small-eye phenotype”, a hypocellular retina and a
reduced proliferation capacity. In addition, in the PTP-Meg2 deficient retina
amacrine cell differentiation was inhibited. In contrast, we present evidence that
progenitors of PTP-Meg2 KO animals adopt bipolar cell properties. Most
interestingly, based on microarray analysis using retinae of newborn PTP-Meg2
KO and wildtype (WT) control littermates, we verified a dysregulation of several
genes highly associated with early synaptogenesis.
Conclusions: In conclusion, our results suggest that PTP-Meg2 plays different
roles, at defined time points of retinal development. Our data provide an important
basis for future studies regarding the functional importance of PTP-Meg2 during
retinal and CNS development.
Commercial Relationships: Jacqueline Reinhard, None; Andrea HorvatBröcker, None; Tina Paech, None; Yingchun Wang, None; Bernd Denecke,
None; Pjotr Knyazev, None; Axel Ullrich, None; Gregory Downey,
None; Andreas Faissner, None
Support: German Research Council (DFG, SFB 509/TPA10 and Fa 159/14-1);
Ruhr-University Research School funded by Germany`s Excellence Initiative (DFG
GSC 98/1)
Program Number: 3957 Poster Board Number: D821
Presentation Time: 3:45 PM - 5:30 PM
Examination of retinal cell type degeneration in embryonic Smoky Joe
Thanh T. Tran1, Gregoy Y. Bedecarrats2, Vivian Choh3. 1Optometry, Univ of
Waterloo Sch of Optometry, Waterloo, ON, Canada; 2Poultry and Animal Science,
University of Guelph, Guelph, ON, Canada; 3School of Optometry, University of
Waterloo, Waterloo, ON, Canada.
Purpose: A genetic mutation in the pigmented White-Leghorn strain of chicken
called Smoky Joe (first describe Salter et al, 1997:J Vet Diagn Invest,9: 407-9),
causes inherited ocular anomalies showing varying levels of retinal degeneration
and blindness at hatch. By 8 weeks post-hatch all homozygous birds are completely
blind. The purpose of this study is to determine the characteristics of retinal cell
degeneration in embryonic Smoky Joe (SJ) chickens.
Methods: Embryos were obtained at 5 time points: embryonic day 4 (E4), E6, E8,
E14, and E18. Parent phenotypes and genotypes were used to predict sight and
blindness of embryos. Entire embryos, entire globes or eyeycups were processed
for sectioning. Immunohistochemistry using cell type-specific markers (Ap2α,
Lim1+2, Brn3a, protein kinase Cα, glutamine synthetase, visinin, lectin wheat germ
agglutinin) was used to label cells of interest (amacrine, horizontal, ganglion,
bipolar, Müller, cone and rod cells, respectively), while DAPI was used to
counterstain cell nuclei. Deconvolution microscopy-imaged retinas were obtained
and the various cell types in the neuroblastic (outer neuroblastic [ONBL] and inner
neuroblastic [INBL]) layers or nuclear (ganglion cell [GCL], inner nuclear [INL],
and outer nuclear [ONL]) layers were counted.
Results: Comparisons of the total means of cells showed significantly lower
numbers in blind SJ embryos than sighted across all time points (p<0.0001; E4,
blind vs sighted: 19555 ± 9347 vs 211019 ± 14285 cells/mm²; E18, blind vs
sighted: 309157 ± 4,075 vs 384986 ± 18651 cells/mm²). Nuclear layers were
formed at E14, and by E18 were clearly distinguishable in both blind and sighted.
No differences in cell numbers were observed in the ONL or GCL across all time
points but starting at E14, the number of cells in the INL was significantly lower in
blind embryos (p=0.0047: blind vs sighted: 235926 ± 18162 vs 280185 ± 12534
cells/mm²). The percentages of amacrine, bipolar, and ganglion cells were on
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
average slightly lower in blind embryos compared to sighted but no significant
differences in numbers of any retinal cell types were detected across all time points
(p>0.0958). Interestingly, amacrine cells developed at a later stage (E14) in blind
embryos than in sighted.
Conclusions: Blind SJ embryos have significantly less cells during development
and the INL was the site for significant cell loss. Percentages of the different retinal
cells were lower in blind embryos suggesting incomplete development or possibly
degeneration of these cells. This work is consistent with the finding that cell
numbers in the INL are lower at hatch in blind birds than in sighted birds (Tran et
al, 2010: ARVO E-Abstract 1817). It remains to be determined how these cells are
affected in post-hatch birds.
Commercial Relationships: Thanh T. Tran, None; Gregoy Y. Bedecarrats,
None; Vivian Choh, None
Support: NSERC
Program Number: 3958 Poster Board Number: D822
Presentation Time: 3:45 PM - 5:30 PM
Retinal Developmental Defects In The good effortnt2 Mutant Zebrafish Are
Correlated With Elevated Cell Death
Travis J. Bailey, Catherine M. Groden, Kristin M. Ackerman, David R. Hyde.
Biological Sciences, University of Notre Dame, Notre Dame, IN.
Purpose: In a screen to identify mutations resulting in eye defects, we found the
good effortnt2 (gef) mutant. The retina of gef embryos is characterized by the
successful initiation of the optic primordium and normal retinal development over
the first two days post fertilization. The mutant retina, however, fails to continue to
grow. This study further characterized the gef mutation to gain a molecular
understanding of cause of the gef phenotype.
Methods: Meiotic mapping was used to localize the mutation interval. Embryos
from gef heterozygous incrosses were analyzed for cell death by acridine orange
and by TUNEL labeling at 2 days post fertilization. Genes found in the linkage
interval were analyzed by quantitative real-time PCR and in situ hybridization.
One-cell embryos were injected with morpholino oligonucleotides against the
candidate gene with or without in vitro transcribed mRNA of the human
orthologous gene to either mimic or rescue the gef mutant phenotype, respectively.
Results: The number of dying cells in gef mutant embryos at 2 days post
fertilization that labeled with TUNEL and acridine orange was significantly greater
than wild type embryos. This time was earlier than any observable gross
morphological differences, which suggested that this cell death led to the gross
morphological defects. Meiotic linkage mapping found a one-megabase interval on
chromosome 9 that contained eleven potential genes. Of these eleven genes, only
itgbl1 transcripts were found at reduced levels in acridine orange labeled embryos
at 2 days post fertilization. The expression of itgbl1 was detected in the brain and
retina with weak or no expression in the lens. High itgbl1 expression was also
found in the developing meninges and vasculature cells. The gef mutants also
exhibited reduced circulation relative to wild-type siblings. Morpholino inhibition
of Itgbl1 protein expression in wild-type embryos phenocopied the gef mutant and
this phenotype was partially rescued by injection of in vitro transcribed human
Conclusions: The failure of the gef mutant retina to increase in size was due to
elevated retinal cell death, which resulted from decreased levels of itgbl1 and
abnormal vasculature. This suggests that the requirement for Itgbl1 activity in
proper zebrafish retina development might be due to effects on patterning of the
periocular and brain vasculature. This will be further examined using a variety of
zebrafish mutants and transgenic lines.
Commercial Relationships: Travis J. Bailey, None; Catherine M. Groden,
None; Kristin M. Ackerman, None; David R. Hyde, None
Support: None
Program Number: 3959 Poster Board Number: D823
Presentation Time: 3:45 PM - 5:30 PM
Olfactomedin 1 Is A New Novel Ligand Of Nogo Receptor 1 In The Retinal
Ganglion Cells
Naoki Nakaya1, Afia Sultana1A, Stanislav I. Tomarev1A. ALRCMB/MMGS,
National Eye Institute, Bethesda, MD.
Purpose: Olfactomedin 1 (Olfm1), also known as noelin and pancortin, is a
secreted glycoprotein that is highly conserved in vertebrates. The functions of
Olfm1 are largely unknown. Here, we investigated the expression pattern of the
Olfm1 protein in the mouse retina and looked for membrane receptor proteins
interacting with Olfm1.
Methods: The distribution of Olfm1 and several retinal markers was investigated
by immunofluorescent labeling using a confocal laser microscope. COS7 cells were
transfected with plasmids encoding candidate membrane proteins and treated with
conditioned medium produced by another batch of COS7 cells transiently
transfected with the Olfm1-alkaline phosphatase (AP) fusion construct. After
incubation at 4°C, cells were washed and bound Olfm1-AP was detected by adding
AP substrate solution. Co-immunoprecipitation and immunofluorescence colocalization analyses were also used to confirm binding of Olfm1 to membrane
Results: Expression of Olfm1 in the retina was first detected at postconception day
13. Olfm1 was found mainly in post mitotic cells in the retinal ganglion cell layer.
In adult mouse retina, most of the Olfm1-producing cells were also located in the
retinal ganglion cell layer. Olfm1 protein was also detected in the nerve fiber layer
and throughout the optic nerve including the corresponding brain target regions.
Among 38 candidate proteins tested by AP assay, only NOGO receptor 1 (NgR1)
showed highly positive binding to Olfm1-AP. Other receptors tested, including
NgR2 and NgR3 that are similar to NgR1, were completely negative in this assay.
NgR1 and Olfm1 were co-localized in the ganglion cell layer and nerve fiber layer
of the retina as judged by immunofluorescence. Binding of Olfm1 to NgR1 was
more efficient than binding of the well-known ligand of NgR1, myelin associated
glycoprotein, as judged by the intensity of AP-staining with corresponding
conditioned media. Olfm1 without the olfactomedin domain interacted well with
NgR1, while Olfm1 with a deletion in the N-terminal domain failed to bind. These
results indicated that the N-terminal domain of Olfm1 is essential for NgR1
binding. Binding of Olfm1 and NgR1 was further confirmed by coimmunoprecipitation in cell culture.
Conclusions: Results strongly suggest that Olfm1 is a ligand of NgR1 that may
modify the function of NgR1 in retinal ganglion cell axon growth and regeneration.
Commercial Relationships: Naoki Nakaya, None; Afia Sultana,
None; Stanislav I. Tomarev, None
Support: the Intramural Research Program of the National Eye Institute, NIH
Program Number: 3960 Poster Board Number: D824
Presentation Time: 3:45 PM - 5:30 PM
Retinal Abnormalities in a Zebrafish Mutant Lacking Retinal and Hyaloid
Craig B. Stevens, Seth Adamson, Deborah L. Stenkamp. Biological Sciences,
University of Idaho, Moscow, ID.
Purpose: Human retinal disorders are often associated with cardiovascular
abnormalities or risk profiles. Interestingly, many genetic and pharmacological
zebrafish models for retinal dysgenesis also display cardiovascular defects, raising
questions about the role of the embryonic vascular supply to the eye in supporting
or regulating retinal development. In the present study, we examine embryonic
retinal development in cloche mutant zebrafish embryos, which fail to develop
hematopoietic, endothelial and endocardial cells. We test the hypotheses that the
cloche locus is required for embryonic development of the hyaloid and retinal
vasculature, as well as for that of the neural retina.
Methods: Eyes, retinas, and retinal and hyaloid vasculature of cloche (m39)
mutants and their wild-type siblings were examined using histological techniques,
in situ hybridization, and morphometric methods. Some embryos were treated with
retinoic acid (RA) in rescue experiments.
Results: Cloche mutants failed to develop hyaloid and retinal vasculature. As
compared to their wild-type siblings, cloche mutants became microphthalmic and
displayed significant defects in histology of the neural retina and in rod and cone
photoreceptor cell differentiation. Defects in rod, but not cone differentiation were
partially rescued by RA treatment. A search for possible causes of retinal
differentiation defects revealed that retinal transcription factors essential for retinal
development were expressed in cloche mutants, although the patterns of expression
of NeuroD and pax6 were abnormal. Expression of Hsp27, a marker of cell stress,
was observed in embryonic cloche retinas but not in wild-type siblings.
Conclusions: The cloche locus is essential for normal retinal development, and for
development of the hyaloid and retinal vasculature. Defects in rod development
related to the absence of cloche function can be partially rescued by RA treatment.
One explanation for these results is that early hyaloid and retinal vasculature
provides factors required for normal retinal development. Alternatively, genes
altered by the genomic deletion in cloche, may play specific, novel, roles in retinal
development. Blastomere transplant experiments are underway to test for tissueautonomous vs. non tissue-autonomous roles of the cloche locus.
Commercial Relationships: Craig B. Stevens, None; Seth Adamson,
None; Deborah L. Stenkamp, None
Support: NIH Grant RO1-EY012146 NIH Grant P20-RR0116454
Program Number: 3961 Poster Board Number: D825
Presentation Time: 3:45 PM - 5:30 PM
Functional Characterization of Prickle 2, a core Planar Cell Polarity protein,
in mouse retina
Samelia Okpodu1, Chunqiao Liu1, Alexander Bassuk2, Helen May-Simera3, Vinit B.
Mahajan4, Werner Graf5, Anand Swaroop1, Tiansen Li1. 1N-NRL, Bldg 6, National
Eye Institute, Bethesda, MD; 2Department of Pediatrics, University of Iowa Carver
College of Medicine, Iowa City, IA; 3National Institutes on Deafness and Other
Communication Disorders, NEI, Bethesda, MD; 4Ophthalmology, University of
Iowa, Iowa City, IA; 5Department of Physiology and Biophysics, Howard
University, Washington, DC.
Purpose: Planar cell polarity (PCP) refers to an asymmetry along the plane of
tissue that is perpendicular to the baso-apical axis. PCP is critical for many aspects
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
of development such as convergent extension of cell movement during gastrulation
and cochlea duct formation in the inner ear. To understand functions of PCP
pathway in retina we focused on functional characterization of Prickle 2, a core
player of PCP pathway.
Methods: In situ hybridization and qRT-PCR was used to study Prickle 2 gene
expression during retinal development. Polyclonal antibodies against Prickle2 were
generated to study Prickle 2 protein expression by immunoblotting and Prickle 2
subcellular localization by immunohistochemistry. Cochlea hair cell formation was
examined by phalloidin staining for actin filament in wildtype and prickle2 null
mice at postnatal day 1. Retinal functions of Prickle 2 knockout mice were assessed
by electroretinography (ERG).
Results: Prickle 2 expression in retina is detected as early as at embryonic day 13.5
(E13.5) by in situ hybridization and is present throughout the developing retina. In
adult retina, it is moderately expressed in the inner nuclear layer, weakly expressed
in ganglion cells, and minimally expressed in photoreceptor layer. Immunoblotting
shows Prickle2 to be a single band of 90 kDa which is absent in the KO retina.
Prickle 2 protein is upregulated in NRL knockout mouse retina both by
immunoblotting and by qRT-PCR. ERG at one month shows that loss of PK2
protein does not lead to a major deficit in photoreceptor development or function.
No gross abnormality in stereocilia bundle formation and orientation was detected
in the knockout mice. Further work is in progress, which will investigate the inner
retinal neurons and circuitry in addition to the PK2 subcellular localization.
Conclusions: Sensory neurons (photoreceptors and cochlear hair cells) appeared to
develop normally in mice lacking Prickle 2. Functional redundancy with Prickle1
may offer an explanation. PK2 expression appears to be influenced by NRL, the
significance of which is under investigation.
Commercial Relationships: Samelia Okpodu, None; Chunqiao Liu,
None; Alexander Bassuk, None; Helen May-Simera, None; Vinit B. Mahajan,
None; Werner Graf, None; Anand Swaroop, None; Tiansen Li, None
Support: None
Program Number: 3962 Poster Board Number: D826
Presentation Time: 3:45 PM - 5:30 PM
DL-Alpha Aminoadipate Disrupts Cholinergic Stratification and Increases
Inner Retinal Cells in 3D Chick Retinospheroids
Paul G. Layer, Gesine Bachmann, Florian Frohns, Gopenath Thangaraj. Biology,
Technische Universitaet Darmstadt, Darmstadt, Germany.
Purpose: To elucidate in vitro developmental roles of Müller glial cells (MCs) in
the histotypical organization of retinal cells using reaggregated retinospheroids as a
suitable 3-dimensional model system.
Methods: Enzymatically dissociated retinal cells from embryonic day 6 chicks
were cultured on a rotating shaker to produce histotypic retinal spheroids (1). To
depict the role of MCs in the organization of laminar areas, we ablated or damaged
them partially by applying 1 and 0.4 mM of the specific gliotoxin DL-alpha
aminoadipate (AAA). Cryostat sections of fixed spheres were stained by DAPI, or
were subjected to immunohistochemistry for choline acetyltransferase (ChAT),
Pax6, axonin1, glutamine synthetase (GS), CERN901 and CERN906.
Results: Our reaggregated retinal spheroids present laminar areas corresponding to
an ONL (rosettes) and an INL, and neuropil-rich areas corresponding to an IPL
(called IPLcircles), which are comprised mainly of processes from amacrine cells
and from MCs. Pairs of starburst amacrine cells (SACs) differentiate first, their
processes forming 1-2 cholinergic subbands in IPLcircles. AAA at 1 mM proved to
be highly toxic to the spheres, while at 0.4 mM AAA, the shape of the spheres was
highly irregular, compared with controls. MCs appeared swollen, presenting
shortened processes, as revealed by GS immunostaining. IPLcircles were still
formed, type II SACs migrated into the IPLcircles, but neuropil there was nonorganized and cholinergic subbands were absent. Immunostaining for Pax6 and
axonin1, both specific for inner retinal cells, showed that AAA treatment increased
their numbers. Concomitantly, formation of photoreceptor rosettes was negligible,
as revealed by CERN901 and CERN906 antibodies.
Conclusions: MCs and their processes - possibly in a premature state - play a
major role in IPL substratification, since a subtoxic concentration of AAA disrupts
it. Further, MCs affect retinal cell lineages and photoreceptor differentiation.
References: (1). Frohns F, Mager M, Layer PG (2009). FGF-2 increases the
precursor pool of photoreceptors, but inhibits their differentiation and apoptosis in
chicken retinal reaggregates. Eur J Neurosci 29, 1931-1942.
Commercial Relationships: Paul G. Layer, None; Gesine Bachmann,
None; Florian Frohns, None; Gopenath Thangaraj, None
Support: ESA-IBER08, DAAD
Program Number: 3963 Poster Board Number: D827
Presentation Time: 3:45 PM - 5:30 PM
Laminin β2 and γ3 chains are critical for retinogenesis and homeostasis of
adult retina
Shweta Varshney1,2, Galina Bachay1, William J. Brunken1,2. 1Ophthalmology and
Cell Biology, SUNY Downstate Medical Center, Brooklyn, NY; 2SUNY EYE
Institute, Brooklyn, NY.
Purpose: Attachment to laminins regulates proliferation, differentiation and
survival of cells. The laminin β2 and γ3 chains are important structural components
of the inner limiting membrane (ILM) of the retina. This study investigates the role
of the laminin β2 and γ3 chains in retinal histogenesis and maintenance of
homeostasis in the adult retina.
Methods: Retinas collected from postnatal day (P) 0 to 15 wild type (WT) and β2/-;γ3-/- mice were used for this study. The phenotypes of retinal cells were analyzed
by immunohistochemistry using cell specific markers. Gene expression was
analyzed using real time PCR and protein expression was analyzed by
Results: Retinal progenitor cells (RPC) and their last progeny, Müller cells (MC),
adhere to the ILM at their basal surface. Our data suggest that deletion of Lamb2
and Lamc3 leads to disruption of the ILM as early as P0. Concurrent with the ILM
disruption, retinal cells lose two key laminin receptors: β-dystroglycan and β1integrin. In tandem with this disruption, RPCs proliferate abnormally. In the P0 β2/-;γ3-/- retina, proliferation assessed using markers such as phosH3 and PCNA is
increased compared to WT; in contrast, by P5 in the β2-/-;γ3-/- retina, the rate of
proliferation declines by 1.5 fold compared to WT. The changes in proliferation in
the β2-/-;γ3-/- retina suggest changes in retinogenesis, consistent with our previous
report of the presence of rosettes in the P15 β2-/-;γ3-/- retina. Experiments are
currently underway to evaluate the precise changes in cell fate specification
corresponding with the changes in proliferation in the β2-/-;γ3-/- retina.
Additionally, in MC of the P15 β2-/-;γ3-/- retina, GFAP expression is increased,
indicating gliosis, whereas Kir4.1 and Aqp4 channel expression are decreased,
indicating dysregulation in ion homeostasis and MC hypertrophy. These changes in
MCs are also accompanied by remodeling of the second and third order neurons.
Conclusions: These data suggest that interactions of RPCs and MCs with the ILM
play important roles in retinal histogenesis and help to maintain normal cellular
architecture, promoting functional stability in the adult retina.
Commercial Relationships: Shweta Varshney, None; Galina Bachay,
None; William J. Brunken, None
Support: EY12676
Program Number: 3964 Poster Board Number: D828
Presentation Time: 3:45 PM - 5:30 PM
Role Of Glast-positive Glial Cells During Photoreceptor Development
Sarah Decembrini, Yvan Arsenijevic. Unit of Gene Therapy & Stem Cell Biology,
Jules-Gonin Eye Hospital, UNIL Lausanne, Switzerland.
Purpose: Taking advantage of two transgenic lines, glast.DsRed and crx.gfp, that
express fluorescent proteins in glial and photoreceptor cells respectively, we
investigate the role of glast-positive glial cells (GPCs) in the
survival/differentiation/proliferation of age-matched photoreceptor cells.
Methods: Primary retinal cells were isolated from newborn transgenic mouse
retina (glast.dsRed::crx.gfp) at postnatal day (P0/P1) and propagated in defined
medium containing epidermal growth factor (EGF) and fibroblast growth factor 2
(bFGF). By flow-sorting another population of pure GPCs was isolated. Both
populations were expanded and analyzed for the presence of specific retinal cell
markers. Notably, the primary cell culture collected from the transgenic line
glast.dsRed::crx.gfp showed a conspicuous presence of immature photoreceptors
growing on top of GPCs. In order to reveal the role of such cells in the
survival/differentiation/proliferation of photoreceptors we set up in vitro cultures of
retina-derived cells that allowed long-term time-lapse recordings charting every
cell division, death and differentiation event. To assess the regenerative potential of
GPCs we challenged them with compounds mimicking retinal degeneration (NMU,
NMDA, Zaprinast). Mass spectrometry (MS), immunostainings and other
molecular approaches were performed to reveal adhesion molecules involved in the
relationship between glial cells and photoreceptors.
Results: Both primary cell lines were highly homogenous, with an elongated
morphology and the majority expressed Müller glia markers (MG) such as glast,
blbp, glt-1, vimentin, glutamine synthetase (GS), GFAP, cd44, mash1 and markers
of reactive Müller glia such as nestin, pax6. Conversely, none of them were found
positive for retinal neuron markers like tuj1, otx2, recoverin.
Primary cultures of GPCs show the incapability of glial cells to give rise to
photoreceptors in both wild type or degenerative environment. Furthermore,
primary cultures of pure GPCs challenged with different compounds did not
highlight the production of new glial cell-derived photoreceptors. Adhesion
molecules involved in the contact between photoreceptors and glial cells are still
under investigation.
Conclusions: Primary glia cells do not give rise to photoreceptor cells in wt and
degenerative conditions at least in vitro. The roles of glial cells seem to be more
linked to the maintenance/proliferation of photoreceptor cells.
Commercial Relationships: Sarah Decembrini, None; Yvan Arsenijevic, None
Support: None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 3965 Poster Board Number: D829
Presentation Time: 3:45 PM - 5:30 PM
Spatiotemporal Development of Late-Born Rods and Bipolar Cells and Their
Synapses in Mice with Gestational Lead Exposure
Shawntay Chaney1, Shradha Mukherjee1, Anand Giddabasappa1, Jerry E.
Johnson2, Donald A. Fox1. 1University of Houston, Houston, TX; 2University of
Houston-Downtown, Houston, TX.
Purpose: Gestational lead exposure (GLE) produces supernormal ERGs in children
(Rothenberg et al., IOVS 2002) and increases and prolongs mouse retinal
progenitor cell (RPC) proliferation during development. This results in a selective
25-30% increase in the number of rod photoreceptors and bipolar cells (BCs) in
adult mice (Giddabasappa et al., EHP 2011). The current goal was to investigate
the spatiotemporal development of late-born rods and BCs and their synapses in
GLE retinas.
Methods: C57BL/6 female mice were exposed to water or lead throughout
gestation (E) and until postnatal day 10 (PN10). Fixed-frozen vertical sections from
E18.5-PN10 central retina were processed for confocal immunohistochemistry
using well-characterized antibodies.
Results: The spatiotemporal expression of OTX2-immunoreactivity (IR), a rod and
BC transcription factor, was similar in control and GLE retinas, although the
number of OTX2-IR cells significantly increased in GLE retinas. In GLE retinas
the onset of rhodopsin-IR, but not recoverin-IR, was delayed 2-3 days. By PN3, the
number of rhodopsin-IR and recoverin-IR rods significantly increased in GLE
retinas. The number of Chx10-IR RPCs significantly increased in GLE retinas
during early postnatal development. Chx10-IR localized to BCs at PN3 in controls
and at PN5 in GLE retinas. At PN5, significantly more BCs were Chx10-IR in GLE
than controls. In controls, protein-kinase C alpha (PKC)-IR was present in the
proximal inner nuclear layer (INL) at PN5 and localized to rod BCs by PN7. In
GLE retinas, PKC-IR in the proximal INL significantly increased at PN5, however,
by PN7 PKC-IR was not yet completely localized to rod BCs. In control and GLE
retinas, plasma membrane calcium ATPase (PMCA)-IR appeared in the outer
plexiform layer (OPL) at PN5. Vesicular glutamate transporter 1 (VGluT1)-IR first
labeled the OPL at PN5 in control and GLE, but significantly decreased in GLE
through PN10. In controls, VGluT1 labeled inner plexiform layer (IPL) sublaminae
a (IPLa) weakly at PN7, and IPLa and IPLb strongly at PN10. In GLE retinas,
VGluT1 labeled IPLa minimally at PN7 and normally at PN10, and IPLb
minimally at PN10.
Conclusions: GLE did not alter the onset of rod and BC cell fate (OTX2-IR) or
cone (recoverin) differentiation. However, GLE delayed the onset of rod and BC
differentiation (rhodopsin-, Chx10- and PKC-IR) and increased the number of these
late-born neurons. GLE did not alter rod spherule development, assessed by
PMCA-IR, although it delayed OPL and IPL synaptic VGluT1 expression. These
delayed differentiation results are consistent with the increased and prolonged RPC
proliferation in GLE retinas that underlie the increased rod and BC cellularity in
adult mice.
Commercial Relationships: Shawntay Chaney, None; Shradha Mukherjee,
None; Anand Giddabasappa, None; Jerry E. Johnson, None; Donald A. Fox,
Support: NIH Grants ES012482, EY07551 and EY07024
Program Number: 3966 Poster Board Number: D830
Presentation Time: 3:45 PM - 5:30 PM
Cystathionine Beta Synthase (cbs) Expression In Mouse Retina
Shanu Markand1A, Amany M. Tawfik1A, Yonju Ha1A, Jaya Gnana-Prakasam1B, Cory
Williams1A, Srinivas Sonne1B, Vadivel Ganapathy1B, Sylvia B. Smith1A. ACellular
Biology and Anatomy, BBiochemistry, 1Georgia Health Science University,
augusta, GA.
Purpose: CBS is a key enzyme in the transsulfuration metabolic pathway that
converts homocysteine to cystathionine, which is further converted to cysteine
required for synthesis of glutathione (GSH), a major antioxidant in retina. The
transsulfuration pathway has been characterized in liver, kidney, CNS, small
intestine and pancreas; however studies in the eye have been limited. Enzyme
activity assays suggest that CBS is present in human and pig retina, however recent
immunohistochemical studies reported that CBS is not present in mouse retina
(Mikami et al, 2011). We found this species difference puzzling. Given the plethora
of studies using mouse retina as a model system, coupled with the importance of
GSH in retina, we investigated CBS expression in mouse retina at the molecular
and cell biological level.
Methods: Wildtype (WT) mice or mice lacking the gene encoding CBS (cbs-/-)
were used in these studies. RNA and protein were isolated from retinas and liver
(positive control) for analysis of: (1) cbs gene expression by qRT-PCR using
primers specific for mouse cbs, and (2) CBS protein expression by Western blotting
using rabbit polyclonal anti-CBS antibody. Eyes were harvested from these mice
and used for immunodetection of CBS in retina. Three retinal cell types (ganglion,
Müller and RPE) were isolated from WT mice for immunocytochemical analysis of
Results: qRT-PCR revealed robust cbs expression in WT liver, and expression
also, albeit much lower, in WT retina. There was no cbs expression in cbs-/- mouse
retina (or liver). Western blotting detected a band of the expected size (~60 kD) in
retina and liver of WT mice, but not cbs-/- mice. In immunohistochemical studies of
the intact retina, CBS was present most abundantly in the ganglion cell layer of WT
retina. It was detected also in primary isolations of WT Müller, RPE and ganglion
Conclusions: We have compelling molecular evidence that CBS is indeed
expressed in mouse retina at the gene and protein level. Our immunofluorescence
data suggest that it is present in several retinal cell types. These findings set the
stage to investigate the role of CBS and the transsulfuration pathway in generation
of GSH in mouse retina.
Commercial Relationships: Shanu Markand, None; Amany M. Tawfik,
None; Yonju Ha, None; Jaya Gnana-Prakasam, None; Cory Williams,
None; Srinivas Sonne, None; Vadivel Ganapathy, None; Sylvia B. Smith, None
Support: NIH R01 EY012830 and EY014560
Program Number: 3967 Poster Board Number: D831
Presentation Time: 3:45 PM - 5:30 PM
SOX2 Maintains the Quiescence of Müller Glial Cells
Tessa Crowl1, Natalia Surzenko2, Larysa Pevny1. 1Genetics and Molecular Biology,
University of North Carolina- Chapel Hill, Chapel Hill, NC; 2Harvard University,
Boston, MA.
Purpose: Although the primary function of Müller glia is to provide trophic and
structural support to the retina, they also preserve progenitor cell morphology and
gene expression. In addition, Müller glia can re-enter the cell cycle in response to
retinal injury in order to replace neurons in the damaged tissue. Because of the
shared qualities of Müller glia and retinal progenitors, we propose to determine
how a gene critical for progenitor cell competence, SOX2, is important for Müller
glial progenitor cell characteristics. SOX2 is a HMG-box transcription factor that is
associated with the maintenance of progenitor cell populations. As neural retinal
progenitor cells differentiate into mature neurons, SOX2 is downregulated.
However, SOX2 is maintained in mature Müller glia, suggesting that it is involved
in preserving their proliferative potential.
Methods: In order to study the function of SOX2 in Müller glia, we used mice
carrying a conditional Sox2 allele (Sox2c) to ablate its function in a spaciotemporalspecific manner. These mice also contain one of the tamoxifen-inducible alleles,
CAG-CreERTM or Glast-CreERT2, in order to ablate Sox2 ubiquitously or in a
Müller glia-specific manner, respectively. Isolated retinal tissue from various ages
are exposed to 4-OH-tamoxifen and cultured for 5 days using an in vitro retinal
explant system. After fixation, retinas are cryosectioned and immunostained with
an array of antibodies.
Results: The conditional postnatal ablation of Sox2 in the early postnatal retina
results in the disruption of structural integrity of the retinal cell layers. In contrast
to the mitotically quiescent Müller glia in wild type retinas, a subset of SOX2deficient Müller glia re-enter the cell cycle. These cells upregulate the expression
of proliferation genes, and their nuclei travel to the apical retinal surface to divide.
Time lapse imaging reveals the nuclear migration and mitotic separation into two
daughter cells in the SOX2-deficient Müller glia.
Conclusions: These data suggest that SOX2 maintains Müller glial cell structure
and mitotic quiescence, possibly via the regulation of cell cycle components. In
addition, SOX2 maintains the progenitor cell characteristics of Müller glia by
preventing cell cycle progression.
Commercial Relationships: Tessa Crowl, None; Natalia Surzenko,
None; Larysa Pevny, None
Support: None
Program Number: 3968 Poster Board Number: D832
Presentation Time: 3:45 PM - 5:30 PM
Developmental Distribution of Mitochondria in Retinal Ganglion Cell Axons
Jessica E. Weinstein, Michael B. Steketee, Jeffrey L. Goldberg. Ophthalmology,
Bascom Palmer Eye Institute/University of Miami Miller School of Medicine,
Miami, FL.
Purpose: Retinal ganglion cells (RGCs) lose their regenerative capacity through
early postnatal development. Mitochondria are key players in RGC survival and
potentially in axon regeneration. In order to further understand how mitochondria
regulate survival and regeneration, we investigated the differences in mitochondrial
size and mass throughout specific developmental stages.
Methods: Retinal wholemounts and optic nerves were obtained from transgenic
Thy1/cox8A-mtCFP mice at embryonic (E) and postnatal (P) days, E18 through
adult. Samples were perfused or fixed by immersion for 2-3 hours with 4% PFA at
room temperature and then mounted and imaged by confocal microscopy,
examining RGC axons in the nerve fiber layer and optic nerve. Images were
analyzed using ImageJ. A limited age range was also examined using electron
microscopy (EM) and analyzed in Axiovision.
Results: Mitochondria were detected in RGC axons in the nerve fiber layer and
optic nerve using both fluorescence and EM. Mitochondrial size (per mitochondria)
and mass (per axon area) in the nerve fiber layer and optic nerve increased
significantly from P9 until adulthood, with a particularly abrupt increase from P13-
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Conclusions: Mitochondrial size and mass in RGC axons in the retina and optic
nerve increase significantly during a short postnatal window coincident with eye
opening. This increase may be light- or activity-dependent, and further experiments
manipulating light and activity are planned to investigate this phenomenon.
Determining mitochondrial dynamics and distribution may lead to new approaches
to enhance RGCs’ survival and regeneration.
Commercial Relationships: Jessica E. Weinstein, None; Michael B. Steketee,
None; Jeffrey L. Goldberg, None
Support: NINDS (NS061348); NEI (P30-EY014801) and an unrestricted grant and
Medical Student Fellowship from Research to Prevent Blindness
Program Number: 3969 Poster Board Number: D833
Presentation Time: 3:45 PM - 5:30 PM
A Zebrafish Model Of Joubert Syndrome: Inositol Phosphatase Inpp5e In
Primary Cilia Development
Na Luo1A, Callah West1A, Ryan Anderson1B, Yang Sun1A. AOphthalmology,
Pediatrics, 1Indiana University, Indianapolis, IN.
Purpose: To investigate the function of inositol 5-phosphatase INPP5E in cilia
development in the retina. Mutations of INPP5E cause Joubert Syndrome
(MIM:213300), which is characterized by retinal degeneration, renal cysts,
polydactyly, and mental retardation (Bielas, SL, et al. Nat Genet. 2009). Although
INPP5E has been implicated in cilia development, its function in retina
development is not known.
Methods: Using antisense morpholino oligonucleotides (MOs), knockdown of
INPP5E was performed in zebrafish and rescue experiments were done using
human wild type mRNA. Immunofluorescence of hTERT-RPE1 cells was
performed to determine the subcellular localization of human INPP5E and ciliary
Results: In zebrafish INPP5E morphants, embryos showed a dose-dependent
phenotype of retinal degeneration, microphthalmia, and body axis asymmetry. The
phenotypes were rescued by concomitant injection of human INPP5E mRNA.
Kupffer vesicle cilia in the INPP5E morphants were markerly shorter than the wild
type controls. Immunofluorescence experiments showed the co-localization of
INPP5E, gamma tubulin, and Rab20 in the basal body in RPE and normal human
Conclusions: Our data support an important role of INPP5E in ciliary development
and maintenance and provide a novel animal model to examine the function of
inositol phosphatases in retinal development.
Commercial Relationships: Na Luo, None; Callah West, None; Ryan
Anderson, None; Yang Sun, None
Support: Knights Templar Eye Foundation, American Glaucoma Foundation
Program Number: 3970 Poster Board Number: D834
Presentation Time: 3:45 PM - 5:30 PM
Retinal Abnormalities in Brown Norway Rat
Min Zhao1, Charlotte Andrieu-Soler1, Michèle Savoldelli2, Brigitte Goldenberg1,
Firas Jammoul1, Laura Kowalczuk1, Francine Behar-Cohen1,2. 1CRC, Inserm
U872, Team 17, Paris, France; 2AP-HP, Hôtel-Dieu of Paris, Department of
Ophthalmology, Paris, France.
Purpose: Brown Norway (BN) rat is one of the commonly used laboratory rats in
ophthalmic research. We report here its retinal abnormalities.
Methods: BN rats from 3 suppliers as well as Lewis rats were used to compare
their retinal morphology, in vivo angiography and retinal function (assessed by
electroretinogram, ERG). Retinal Muller glial cell and neurons were characterized
using immunofluorescence.
Results: BN rats from Janvier and Charles River showed retinal lesions beginning
at postnatal day 15 with irregular formation of photoreceptor outer segments,
followed by disorganization of outer nuclear layer and inner nuclear layer in young
rats and retinal dystrophy and cysts in older rats (6 months). Immunohistochemical
analysis showed Muller cell gliosis and hypertrophy, loss of cones, rods and their
synapses, and disorganization of bipolar and amacrine cells in the inner nuclear
layer. These abnormalities could not be prevented by protection of animals from
light. In vivo fluorescein angiography showed punctate diffusions and base ERG
demonstrated severe reduction of b wave amplitude. BN rats from Harlan
Laboratories also showed mild retinal morphological and vascular alterations, but
at older age.
Conclusions: The pigmented BN rats are widely used for pharmacologic and
toxicologic studies. However, we found that BN rats from different origins present
various severity of progressive retinal lesions that would certainly interfere with
such studies. The causes of the retinal abnormalities in BN rats are under
Commercial Relationships: Min Zhao, None; Charlotte Andrieu-Soler,
None; Michèle Savoldelli, None; Brigitte Goldenberg, None; Firas Jammoul,
None; Laura Kowalczuk, None; Francine Behar-Cohen, None
Support: None
Program Number: 3971 Poster Board Number: D835
Presentation Time: 3:45 PM - 5:30 PM
Evaluation of the Effect of Polymer Stiffness on Encapsulated Photoreceptor
Chris J. Lin1, Winnette McIntosh Ambrose2, Anand Swaroop3. 1NeurobiolNeurodegnrtn & Repair, NEI, Bethesda, MD; 2Neurobiol Neurodegeneration &
Repair, National Eye Institute (NEI) - NIH, Bethesda, MD; 3N-NRL, Bldg 6,
National Eye Institute, Bethesda, MD.
Purpose: To optimize and evaluate an in vitro, three dimensional, retina culture
model using primary photoreceptor cells encapsulated in polymer-based scaffolds.
We hypothesize that increased stiffness of the hydrogel constructs may increase
cell viability and photoreceptor specific protein expression of retinal cells.
Methods: Cells were isolated from the retina of Nrl-GFP mice at post natal stage
P4 and encapsulated in poly(ethylene glycol) polymer (PEG). Ultraviolet light was
used to photopolymerize the polymer to form a solid gel. The constructs used a
range of 10% to 20% weight to volume ratio of PEG in conjunction with 5% to
10% laminin, known for aiding neural development. Cultured for up to 10 days, the
gels were evaluated using live/dead viability assays at two time points. GFP
expression was also measured at these time points.
Results: Gel constructs with a higher weight to volume ratio of polymer exhibited
increased cell viability during the culturing period compared to gels with lower
weight to volume ratio of polymer. The GFP expression, at earlier time points, was
increased in gels with higher weight to volume polymer ratios. Regardless of the
amount of polymer, gel constructs that included laminin showed an increase in cell
viability in comparison to those without laminin.
Conclusions: Increased cell survival in PEG constructs with higher weight to
volume polymer ratio and the increase in GFP expression at the earlier time point
suggest that stiffer scaffolding may provide a better environment for photoreceptor
growth. These results indicate potential benefits of stiffer polymer-based scaffolds
for the in vitro culture of photoreceptors.
Commercial Relationships: Chris J. Lin, None; Winnette McIntosh Ambrose,
None; Anand Swaroop, None
Support: Intramural NIH, UNCF-Merck
Program Number: 3972 Poster Board Number: D836
Presentation Time: 3:45 PM - 5:30 PM
Adult Müller Glia Cells Are Efficient Progenitor Cells For Starburst
Amacrine Cells
Jan Wijnholds1, Pete Quinn1, Ditte M. Lundvig1, Lucie P. Pellissier1, Berend
Hooibrink2, Robert M. Hoek1. 1Neuromedical Genetics, Netherlands Institute for
Neuroscience, Amsterdam, The Netherlands; 2Cell Biology and Histology,
Academic Medical Center (AMC), Amsterdam, The Netherlands.
Purpose: Müller glia cells in fish and birds are potential progenitor cells for retinal
neurons, but mammalian Müller glia cells are limited in generating neurons in situ.
We have studied the capacity of highly purified Müller glia cells to form, upon
retinal transplantation, retinal neurons.
Methods: Mouse Müller glia cells were dissociated, purified by fluorescenceactivated cell sorting (FACS) by use of cell surface antigens and vital dye cell
trackers, and transplanted subretinally or intravitreously into the mouse retina.
Immunohistochemistry was performed on retinal sections and whole-mount
Results: Immunocytochemical analysis of sorted Müller glia cells showed that
these cells express markers for Müller glia cells immediately after sort. Upon
subretinal or intravitreal transplantation of unmanipulated cells in Crb1-/- or wildtype recipients, these cells efficiently migrate towards the inner face of the inner
nuclear layer (INL) and to the ganglion cell layer (GCL), within 9 days. The
transplanted Müller glia cells change cell fate; they re-differentiate into cholineacetyl transferase (ChAT) positive starburst amacrine cells (SAC). Whole mount
staining and mosaics analysis indicated that the projection and mosaics pattern of
the transplanted cells is similar to that of resident SACs. The transplanted cells
project to the sub-layer s1/2 and s4/5 in the inner plexiform layer (IPL).
Furthermore, we showed that transplantation into wild type mice also led to the redifferentiation of these cells into SACs and efficient migration into the retina. In
addition, we have initiated experiments to culture these cells enabling their
manipulation by applying factors to guide their differentiation behavior, in order to
obtain other retinal cell types upon transplantation.
Conclusions: Mammalian Müller glia cells can be purified by FACS using cell
surface antigens. Upon retinal transplantation, the mouse Müller glia cells
efficiently re-differentiate into retinal neurons. Experiments to repeat the results
with human Müller glia cells are ongoing.
Commercial Relationships: Jan Wijnholds, None; Pete Quinn, None; Ditte M.
Lundvig, None; Lucie P. Pellissier, None; Berend Hooibrink, None; Robert M.
Hoek, None
Support: EC HEALTH-F2-2008-200234
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
401 Retinal Vascular Biology
Wednesday, May 9, 2012, 8:30 AM - 10:15 AM
Floridian A Paper Session
Program #/Board # Range: 4117-4122
Organizing Section: Retinal Cell Biology
Program Number: 4117
Presentation Time: 8:30 AM - 8:45 AM
Retinal Vascular Dysfunction Induced By Mutations In Per2 Clock Gene Is
Mediated By Upregulation Of TGF-β1 Pathway
Ashay D. Bhatwadekar1, Jeffrey S. Thinschmidt1, Yuanqing Yan1, James M.
Dominguez1, Choogon Lee2, Julia V. Busik3, Maria B. Grant1. 1Pharmacology and
Therapeutics, University of Florida, Gainesville, FL; 2Florida State University,
Tallahassee, FL; 3Physiology, Michigan State University, East Lansing, MI.
Purpose: Diabetic retinopathy is associated with upregulation of transforming
growth factor β1 (TGF-β1) and its downstream effectors. Mutations of clock gene
Per2 induce endothelial dysfunction and inadequate vascular repair. In this study,
we hypothesized that retinal vascular dysfunction in Per2 mutant mice is associated
with modulation of TGF-β1 and an imbalance of nitric oxide (NO) which could
mimic the retinal vascular dysfunction seen in diabetes.
Methods: Per2 (Per2tm1Brd/J) or wild type (WT) mice of 4 and 12 months of age
were utilized for the study. Retinas were processed for mRNA expression of TGFβ1, DNA-binding protein inhibitor (ID-1), connective tissue growth factor (CTGF),
fibronectin (FN) and plasminogen activator inhibitor (PAI-1), eNOS and iNOS by
RT-PCR. Retinal vascularity was assessed on flat mounted retinas following the
injection of rhodamine conjugated BS-1 isolectin. Acellular capillaries were
evaluated using trypsin digestion. In parallel studies bone marrow progenitor cells
(BMPCs) were evaluated for NO levels.
Results: Per2 mutant retinas showed a 2 fold increase in TGFβ1 mRNA,
accompanied by upregulation in PAI-1 (2 fold; p<0.05) and Id-1 expression (2 fold
p<0.05). CTGF, an important downstream mediator of pro-fibrotic effect of TGFβ1
was upregulated by 2.5-3 folds (p<0.05) while FN expression was increased 2.5
fold (p<0.05) in Per2 mutant mice. There was no change in number of branch
points of retinal vasculature of Per2 mutant mice, however there was a 40 %
increase (p<0.05) in number of acellular capillaries in Per2 mutant mice when
compared to WT. BMPCs obtained from Per2 mutant mice showed reduced
proliferative response with 1.5 fold (p<0.05) decrease in NO levels.
Conclusions: In conclusion, retinal vascular dysfunction in Per2 mutant mice is
mediated through upregulation of TGF-β1 and enhanced oxidative stress, this
together with reduced contribution by BMPCs and increased acellular capillaries
results in pathologic alterations of retina similar to diabetic retinopathy.
Commercial Relationships: Ashay D. Bhatwadekar, None; Jeffrey S.
Thinschmidt, None; Yuanqing Yan, None; James M. Dominguez,
None; Choogon Lee, None; Julia V. Busik, None; Maria B. Grant, None
Support: Thomas H. Maren Junior Investigator Award (ADB); NEI RO1EY007739, EY012601(MBG)
Program Number: 4118
Presentation Time: 8:45 AM - 9:00 AM
A Mutation In βA3/A1 Crystallin Disrupts Astrocyte Template Formation
And Retinal Vascularization
Mallika Valapala, Samuel J. Zigler, Jr., Stacey Hose, Gerard A. lutty, Debasish
Sinha. Ophthalmology, Johns Hopkins Univ Sch of Med, Baltimore, MD.
Purpose: The primary vascular layer in the retina is intimately associated with the
underlying meshwork of astrocytes that emerge from the optic nerve. Although, it
has been suggested that the astrocyte template guides the endothelial cells to
facilitate vessel growth, the molecular mechanisms underlying this process remain
elusive. Here, we propose that Notch signaling is important for proper astrocyte
template formation and maintenance of vascular homeostasis in the retina. We also
report a novel role for βA3/A1-crystallin in regulating the activity of the Notch
Methods: The function of βA3/A1-crystallin was studied by utilizing the Nuc1
spontaneous mutant rats in which βA3/A1-crystallin gene is mutated. Optic nerve
astrocytes were isolated from the postnatal day 3-5 wild type and Nuc1 rats.
Quantitative real time PCR and luciferase assays were used to measure the activity
of the Notch pathway. The localization of Notch pathway proteins was studied
using subcellular fractionation, confocal microscopy and transmission electron
Results: In Nuc1 astrocytes, the expression of active Notch was greatly reduced
with a concomitant reduction in the levels of Notch downstream proteins Hey and
Hes and secretory Vascular endothelial growth factor (VEGF) compared to the wild
type cells. The expression of total Notch1 receptor was however elevated in the
Nuc1 cells. Our results show that neither the proteolytic processing of the Notch
receptor nor the nuclear translocation of the active Notch was altered in the mutant
astrocytes. Our data strongly suggests that a defect in the degradation of the Notch1
receptor leads to the inefficient removal of inactive Notch from the plasma
membrane, resulting in impaired ligand-induced Notch signaling in the Nuc1
mutant astrocytes. We also show that βA3/A1-crystallin is localized to the
lysosomes in optic nerve astrocytes and that the protein is important for the proper
functioning of the lysosomal degradative pathway
Conclusions: Our studies provide evidence that impaired Notch signaling pathway
could disrupt the normal cross talk between Notch and VEGF pathways. We also
propose a novel function for βA3/A1-crystallin in regulating the crucial molecular
pathways that mediate astrocyte-endothelial cell communication during vascular
development in the retina
Commercial Relationships: Mallika Valapala, None; Samuel J. Zigler, Jr.,
None; Stacey Hose, None; Gerard A. lutty, None; Debasish Sinha, None
Support: NIH Grant EY018636
Program Number: 4119
Presentation Time: 9:00 AM - 9:15 AM
Knockdown Of Splicing Factor Fbp21 Alters Pro - To Anti-angiogenic Vegf
Protein Ratio In Primary Retinal Pigment Epithelial Cells
James G. Carter1A, Amanda J. Churchill1B, David O. Bates1A. APhysiology &
Pharmacology, BAcademic Unit of Ophthalmology, 1University of Bristol, Bristol,
United Kingdom.
Purpose: Development of angiogenic eye disease is mediated by changes in the
balance of Vascular Endothelial Growth Factor (VEGF) isoforms between proangiogenic VEGFxxx and anti-angiogenic VEGFxxxb families. Anti-angiogenic
borrelidin derivatives target the splice factor FBP21, which may be involved in the
splicing switch between pro- and anti-angiogenic VEGF isoforms[1]. This study
was to determine the effect of FBP21 knockdown on VEGF isoform expression.
Methods: Primary retinal pigment epithelial (RPE) cells were treated with siRNA
targeted to FBP21. mRNA was extracted to confirm knockdown of FBP21
compared with scrambled siRNA by qRT-PCR. Protein was assessed for changes
in VEGF isoform expression by ELISA. The same knockdown experiments were
replicated in a luciferase reporter assay in HEK293 cells to determine if changes in
VEGF expression profiles were pre- or post- transcriptional. The cells were
transfected with reporter constructs containing VEGF165 or VEGF165b 3’-UTRs and
the effect of FBP21 siRNA knockdown compared with scrambled siRNA was
assessed by changes in luciferase/TK renilla expression.
Results: Knockdown of FBP21 by siRNA was confirmed by qRT-PCR (p<0.01).
This siRNA treatment caused a decrease in overall VEGF protein production
(685pg/mg vs. 975pg/mg, p<0.01), but critically a significant decrease in the
proportion of VEGFxxxb protein being produced in 1° RPE cells (48.8% vs. 64.8%,
p<0.01). There were no significant changes in luciferase expression under FBP21
knockdown for either VEGF165 or VEGF165b constructs in the reporter assay. This
shows that these changes in VEGF expression ratios were mediated at the point of
transcription, rather than a post-transcriptional/translation regulation event.
Conclusions: Inhibition of FBP21 alters VEGF isoform expression at the splicing
level. Identifying mechanisms involved in FBP21-mediated splice site selection
and assessing the possible therapeutic role of borrelidins on these splicing
complexes may give an alternative to current anti-VEGF treatments in ocular
1.Woolard, J., et al., Borrelidin modulates the alternative splicing of VEGF in
favour of anti-angiogenic isoforms. Chemical Science, 2011. 2(2): p. 273-278.
Commercial Relationships: James G. Carter, None; Amanda J. Churchill,
None; David O. Bates, None
Support: National Eye Research Centre, UK
Program Number: 4120 Presentation Time: 9:15 AM - 9:30 AM
Microrna-132 Antagonism Potently Limits Neovascularization In Multiple
Disease Models
Peter D. Westenskow1, Toshihide Kurihara1, Lea Scheppke1, Stacey Moreno1, Edith
Aguilar1, Sudarshan Anand2, Iacovos Michael3, Andras Nagy3, David A. Cheresh2,
Martin Friedlander1. 1Cell Biology, Scripps Research Institute, La Jolla, CA;
Moores UCSD Cancer Center, UCSD, San Diego, CA; 3Samuel Lunenfeld
Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
Purpose: MicroRNAs (miRs) can be activated by various stimuli to refine the
activity of molecular signaling pathways. We recently reported that miR-132
directly negatively regulates RasGAP to drive vascular proliferation. In the eye,
anti-miR-132 injections severely retard ocular angiogenesis. If miR-132 also
controls pathological angiogenesis anti-miR-132 therapies may be useful for
treating multiple blinding disorders in humans.pathological angiogenesis anti-miR132 therapies may be useful for treating multiple blinding disorders in humans.
Methods: We utilized RT-PCR, in-situ hybridization, and immunohistochemistry
to measure levels of miR-132 and RasGAP expression during development and in
two relevant animal disease models, oxygen-induced retinopathy (OIR) and
VLDLR-/- mice. Anti-miR-132 was injected intravitreally to determine if
progression of each of these conditions could be limited. The functions of antimiR-132 were directly compared with a potent anti-angiogenic VEGF-trap in each
model. We also utilized microarrays and multiplex ELISAs to determine if any offtarget or compensatory effects may be induced by targeting miR-132.
Results: We demonstrate in this study that miR-132 and RasGAP have inverse
expression patterns during critical early postnatal stages of retinal angiogenesis. In
both models of pathological angiogenesis we determine that miR-132 expression is
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
dysregulated; injections of anti-miR-132 restore RasGAP to normal levels and very
effectively prevent significant neovascularization. Furthermore, in our studies antimiR-132 consistently outperforms VEGF-trap at limiting neovascularization.
Lastly, both the microarray and ELISA results demonstrate that significantly more
compensatory pro-angiogenic genes and proteins are upregulated due to VEGF-trap
injections than by anti-miR-132.
Conclusions: We demonstrate that miR-132 controls normal and pathological
ocular angiogenesis. Injections of anti-miR-132 limit neovascularization in OIR
and VLDLR-/- mice more effectively than VEGF-trap. Additionally, anti-miR-132
appears to induce fewer off-target effects and limits compensatory upregulation of
several other pro-angiogenic factors. Therefore, these results provide a strong
molecular basis for targeting miR-132 as a novel anti-angiogenic therapeutic
Commercial Relationships: Peter D. Westenskow, None; Toshihide Kurihara,
None; Lea Scheppke, None; Stacey Moreno, None; Edith Aguilar,
None; Sudarshan Anand, None; Iacovos Michael, None; Andras Nagy,
None; David A. Cheresh, None; Martin Friedlander, None
Support: NIH grant EY11254
Program Number: 4121
Presentation Time: 9:30 AM - 9:45 AM
Suppression Of Timp3 Expression In Retinal Endothelial Cells By Hypoxiainduced Mir-21
Chaunte E. Stampley1A, Priyanka Thakur1, Folami Lamoke1B, Stephanie Goei1,
Manuela Bartoli1A. AOphthalmology, BPharmacology and Toxicology, 1Georgia
Health Sciences University, Augusta, GA.
Purpose: Suppression of tissue inhibitor of matrix metallo proteinase 3 (TIMP3)
has been shown to exacerbate retinal neovascularization (RNV). We have
previously shown that MicroRNA-21 (MiR-21) is up-regulated in retinal
microvascular endothelial cells (RECs) exposed to hypoxia. Because TIMP3 is a
potential target of miR-21, in this study we determined whether up-regulation of
miR-21resulted in suppression of TIMP3 gene expression in RECs exposed to
Methods: RECs of human and bovine origins were exposed to hypoxic conditions
(pO2≤ 2%) for 2, 4 and 6 hours. TIMP3 expression was measured at mRNA and
protein levels by quantitative PCR (qPCR) and Western blotting analysis,
respectively. MiR-21 expression was assessed by qPCR. Inhibition of miR-21
expression in hypoxic RECs was achieved by overexpression of antagomiR-21.
Results: The expression of miR-21 was significantly up-regulated in RECs
exposed to hypoxia (peak at 2hours), whereas TIMP3 was down-regulated at
mRNA and protein level at 4 and 6 hours of hypoxia, respectively. Overexpression
of antagomiR-21 blocked hypoxia-induced suppression of TIMP3 gene expression
and also restored protein level.
Conclusions: Our data show that hypoxia inhibits TIMP3 expression in retinal
endothelial cells through a mechanism involving up-regulation of miR-21
expression. Thus, this study suggests that regulation of TIMP3 and
neovascularization in the ischemic retina may involve miR-21 up-regulation and
Commercial Relationships: Chaunte E. Stampley, None; Priyanka Thakur,
None; Folami Lamoke, None; Stephanie Goei, None; Manuela Bartoli, None
Support: Vision Discovery Institute, Georgia Health Sciences University and
Knights Templar Georgia Chapter
Program Number: 4122
Presentation Time: 9:45 AM - 10:00 AM
Hypoxic Stress Induces Accelerated Pathological Angiogenesis and Gliosis in a
Murine Model of Duchenne Muscular Dystrophy
Mollie Friedlander, Toshihide Kurihara, Peter D. Westenskow, Lea Scheppke,
Edith Aguilar, Martin Friedlander. Cell Biology, The Scripps Research Institute,
La Jolla, CA.
Purpose: Duchenne muscular dystrophy (DMD) is carried by an X-linked
recessive gene (dystrophin). DMD is an inherited disease causing severe muscle
degeneration with an incidence of 1 in 3,000 males. Five isoforms of dystrophin
gene products (Dp427p, Dp427c, Dp427m, Dp260, and Dp71) are expressed in the
retina. Dp71 is known to be expressed in glial cells (including retinal Muller glia),
and functions to anchor water (AQP4) and potassium channels (Kir4.1) to cell
membranes. We hypothesized that dystrophin isoforms may function to maintain
fluid homeostasis when stressed. In this study, we examined the retinal phenotypes
of MDX3CV mutant mice, which do not generate any of the dystrophin isoforms,
after exposing them to hypoxic stress.
Methods: Breeding pairs of MDX3CV mutant mice were established to obtain
phenotypic mutant mice and control in the same litters. Control and mutant mice
were exposed to 75% oxygen from postnatal day 7 (P7) to P12 to induce oxygeninduced retinopathy (OIR). Pathological angiogenesis and gliosis were evaluated in
littermates using immunohistochemistry on whole mount retinal preparations and
cryosectioned retinas. Real-time quantitative PCR analyses were performed to
identify any genes dysregulated in the mutants during hypoxic stress.
Results: Significantly more neovascular tufts (p<0.01) and activated Muller glia
(p<0.05) are observed in P17 MDX3CV mice compared with control littermates.
Additionally, a significant downregulation of aquaporin 4 (p<0.05) and Kir4.1
(p<0.01) genes was observed in mutants compared to controls.
Conclusions: These results suggest that dystrophin isoforms in the retina maintain
proper water balance, suppress Muller glia activation, and prevent pathological
angiogenesis under condition of hypoxic stress.
Commercial Relationships: Mollie Friedlander, None; Toshihide Kurihara,
None; Peter D. Westenskow, None; Lea Scheppke, None; Edith Aguilar,
None; Martin Friedlander, None
Support: NIH Grant EY-11254
415 Lipids: Retinal Friends or Foes?
Wednesday, May 9, 2012, 8:30 AM - 10:15 AM
Hall B/C Poster Session
Program #/Board # Range: 4271-4302/A461-A492
Organizing Section: Retinal Cell Biology
Program Number: 4271 Poster Board Number: A461
Presentation Time: 8:30 AM - 10:15 AM
Docosahexaenoic Acid Potentiates Pigment Epithelium Derived FactorInduced Protection in ARPE-19 Cells
William C. Gordon, Eric J. Knott, Nicolas G. Bazan. Ophthalmology &
Neuroscience Center, LSU Health Sciences Center, New Orleans, LA.
Purpose: The over expression of the serpin anti-angiogenic neurotrophic Pigment
Epithelium Derived Factor (PEDF) protects photoreceptors from light damage, and
ARPE-19 cells from H2O2-induced apoptosis. We have shown that PEDF is an
agonist of docosahexaenoic acid (DHA)-derived neuroprotectin D1 (NPD1)
synthesis, and that this docosanoid, in turn, elicits potent anti-inflammatory and
anti-apoptotic actions. Therefore, the purpose of this study was to determine if
PEDF’s mediated protection, which is known to engage NPD1, persists after
removal of PEDF treatment, and if DHA supplementation potentiates this
Methods: ARPE-19 cells where cultured for 72 h to achieve ~100% confluencey.
Cells were serum starved for 18 hours in 0.5% FCS, DMEM/F12 1:1 media, after
which 50 ng/ml PEDF, 200nM DHA + 50ng/ml PEDF, or ETOH (equal volume)
was added to the media. After 6 hours of incubation, media was replaced with fresh
0.5% FCS, DMEM/F12 1:1 media for 24h. Cells were then challenged with 675,
700, and 725 µM H2O2 for 16 hours. Cells were fixed with neutral buffered
formalin and stained with Hoestch 33258 in 1% triton X100. Cells were counted
using an automated unbiased image analysis of nuclear morphology. Cell death was
determined via nuclear chromatin condensation (µm2).
Results: Control cells display a survival level of 92 ± 2%. Cells treated with 675,
700, and 725µM H2O2 for 16 hours resulted in nuclear chromatin condensation
and a cell survival level of 87± 4%, 45 ± 5, and 42%±9%, respectively. Cells
pretreated with 50ng/ml PEDF for 6 hours, then DMEM/F12 for 24 hours prior to
stress, exhibit protection with a survival level of 93 ± 6%, 90% ± 7%, and
50%±12%, respectively. Cells pretreated with PEDF plus 200nM DHA show
protection with a survival level of 97±2%, 94±3%, and 74±10%, respectively.
Conclusions: Our results demonstrate that PEDF pretreatment elicits protection of
human-ARPE-19 cells from oxidative stress-induced cell death up to 24 hours post
attenuation of the treatment with PEDF, and when DHA is supplemented
simultaneously this protection is bolstered. This data suggests protection via DHAderived NPD1 synthesis. Thus, application of DHA/NPD1 and/or PEDF could be
used as a potential therapeutic approach for the prevention or attenuation of the
initiation or early progression of retinal degeneration.
Commercial Relationships: William C. Gordon, None; Eric J. Knott,
None; Nicolas G. Bazan, None
Support: NEI EY005121; Research to Prevent Blindness, Inc.
Program Number: 4272 Poster Board Number: A462
Presentation Time: 8:30 AM - 10:15 AM
Analysis Of The Components And Clearance Of Retinal Lipofuscin
Radouil T. Tzekov1A, Lei Lei1A, Huapeng Li1B, J Hugh McDowell2, Guangping
Gao1B, W. Clay Smith2, Shibo Tang3, Shalesh Kaushal1A. AOphthalmology, BGene
Therapy Center, 1University of Massachusetts Medical School, Worcester, MA;
Ophthalmology, University of Florida, Gainesville, FL; 3Zhongshan Ophthalmic
Center, Sun Yat-sen University, Guangzhou, China.
Purpose: To evaluate several aspects of lipofuscin-like material (LLM)
accumulation in retinal pigment epithelial (RPE) cells, including time course,
natural clearance, effects of phagocytosis rate, effects of rod outer segment (ROS)
oxidation, contribution of different ROS components and pharmacological
manipulation of autophagy.
Methods: Post-confluent ARPE-19 cells were cultured in 24 well plates and fed
with different types of bovine ROS for 7 to 14 days. Additionally, several ROS
components were added to the RPE cells separately: 11-cis retinal, all-trans retinal,
9-cis retinal, opsin, liposomes containing phosphatidylethanolamine and
phosphatidylcholine. Lipofuscin-like autofluorescence (LLAF) was determined at
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
two wavelengths (530 and 585 nm) by FACS. Cells were also treated with
lysosomal (chloroquine, NH4Cl) or autophagy inhibitors (3-MA), or with
autophagy inducers (Ku-0063794, PI-103, PIK-90) to evaluate the effect on LLAF.
Additionally, rapamycin (10 and 20 µM) was added over a 2-day period or as a
single application for live cell imaging. Autophagy inhibition was also performed
by siRNA and shRNA knockdown of Atg5 and Atg7.
Results: Application of ROS indicates that the rate of LLM accumulation is linear
over a 14-day period with higher rate of increase for the first 7 days. The rate of
lipofuscin accumulation was proportional to the rate of phagocytosis of the ROS;
LLM appears relatively stable once it accumulates in the RPE cell with only a
limited loss (10-15%) over a 7-day period. Feeding with oxidatively modified ROS,
and individual retinoids such as 11-cis and all-trans retinal all enhanced LLAF,
with a more pronounced increase at 585 nm. Application of lysosomal or
autophagy inhibitors and suppression of Atg5 or Atg7 increased LLAF, while
application of autophagy inducers decreased LLAF. Rapamycin decreased LLAF in
a dose-dependent way and most of the decrease occurred within the first 30 min
after application.
Conclusions: These results emphasize the role of retinoids, lipids and oxidation in
LLM accumulation in RPE cells. Furthermore, it confirms the possibility for a
pharmacological clearance of RPE LLM by small molecules modulating the
mTOR/autophagy pathway, thus opening new avenues for the treatment of dry
ARMD and other lipofuscinopathies.
Commercial Relationships: Radouil T. Tzekov, None; Lei Lei, None; Huapeng
Li, None; J Hugh McDowell, None; Guangping Gao, None; W. Clay Smith,
None; Shibo Tang, None; Shalesh Kaushal, None
Support: University of Massachusetts Department of Ophthalmology Research
and Education fund
Program Number: 4273 Poster Board Number: A463
Presentation Time: 8:30 AM - 10:15 AM
The Ratio of Melanosome to Lipofuscin Granule Number, Not Lipofuscin
Content Alone, Determines the Susceptibility of Individual RPE Cells to
Lethal Photic Stress in vitro
Mariusz Zareba1, Tadeusz J. Sarna2, Janice M. Burke1. 1Ophthalmology, Medical
College of Wisconsin, Milwaukee, WI; 2Biophysics, Jagiellonian University,
Krakow, Poland.
Purpose: Autofluorescent lipofuscin is believed to predispose the aging RPE to
photic stress. Since RPE cells vary in the content of both photo-reactive lipofuscin
granules and photo-protective melanosomes, we asked whether individual RPE cell
susceptibility to light-induced injury depended on lipofuscin abundance alone, or
could be modulated by coincident melanosome content.
Methods: Primary cultures of human RPE containing varying numbers of
lipofuscin granules were allowed to phagocytize isolated porcine melanosomes
(pigment granules unmodified by age) or control black latex beads. Individual cells
in sub-confluent cultures were selected for their granule number and their ratio of
granule types using absorbance to quantify total granule number per cell,
autofluorescence to identify lipofuscin, and latex fluorescence (635-685, excitationemission) to detect beads. Cells were irradiated with blue light (490 nm) and the
time of cell death after light onset was determined by real-time live cell imaging
using image capture at 30 s intervals over 8 hrs.
Results: Cell death induced by blue light varied with lipofuscin content; cells with
abundant endogenous lipofuscin died first while those with fewer lipofuscin
granules showed delayed death or survived irradiation. The presence of
melanosomes within cells in addition to lipofuscin delayed cell death as compared
to cells with comparable amounts of lipofuscin alone. Preliminary results showed
no significant death delay in cells containing beads, suggesting that the protective
effect of melanosomes was at least partly due to properties of the pigment (e.g.,
acting as an antioxidant) aside from optical screening.
Conclusions: RPE cells are exposed throughout life to blue light.The results here
suggest that the content of autofluorescent lipofuscin alone may not be an effective
predictor of the photic stress susceptibility of the tissue. Since the vulnerability to
stress differs among individual cells, a better predictor of an at-risk monolayer may
be the frequency of RPE cells with both high lipofuscin content and low
melanosome number.
Commercial Relationships: Mariusz Zareba, None; Tadeusz J. Sarna,
None; Janice M. Burke, None
Support: NEI grants R01 EY019664 and P30 EY01931; RPB; CO6 RR016511
Program Number: 4274 Poster Board Number: A464
Presentation Time: 8:30 AM - 10:15 AM
Effect of Pigment Epithelium-Derived Factor on Hydrogen Peroxide-Exposed
Human Retinal Pigment Epithelial Cells
Yao Wang, Piyush Kothary, Monte Del Monte. University of Michigan Medical
School, Ann Arbor, MI.
Purpose: Oxidative injury results in human retinal pigment epithelial (hRPE) cell
damage, which has been postulated to be involved in the pathogenesis of agerelated macular degeneration (AMD). We hypothesize that pigment epithelium-
derived factor (PEDF) can serve as an anti-angiogenic factor and a neuroprotective
factor in hRPE cells. This study examines the effects of PEDF on hRPE cell
proliferation and morphology under oxidative stress induced by hydrogen peroxide
(H2O2). We also investigated the effects of H2O2 on endogenous PEDF production.
Methods: Human RPE specimens were cultured from postmortem nonpathological eyes. Cellular proliferation and viability in the presence of increasing
concentrations of H2O2 and PEDF were measured by [3H]thymidine incorporation
and by the trypan blue exclusion method, respectively. After exposure to varying
concentrations of H2O2, synthesis of PEDF was measured quantitatively by
utilizing [14C]methionine immunoprecipitation with anti-PEDF and qualitatively
via immunocytochemical methods.
Results: Fetal bovine serum (FBS) stimulated hRPE cell proliferation in a dosedependent manner. In contrast, H2O2 (0.1mM-0.5mM) decreased hRPE cell
proliferation and viability, again in a dose-dependent manner as measured by
[3H]thymidine incorporation and trypan blue exclusion method, respectively. The
addition of PEDF (10ng/mL) to H2O2-exposed cells resulted in cell rescue,
indicated by increased hRPE cell proliferation (358.43±57.03, n=12 vs.
1,087.22±235.58, n=6, CPM±SEM, p<0.05) and increased cell viability
(60,000±10,700 vs. 164,500±20,100, n=28, cells±SEM, p<0.05). H2O2 also
stimulated endogenous [14C]methionine-PEDF synthesis in hRPE cells in a dose
dependent manner. Immunocytochemical studies confirmed endogenous production
of PEDF in H2O2-exposed hRPE cells as well as nuclear rescue after the addition of
PEDF to H2O2-induced hRPE cells.
Conclusions: These studies suggest that endogenous synthesis of PEDF is
stimulated by H2O2. PEDF rescues H2O2-exposed hRPE cells, potentially by
stimulating DNA synthesis and reversing nuclear damage induced by H2O2.
Therefore, PEDF may have therapeutic value in treating AMD.
Commercial Relationships: Yao Wang, None; Piyush Kothary, None; Monte
Del Monte, None
Support: University of Michigan Summer Biomedical Research Program funded
by NIH
Program Number: 4275 Poster Board Number: A465
Presentation Time: 8:30 AM - 10:15 AM
Endoplasmic Reticulum Stress In Environmental And Genetic Models Of
Retinal Degeneration
Jonathan H. Lin1, Heike Kroeger1, Carissa Messah1, Kelly Ahern2, Jason Gee2,
Victory Joseph1, Michael T. Matthes2, Douglas Yasumura2, Marina S. Gorbatyuk3,
Matthew M. LaVail2. 1Pathology, Univ of CA San Diego Sch of Med, La Jolla, CA;
Beckman Vision Center, UCSF School of Medicine, San Francisco, CA; 3Cell
Biology and Anatomy, Univ of North Texas Health Sci Ctr, Fort Worth, TX.
Purpose: Endoplasmic reticulum (ER) stress has been observed in animal models
of retinitis pigmentosa arising from P23H rhodopsin. We asked if ER stress was
found in other models of retinal degeneration arising from environmental or genetic
causes. We measured ER stress levels in mice in a constant light (CL)-induced
model of retinal degeneration; in transgenic rats expressing mutant S334ter
rhodopsin; and in Royal College of Surgeons (RCS) rats bearing mutant Mertk.
Methods: BALB/c albino mice were exposed to 15,000 lux of fluorescent CL for
0,1,2,4, or 8 hours. S334ter transgenic rats (lines 3, 4, and 5), RCS and RCS-p+ rats
with inherited retinal dystrophy were collected at postnatal (P) days 10, 12, 20, 30,
40, 60, 90, and 120. Retinal tissues from 3-10 animals per experimental condition
were collected for histologic and molecular analyses to quantify retinal
degeneration and mRNA levels of two well-studied ER stress-induced genes, BiP
and Chop.
Results: BALB/c mice revealed 1.5x (P=0.0324) more BiP and 1.3x (P=0.0285)
more Chop mRNA levels after 4 hours of CL. These findings correlated with
photoreceptor layer cell loss by histology. S334ter-3 retinas revealed 3.3x (P=1E06) more BiP and 1.3x (P=0.03) more Chop mRNA levels at P15; S334ter-4
showed 4x (P=9E-06) more BiP mRNA at P60 and 1.5x (P=0.005) more Chop
mRNA at P30; retinal tissue from S334ter-5 had 2.2x (P=0.008) more BiP mRNA
at P90, but no significant increase in Chop mRNA was found when compared to
age-matched controls. P23H-3 rats demonstrated 2.3x (P=0.0048) more BiP mRNA
levels and 1.6x (P=0.0073) more Chop mRNA levels at P60 compared to controls.
RCS and RCS-p+ rats showed a significant increase of BiP mRNA at 2.4x at P60
and 1.8x at P20, respectively (RCS, P=0.0006; RCS-p+, P=0.005) without changes
in Chop mRNA levels.
Conclusions: Our study revealed statistically significant increases in ER stress
markers BiP in CL-induced retinal degeneration, S334ter rats, P23H rats, and RCS
rats; and Chop in CL-induced retinal degeneration, two S334ter rat lines, and P23H
rats. Increase in ER stress correlated with histologic evidence of retinal
degeneration in all models. We propose that manipulation of ER stress responses
may be helpful in treating many environmental and heritable forms of retinal
Commercial Relationships: Jonathan H. Lin, None; Heike Kroeger,
None; Carissa Messah, None; Kelly Ahern, None; Jason Gee, None; Victory
Joseph, None; Michael T. Matthes, None; Douglas Yasumura, None; Marina S.
Gorbatyuk, None; Matthew M. LaVail, None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Support: NIH grants EY001919, EY006842, EY018313, EY020846, EY020905,
and the Foundation Fighting Blindness
Program Number: 4276 Poster Board Number: A466
Presentation Time: 8:30 AM - 10:15 AM
Hydroquinone Induces Autophagy in Human Retinal Pigment Epithelial Cells
in vitro
Grace B. Woo, Laura S. Woo, Claudio Ramirez, Khoa Pham, Sarah Hashmi,
Marilyn Chwa, Baruch D. Kuppermann, Maria C. Kenney. Ophthalmology,
University of California, Irvine, Irvine, CA.
Purpose: We explored the effects of hydroquinone (HQ), a toxic component found
in cigarette tar, on human retinal pigment epithelial cells (ARPE-19) in order to
understand the mechanism(s) of cell death.
Methods: ARPE-19 cells were grown in vitro and treated for 24 hours with four
different concentrations of HQ (50µM, 100µM, 200µM, 500µM). Caspase 3/7
activity was detected using the Carboxyfluorescein FLICA Apoptosis Detection kit.
Proteins were extracted and subjected to Western blot analyses probed with rabbit
anti-microtubule-associated protein light chain 3 (LC3-II), mouse antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH), mouse anti-HSP60 (Heat
Shock Protein 60), rabbit anti-HSP70 (Heat Shock Protein 70), and mouse antiNMDAR1 (N-methyl-D-aspartate receptor 1). The resulting bands were visualized
via colorimetric detection, quantified using ImageJ, and subjected to statistical
analysis using GraphPad Prism software.
Results: After treatment with HQ, there was no change in caspase 3/7 activities at
any of the concentrations tested. Both HSP60 and NMDAR1 levels were
significantly decreased at 200µM HQ (5860±601 vs 11394±1670, p=0.0110;
1814±218 vs 7717±855, p=0.0478, respectively) while the HSP70 levels increased
(24209±2298 vs 13605±2582, p=0.049) at 100µM HQ compared to the dimethyl
sulfoxide (DMSO) treated controls. The LC3-II band (14kDa) was present in the
cultures treated with 100µM and 200µM HQ.
Conclusions: Our findings suggest that the mechanism of cell death for ARPE-19
cells exposed to HQ does not involve apoptosis but occurs via autophagy, as
confirmed by the presence of LC3-II, and other cell-signaling molecules. The
HSP70, which has been implicated as an anti-apoptotic and pro-autophagic
signaling molecule, was elevated and HSP60, which has been implicated as proapoptotic, was decreased following HQ exposure. The reduced NMDAR1 levels
suggest that the disruption of glutamate homeostasis may also play a role in the
HQ-induced cell death.
Commercial Relationships: Grace B. Woo, None; Laura S. Woo,
None; Claudio Ramirez, None; Khoa Pham, None; Sarah Hashmi,
None; Marilyn Chwa, None; Baruch D. Kuppermann, None; Maria C. Kenney,
Support: Discovery Eye Foundation, Polly & Michael Smith Foundation,
Beckman Initiative for Macular Research, Lincy Foundation, Iris & B. Gerald
Cantor Foundation, Research to Prevent Blindness
Program Number: 4277 Poster Board Number: A467
Presentation Time: 8:30 AM - 10:15 AM
The small GTPase Rap1 regulates intracellular ROS generation in RPE
Haibo Wang1, Erika Wittchen2, M. Elizabeth Hartnett1. 1John A Moran Eye Ctr,
Ophthalmology, University of Utah, Salt Lake City, UT; 2Department of Cell and
Developmental Biology, University of North Carolina, Chapel Hill, NC.
Purpose: We have previously shown that NADPH oxidase-mediated generation of
reactive oxygen species (ROS) is involved in pathologic steps of neovascular
AMD. Activation of NADPH oxidase involves assembly of the cytosolic (p47phox,
p67phox, and Rac1) and membrane-associated components (NOX and p22phox).
We recently showed that activation of a GTPase, Rap1, reduced CNV size in a laser
injury model. We now tested the hypothesis that inhibition of Rap1 increases ROS
generation in RPE cells by interfering with NADPH oxidase subunit assembly and
Methods: Using CM-H2DCFDA, intracellular ROS generation was measured in
RPE cells infected with an adenoviral vector expressing GTPase-activating proteins
(Ad-RapGAP) to inhibit Rap1 activity or control vector expressed with GFP (AdGFP). Protein interaction of Rap1 and NADPH oxidase subunit, p22Phox, in RPE
cells was determined by co-immunoprecipitation of Rap1 and p22phox in RPE
cells infected with the Ad-RapGAP or control Ad-GFP. To activate Rap1, RPE
were exposed to an Epac-specific cAMP analogue, 8-pCPT-2_OMe-cAMP (8CPT, Tocris Bioscience). Statistics were performed using ANOVA.
Results: Compared with control Ad-GFP, ROS generation was increased in RPE
cells infected with the Ad-RapGAP (P=0.003) and decreased in RPE cells
incubated with 8-CPT (P=3.3464E-15). Compared to RPE infected with control
Ad-GFP without 8-CPT treatment, infection of RPE with Ad-RapGAP decreased
endogenous Rap1 bound to p22phox (P=0.000036), while incubation of RPE with
8-CPT restored the binding affinity of endogenous Rap1 to p22phox.
Conclusions: Activated Rap1 prevented ROS generation in RPE cells. The
molecular mechanism may involve inhibition of NADPH oxidase activation by
interfering with NADPH oxidase subunit assembly.
Commercial Relationships: Haibo Wang, None; Erika Wittchen, None; M.
Elizabeth Hartnett, None
Support: R01 R01EY015130 MEH
Program Number: 4278 Poster Board Number: A468
Presentation Time: 8:30 AM - 10:15 AM
Pigment Epithelium-derived Factor Inhibits Neuroretinal Apoptosis in a
Murine Model of Focal Retinal Degeneration
Yujuan Wang1A,2, Defen Shen1A, Jingsheng Tuo1A, Preeti Subramanian1B, S.
Patricia Becerra1B, Chi-Chao Chan1A. ALaboratory of Immunology, BLaboratory of
Retinal Cell and Molucular Biology, 1NEI, Bethesda, MD; 2Zhongshan Ophthalmic
Center, Sun Yat-sen University, Guangzhou, China.
Purpose: Age-related macular degeneration (AMD) is a neurodegenerative disease
that causes irreversible central vision loss in the elderly. Apoptosis of retinal cells
plays a critical role in the pathogenesis of AMD. The Ccl2-/-/Cx3cr1-/- mice on rd8
background (DKO) are generated as an AMD model, developing AMD-like retinal
lesions. Pigment epithelium-derived factor (PEDF) is a potent neurotrophic and
neuroprotective glycoprotein that protects the RPE, photoreceptors and neurons
against cell death by a variety of pathological insults. In this study, we evaluated
the effects of PEDF on the retinal lesions of the DKO mice.
Methods: The right eyes of 6-week-old DKO mice were intravitreously injected
with highly purified recombinant human PEDF protein (1 µg), followed by a
second subconjunctival injection of PEDF (3 µg) 4 weeks later. The left eyes were
untreated and served as controls. Fundoscopic pictures were taken and scored
before injection and 4 weeks after each injection. The mice were sacrificed 4 weeks
after the second injection. Ocular histopathology was examined. Retinal A2E, a
major component of lipofuscin, was measured by HPLC. Apoptosis related
transcripts were measured by quantitative RT-PCR and compared between the
treated and control eyes.
Results: Two independent experiments were performed and yielded similar data.
The series fundoscopy for 8 weeks showed a tendency of slower progression or
attenuation of the focal, deep retinal lesions in the PEDF treated eyes compared
with the control eyes. Among 24 pairs of eyes, average clinical scores of
progression are 1.14±0.12 in the treated eyes and 1.32±0.14 in the control (p=0.08),
respectively. This observation was confirmed by histology illustrating fewer and/or
smaller photoreceptor lesions in the treated eyes than the control. A2E levels were
significantly lower in the treated eyes than the control eyes (14±1.3 vs 21.3±1.6,
p<0.01). Quantitative RT-PCR showed up-regulated expression of FasL and Fas
transcripts in the treated eyes compared with the control eyes.
Conclusions: PEDF potently stabilizes photoreceptor degeneration in DKO mice.
One of the protective mechanisms may be via antiapoptotic pathway. The
neuroprotective effect of PEDF represents a novel approach for potential treatment
of AMD.
Commercial Relationships: Yujuan Wang, None; Defen Shen,
None; Jingsheng Tuo, None; Preeti Subramanian, None; S. Patricia Becerra,
None; Chi-Chao Chan, None
Support: National Eye Institute Intramural Research Program
Program Number: 4279 Poster Board Number: A469
Presentation Time: 8:30 AM - 10:15 AM
Calpain Protease Causes Hypoxia-Induced Proteolysis in Cultured Human
Mitsuyoshi Azuma1,2, Katherine B. Hammond1,2, Emi Nakajima1,2, Thomas R.
Shearer2. 1Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co, Ltd.,
Beaverton, OR; 2Integrative Biosciences, Oregon Health & Science University,
Portland, OR.
Purpose: Decreased blood flow in the choroid or retinal arteries leads to rapid cell
death and blindness in age-related macular degeneration, glaucoma from high
intraocular pressure, and diabetic retinopathy. Calpain and caspase proteases are
known to be involved in retinal cell death in a number of animal models.
Interestingly, in monkey retinal cells cultured under hypoxia, calpain protease
caused the cell damage. This is not always the case because most tissues contain
significant amounts of the highly specific, endogenous calpain inhibitor calpastatin.
Thus, the clinically relevant purpose of the present study was to test for calpain
activation in human retinas cultured under hypoxic conditions.
Methods: Human and monkey retinas were quartered and incubated in DMEM.
Hypoxia was imposed using the GasPack pouch system. Retinal cell marker
proteins, activation of calpains, and proteolysis of α-spectrin substrate were
detected by immunoblotting. Calpain inhibitor SNJ-1945 was used to confirm
activation of calpain.
Results: In cultured human retina, hypoxia caused autolysis of calpain (indicative
of calpain activation) and accumulation of the 145 kDa calpain-specific, α-spectrin
breakdown product. Opsin (photoreceptor marker) and vimentin (Müller cell
marker) were also degraded. SNJ-1945 inhibited calpain activation and degradation
of opsin and vimentin in a dose dependent manner. Retinas cultured under
normoxic conditions showed a lower level of calpain activation and substrate
proteolysis. Similar results were observed in monkey retina.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Conclusions: The most important finding was that human retina shows calpain
activation when cultured under hypoxia. Activated calpain proteolyzes critical
substrates such as opsin, vimentin and α-spectrin; leading to cell death. Synthetic
calpain inhibitor SNJ-1945 may be a useful drug to protect against proteolysis in
photoreceptor and Müller cells from patients with ischemic retinal diseases.
Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are
employees of, Senju Pharmaceutical Co, Ltd.
Commercial Relationships: Mitsuyoshi Azuma, Senju Pharmaceutical Co, Ltd.
(E); Katherine B. Hammond, Senju Pharmaceutical Co, Ltd. (E); Emi Nakajima,
Senju Pharmaceutical Co, Ltd. (E); Thomas R. Shearer, Senju Pharmaceutical Co,
Ltd. (C)
Support: None
Program Number: 4280 Poster Board Number: A470
Presentation Time: 8:30 AM - 10:15 AM
Contribution of Calpain and Caspases to Cell Death from Zinc Depletion in
Cultured Monkey RPE
Katherine B. Hammond1,2, Emi Nakajima1,2, Thomas R. Shearer2, Mitsuyoshi
Azuma1,2. 1Senju Laboratory of Ocular Sciences, Senju Pharmaceutical Co. Ltd.,
Beaverton, OR; 2Integrative Biosciences, Oregon Health & Science University,
Portland, OR.
Purpose: Low levels of macular zinc have been reported in age-related macular
degeneration (AMD), and oral zinc supplementation ameliorated some symptoms.
We previously reported that the membrane-permeable zinc chelator TPEN caused
cell death in cultured, primary, human retinal pigment epithelium (RPE) cells due
to activation of calpain protease. However, we did not determine if the other major
executor of cell death, caspase, was also involved in TPEN-dependent RPE cell
death. Thus, the purpose of the present study was to investigate caspase and calpain
activation during cell death, using more readily available monkey RPE cells.
Methods: Monkey primary RPE cells were cultured with TPEN, and cell viability
was assessed by the MTS assay. Activation of calpains and caspases and
proteolysis of their substrate, α-spectrin, were detected by immunoblotting for
activation-specific fragments. Calpain inhibitor, SNJ-1945, and caspase inhibitor,
z-VAD-fmk, were also used to confirm activation of the proteases.
Results: TPEN caused a time dependent decrease in viable RPE cells. Activation
of calpain 1 (presence of 78/76 kDa active fragments), mitochondria-dependent
caspase-9 (37/34 kDa fragments) and executer caspase-3 (17 kDa fragments)
occurred during TPEN-induced cell death. Immunoblots showed the calpainspecific spectrin breakdown product (SBDP) at 145 kDa and the caspase-3 SBDP
at 120 kDa. Calpain inhibitor, SNJ-1945, inhibited calpain activation and slightly
inhibited caspase-9 activation. Pan-caspase inhibitor, z-VAD-fmk, inhibited
caspase and calpain 1 activation. TPEN did not cause endoplasmic reticulum (ER)
stress or caspase-12 activation, while the positive control thapsigargin did.
Conclusions: Relative zinc deficiency in monkey RPE cells causes activation of
the cytosolic calpain and mitochondrial caspase pathways, but ER stress appears
not to be involved. Caspase activation generally indicates apoptotic cell death.
Further investigation is necessary to determine whether the observed calpain
activation also induces necrosis/apoptosis in the zinc deficient macula.
Dr. Shearer receives consulting fees from, and Drs. Azuma and Nakajima are
employees of, Senju Pharmaceutical Co. Ltd.
Commercial Relationships: Katherine B. Hammond, Senju Pharmaceutical Co.
Ltd. (E); Emi Nakajima, Senju Pharmaceutical Co. Ltd. (E); Thomas R. Shearer,
Senju Pharmaceutical Co. Ltd. (C); Mitsuyoshi Azuma, Senju Pharmaceutical Co.
Ltd. (E)
Support: None
Program Number: 4281 Poster Board Number: A471
Presentation Time: 8:30 AM - 10:15 AM
Age-dependent Differences In The Activation Of Autophagy In Retinal
Matus Rehak1,2, Sindhu Saraswathy2, Peter Wiedemann1, Narsing A. Rao2.
Department of Ophthalmology, University of Leipzig, Leipzig, Germany;
Department of Ophthalmology, Doheny Eye Institute, University of Southern
California, Los Angeles, CA.
Purpose: Recent studies suggested that autophagy plays a role in a retinal damage
resulting from ischemia-reperfusion (IR). The ageing might influence the total
autophagic activity as well as the pathway which leads to the activation of the
autophagy. We investigated the age-dependent changes in the activation of
autophagy in the murine model of IR.
Methods: Two age groups of C57BL/6 mice were investigated. In 34 young mice
(4 weeks ) and in 34 old mice (14 months) the unilateral retinal IR was induced by
cannulating the anterior chamber of the globe and transiently elevating the
intraocular pressure for 60 min. Untreated eyes served as controls. After 1, 4, 12,
24 and 72 hours of reperfusion the animals were sacrificed and the markers of
autophagy as light chain 3 (LC3-I and II), beclin-1 and autophagy-related gene 5
(atg-5) as well as calpains and caspase-3 were investigated using western blot
techniques and immunohistochemistry. Further, the retinal thickness and
histological changes were determined on day 45 after IR.
Results: In both age groups IR resulted in a rapid activation of autophagy
revealednby significant increase of LC3-II, beclin-1 and atg-5 detected by western
blotting. The highest amount of LC3-II and atg-5 was found in the young mice 12
hours after IR whereas in the old mice 24 hours after IR. The presence of beclin-1
protein was significantly higher in the young mice 4 and 12 hours after IR when
compared with old animals. Calpains (1 and 4 hours after IR) and casase-3 (24
hours after IR) showed significantly higher activity in the old animals. On day 45
after IR the reduction of the total retinal thickness was significantly higher in the
old mice (145.7±23.8 µm) compared to young mice (202.0±30.8 µm).
Conclusions: Our study showed the age-dependent differences in the activation of
the autophagy after retinal ischemia-reperfusion injury. In the young animals the
beclin-dependent way leads to significantly faster activation of the autophagy
whereas in the old animals the beclin-independent activation seems to play much
important role. In the old animals IR leads to the significantly higher activation of
the caspase-3 and the retinal atrophy.
Commercial Relationships: Matus Rehak, None; Sindhu Saraswathy,
None; Peter Wiedemann, None; Narsing A. Rao, None
Support: None
Program Number: 4282 Poster Board Number: A472
Presentation Time: 8:30 AM - 10:15 AM
Endoplasmic Reticulum Stress-associated Cone Degeneration in Cyclic
Nucleotide-gated Channel Deficiency
Arjun Thapa1, Lynsie Morris1, Jianhua Xu1, Stylianos Michalakis2, Martin Biel2,
Xi-Qin Ding1. 1Cell Biology, Univeristy of OKlahoma, Oklahoma City, OK;
Pharmacology Center for Drug Research, Ludwig-Maximilians-Universität,
München, Germany.
Purpose: Cyclic nucleotide-gated (CNG) channels play a pivotal role in
phototransduction. Mutations in the cone CNG channel subunits CNGA3 and
CNGB3 are associated with cone dystrophies and account for over 70% of all
known cases of achromatopsia. Cones degenerate in achromatopsia and cone
dystrophy patients and in CNGA3-/- and CNGB3-/- mice. This work investigates the
molecular basis of cone degeneration resulting from CNG channel deficiency.
Methods: As cones comprise only 3-5% of the total photoreceptor population in a
wild-type mouse retina, we generated the mouse lines with CNG channel
deficiency on a cone-dominant background, i.e., CNGA3-/-/Nrl-/- and CNGB3-/-/Nrl-/mice. The retinal phenotype and potential cell death pathways were examined by
functional, biochemical and immunohistochemical approaches.
Results: CNGA3-/-/Nrl-/- and CNGB3-/-/Nrl-/- mice showed impaired cone function,
cone degeneration and cell death, and opsin mislocalization as in their respective
single knockout mice. The significant elevations of the endoplasmic reticulum
stress markers, including Grp78/BiP, phospho-eIF2α, phospho-IP3R and CHOP,
were detected in the retinas of CNGA3-/-/Nrl-/- and CNGB3-/-/Nrl-/- mice, compared
to the age-matched Nrl-/- mice (at postnatal 30 days). Along with these findings, upregulation of the cysteine protease calpains and cleavage of caspase-7 and caspase12 were observed in the CNGA3-/-/Nrl-/- and CNGB3-/-/Nrl-/- retinas, suggesting an
ER stress-associated apoptosis. In addition, we observed a nucleus translocation of
AIF and Endo G in the channel-deficient retina, implying a mitochondrial insult in
the ER stress-activated cell death process.
Conclusions: By using CNGA3-/-/Nrl-/- and CNGB3-/-/Nrl-/- mouse lines we
demonstrate a primary role of the ER stress in cone degeneration in CNG channel
Commercial Relationships: Arjun Thapa, None; Lynsie Morris,
None; Jianhua Xu, None; Stylianos Michalakis, None; Martin Biel, None; XiQin Ding, None
Support: The National Center for Research Resources (P20RR017703), the
National Eye Institute (P30EY12190 and R01EY019490), and the Deutsche
Forschungsgemeinschaft (DFG).
Program Number: 4283 Poster Board Number: A473
Presentation Time: 8:30 AM - 10:15 AM
Role of Endoplasmic Reticulum Stress in Retinal Degeneration in S334ter
Rhodopsin Rats
Vishal M. Shinde1, Olga S. Sizova1, Jonathan H. Lin2, Matthew M. LaVail3, Marina
S. Gorbatyuk1. 1Cell Biology and Anatomy, University of North Texas Health
Science Center, Fort Worth, TX; 2Pathology, Univ of CA San Diego Sch of Med,
La Jolla, CA; 3Beckman Vision Center, UCSF School of Medicine, San Francisco,
Purpose: The S334ter rhodopsin (Rho) rat (line 4) bears the rhodopsin gene with
an early termination codon at residue 334 that is similar to the one found in human
patients with autosomal dominant retinitis pigmentosa (ADRP). The Unfolded
Protein Response (UPR) or Endoplasmic Reticulum (ER) is implicated in the
pathophysiology of several retinal disorders including ADRP in P23H Rho rats.
The aim of this study was to examine the onset of UPR gene expression in S334ter
Rho retinas to determine if UPR is activated in ADRP animal models and to
investigate how the activation of UPR molecules leads to the final demise of
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
S334ter Rho photoreceptors.
Methods: We have collected retinas of SD (control) and S334ter Rho
(Experimental) rat and RT-PCR was performed on CDNA prepared from these
retinas to evaluate the gene expression profiles for postnatal day (P)10, P12, P15,
P18 and P21 stages of the development and progression of ADRP in S334ter Rho
photoreceptors. We have also performed Western Blots for few proteins to confirm
the results.
Results: We found that during the P12-P15 period, ER stress-related genes are
strongly upregulated in transgenic retinas resulting in the activation of the unfolded
protein response (UPR) that was confirmed using western blot analysis. The
activation of UPR was associated with the increased expression of JNK, Bik, Bim,
Bid, Noxa and Puma genes that together with activated calpains compromise the
integrity of the mitochondrial MPTP(Mitochondrial permeability transition pore),
leading to the release of pro-apoptotic AIF1 into the cytosol of S334ter Rho
photoreceptor cells.
Conclusions: Two major cross-talking pathways, the UPR and mitochondrial
MPTP pathways communicate closely with each other in S334ter Rho retinas and
eventually promote the death of the photoreceptor cells.
Commercial Relationships: Vishal M. Shinde, None; Olga S. Sizova,
None; Jonathan H. Lin, None; Matthew M. LaVail, None; Marina S.
Gorbatyuk, None
Support: FFB (TA-GT-0409-0508-NTERI), NIH (RO1EY020905)
Program Number: 4284 Poster Board Number: A474
Presentation Time: 8:30 AM - 10:15 AM
Effects of Bevacizumab on Bcl-2 Expression and Apoptosis in Retinal Pigment
Epithelial Cells under Oxidative Stress
Suk Jin Kim, Kyong Sil Kim, Myung Hun Yoon, Na Rae Kim, Hee Seung Chin.
Department of Ophthalmology and Inha Vision Science Laboratory, Inha
University School of Medicine, Incheon, Republic of Korea.
Purpose: To evaluate the effects of bevacizumab on expression of Bcl-2, Bcl-XL
and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress.
Methods: RPE cells were treated with H2O2 (50,100,200,300,400µM) and
bevacizumab at or above the dose normally used in clinical practice (0.33, 0.67,
1.33, 2.67mg/ml). Cell death was measured by flow cytometry with annexin VFITC. The expression of Bcl-2 and Bcl-XL mRNA was determined by RT-PCR.
Results: Under low oxidative stress conditions (50 and 100 µM), cell apoptosis
was not significantly different at all concentrations of bevacizumab. Bcl-2 mRNA
expression were decreased by increasing concentrations of bevacizumab (0.33,
0.67, 1.33, 2.67mg/ml) under low oxidative stress. Under moderate oxidative stress
conditions (200 µM), cell survival was increased. Cell survival and Bcl-2 mRNA
expression were decreased by increasing concentrations of bevacizumab (0.33,
0.67, 1.33, 2.67mg/ml) under moderate oxidative stress. Under high oxidative
stress (300 µM) conditions, cell survival increased at high concentrations of
bevacizumab(1.33, 2.67mg/ml), but it did not correlate to Bcl-2 expression. No
statistically significant results in the expression of Bcl-XL were observed under any
Conclusions: Withdrawal of VEGF, as in eyes with age-related macular
degeneration, leads to RPE cell apoptosis and influences the expression of antiapoptotic genes such as Bcl-2. Anti-VEGF therapies like bevacizumab should be
administered with caution until a definite conclusion can be drawn regarding its
Commercial Relationships: Suk Jin Kim, None; Kyong Sil Kim, None; Myung
Hun Yoon, None; Na Rae Kim, None; Hee Seung Chin, None
Support: None
Program Number: 4285 Poster Board Number: A475
Presentation Time: 8:30 AM - 10:15 AM
Advance Glycation End Product and its Role in Age-related Degenerative
Diseases of the Eye
Tony Lin1, Jing Z. Cui2, Joanne A. Matsubara2. 1University of Western Ontario,
London, ON, Canada; 2Ophthalmology and Visual Sciences, University of British
Columbia, Vancouver, BC, Canada.
Purpose: Advanced glycation endproducts (AGE), a result of covalent
modifications of proteins by glycosylation, accumulate with aging and are linked to
several age-related retinal disorders. AGE deposits are found in drusen and in
Bruch's membrane of aged and diseased eyes, but what cellular pathways are
activated by their presence in outer retina is not fully known. This study
investigates the effects of AGE stimulation on global gene transcription, cellular
pathways and protein secretion in primary culture of human retinal pigment
epithelial cells (RPE). Revealing the effects of AGE on RPE function will help
elucidate the pathological changes that occur in outer retina with accumulation of
AGE and may allow new insight into the effects of drusen on age-related retinal
changes and the pathogenesis of AMD.
Methods: Primary cultures of human RPE cells were stimulated with 10 µg/ml
AGE for 4h, and genome-wide changes in gene expression were studied with
Agilent Oligo microarrays. Primary genes of interest were detected in RNA
samples using qRT-PCR with primers designed from Primer Express 2.0 software.
Western blot analysis of protein extract was performed with select primary
autoantibodies. RPE gene differential expression was analyzed using gene set
enrichment analysis (GSEA). Protein levels of secreted cytokines and growth
factors were studied using Bio-Plex Luminex arrays.
Results: A total of 41 up- and 18 down-regulated RPE genes were differentially
expressed. These genes fell into three main categories as assessed by GSEA:
inflammation (interferon-induced, immune response and chemokine expression),
proteasome degradation and caspase pathways. The highest upregulated gene was
CXCL11, a chemokine. Its production in AGE stimulated RPE cells was confirmed
using western blot analysis. Cytokine assay of supernatant using Bio-Plex Luminex
assay showed the highest increase in pro-inflammatory cytokine GM-CSF secretion
along with several anti-inflammatory cytokines including IL-10, IL-1ra and IL-9,
while many pro-inflammatory cytokines remained unchanged or underexpressed
after AGE stimulation.
Conclusions: Our studies provide new insights into the cellular pathways triggered
by the accumulation of AGE in the outer retina. RPE cells respond to AGE
stimulation by activation of two major cell signaling pathways: the NF-kB pathway
and the JAK-STAT signaling pathway. Our results suggest novel mechanisms for
AGE modulation of RPE cellular pathways and identify novel targets for drug
development that can minimize the progression of age-related retinal diseases.
Commercial Relationships: Tony Lin, None; Jing Z. Cui, None; Joanne A.
Matsubara, None
Support: CIHR Grant (MOP-97806)
Program Number: 4286 Poster Board Number: A476
Presentation Time: 8:30 AM - 10:15 AM
Retinal Degeneration in a Rat Model of Smith-Lemli-Opitz Syndrome is
Caspase-Independent, and Involves Cathepsin D Upregulation
Matthew D. Behringer1,2, Bruce A. Pfeffer1, Steven J. Fliesler1,2. 1Research Service,
VAWNYHS, Amherst, NY; 2Ophthalmology and Biochemistry, University at
Buffalo/SUNY and SUNY Eye Institute, Buffalo, NY.
Purpose: Rats treated with AY9944, an inhibitor of the final step of cholesterol
biosynthesis, undergo progressive retinal degeneration and provide an animal
model of Smith-Lemli-Opitz syndrome (SLOS). Prior proteomic and Western blot
analysis (Tu et al., ARVO 2011) revealed increased cathepsin D (CathD) levels in
AY9944-treated retinas, relative to controls; in contrast, cleaved caspase-3 (Casp3)
was not detected, despite marked TUNEL-positive outer nuclear layer labeling.
Here, we performed correlative immunohistochemical localization of CathD and
cleaved Casp3 in the retinas of control and AY9944-treated rats.
Methods: Sprague-Dawley rats were treated with AY9944 as previously described
(Fliesler et al., Pediatr. Res., 2007); untreated age-matched rats served as controls.
Eyes were harvested at ca. 2 mo of age, fixed in buffered formalin, and processed
for paraffin embedment; following antigen retrieval, tissue sections were incubated
with primary antibody and subjected to secondary immunofluorescence detection.
For CathD, summed z-stacks obtained by confocal microscopy were used to
compare fluorescence signal intensity and distribution in control and experimental
retinal sections of equivalent thickness. Etoposide-treated, paraffin-embedded
Jurkat cells served as a cleaved Casp3 positive control.
Results: In control retinas, CathD immunolabeling was detected in the RPE, as
expected; in addition, the perinuclear cytoplasm of cells in the inner nuclear and
ganglion cell layers was labeled. Punctate CathD immunolabeling in the outer
nuclear layer and outer limiting membrane, but not the plexiform layers, also was
observed. A similar, but more robust, immunolabeling pattern was observed in
retinas from AY9944-treated rats, particularly in ganglion cells; in addition,
labeling of the plexiform layers and the interphotoreceptor space was observed.
CathD immunolabeling did not colocalize with glutamine synthetase
immunoreactivity, excluding Müller cells as a significant CathD source in retinas.
Cleaved Casp3 immunoreactivity was not detected in either control or treated
retinas, whereas etoposide-treated Jurkat cells were robustly immunopositive.
Conclusions: Retinal degeneration in the AY9944-induced SLOS rat model is
Casp3-independent and involves CathD upregulation. The latter may be indicative
of an autophagic response to oxidative stress, particularly in the inner retina.
Commercial Relationships: Matthew D. Behringer, None; Bruce A. Pfeffer,
None; Steven J. Fliesler, None
Support: NIH Grant EY007361 (SJF); RPB Unrestricted Grant (SJF)
Program Number: 4287 Poster Board Number: A477
Presentation Time: 8:30 AM - 10:15 AM
miR-429 Negatively Regulates Autophagy in ARPE19 Cells
Sayak K. Mitter, Chunjuan Song, Haripriya V. Rao, Xiaoping Qi, Jun Cai, William
A. Dunn, Jr., Michael E. Boulton. Anatomy and Cell Biology, University of
Florida, Gainesville, FL.
Purpose: Autophagy plays a critical role in cellular housekeeping in the RPE.
MicroRNAs have recently been shown to play a critical role in targeting autophagic
proteins and thus regulating the autophagic response in cells. The aim of this study
was to investigate whether miR-429, which has been previously reported to be
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
downregulated by stimulation of autophagy in human cancer cell lines, regulates
autophagy in the RPE.
Methods: ARPE19 cells were reverse transfected with 80nM of miR-429 mimic
(Applied Biosystems Inc.) or miR negative control with SiPort NeoFX transfection
reagent. 24 and 48 hours after transfection the cells in the presence or absence of
bafilomycin A1 (autophagosome and lysosome fusion inhibitor) were harvested to
immunoblot for p62 and LC3 lipidation levels (markers of autophagy flux). In
parallel, cells expressing tandem tagged RFP-GFP-LC3 plasmid were transfected
with the mimic and the number of autophagosomes counted. In order to assess the
regulation of starvation-induced autophagy, the above experiments were repeated
with 3 hours of starvation prior to harvest. Quantitative RT-PCR and Western blot
were used to assess the levels of Unc51-like kinase 2 (ULK-2) and
autophagy/beclin-1 regulator 1 (AMBRA1), the in silico predicted autophagy
pathway targets (TargetScan) of miR-429, after transfection with microRNA
Results: Western Blot showed that that LC3 lipidation (as calculated by the ratio of
LC3 II to LC3 I) significantly decreased while p62 levels increased in the RPE
under both basal and starvation conditions when miR-429 mimic was
overexpressed for 24 and 48 hours compared to the miR negative control. This
result was supported by the observation that the number of autophagosomes
showed a significant decline in miR-429 mimic transfected cells when compared to
untransfected control or the negative control miR transfected cells. Our previous in
silico prediction that ULK-2 and AMBRA1 are putative targets of miR-429, was
confirmed by qRT-PCR and Western blot which showed significant decline in the
messenger RNA and protein levels of ULK-2 and AMBRA1 in RPE
overexpressing miR-429.
Conclusions: miR-429 plays a critical role in regulating autophagy in the RPE. Our
data suggests that miR-429 acts by downregulating two important autophagic
initiation factors, ULK-2 and AMBRA1.
Commercial Relationships: Sayak K. Mitter, None; Chunjuan Song,
None; Haripriya V. Rao, None; Xiaoping Qi, None; Jun Cai, None; William A.
Dunn, Jr., None; Michael E. Boulton, None
Support: NIH Grants EY018358 and EY021626
Program Number: 4288 Poster Board Number: A478
Presentation Time: 8:30 AM - 10:15 AM
Hypoxia-Induced Autophagy in Human Retinal Endothelial Cells
Andrew J. Stempel, Yuzhen Pan, Justine R. Smith, Binoy Appukuttan. Casey Eye
Institute, Oregon Health & Sciences University, Portland, OR.
Purpose: The autophagy-lysosomal pathway allows a cell to degrade non-essential
components and potentially survive exposure to physiological or pathological
stress. Hypoxic stress is one initiator of autophagy that has been studied extensively
in tumor pathobiology. We investigated the ability of human retinal endothelial
cells to develop an autophagy response in the setting of oxygen starvation, which is
a key feature of human retinal ischemic vasculopathies, including diabetic
retinopathy and retinopathy of prematurity.
Methods: Confluent immortalized human retinal endothelial cells were cultured
under conditions of normoxia (O2 = 21%) or hypoxia (O2 < 1%), achieved in an airtight Modulator Incubator Chamber (Billups-Rothenberg, Del Mar, CA) flushed
with hypoxic gas every 8 hours, for 48 hours. Subsequently, total RNA was
isolated with the RNeasy Mini Kit, and cDNA was synthesized from RNA using
the RT2 First Strand Kit (both from Qiagen). Relative expression of 84 transcripts
with established roles in autophagy, as well as 5 house keeping genes, was
determined in quintuplicate using the human RT² Profiler™ PCR Array (Qiagen)
and the Chromo4 Thermocycler (Bio-Rad) according to manufacturer’s protocol.
Data analysis was performed with the aid of RT2 Profiler PCR Array Data Analysis
software (Qiagen). Expression of vascular endothelial growth factor (VEGF)165 and
VEGF121 by endothelial cells was determined in parallel to verify hypoxic stress.
Results: An approximate 6-fold increased expression of VEGF165 and VEGF121
was measured for hypoxic human retinal endothelial cells versus control cells
(p<0.001), confirming that treated cells had experienced hypoxic stress. After
normalization to house keeping genes, 21 autophagy-related transcripts (25%) were
significantly (p < 0.05) up-regulated and 11 transcripts were significantly downregulated in the endothelial cells as a result of exposure to hypoxia, by PCR array.
However, increases were greater than 1.5-fold in just 4 genes (i.e., CXCR4,
Cathepsin S, DNA-damage regulated autophagy modulator 1 and tumor necrosis
factor). Down-regulated transcripts included ATG3, ATG4A, ATG4C and ATG9B,
which are involved in autophagic vacuole formation.
Conclusions: Our results suggest that while human retinal endothelial cells may
activate autophagy in the presence of hypoxic stress, the response appears to be
limited. Overcoming the diminished capacity of these endothelial cells to use
autophagy may have therapeutic potential in patients with retinal ischemic
Commercial Relationships: Andrew J. Stempel, None; Yuzhen Pan,
None; Justine R. Smith, None; Binoy Appukuttan, None
Support: NIH R01 EY019875 & R01 EY019042, Research to Prevent Blindness
(unrestricted grant to Casey Eye Institute)
Program Number: 4289 Poster Board Number: A479
Presentation Time: 8:30 AM - 10:15 AM
Eicosapentaenoic Acid Is Metabolized To Docosahexaenoic Acid In Retina
Neurons To Protect Photoreceptors From Apoptosis Induced By Oxidative
Nora P. Rotstein1, Daniela L. Agnolazza1, Luis E. Politi1, Martin-Paul G.
Agbaga2A, Robert E. Anderson2B. 1UNS-CONICET, Inst de Investigaciones
Bioquimicas, Bahia Blanca, Argentina; AOphthalmology, BOphthalmology/Cell
Biology, 2Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK.
Purpose: Oxidative stress triggers apoptosis of photoreceptors (PHRs) and is
involved in several retinal neurodegenerative diseases. We have previously
established that docosahexaenoic acid (DHA), the major polyunsaturated fatty acid
in the retina, promotes PHR differentiation and protects PHRs from oxidative
stress-induced apoptosis. We recently demonstrated that eicosapentaenoic acid
(EPA), a DHA metabolic precursor, had a similar protective effect. We here
investigated if EPA also enhanced PHR differentiation and whether it exerts its
effects by itself or through its metabolization to DHA.
Methods: Pure neuronal cultures prepared from rat retinas were supplemented with
or without EPA at day 1. Desaturation of EPA was inhibited by adding CP-24879
hydrochloride, a ∆5/∆6 desaturase inhibitor, one hour before EPA supplementation.
Oxidative stress was induced at day 3 with paraquat (PQ) or hydrogen peroxide
(H2O2). Differentiation was determined by analyzing opsin expression. Apoptosis
was evaluated by TUNEL assay and DAPI labeling and cellular viability with
propidium iodide. Mitochondrial integrity was determined with the fluorescent
probe Mitotracker. Fatty acid composition of cultured neurons was evaluated by
Results: Supplementation with EPA increased opsin expression and protected
PHRs from PQ and H2O2-induced apoptosis, preserving their mitochondrial
membrane potential. EPA addition did not modify EPA content in retina neurons,
but significantly increased DHA levels, compared to cultures lacking EPA. Adding
CP-24879 to the cultures prior to EPA supplementation prevented the increase in
DHA levels, in spite of EPA addition. Moreover, inhibition of DHA synthesis
completely blocked EPA protective effect on PHRs from oxidative stress-induced
Conclusions: Our results suggest that EPA promoted PHR differentiation and
rescued PHRs from apoptosis induced by oxidative stress through its elongation
and desaturation to DHA, implying that retina neurons, at least in culture, are able
to synthesize this fatty acid.
Commercial Relationships: Nora P. Rotstein, None; Daniela L. Agnolazza,
None; Luis E. Politi, None; Martin-Paul G. Agbaga, None; Robert E.
Anderson, None
Support: PIP CONICET 112-200801-021-105, PGI UNS, 24ZB26 (NP, LP),
FONCyT PICT 711 (LP) (Argentina); NIH EY04149, EY00871, and EY12190,
RP; Foundation Fighting Blindness (REA);, Hope for Vision (MPA)
Program Number: 4290 Poster Board Number: A480
Presentation Time: 8:30 AM - 10:15 AM
Omega-3 Polyunsaturated Fatty Acids Preserve Retinal Function In Type II
Diabetic Mice
Dorothy T. Pei1, Przemyslaw Sapieha2, Jing Chen1, Andreas Stahl3, Aimee M.
Juan1, Colman J. Hatton1, Christian G. Hurst1, Timothy S. Kern4, James D. Akula1,
Lois E. H. Smith1. 1Children's Hospital Boston/Harvard Medical School, Boston,
MA; 2Maisonneuve-Rosemont Hospital Research Centre/ University of Montreal,
Montreal, QC, Canada; 3University Eye Hospital, Freiburg, Germany; 4Louis
Stokes Veterans Hospital/ Case Western Reserve University, Cleaveland, OH.
Purpose: Due to the obesity epidemic, diabetic retinopathy (DR), the most
common complication of diabetes, affects a growing number of Americans each
year. Current ablation treatments are invasive, costly and do not improve vision.
Consequently, less invasive, preventative measures are increasingly necessary.
Characterized by vessel loss and subsequent pathological neovascularization (NV)
in the retina, DR is associated with hyperglycemia as well as dyslipidemia. We
previously found that ω-3 long chain polyunsaturated fatty acids (PUFAs) reduced
retinal NV compared to ω-6PUFAs in a mouse model of oxygen-induced
retinopathy (OIR), which mimics the proliferative stage of DR. The link between
DR and abnormal lipid metabolism raises the possibility that the protective effect
of an ω-3PUFA-rich diet may extend to a mouse model of type II diabetes mellitus
(T2DM), which is the aim of this study.
Methods: Leptin-receptor deficient type II diabetic mice (db/db) were fed a
defined diet with a 2% enrichment in either ω-3 or ω-6PUFAs for 22 weeks. Visual
function was measured at 9, 14, and 26 weeks by electroretinography (ERG).
Retinal capillary, neuronal integrity, and glucose stress response were assessed for
each diet. Expression of inflammatory mediators was analyzed with RT-PCR.
Results: ERG measurement showed that ω-3PUFAs significantly preserved retinal
function in the mouse model of type II diabetes to levels similar to those observed
in nondiabetic control mice on normal chow. Conversely, retinal function gradually
deteriorated in db/db mice on a ω-6PUFA rich diet. In addition, there was an
enhanced ability for ω-3PUFA-fed mice to respond to glucose challenge. There
were no significant differences in retinal vasculature, retinal ganglion cell (RGC)
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
numbers, or inflammatory mediatory levels between the ω-3PUFA-fed and ω6PUFA-fed mice, suggesting that the protection of visual function was independent
of cytoprotective or anti-inflammatory effects of ω-3PUFAs.
Conclusions: The results from this study suggest that an ω-3PUFA-rich diet is
beneficial in preserving retinal function in a mouse model of T2DM. Moreover,
dietary supplementation of ω-3PUFAs may represent a safe, inexpensive means of
slowing the progression of vision loss in T2DM.
Commercial Relationships: Dorothy T. Pei, None; Przemyslaw Sapieha,
None; Jing Chen, None; Andreas Stahl, None; Aimee M. Juan, None; Colman
J. Hatton, None; Christian G. Hurst, None; Timothy S. Kern, None; James D.
Akula, None; Lois E. H. Smith, None
Support: NIH (EY017017, EY017017-04S1, EY017017-05) and Research to
Prevent Blindness Senior Investigator Award (LEHS), CIHR and CDA (PS), JDRF
and Charles H. Hood Foundation (JC).
Program Number: 4291 Poster Board Number: A481
Presentation Time: 8:30 AM - 10:15 AM
Aryl Hydrocabron Receptor (AhR) Activation Regulates Lipid Synthesis in
Retinal Pigment Epithelial (RPE) Cells
Goldis Malek1A, Amanda Bednar1B, Peng Hu1B. AOphthalmology and Pathology,
Ophthalmology, 1Duke University, Durham, NC.
Purpose: AhR is a nuclear receptor activated by environmental contaminants such
as dioxin and benzo(a)pyrene. These toxins have been shown to promote the
development of atherosclerosis-related cardiovascular disease and lipid
accumulation in macrophages. Previously we demonstrated that the AhR signaling
pathway is activated in human RPE cells after treatment with cigarette smoke
extract and dioxin. Given that cigarette smoking is an established risk factor for
age-related macular degeneration, we investigated the molecular mechanisms
underlying AhR activation in RPE cells.
Methods: Expression of AhR-regulated genes in dioxin and cigarette smoke
extract treated ARPE19 cells transfected with and without AhR siRNA was
determined through microarray and quantitative real-time PCR. Gene pathways
were clustered using GoRilla. The effect of cholesterol on AhR activation was
evaluated using luciferase based activity assays, qPCR and Western blot analysis.
Cellular lipid content was determined based on oil red staining and
cholesterol/cholesterol ester quantitation assays.
Results: Microarray analysis revealed increased expression of genes involved in
lipid metabolism in ARPE19 cells, after exposure to dioxin and regulated through
AhR. These included but are not limited to CYP2J2, ABCA1, FADS, PPARdelta
and LDLR. Treatment with cholesterol and 25 hydroxy cholesterol activated AhR
and stimulated expression of AhR specific phase I toxin-metabolizing genes
CYP1A1, CYP1B1, IL1-B and AhRR. Dioxin triggered lipid accumulation in cells.
Conclusions: Environmental toxins regulate lipid metabolism and accumulation in
RPE cells through the AhR. Cholesterol and 25 hydroxy cholesterol, which is
formed after a dietary meal from endogenous cholesterol, are activating ligands of
AhR. Activation of the AhR signaling pathway may play a role in pathogenic
accumulation of the lipid-protein rich deposits under the RPE in aging and
characteristic of dry age-related macular degeneration.
Commercial Relationships: Goldis Malek, None; Amanda Bednar, None; Peng
Hu, None
Support: NIH EY02868, AHAF-Macular Degeneration RPB Core
Program Number: 4292 Poster Board Number: A482
Presentation Time: 8:30 AM - 10:15 AM
Characterization Of Primary Cultures From Adult Retinal Pigment Epithelial
Cells (hrpe) And Protection Mediated By Docosahexaenoic Acid (dha)
Lynn A. Caviness1, Pranab K. Mukherjee2A, Eric J. Knott, Jr.3, Nicolas G. Bazan2B.
Neuroscience, LSU Health Science Center, New Orleans, LA; ANeuroscience
Cntr/Ophthalmology, BOphthal & Neuroscience, 2LSU Health Sciences Center,
New Orleans, LA; 3Neuroscience Center, Louisiana State Univ Hlth Sci Ctr, New
Orleans, LA.
Purpose: Culturing adult primary hRPE cells has proven challenging. These cells
grow slowly, have a tendency to adopt a fusiform morphology, and detach in the
presence of oxidative stress (OS). Thus in this study, we first established optimal
culture conditions and then asked if docosahexaenoic acid (DHA) pretreatment
induces protection against H2O2-mediated cell death, as seen in ARPE-19 cells.
Methods: Cells were cultured in Eagle Minimum Essential Medium 10% Fbs with
FGF-β (10ng/mL) in the presence of Rho kinase inhibitor, retinoic acid, or Poly-LLysine coated plates. Once cuboidal morphology was attained, the cells were
characterized with RPE cell markers ZO-1, RPE65, and Neu-N via Western blot
and immunocytochemical analysis. All cellular culture conditions were tested using
varying levels of OS. Using Poly-L-lysine coated plates, cells were pretreated with
600 nM DHA for 6h then placed in DHA free medium for 24h before challenge
with H2O2+TNF-α. Apoptosis was assessed using Hoechst staining and confocal
Results: hRPE cells cultured with Rho kinase inhibitor were most resilient to OSinduced apoptosis, yet they failed to achieve the cuboidal morphology
characteristic of RPE cells. When Rho kinase inhibitor was combined with Poly-LLysine-coated plates, this effect was enhanced (J.Neuroscience.Res.,86,27080,2008). Cells cultured on Poly-L-Lysine-coated plates without Rho kinase
inhibitor achieved the epithelial-like morphology and were slightly more sensitive
to OS than Rho kinase inhibitor alone, but this difference was not significant. In
both the control and retinoic acid conditions, cells detached at 150µM OS. Western
blot and immuncytochemical analysis of Poly-L-Lysine coated plates indicate that
these cells express ZO-1, RPE65, and Neu-N. In addition, when challenged with 0,
150, 250, and 350 µM OS +TNF-α these cells displayed survival levels of
99.6±0.1% 95.5±1.5%, 69.6±12.5%, 59.2±8.8%, respectively, while DHA
exhibited protection with survival levels of 97.6±0.9%, 96.3±0.9%, 95.4±3.4%, and
87.1±10.3% respectively.
Conclusions: Our results demonstrate that hRPE cells (expressing ZO-1, RPE65,
and Neu-N), grown on Poly-L-Lysine-coated plates in the presence of FGF-β,
result in high densities of cuboidal cells, which remain attached in the presence of
OS, and provide better cell morphology than Poly-L-Lysine-coated plates with the
Rho inhibitor. Also, when pretreated with DHA, these cells are protected from
H2O2-induced cell death, implicating the importance of DHA levels in hRPE cell
Commercial Relationships: Lynn A. Caviness, None; Pranab K. Mukherjee,
None; Eric J. Knott, Jr., None; Nicolas G. Bazan, None
Support: Support: NEI EY005121; Research to Prevent Blindness, Inc.
Program Number: 4293 Poster Board Number: A483
Presentation Time: 8:30 AM - 10:15 AM
Sphingolipid Signaling in Mammalian Photoreceptor Cells
Md Nawajes A. Mandal1,2, Nicole Prabhu1,2, Carolina Abrahan3, Nora Rotstein3,
Joel McRae1,2, Annette Eckerd1,2, Rishard S. Brush1,2. 1Ophthalmology, Univ of
Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Dean McGee Eye Institute,
Oklahoma City, OK; 3Instituto de Investigaciones Bioquímicas de Bahía Blanca
(INIBIBB), Universidad Nacional del Sur (UNS)-CONICET, Bahía Blanca,
Purpose: Sphingolipids are essential components of every cell membrane.
Recently, a signaling role of bioactive sphingolipids such as ceramide and
sphingosine-1-phosphate (S1P) has been established. Ceramide induces apoptosis
and S1P plays an anti-apoptotic role. S1P signals through G-protein coupled
receptors. Here we investigated ceramide and S1P signaling in photoreceptor cells.
Methods: Expression of S1P biosynthetic enzymes sphingosine kinase (SPHK1
and 2) and their receptors S1P1-5 in rat retina was tested by quantitative RT-PCR,
Western blotting (WB) and by Immunohistochemistry (IHC). Developing rat
photoreceptors were isolated and used for immunocytochemistry. Rod outer
segments (ROS) were prepared from light-adapted and dark-adapted rat retinas by
sucrose density gradient centrifugation and subjected to WB with antibodies of the
above proteins. Further, rat retinas were light stressed in vivo at 2700 lux for 6 h,
which specifically affects the photoreceptor cells, and the signaling of ceramide and
S1P was measured by assaying gene expression, activity of bio-synthetic enzymes,
and levels of bio-synthetic intermediates of ceramide and S1P.
Results: We detected expression of both SPHK1 and 2 in photoreceptor cells by
RT-PCR; we detected localization of both the proteins in different layers of the
retina; SPHK1 was not detected in ROS. Photoreceptor cells expressed S1P
receptors S1P1, S1P2 and S1P3 with very prominent expression of S1P2 in ROS.
S1P1 expression in ROS was reduced in light-adaptation. In immunohistochemical
sections, S1P1 had intense labeling at RPE apical membrane. In isolated rod
photoreceptors S1P3 was located on plasma membrane and S1P2 on developing
ROS. Intense light exposure increased retinal ceramide and S1P levels well before
the onset of apoptosis. In the light-damage model, ceramide increased by de novo
biosynthesis whereas increased S1P blocked ceramide formation from acid
sphingomyelinase (aSMase). Intense light also increased expression of S1P2 and
S1P3 indicating S1P signaling in photoreceptors.
Conclusions: Ceramide and S1P play a critical role in photoreceptor apoptosis
induced by light stress. Expression and localization of SPHKs and G-proteincoupled S1P receptors on photoreceptor and RPE cells suggest their functions in
these cells, which may include visual signal transduction, membrane turnover and
transport or trafficking of proteins in the RPE and ROS membrane, and stress
Commercial Relationships: Md Nawajes A. Mandal, None; Nicole Prabhu,
None; Carolina Abrahan, None; Nora Rotstein, None; Joel McRae,
None; Annette Eckerd, None; Rishard S. Brush, None
Support: NIH/NCRR COBRE (RR17703), Research to Prevent Blindness Inc.
Program Number: 4294 Poster Board Number: A484
Presentation Time: 8:30 AM - 10:15 AM
Sterculic Acid Inhibits 7-ketocholesterol-mediated Unfolded Protein Response
In ARPE-19 Cells
Jiahn-Dar Huang, Jung Wha Lee, Ignacio R. Rodriguez. LRCMB, National Eye
Institute, Bethesda, MD.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Purpose: 7-ketocholesterol (7KCh) is a potent inducer of unfolded protein
response (UPR) and a major oxysterol in oxidized lipoprotein deposits in sub-RPE
region. The prolonged UPR mediated by 7KCh may lead to local chronic
inflammation which in turn may contribute to the development of age-related
macular degeneration (AMD). We have previously shown that sterculic acid shuts
down 7KCh-mediated inflammatory responses. Here we examine the effect of
sterculic acid on 7KCh-mediated UPR in ARPE-19 cells.
Methods: Cultured ARPE-19 cells were treated with 7KCh in serum-free medium
with or without sterculic acid. Stearic acid was used as a structurally similar
control. After 24hr treatment, the mRNA expression of the UPR components
including PERK, EIF2α, ATF4, CHOP, IRE1, XBP1, GRP78, and P58IPK in
ARPE-19 cells were determined by quantitative RT-PCR. The protein levels of
phosphorylated-EIF2α, EIF2α, phosphorylated-IRE1α, IRE1α, CHOP, GRP78, and
P58IPK were visualized using immunoblot. The 7KCh-mediated disruption of
cytoplasmic calcium level was evaluated using Fluo-4 assay with an EnVision
Multilabel Reader.
Results: Sterculic acid significantly attenuated 7KCh-mediated mRNA expressions
of UPR components in ARPE-19 cells while stearic acid did not show any effect.
Immunoblot showed that sterculic acid suppressed the phosphorylation of EIF2α
and IRE1α as well as the induction of CHOP and GRP78 by 7KCh treatment. The
protein expression of P58IPK did not change by 7KCh treatment either with or
without sterculic acid. In ARPE-19 cells, a transient increase of cytoplasmic
calcium level was observed immediately after 7KCh treatment. Adding a calcium
ion chelator (EGTA) or a calcium channel (i.e. IP3 receptor) inhibitor (2-APB)
suppressed the transient calcium increase. However, adding sterculic acid to 7KChtreated cells did not suppress the transient calcium increase.
Conclusions: Sterculic acid significantly inhibits all three UPR pathways induced
by 7KCh. This antagonist effect is not due to the induction of P58IPK, an inhibitor
of the PERK pathway. This antagonist effect is also not caused by suppressing the
transient calcium flow from extracellular sources or the endoplasmic reticulum. In
summary, our results suggest that sterculic acid did not antagonize 7KCh-mediated
inflammation by inhibiting specific UPR pathways. Sterculic acid seems to inhibit
upstream to UPR pathways and possibly downstream of the calcium response.
Commercial Relationships: Jiahn-Dar Huang, None; Jung Wha Lee,
None; Ignacio R. Rodriguez, None
Support: NEI intramural program
Program Number: 4295 Poster Board Number: A485
Presentation Time: 8:30 AM - 10:15 AM
Human Retinal Pigment Epithelium Cells As Functional Models for Retinoid
Lucero J. Vivar1A, Yueying Liu1A, Patrice Goletz1A, Mohammad Dahrouj1A, Peter
H. Tang1B, Zsolt Ablonczy1A. AOphthalmology, BNeurosciences - Ophthalmology,
Medical University of South Carolina, Charleston, SC.
Purpose: A culture model of the retinal pigment epithelium (RPE) is fetal human
RPE (fhRPE) cells. Under correct culture conditions, these cells express
considerable levels of RPE markers and can develop monolayers of highly
pigmented cells that are uniform in size and shape. They also develop
transepithelial potential and high (> 400 Ωcm2) transepithelial resistance (TER).
Although these cells exhibit many functional charateristics of the native RPE, their
ability to participate in retinoid metabolism and convert vitamin-A into 11-cis
retinal has received only limited attention.
Methods: fhRPE cells were cultured on transwell filters by established methods
until they formed monolayers of cuboid pigmented cells with a TER > 400 Ωcm2.
The expression of RPE65, CRALBP, and CRBP were examined by
immunoblotting and immunohistochemical analysis. mRNA expression levels of
RDH 5, RDH 8, RDH 10, RDH were determined by RT-PCR. Some cultures were
administered vitamin-A or all-trans retinal in phosphatidylcholine vesicles at a
concentration of 5nM/L for 2 days, then the cells and the media were harvested and
analyzed by HPLC to determine the respective retinoid profiles. Select cultures
received bleached rod outer segments (ROS), or human albumin after the vesicles
were absorbed.
Results: fhRPE cells express the full complement of RPE proteins, which facilitate
the isomerization of 11-cis retinal. Western blots provided evidence on the presence
of substantial quantities of RPE65, CRALBP, and CRBP. RT-PCR showed the
expression of RDH5, RDH8, RDH10, and RDH12. Immuno-histochemistry
indicated that the proteins were localized in their native compartments. Feeding the
cells with all-trans retinal or vitamin-A encapsulated in phosphatidylcholine
vesicles resulted in the appearance of mainly retinyl esters and a small quantity of
11-cis retinal in the cells, while the administered retinoid disappeared from the
media. Administering bleached rod outer segments or human albumin after the
retinoid-filled vesicles were absorbed by the cells enhanced the quantity of detected
11-cis retinal.
Conclusions: Fully differentiated fhRPE cells not only express all the enzymes
which facilitate the visual cycle, but these enzymes are in the correct cellular
compartments, and together, they can also functionally convert vitamin-A to 11-cis
retinal. The efficiency of the conversion is relatively little compared to the eye, but
it is mostly a direct consequence of missing photoreceptors. When a sink, which
can absorb 11-cis retinal, is introduced the conversion speeds up. These data
demonstrate that fhRPE cells are a suitable functional model for vitamin-A
Commercial Relationships: Lucero J. Vivar, None; Yueying Liu,
None; Patrice Goletz, None; Mohammad Dahrouj, None; Peter H. Tang,
None; Zsolt Ablonczy, None
Support: NIH grants EY019065 (ZA), the South Carolina Lions Foundation, and
an unrestricted grant from RPB (MUSC)
Program Number: 4296 Poster Board Number: A486
Presentation Time: 8:30 AM - 10:15 AM
Expression of Matriptase2 (TMPRSS6), a Proteolytic Regulator of Iron
Homeostasis, in Retina, and Expression of Iron-regulatory Genes in
Matriptase2-null Mouse Retina
Brooke R. Bozard1A, Sudha Ananth1A, Jaya P. Gnana-Prakasam1A, Pamela M.
Martin1A, Sylvia B. Smith1B, Vadivel Ganapathy1A. ABiochemistry and Molecular
Biology, BCellular Biology and Anatomy, 1Georgia Health Sciences University,
Augusta, GA.
Purpose: Retina expresses a complete set of iron-regulatory proteins essential for
iron homeostasis. Important among these are HFE, hemojuvelin (HJV), hepcidin,
ferroportin, and transferrin receptors, which are expressed in retina in a cell typespecific manner. Matriptase2 (TMPRSS6) is a protease that cleaves hemojuvelin on
the cell surface and consequently regulates relative levels of soluble HJV and
membrane-bound HJV. Since cell signaling by bone morphogenic proteins is
controlled differentially by soluble HJV and membrane-bound HJV, this process is
also regulated by matriptase2. Deletion of matriptase2 leads to systemic iron
deficiency. Here the retinal expression of this proteolytic regulator of iron
homeostasis in wild type mice, and alterations in retinal expression pattern of ironregulatory genes in matriptase2-null mice were investigated.
Methods: Expression of matriptase2 in wild type mouse retina was monitored by
RT-PCR and immunofluorescence, and its colocalization with HJV was studied by
double labeling. Iron levels in wild type and matriptase2-null retina were measured
indirectly by quantifying tissue levels of ferritin by ELISA. Retinal morphology
was compared between wild type and matriptase2-null mice by H & E staining.
Expression of various iron-regulatory genes in wild type and matriptase2-null
mouse retinas was monitored by RT-PCR and immunofluorescence.
Results: RT-PCR and immunofluorescence studies showed that matriptase2 is
expressed widely in the retina and its expression pattern is similar to that of HJV.
In RPE, which is a polarized cell, matriptase2 and HJV colocalize at the apical
membrane. The levels of ferritin are much lower in matriptase2-null mouse retina
than in wild type mouse retina, indicating reduced levels of iron in the knockout
mouse retina. RT-PCR showed that the levels of HJV mRNA and hepcidin mRNA
were markedly elevated in matriptase2-null mouse retina compared to wild type
mouse retina. However, there was no evidence of any morphological change in the
null mouse retina.
Conclusions: Retina expresses matriptase2, the proteolytic regulator of iron
homeostasis. The expression pattern of matriptase2 is similar to that of HJV, its
proteolytic substrate. Iron deficiency is evident in matriptase2-null mouse retina,
which is a consequence of increased levels of HJV and hepcidin within the retina.
Commercial Relationships: Brooke R. Bozard, None; Sudha Ananth,
None; Jaya P. Gnana-Prakasam, None; Pamela M. Martin, None; Sylvia B.
Smith, None; Vadivel Ganapathy, None
Support: EY019672
Program Number: 4297 Poster Board Number: A487
Presentation Time: 8:30 AM - 10:15 AM
Protection Effect Of Astaxanthin Against Light-induced Retinal Damage In
Atsushi Yamamoto, Mitsuko Yuzawa. Department of Ophthalmology, Nihon
University School of Medicine, Tokyo, Japan.
Purpose: To investigate the protection effect of Astaxanthin (ASX), a xanthophyll
carotenoid from the marine algae Hematococcus pluvialis, against light-induced
retinal damage.
Methods: Albino rats were divided into three groups including control, ASX
treated(100mg/kg), ASX treated (1mg/kg). Rats were treated with ASX
approximately 1 hour before 12-hours exposure to 3000-lux white LED light. After
7 days exposure, protective effect of ASX was evaluated functionally by
electroretinogram(ERG) and histologically by counting retinal apoptosis.
Results: After exposure to light, control rats had a 81% loss of ERG A-wave
amplitudes. Compared with control rats, ERG A-wave amplitudes were
significantly higher in 100mg/kg(P<0.005), and 1mg/kg(P<0.01) ASX-treated rats.
Retinal TUNEL staining showed apoptosis was observed in all groups; however
ASX treatment significantly decreased the percent of apoptotic cells when
compared to non treated group.
Conclusions: Astaxanthin might have protective effect against light-induced retinal
damage in rats.
Commercial Relationships: Atsushi Yamamoto, None; Mitsuko Yuzawa, None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Support: None
Program Number: 4298 Poster Board Number: A488
Presentation Time: 8:30 AM - 10:15 AM
Lipoprotein(a) Protects against Oxidized Phospholipid in the Retina
Mizuki Tagami1, Anna Janiak1, Sotirios Tsimikas2, Joseph L. Witztum2, James T.
Handa1. 1Ophthalmology-Retinal Vascular Service, Johns Hopkins Wilmer eye
Inst, Baltimore, MD; 2Dept of medicine, division of cardiology, UC SAN DIEGO,
San diego, CA.
Purpose: Oxidative stress has been hypothesized to contribute to Age-related
macular degeneration (AMD). Oxidized phosphatidylcholine (OxPL), results from
chronic oxidative stimulation that occurs in AMD. In human plasma, OxPL is
specifically bound by lipoprotein(a) (Lp(a)). We previously demonstrated the
accumulation of oxidized apolipoprotein B100 lipoproteins (apoB) in drusen of
AMD. Lp(a) is composed of apoB100, but it also contains the apo(a) protein. While
Lp(a) is a risk factor for cardiovascular disease, its function in the retina is
unknown. The purpose herein was to determine whether Lp(a) influences the
phenotype of the retina.
Methods: Human AMD maculas were surveyed for Lp(a) and OxPL by IHC. Mice
were fed a regular chow diet and exposed to a 12 hour on/off light cycle. Mice
transgenic for human Lp(a) (containing both human apo(a) and human apoB100)
and mice transgenic for a mutant Lp(a) unable to bind OxPL (muLp(a)- containing
a human mutated apo(a) and human apoB100) were studied along with wild-type
C57Bl6 and mice transgenic for human apoB were evaluated for OxPL using E06
antibody, and for AMD phenotype by electron microscopy (TEM).
Results: Human AMD maculas (n=14) and age-matched controls (n=5) showed
staining for Lp(a) and OxPL was confined to outer Bruch’s membrane and choroid.
In AMD eyes, drusen stained for both Lp(a) and OxPL. While apo(a) was
expressed in HepG2 cells, it was not expressed by human ARPE-19 cells using
either RT-qPCR or Western blot analysis. We next compared the phenotype of
Lp(a) to muLp(a) mice, which do not bind OxPL. Compared to muLp(a) mice, 12
mo Lp(a) mice had reduced OxPL in the retina, RPE, Bruch’s membrane, and
choroid (n=8 per group). Semi-thin sections (n=10 per group) showed prominent
intra-RPE vacuoles in 100% of muLp(a) and 10% of Lp(a) mice, and none in WT
or ApoB mice. Drusen were seen in 30% of muLp(a) eyes only. TEM showed a
marked increase in intracellular membranous vacuoles (p<0.006), basal laminar
deposits (p=0.056), and outer collagenous layer deposits (p<0.003) in muLp(a)
mice over all other groups.
Conclusions: Lp(a) localizes with oxPL to the outer macula. Our data support the
hypothesis that Lp(a) removes OxPL from the posterior pole of retina, while
impaired removal of oxPL -as in the muLp(a) mice- is associated with an early
AMD phenotype.
Commercial Relationships: Mizuki Tagami, None; Anna Janiak,
None; Sotirios Tsimikas, None; Joseph L. Witztum, None; James T. Handa,
Support: EY14005, EY019904, Thome Foundation, Beckmann Foundation,
Unrestricted RPB award to the Wilmer Eye Institute, Robert Bond Welch
Program Number: 4299 Poster Board Number: A489
Presentation Time: 8:30 AM - 10:15 AM
The Further Opening Of The DHA Metabolome In Retinal Pigment Epithelial
(RPE) Cells: Maresin Synthesis Diverges From NPD1 Synthesis By Enhanced
Oxidative Stress
Bokkyoo Jun, Eric J. Knott, Changde Zhang, Nicolas G. Bazan. Neuroscience
Center, LSU Health Sciences Center, New Orleans, LA.
Purpose: Endogenous bioactive mediators synthesized in RPE cells in response to
enhanced oxidative stress are important for photoreceptor function and integrity
(IOVS:48,4866-81,2007). DHA is concentrated and avidly retained in
photoreceptors. Also DHA traffics daily through RPE cells. Thus, following the
discovery of neuroprotectin D1 (PNAS:101,8491-6,2004), we now report the
induction of a 14S-hydroxydocosa-4Z,7Z,10Z,12E,16Z,19Z-hexaenoic acid
pathway from DHA in RPE cells. Addition of deuterium-labeled DHA in the
presence of oxidative stress led to the synthesis of these novel docosanoids - the
maresins - reported in activated human macrophages (J.Exp.Med.:206,15-23,
2009). We found divergent induction of the 14S- (maresins) and 17S- (NPD1)
pathways by enhanced oxidative stress in RPE cells.
Methods: Human ARPE-19 cells were incubated in the medium with DHA-d5
(4Z,7Z,10Z,13Z,16Z,19Z-docosahexaenoic-21,21,22,22,22-d5 acid) supplemented
for 6h, and the medium was replaced with DHA-d5 free medium. The cells were
under oxidative stress with H2O2 (50, 400, 500 and 600µM) and TNF-α. Lipids
were extracted and loaded onto an Eclipse Pluse C18 column (Agilent
Technologies, Santa Clara, CA) and run gradient for 12min by changing solvent A
(100:0.01 MeOH:CH3COOH) from 70% to 100% while decreasing solvent B
(100:0.01 H2O: CH3COOH), 6min of 100% B before changing to A:B=70:30 for
5min. TSQ Quantum Ultra (Thermo-Finnigan, San Jose, CA) triple quadrupole
tandem mass spectrometer (with HESI probe) was used for the analysis. Selected
parent and daughter ion pairs of maresin and maresin-d5 were 359/250 m/z and
364/250 m/z, respectively.
Results: Endogenous maresin from DHA incorporated in the cellular membrane
was found 2 ~ 5 times smaller than maresin-d5 from supplemented DHA-d5,
depending on the amount of oxidative stress. One of the maresin isomers, 7Smaresin, was also increased from supplemented DHA. The maresin synthesis
decreased as oxidative stress was enhanced, which is the opposite of what occurs
for NPD1 synthesis.
Conclusions: We show here for the first time the ability of RPE cells to activate a
14S-pathaway for the synthesis of maresins. Maresin synthesis from DHA is
activated by low oxidative stress conditions (50 µM H2O2). These new RPE
docosanoids may contribute to cope with inflammatory resolution. The significance
of the presence of maresins in macrophages also is being explored in RPE cells for
their potential involvement in outer segment phagocytosis.
Commercial Relationships: Bokkyoo Jun, None; Eric J. Knott, None; Changde
Zhang, None; Nicolas G. Bazan, None
Support: NEI EY005121, Research to Prevent Blindness, Inc.
Program Number: 4300 Poster Board Number: A490
Presentation Time: 8:30 AM - 10:15 AM
7-Dehydrocholesterol-derived Oxysterols are Differentially Cytotoxic to 661W
Photoreceptor Cells
Bruce A. Pfeffer1, Priyanka H. Patel1, Libin Xu2, Ned A. Porter2, Steven J.
Fliesler1,3. 1Research Service, VAWNYHS, Amherst, NY; 2Chemistry and
Vanderbilt Inst of Chem Biol, Vanderbilt University, Nashville, TN;
Ophthalmology and Biochemistry, University at Buffalo/SUNY and SUNY Eye
Institute, Buffalo, NY.
Purpose: 7-dehydrocholesterol (7DHC), an immediate precursor of cholesterol
(CHOL), accumulates as the dominant sterol in tissues and bodily fluids of humans
afflicted with Smith-Lemli-Opitz Syndrome (SLOS), a recessive metabolic disease.
7DHC is ca. 200-fold more labile to peroxidation than is CHOL, and gives rise to a
variety of oxysterol products, some of which are cytotoxic (Korade et al., JLR
2010). Retinal degeneration (involving progressive photoreceptor death), along
with 7DHC and 7DHC-derived oxysterol formation, occur in the AY9944-induced
rat model of SLOS (Fliesler et al., Arch Ophthalmol. 2004; Xu et al., ARVO 2011).
Here, we evaluated the relative susceptibility of cultured 661W cells (a
photoreceptor-derived cell line) to the cytotoxic effects of 7DHC-derived
Methods: 661W cells were incubated with 7-ketocholesterol (7kCHOL), 3β,5αdihydroxycholest-7-en-6-one (DHCEO), 4β-hydroxy-7DHC (4HDHC), 5,9endoperoxy-cholest-7-en-3β,6β-diol (EPCD), or CHOL, each at 0.1 to 30 µM,
solvated with hydroxypropyl-β-cyclodextrin (HPCD, 0.96 mM final conc’n), in
DMEM/F12 culture media containing 0.2% (v/v) serum (1.9 µM CHOL). After 48
h, cellular metabolic competence was assessed by resazurin reduction (RzR) assay,
while cell death was assayed by Sytox Orange (SO) binding; SO values were
normalized to RzR values (the latter proportional to cell number). Cell morphology
was monitored by indirect photomicroscopy.
Results: At 30 µM, both 7kCHOL and DHCEO were markedly cytotoxic, causing
cell rounding/blebbing and detachment, precluding quantitative SO measurements,
whereas 30 µM 4HDHC was moderately well-tolerated. CHOL was not soluble at
30 µM in HPCD/medium. At 3 and 10 µM, the order of cytotoxicity was: EPCD >>
7kCHOL > DHCEO > 4HDHC >> CHOL (CHOL was comparable to medium
alone, +/- HPCD). EPCD exhibited toxicity even at 1 µM.
Conclusions: 661W cells exhibit differential cytotoxicity to 7DHC-derived
oxysterols. Our results are consistent with the hypothesis that such oxysterols are
involved in the progressive photoreceptor degeneration observed in the SLOS rat
model, and potentially in SLOS as well.
Commercial Relationships: Bruce A. Pfeffer, None; Priyanka H. Patel,
None; Libin Xu, None; Ned A. Porter, None; Steven J. Fliesler, None
Support: NIH Grants EY007361 (SJF), ES013125 and HD064727 (NAP and LX);
NSF Grant CHE 0717067 (NAP and LX); and RPB Unrestricted Grant (SJF)
Program Number: 4301 Poster Board Number: A491
Presentation Time: 8:30 AM - 10:15 AM
7-Ketocholesterol Is Metabolized by Acyl-coenzyme A: Cholesterol
Acyltransferase 1 (ACAT1/ SOAT1) in ARPE-19 Cells
Jung W. Lee, Jiahn-Dar Huang, Ignacio R. Rodriguez. Mechanisms of Retinal
Diseases Section, LRCMB, National Eye Institute/NIH, Bethesda, MD.
Purpose: Acyl-coenzyme A: cholesterol acyltransferase (ACAT1/SOAT1) is an
endoplasmic reticulum enzyme that generally catalyzes the formation of cholesteryl
esters from cholesterol (Ch) and long-chain fatty acids. The purpose of this study is
to investigate the involvement of ACAT1 in the metabolism of 7-ketocholesterol
(7KCh) in ARPE-19 cells.
Methods: ACAT1 mRNA was measured in human tissues and ARPE-19 cells by
real time qRT-PCR. A rabbit polyclonal affinity purified anti-ACAT1 antibody was
used for immnublot and immunohistochemistry. Immunolocalization of ACAT1
was performed on monkey retina vibrotome sections (100 µm). ARPE-19 cells
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
were preincubated with either ACAT1 inhibitor or cytosolic phospholipase A2a
(cPLA2a) inhibitor prior to 7KCh treatment. Cells were collected and analyzed for
7KCh, Ch and 7KCh-fatty acid esters (7KFAEs) 24 h after 7KCh treatment. Cell
viability was determined by celluar dehydrogenase activity. Identification and
quantification of 7KCh, Ch and 7KFAEs was performed by HPLC-UV and LCMS.
Results: qRT-PCR analyses and immunoblots detected a significant level of
ACAT1 mRNA and protein in human retina and ARPE-19 cells. In monkey retina
ACAT1 localized mainly to the photoreceptor outer segments, outer plexiform and
ganglion cell layers. A lower level of expression was observed in the RPE and
choroid. HPLC-UV analyses of extracts from 7KCh-treated ARPE-19 cells
detected 4 main 7KFAEs. Preliminary LCMS results indicate these esters are to
16:1 (palmitoleic), 18:3 (linolenic), 20:4 (arachidonic), and 20:3 (eicosatrienoic).
The 7KFAEs accounted for 15-25% of the internalized 7KCh. No hydroxylated or
sulfated forms of 7KCh were detected by LCMS. Inhibition of ACAT1 ablated the
7KFAEs synthesis but did not prevent cytotoxicity. By contrast, inhibition of
cPLA2a while also ablating the 7KFAE synthesis provided some protection from
7KCh cytotoxicity.
Conclusions: Our results indicate that the only significant metabolism of 7KCh in
these cultured cells is via fatty acid esterification catalyzed by ACAT1. Inhibition
of ACAT1 provided no protection from cytotoxicity suggesting the 7KFAEs are
not involved in the 7KCh-mediated inflammatory pathways. Inhibition of cPLA2a
did provide some protection to 7KCh-mediated cytotoxicity suggesting that the
fatty acid released by cPLA2 while serving as substrates for ACAT1 may also be
involved in the 7KCh-mediated inflammation.
Commercial Relationships: Jung W. Lee, None; Jiahn-Dar Huang,
None; Ignacio R. Rodriguez, None
Support: NEI intramural Research Program
Program Number: 4302 Poster Board Number: A492
Presentation Time: 8:30 AM - 10:15 AM
A Rat Model for 7-Ketochlesterol (7KCh) Induced Inflammation and
Juan Amaral1A, Maria M. Campos1B, Joshua Chou1A, Ignacio R. Rodriguez1A.
Mechanism of Retinal Diseases / LRCMB, BBiological Imaging Core, 1National
Eye Institute, Bethesda, MD.
Purpose: Oxidative processes have been proposed to play a causative or
contributing role in a steadily growing number of diseases, such as heart disease,
certain types of cancers, neurodegenerative disorders, cataract, and age-related
macular degeneration (AMD). 7KCh, a toxic oxysterol that accumulates in the
RPE/choriocapillaris complex, is known to induce several inflammatory pathways.
The purpose of this study is to describe a novel method to study 7KCh-induced
angiogenesis in vivo.
Methods: Poly (2-hydroxyethylmethacrylate) and polyethylene glycol (20,000)
were mixed in equal portions and dissolved in 100% ethanol. 7KCh was added to
the mixture and allowed to dissolve. The final concentrations for 7KCh were 10%,
15% and 20% (w/w) and for Cholesterol (Ch) 20%. The dried solids were grounded
to a fine powder in a mortar and pestle. The powder (50 mg) was placed into a 20
mm die and subjected to 25 metric tons of pressure to form a wafer. Small 0.5 mm
discs were punched from the main wafer using a trephine. A corneal incision was
made to allow access to the anterior chamber for implantation of the 0.5 mm
wafers. After implantation, corneal vessel growth was evaluated for up to 21 days
using sodium fluorescein injections, fluorescent microscopy and analysis software
to quantify neovessel area. In-vivo release of 7KCh from wafers was evaluated by
HPLC analysis. Animals were perfused with FITC Dextran, and anterior segment
cryosections were stain with Isolectin IB4 for histologic evaluation.
Results: Approximately 98% of 7KCh was released from the wafer by day 21.
Neovessel formation peaked 14 days after 7KCh implantation with median areas
(mm2) of 1121 (10%), 1362 (15%) and 1829 (20%) while Ch wafers didn’t induce
neovessel growth. Immunostaining demonstrated mobilization of Isolectin positive
cells by day 2 and perfused corneal neovessels by day 4.
Conclusions: We have developed a simple and reliable alternative to corneal
pockets to study pro-angiogenic molecules. Our results show that 7KCh induces
robust neovessel growth while Ch has no angiogenic effects. These results further
support the hypothesis that accumulation of 7KCh and other oxidized lipids may be
involved in the development of exudative AMD.
Commercial Relationships: Juan Amaral, None; Maria M. Campos,
None; Joshua Chou, None; Ignacio R. Rodriguez, None
Support: None
453 AMD: Stress in the Retina and RPE
Wednesday, May 9, 2012, 1:45 PM - 3:30 PM
Hall B/C Poster Session
Program #/Board # Range: 4750-4785/A425-A460
Organizing Section: Retinal Cell Biology
Program Number: 4750 Poster Board Number: A425
Presentation Time: 1:45 PM - 3:30 PM
Retinal Function changes in IDE KO mice
Kumar Sambamurti1A, Eliza Barnwell1A, Beth Coughlin1A, Baerbel Rohrer2, Jeffrey
Crosson1A, Craig E. Crosson3, Zsolt Ablonczy1B, Annamalai Prakasam1A,
Padmaraju Vasudevaraju1A. ANeurosciences, BOphthalmology, 1Medical
University of South Carolina, Charleston, SC; 2Ophthalmology, Med Univ of South
Carolina, Charleston, SC; 3Ophthalmology, Medical Univ of South Carolina,
Charleston, SC.
Purpose: Insulin degrading enzyme (insulysin) has been identified as the major
amyloid beta protein (AB)- degrading enzyme. Several studies have linked retinal
degeneration with amyloid deposition in animal models and it has been reported
that amyloid deposits accumulate in the eyes of Age related Macular degeneration
and Glaucoma subjects. In pursuit of our goal to understand the role of the
Alzheimer’s amyloid protein precursor and its processing pathways on retinal
function, we have examined retinal sensitivity in insulysin-KO mice.
Methods: We have generated insulysin-KO mice expressing human Alzheimer’s
amyloid protein precursor (APP) in the C57BL6 background and have determined
their effects on scotopic ERGs.
Results: : Although APP transgenic mice showed a significant increase in
sensitivity to light as measured by scotopic ERG, we did not observe a further
increase in Insulysin-KO mice expressing APP. Interestingly, we did observe a
trend towards an increase in a wave and a reduction in b wave in amyloid beta
protein precursor (APP) knockout mice an in a homologue, APLP2-KO mice.
Conclusions: APP transgenic mice exhibit increased photoreceptor and bipolar cell
responses. The data are consistent with several reports showing that Aβ facilitates
synaptic activity; it is unlikely that these mice show substantial Aβ accumulation at
this young age. We plan to age the animals to see if insulysin KO affects ERG
signal. Both APP KO mice and APLP2 KO mice show a trend towards reduction in
light sensitivity. If this trend holds up and reaches significance, we may conclude
that regions other than AB play a role in increasing light sensitivity, given that the
AB sequence is not conserved between the two homologues.
Commercial Relationships: Kumar Sambamurti, None; Eliza Barnwell,
None; Beth Coughlin, None; Baerbel Rohrer, None; Jeffrey Crosson,
None; Craig E. Crosson, None; Zsolt Ablonczy, None; Annamalai Prakasam,
None; Padmaraju Vasudevaraju, None
Support: AG023055, EY019065, Alzheimer's Association IIRG10-173180
Program Number: 4751 Poster Board Number: A426
Presentation Time: 1:45 PM - 3:30 PM
Cytoskeletal Keratin Phosphorylation Induced by Autophagy Protects Retinal
Pigment Epithelial Cells from Apoptosis during Oxidative Stress
Hyewon Chung1, Soojin Yoon2, Dong-Eun Kim2. 1Ophthalmology, Konkuk
University School of Medicine, Seoul, Republic of Korea; 2Bioscience and
Biotechnology, Konkuk University, Seoul, Republic of Korea.
Purpose: Oxidative stress has been documented as the pathogenesis of
degenerative diseases of the retinal pigment epithelium (RPE) and retina, such as
age-related macular degeneration (AMD). However, details on the adaptive change
or survival mechanism of the RPE in oxidative environment are not well known.
We recently found that increased level of cytokeratin 8 (CK8) and autophagyrelated proteins exists in the aqueous humor of AMD patients through the
proteomic approach. We speculate that increased CK8 and autophagy might be a
cytoprotective mechanism of RPE under oxidative stress. Here we investigated the
potential correlation between the increased activity of CK8 and autophagy and the
oxidative stress in RPE cell cultures.
Methods: ARPE19 cell cultures were prepared as described previously. Cell death
was monitored using flow cytometry. ARPE19 cells were transfected with GFPLC3 plasmid DNA. Immunocytochemistry of GFP-LC3, CK8, and phosphorylated
CK8 (phospho-CK8) and immunoblotting were carried out. Apoptotic assay was
performed by monitoring the activation of caspase-3.
Results: Exposure to peroxide-generating paraquat (400 µM, 24 h) did not cause
apoptosis or cell death in ARPE19 cells. Increased expressions of Atg5/12, Beclin1, and LC3-II indicated that autophagy was activated in ARPE19 cells exposed to
oxidative stress. CK8 and phospho-CK8 began to increase 6 h after exposure to
paraquat and peaked at 12-24 h. Co-localization of GFP-LC3 and phospho-CK8
was observed after exposure to paraquat in immunocytochemistry. Phosphorylation
of CK8 was significantly inhibited by the presence of 3-methyladenine, an inhibitor
of autophagy. Inhibition of autophagy or downregulation of CK8 with siRNA
resulted in decrease of cell viability through the activation of caspase-3. Autophagy
induction by oxidative stress seemed to be mediated by decreased expression and
activity of protein phosphatase 2A (PP2A), whose substrate is known to be
phosphorylated CK8.
Conclusions: We have identified that the PP2A inactivation-mediated autophagy is
an important resistance mechanism to oxidative stress in RPE cells and showed that
CK8 phosphorylation is key to this response. Our findings suggest that CK8 could
be a novel therapeutic target for AMD.
Commercial Relationships: Hyewon Chung, None; Soojin Yoon, None; DongEun Kim, None
Support: None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Program Number: 4752 Poster Board Number: A427
Presentation Time: 1:45 PM - 3:30 PM
Aggregates Of Alpha Crystallin Exhibits Autofluoresence, Implications For
lipofuscin in RPE
Claudio Zuniga1,2, Cesar Diaz1, Tomas Vega-Zuniga3, Alejandro Munizaga4,
Octavio Monasterio1. 1Molecular and structural laboratory, Faculty of Sciences,
Universidad de Chile, Santiago, Chile; 2Fundacion2020-IOARES, Santiago, Chile;
Neurosciences laboratory, Faculty of Sciences, Universidad de Chile, Santiago,
Chile; 4Faculty of Sciences, Universidad Catolica de Chile, Santiago, Chile.
Purpose: to demonstrate the autofluorescense emission of a small heat shock
protein (Alpha crystallin) as part of the autofluorescense emission from lipofuscin
of the RPE at 488 nm of excitation.
Methods: 1) Human alpha crystallin were purified from sonicated human lens
fibers by using fast performance liquid chromatography and detected through
western blotting and mass spectrometry. Also we used commercial available alfa
2) Alfa crystallin autofluorescence in solution with excitation wavelength of 488
nm was recorded in a spectrofluorimeter.
3) Aggregates of alfa crystallin was identified by immunoblots of fractions
belonging to ultracentrifugated discontinuous sucrose gradients in an chemical
denaturant agent, in these fractions was measured the presence of autofluorescence,
as well.
4) The presence of alfa crystallin in lipofuscin was demonstrated like we showed in
"Alpha Crystallin In Lipofuscin From Human Retinal Pigment Epithelium. 2011
Annual Meeting on Visionary Genomics. ARVO 2011 Annual Meeting"
5) The autofluorescence of lipofuscin from RPE cells was detected by
epifluorescence and confocal fluorescence microscopy of specimens from human
cells and ARPE-19 cells.
Results: Alpha crystallin shows autofluorescence at 488 nm of excitation, has
increasing light scattering and autofluorescence in higher concentration of the
protein, wich are dismunished with denaturant agent in the buffer media.
Conclusions: Alpha crystallin has autofluorescence with 488 nm of excitation
wavelength that depends on oligomerization state of the protein and could be
related to the lipofucsin autofluorescence in between others fluorophores in
Commercial Relationships: Claudio Zuniga, None; Cesar Diaz, None; Tomas
Vega-Zuniga, None; Alejandro Munizaga, None; Octavio Monasterio, None
Support: None
Program Number: 4753 Poster Board Number: A428
Presentation Time: 1:45 PM - 3:30 PM
Effect of GAG Removal & LDL Concentration on Hydraulic Conductivity of
and LDL Transport Across Bruch's Membrane
Mark Johnson1A, Zdravka Cankova1B. ABiomedical Engineering, Mechanical
Engineering and Ophthalmology, BBiomedical Engineering, 1Northwestern
University, Evanston, IL.
Purpose: Lipids accumulate with age in Bruch's membrane (BrM) and are
associated with AMD. In atherosclerosis, lipid accumulation in the vessel wall has
been associated with both the presence (Skalen et al., Nature, 2002) and absence
(Pillarisetti et al., JCI, 1997) of glycosaminoglycans. We used GAGases to remove
GAGs from bovine BrM and determined the effects on hydraulic conductivity (Lp)
of the tissue and on low density lipoprotein (LDL) transport across the tissue. We
also separately determined the effect of LDL concentration on its transport through
Methods: 2x2 cm tissue squares of BrM and choroid from non-tapetal regions
were gently separated from post-mortem bovine eyes. The tissue was treated with
chondroitinase ABC, heparinase III, both enzymes or neither for 6 to 24 hours.
Enzymatic activity was confirmed morphologically in control studies. The tissue
was then placed into a Ussing chamber and perfused with buffer or buffer with
fluorescent 3,3'-dioctadecylindocarbocyanine low-density lipoprotein (DiI-LDL:
0.005 or 0.05 mg/ml). The concentrations of DiI-LDL in the upstream and
downstream compartments were determined at the experiment's conclusion. Lp and
the reflection coefficient of BrM/choroid to DiI-LDL were then calculated.
Results: BrM had a significantly higher (p=0.035) reflection coefficient (0.61 ±
0.25, n=15) to the higher concentration of perfused LDL as compared to the lower
concentration (0.39 ± 0.28, n=12). Use of either chondroitinase ABC (n=10) or
heparinase III alone did not significantly alter either Lp of BrM or its reflection
coefficient to LDL. However, use of both enzymes (n=4 in experimental group)
together led to a marginally significant increase in hydraulic conductivity (1.05 ±
0.31 x 10-8 cm2s/g to 1.45 ± 0.54 x 10-8 cm2s/g, p=0.052) and decrease in the
reflection coefficient (0.61 ± 0.26 to 0.34 ± 0.32, p=0.078).
Conclusions: Increased LDL concentration hindered transport of these particles
through BrM. GAGase treatment appeared to facilitate transport of LDL through
BrM, consistent with findings in arterial walls that GAGs bind lipids. These results
are consistent with a hypothesis that an increased level of lipoprotein secreted by
the RPE and/or an increased concentration of GAGs in BrM contribute to the agerelated accumulation of lipids in BrM.
Commercial Relationships: Mark Johnson, None; Zdravka Cankova, None
Support: None
Program Number: 4754 Poster Board Number: A429
Presentation Time: 1:45 PM - 3:30 PM
Accumulation Of Lipofuscine In Retina Pigment Epithelium Cells Increases
Susceptibility To Oxidative Stress
yu yi lin1A, yin chang2, h.c. lee1B. AInstitute of Biomedical Engineering, BInstitute of
Pharmacology, 1National Yang-Ming University, Taipei, Taiwan,, taipei, Taiwan;
Inst of Biomedical Engineering, National Yang-Ming University, Taipei, Taiwan.
Purpose: Age-related macular degeneration (AMD) is the leading cause of
blindness in the developed countries. The etiology is thought to be related to
cumulative lipofuscine (LF) and oxidative stress in the retina pigment epithelium
(RPE). However, the mechanism remains poorly understood. The purpose of this
work is to study whether LF-accumulated and senescent RPE cells will increase
susceptibility to reactive oxygen stress (ROS).
Methods: H2O2 -induced oxidative stress were measure in ARPE-19 cells which
were cultured with four conditions (young, young with LF accumulation,
replicative senescence, and senescence with LF accumulation). Five experiments
were carried out and fluorescence intensity was measured by flow cytometry: (1)
We treated young and replicative senescent RPE cells by loading photoreceptor
outer segments (POS) which were extracted from porcine retinas to induce LF
accumulation;(2) RPE cells cocultured with FITC labeled POS for functional
analysis; (3) mitochondrial mass was measured by labeling 10-n-nonyl-acridine
orange (NAO); (4)Reactive oxygen stress was measured by staining DCFHDA. (5)
Cytotoxicity analyses by staining MTT on cells was analyzed by ELISA reader.
Results: The senescent cells and LF-accumulated senescent cells were found to
have higher levels of reactive oxygen stress (ROS) (young: 100±5, senescent with
LF accumulate:170±10 (arbitrary unit, au), P<0.05), and mitochondrial mass was
greater in the senescent with LF accumulate cells (young: 100±7,:223±8 (au),
P<0.05). In Functional analysis these cells showed that the phagocytic ability was
reduced in the senescent cells and LF-accumulated senescent cells (young: 100±15,
senescent with LF accumulate:10±3 (au), P<0.05). Cytotoxicity analysis showed
that the susceptibility to oxidative stress was higher in senescence cells and LFaccumulated senescent cells (young: 1.0±0.05, senescent with LF
accumulate:0.5±0.1 (au), P<0.05).
Conclusions: These results suggest that accumulation of LF in RPE results in
reduced phagocytic activity and increased susceptibility to ROS, which might be
involved in the etiology of AMD.
Commercial Relationships: yu yi lin, None; yin chang, None; h.c. lee, None
Support: None
Program Number: 4755 Poster Board Number: A430
Presentation Time: 1:45 PM - 3:30 PM
Dose Response Study Of Cytoprotective Effect By Lutein And Zeaxanthin On
Retinal Pigment Epithelium From Oxidative Stress Induced Cytotoxicity
Kavitha Ravi, Ravi Keshavamurthy, S Balaiya, K V. Chalam. Ophthalmology,
University of Florida, Jacksonville, FL.
Purpose: Lutein (LUT) and zeaxanthin (ZEA), naturally occurring antioxidants
and major components in macular pigment, are currently under investigation in
clinical trials as prophylactic nutritional agents for age related macular
degeneration (AMD). However, dose used in these trials is empirical and not been
investigated in in vitro studies. In this study, we investigated the dose response
effect of LUT and ZEA in protecting retinal pigment epithelium (RPE) from
oxidative stress, a common underlying pathology in AMD
Methods: Different concentrations of hydrogen peroxide (H2O2), a common
oxidant, were applied to the cultured human retinal pigment epithelial cells (ARPE19) cells for 24 hours to generate a dose response curve. 3000 cultured human
retinal pigment epithelial cells (ARPE-19) were plated in 72-well plate and after
24hrs, cells were exposed to four different concentrations of LUT (4,2,1 and 0.5
µg/ml) and ZEA (0.8,0.4,0.2 and 0.1µg/ml ). After 24 hours incubation, cells were
subjected to oxidative stress induced with hydrogen peroxide. Cultures containing
saline solution and trichloromethane served as controls. Cell viability was assessed
using the WST assay and cell counts were measured using Vi-cell counter.
Caspase-3 levels were measured using quantitative Western blot analysis.
Results: Using the WST assay, a dose dependent cytoprotective effect was
observed in ARPE-19 cells exposed to hydrogen peroxide after pretreatment with
both LUT and ZEA. (Cell viability as a percentage of control was 81.3, 81.1, and
88.8 % at 4, 2, and 1 µg/ml, respectively of Lutein, p<0.001). LUT at 2 µg/ml and
ZEA at 0.1 µg/ml were optimum concentrations at which protective effect was
observed. At higher doses, there was a reversal of cytoprotective effect. Results
were validated by Vi-cell counting. Mitochondrial mediated apoptosis was
confirmed by increased expression and activity of caspase-3.
Conclusions: Lutein at 2 µg/ml and Zeaxanthin at 0.1 µg/ml provide optimum
cytoprotective effect in retinal pigment epithelium from oxidative stress induced
Commercial Relationships: Kavitha Ravi, None; Ravi Keshavamurthy,
None; S. Balaiya, None; K. V. Chalam, None
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Support: None
Program Number: 4756 Poster Board Number: A431
Presentation Time: 1:45 PM - 3:30 PM
AMD-like Pathology in Klc1-/- Mice
Julian Esteve-Rudd1, Concepcion Lillo2, David S. Williams1,2. 1Jules Stein Eye
Institute, UCLA School of Medicine, Los Angeles, CA; 2Department of
Pharmacology, UCSD School of Medicine, La Jolla, CA.
Purpose: The phagocytosis and digestion of photoreceptor outer segment disk
membranes is a major function of the RPE. We have tested whether kinesin-1 has a
critical role in this process by studying retinas of mice lacking kinesin light chain 1
Methods: Eyes were collected and processed for light and electron microscopy,
and immunocytochemistry. Cryosections were labeled with antibodies against
complement pathway markers, fibronectin and cathepsin D, and images obtained
on a confocal microscope. Eyes for morphological and ultrastructural analysis were
post-fixed in osmium tetroxide, dehydrated and embedded in epon resin. Some
tissues were post-fixed in osmium-tannic acid-paraphenylenediamine (OTAP) to
preserve neutral lipid composition.
Results: Klc1 knock-out mice exhibited a loss of photoreceptors and choroidal
neovascularization, particularly in the central retina. Bruch's membrane was
thicker, mainly due to the accumulation of sub-RPE deposits and extensions of the
inner collagen layer. Membranous debris and neutral lipid aggregates were
observed among the basal deposits. This area also showed increased fibronectin
labeling. The RPE cells exhibited aggregates of undigested phagosomes as well as
accumulation of lysosomes, as indicated by cathepsin D labeling, in their
cytoplasm. They also showed increased autofluorescence in frozen sections. C3
immunolabeling was significantly more intense along the basal side of RPE cells,
and was higher in the central retina when compared to the periphery. C5b-9
immunolabeling was also detected in the choroid-RPE complex of mutant mice,
suggesting activation of the complement terminal pathway.
Conclusions: Our studies indicate that impaired kinesin-1 function, due to lack of
KLC1, results in AMD-like pathology. The indicated defective digestion of
phagosomes suggests that kinesin-1 may function in the transport of phagosomes or
lysosomes, and thus play an important role in phagosome maturation.
Commercial Relationships: Julian Esteve-Rudd, None; Concepcion Lillo,
None; David S. Williams, None
Support: NIH Grant EY07042
Program Number: 4757 Poster Board Number: A432
Presentation Time: 1:45 PM - 3:30 PM
Analysis of the Immunomodulatory Roles of Hsp70 in Experimental
Subretinal Fibrosis
Yang Yang1, Atsunobu Takeda1, Yuji Oshima1, Takeru Yoshimura1, Koh-hei
Sonoda2, Tatsuro Ishibashi1. 1Ophthalmology, Kyushu University, Fukuoka, Japan;
Ophthalmology, Yamaguchi University, Ube, Japan.
Purpose: Subretinal fibrosis often causes destruction of normal retinal structures,
including photoreceptor cells, within and/or around the macular area as seen in
advanced age-related macular degeneration. Previously we reported that the
interactions between active macrophages and retinal pigment epithelial cells
(RPEs) play a key role in the development of subretinal fibrosis due to choroidal
neovascularization (CNV). Heat shock proteins (HSPs), including HSP70, have
recently been shown to act like cytokines to confer cytoprotection in several
diseases via Toll-like receptor 2 (TLR2) and TLR4. Here we report the HSP70induced immunomodulatory property of RPEs in experimental subretinal fibrosis.
Methods: Expressions of TLR2 and TLR4 in PEC-inoculated eyes and in HSP70stimulated cultured RPEs were examined by immunohistochemistry. IL-10
expression by both RPEs and macrophages from WT, TLR2-/- and TLR4-/- mice
were assessed by quantitative real-time PCR and ELISA.
Results: Expressions of TLR2 and TLR4 in WT mice after HSP70 administration
were detected in the RPE layer around the area where PECs were inoculated. IL-10,
known as a potent anti-inflammatory cytokine, was detected in HSP70-stimulated
cultured RPEs from WT mice, but not in those from TLR2-/- and TLR4-/- mice.
Cultured macrophages from WT mice failed to produce IL-10 in response to
Conclusions: RPEs, not macrophages, are the main source of HSP70-mediated IL10 production via TLR2 and TLR4 signaling pathways. HSP70 may have an
important immunomodulatory role in experimental subretinal fibrosis.
Commercial Relationships: Yang Yang, None; Atsunobu Takeda, None; Yuji
Oshima, None; Takeru Yoshimura, None; Koh-hei Sonoda, None; Tatsuro
Ishibashi, None
Support: Grant-In-Aid for Scientific Research, Japan
Program Number: 4758 Poster Board Number: A433
Presentation Time: 1:45 PM - 3:30 PM
Multiple Autofluorescent Peaks In RPE Of Control And ABCA4-/- Mice: A
Role For Perturbed Autophagy?
Ann C. O'Brien-Jenkins1A, Sonia Guha1B, Wennan Lu2, Bardia Nabet1A, Gabriel
Baltazar3, Alan M. Laties4, Claire H. Mitchell1B. AAnatomy & Cell Biology,
Anatomy and Cell Biology, 1University of Pennsylvania, Philadelphia, PA;
Anatomy & Cell Biology, Univ of Pennsylvania, Sch of Dental Med, Philadelphia,
PA; 3Anatomy and Cell Biology, Sch of Dental Med, Univ of Pennsylvania,
Philadelphia, PA; 4Ophthalmology, Univ of Pennsylvania Sch of Med,
Philadelphia, PA.
Purpose: Macular degeneration is a complex disorder wherein initial defects
trigger secondary pathologies. The autofluorescent retinoid A2E accumulates in the
lysosomes of RPE cells as lipofuscin. RPE cells from the ABCA4-/- mouse model
of Stargardt’s degeneration have enhanced A2E levels. A2E alkalinizes RPE
lysosomes, and the lysosomal pH of RPE cells from ABCA4-/- mice is elevated. As
lysosomal alkalinization can impair acid lipase activity, alkalinization itself may
lead to partially degraded autofluorescent lipids, another component of lipofuscin.
Here we examined the ability of lysosomal alkalinization to increase
autofluorescence as part of a detailed analysis of autofluorescence in wildtype and
ABCA4 mice-/-.
Methods: Autofluorescence excited at 488nm in ARPE-19 cells was determined
after 2 wks in chloroquine with FACS analysis. Levels of autophagy marker
LC3BII were assayed with Western blots. TFEN mRNA was quantified using
qPCR. Autofluorescent emission was measured from fixed sections of RPE cells
from wildtype C57 and ABCA4-/- mice on samples from 6, 16 and 24 months old
mice. Emission was determined in 2.5 nm widths using the spectral detector in the
Nikon A1N microscope.
Results: Chloroquine increased the autofluorescence in cultured RPE cells by
60%.Chloroquine also increased levels of LC3BII, consistent with impaired
autophagy leading to autofluorescence. Autofluorescence was detected at multiple
wavelengths in RPE cells from both control C57 and ABCA4-/- mice. Peaks were
Ex (excite) 405 nm; Em (emission) 490, 571, and 651 nm; Ex 488 nm Em 571 and
651 nm; Ex 561 nm, Em 612 and 663 nm; Ex 639 nm, Em 663 and 702 nm. When
excited at 405 nm the largest emission peak in ABCA4-/- mice was at 571nm; this is
very close to the emission for A2E in fixed RPE cells. The 571nm emission peak
was present, but not as prominent, in control mice. Preliminary results suggest that
the autophagy transcription factor TFEN is upregulated in ABCA4-/- mice,
consistent with secondary impairment of autophagy.
Conclusions: Autofluorescence in RPE cells can be triggered either by A2E or
other means of lysosomal alkalinization. Future studies will tell if lysosomal
acidification can reduce one or both of these sources of lipofuscin.
Commercial Relationships: Ann C. O'Brien-Jenkins, None; Sonia Guha,
None; Wennan Lu, None; Bardia Nabet, None; Gabriel Baltazar, None; Alan
M. Laties, None; Claire H. Mitchell, None
Support: EY013434, EY015537
Program Number: 4759 Poster Board Number: A434
Presentation Time: 1:45 PM - 3:30 PM
Impairment of the Ubiquitin-Proteasome Pathway in RPE Causes AMDRelated Lesions in Mice
Fu Shang1, Benchun Miao1, Qingning Bian1, Allen Taylor1, Donald E. Smith1, Janis
Lem2, Paulo Pereira3. 1Human Nutrition Res Ctr on Aging, Tufts University,
Boston, MA; 2Tufts Medical center, Boston, MA; 3Center of Ophthalmology,
University of Coimbra, Coimbra, Portugal.
Purpose: The ubiquitin-proteasome pathway (UPP) plays important roles in
protein quality control and signal transductions. It was reported that activities of the
UPP in retina and RPE decrease upon aging and oxidative stress. The objective of
this work was to test the hypothesis that impairment of the UPP in RPE contributes
to the pathogenesis of AMD-related lesions and to investigate the mechanisms by
which impairment of the UPP triggers the development of AMD-related lesions.
Methods: To impair the function of the UPP in RPE, the RPE-65 promoter was
used to drive the expression of K6W-ubiquitin (a dominant negative inhibitor of the
UPP) in RPE of transgenic mice. The transgenic mice were crossed back to
C57BL/6 background. The retinas of wild type (wt) and transgenic mice were
examined by fundus photography at different ages. Retina sections were examined
morphologically and immunohistochemically. ARPE-19 cells were used to
determine the effects of impairment of the UPP on expression and secretion of proangiogenic and pro-inflammatory factors.
Results: The transgenic mice that express K6W-ubiquitin in RPE developed
normally and there were no detectable lesions until 6 months. By 12 months of age,
some retina pigment abnormalities were detected by fundus photography. By 18
months of age, ~70% of the transgenic mice showed pigment abnormalities
whereas all age-matched wt mice showed normal retinas. Morphological
examination of retinal sections showed that there were focal losses of RPE in
transgenic mice at 12 months of age. By18 months of age, ~70% of the transgenic
mice showed focal choroidal neovascularization. Immunochemical examination of
the sections showed that there was enhanced deposition of C3d onto basal side of
RPE. In cultured ARPE-19 cells, impairment of the UPP increased the expression
and secretion of VEGF and IL-8, but decreased the expression and secretion of
complement factor H and MCP-1.
Conclusions: These data demonstrate that impairment of the UPP in RPE triggers
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
the development of AMD-like lesions in mice, indicating that age-or stress-related
decline in UPP function is a contributing factor to AMD pathogenesis. Altered
expression and secretion of angiogenic and inflammatory factors by impairment of
the UPP appears to be involved in the development of AMD-like lesions.
Commercial Relationships: Fu Shang, None; Benchun Miao, None; Qingning
Bian, None; Allen Taylor, None; Donald E. Smith, None; Janis Lem,
None; Paulo Pereira, None
Support: NIH EY 11717 (to FS), EY 13250 (to AT), USDA AFRI Award 200935200-05014 (to FS), AHAF grant M2010038 (to FS), grant FCT – PTDC/SAUOSM/67498/2006 (to PP), and USDA CRIS 1950- 51000-060-02A (to AT)
Program Number: 4760 Poster Board Number: A435
Presentation Time: 1:45 PM - 3:30 PM
Oxidative Stress Caused By Continuous Fluorescent Lamp Illumination
And/Or Indocyanine Green On Cultured Human Retinal Pigment Epithelial
Tomohito Sato1A, Yoko Karasawa1A, Naoko Kato1A, Masataka Ito1B, Masaru
Takeuchi1A. AOphthalmology, BDepartment of Developmental Anatomy and
Regenerative Biology, 1National Defense Medical Collage, Tokorozawa, Japan.
Purpose: Indocyanine green (ICG) is a photosensitive tricarbocyanine dye, and is
generally used for retinal angiography. However, ICG is known to remain in retinal
pigment epithelium (RPE)-Bruch's membrane complex after angiography. In this
study, we investigated the cellular damage from continuous illumination of visible
rays and/or ICG by assessment of modifier oxide via photo-induced oxidative
Methods: Human retinal pigment epithelial (RPE; ARPE-19) cells maintained in
DMEM/F12 supplemented with 10 % FBS were cultured to confluence in multiwell polystyrene plates. Then, culture medium was changed to a colorless medium
(PBS supplemented with 1% FBS, 1 mg/mL glucose, 1 mg/mL CaCl2 and 1 mg/mL
MgCl2). The cells were incubated with or without ICG (10 µg/mL) for 24 hours in
the dark or under continuous illumination of fluorescent lamp (daylight, 6500 K,
2000 lux). After the incubation, oxidative stresses were evaluated by examining
production of total reactive oxygen species (ROS; 2’, 7’Dichlorodihydrofluorescein assay), protein carbonyl formation (immunoblotting of
dinitrophenylhydrazine), lipid peroxidation (immunostaining of malondialdehyde,
MDA) and mitochondrial membrane potential (fluorescence intensity of
tetramethylrhodamine ester, TRME).
Results: After 24-hour incubation, the levels of total ROS were higher in the
cultures with ICG in dark (B), without ICG under illumination (C) or with ICG
under illumination (D) than the cultures without ICG in dark (A; control).
Molecular weights of protein carbonyls over 240 kDa were detected in C and D but
not in A or B. Furthermore, the molecular weights of protein carbonyls in D were
higher than in C. The values of lipid peroxidation were almost equal in A and B,
higher in C than A (P<0.01), and the highest in D (vs. C; P<0.01). The values of
mitochondrial membrane potential in B and C were almost equal to A, but lower in
D (P<0.01).
Conclusions: Continuous illumination of visible rays produces ROS in cultured
RPE cells. ICG enhanced the ROS production via photo-oxidative stress and
damages RPE cells. Persistent ICG in RPE-Bruch's membrane complex after ICG
administration could enhance production of total ROS and modifier oxide, and
impede functions of mitochondria under the illumination.
Commercial Relationships: Tomohito Sato, None; Yoko Karasawa,
None; Naoko Kato, None; Masataka Ito, None; Masaru Takeuchi, None
Support: None
Program Number: 4761 Poster Board Number: A436
Presentation Time: 1:45 PM - 3:30 PM
AMD Affected Donors Accumulate High Amounts Of Heavy Metal Ions In
Melanosomes Of The Retinal Pigment Epithelium (RPE) And Calcified Areas
Of Bruch’s Membrane
Antje K. Biesemeier1, Ulrich Schraermeyer2, Oliver Eibl3. 1Experimental
Vitreoretinal Surgery, Center for Ophthalmology, Tuebingen, Germany;
Experimental Vitreoretinal Surgery, Center for Ophthalmology, Tuebingen,
Germany; 3Institute of Applied Physics, University of Tuebingen, Tuebingen,
Purpose: To identify differences in the ultrastructure, heavy metal storage ability
and elemental composition of melanosomes of human RPE of five AMD affected
donors compared to five age-matched healthy individuals. RPE melanosomes, lying
above or below different pathological tissues (drusen, basal deposits, thickening
and calcification of Bruch’s membrane or neovascularisation membranes and
retinal damage) were measured.
Methods: Analytical Electron Microscopy (AEM) including Energy-Filtered
Transmission Electron Microscopy (EFTEM), Energy-Dispersive X-ray
microanalysis (EDX) and Electron Energy-Loss Spectroscopy (EELS).
Results: Eyes of AMD affected donors were investigated ultrastructurally and
using AEM. Melanosomes of the RPE of AMD affected donors contained
increased amounts of iron (0.04 - 0.09 at%) compared to controls where Fe was at
or below the detection limit of 0.02 at%. In addition, AMD melanosomes showed a
significant lead mole fraction (0.03 - 0.1 at%), which was undetectable in control
samples. First experiments showed no clear differences in the elemental
composition of melanosomes overlying drusen and basal deposits. Some areas of
Bruch’s membrane were calcified containing CaPO4 crystals in the elastic layer.
These areas could be recognized by AEM, and P elemental maps identified their
ultrastructural location in the tissue. These calcified layers contained heavy metal
depositions with additional Fe and Pb mole fractions. P, Ca and Pb were not
identified in the other areas of Bruch’s membrane.
Conclusions: Iron and lead were significantly increased in the melanosomes of all
AMD donor eyes investigated. They were also found in calcified areas of the elastic
layer of Bruch’s membrane. Heavy metals promote oxidative stress in the retina
and are thought to be potential initiators of AMD. Whether the specific
accumulation in melanosomal stores and Bruch’s membrane has beneficial or toxic
effects on the surrounding tissues must be further elucidated.
Commercial Relationships: Antje K. Biesemeier, None; Ulrich Schraermeyer,
None; Oliver Eibl, None
Support: fortüne 1957-0-0
Program Number: 4762 Poster Board Number: A437
Presentation Time: 1:45 PM - 3:30 PM
Comparison of Cybrids from Different Mitochondrial DNA (mtDNA)
Haplogroups, Implications for Age-related Macular Degeneration (AMD)
Janelle M. Pavlis1, Marilyn Chwa1, Claudio A. Ramirez1, Nitin Udar1, S M.
Jazwinski2, M V. Miceli2, Vishal R. Sharma1, M. C. Kenney1, Rhina M. Piche
Lopez1. 1Gavin Herbert Eye Institute, UC Irvine, Irvine, CA; 2Tulane Center for
Anging, Tulane University, New Orleans, LA.
Purpose: AMD is a leading cause of vision loss in people over 65 years of age.
Mitochondrial genetics is a potentially important area of research that will help us
understand the genetic predisposition toward AMD. Previous studies have shown
that the mtDNA haplogroup J but not haplogroup H is associated with AMD.
However, the functional consequences of this difference is not understood. Cybrids
(cytoplasmic hybrids) are created by introducing the mitochondria into a host cell
line that lacks mitochondria. The resulting cybrids carry the same nuclear genes but
vary only in their mitochondrial content. Mitochondria representing different
haplogroups can therefore be introduced and compared. In this study, some key
biochemical pathways representing mitochondrial function were studied. We
hypothesize that ARPE-19 cells, dissimilar only in their mtDNA haplogroup will
behave differently in vitro.
Methods: Cybrids were created by fusing platelets from individuals with either H
or J mtDNA haplogroups with rho0 ARPE-19 cells which lack mitochondria.
Reactive oxygen and nitrogen species (ROS/RNS) levels were determined using
the 2’, 7’-dichlorodihydrofluorescein diacetate (H2DCFDA) dye assay, ATP
production was measured using the ATPlite luminescent detection assay,
mitochondrial membrane potential (∆Ψm) was quantified using a mitochondrial
membrane potential detection assay, apoptosis and necrosis were measured using
the Membrane Permeability/Dead Cell Apoptosis kit. Values were compared using
relative percentages.
Results: ATP production was 35-62% higher in haplogroup H compared to J (p
<0.002). There was a 25% reduction in apoptosis and necrosis in haplogroup H
versus J (p< 0.02), and haplogroup H produced 22% more ROS/RNS compared to
haplogroup J (p=0.007). There was no statistically significant difference in
mitochondrial membrane potential.
Conclusions: This study examines characteristics of cells that are identical except
for their mtDNA. These preliminary findings suggest that mtDNA haplogroup
differences have functional consequences for cells in vitro. This data may have
implications for understanding why haplogroup J is associated with AMD. More
research is needed to understand unique haplogroup features which relate to the
development of diseases.
Commercial Relationships: Janelle M. Pavlis, None; Marilyn Chwa,
None; Claudio A. Ramirez, None; Nitin Udar, None; S. M. Jazwinski, None; M.
V. Miceli, None; Vishal R. Sharma, None; M. C. Kenney, None; Rhina M.
Piche Lopez, None
Support: Supported by Discovery Eye Foundation, Polly & Michael Smith
Foundation, Beckman Initiative for Macular Research, Lincy Foundation, Iris & B.
Gerald Cantor Foundation, Research to Prevent Blindness
Program Number: 4763 Poster Board Number: A438
Presentation Time: 1:45 PM - 3:30 PM
Retinal Degeneration is Significantly Accelerated in Dark Reared Animals in a
Novel Murine Model of Age-related Macular Degeneration
Stephen Bravo, Toshihide Kurihara, Peter D. Westenskow, Edith Aguilar, Martin
Friedlander. Cell Biology, The Scripps Research Institute, La Jolla, CA.
Purpose: Age-related macular degeneration (AMD) is one of the leading causes of
incurable blindness in industrialized countries and can be caused by retinal pigment
epithelium (RPE) dysfunction or death. We have recently developed a novel
murine model of AMD in which inducible von Hippel-Lindau (VHL) gene deletion
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
in the adult retinal pigment epithelium (RPE) leads to the onset of retinal
degeneration closely resembling human AMD. VHL regulates critical oxygen
sensing molecular pathways in multiple ocular tissues, and hypoxic metabolism
tolerance can be impaired in the elderly. RPE cells perform critical functions
including phagocytosis of shed photoreceptor outer segments and maintenance of
the visual cycle. Typically, inherited retinal degenerations in animal models can be
slowed by restricting their exposure to light. Excessive light exposure induces
retinal degeneration, even in wild-type animals. Therefore, we examined the
dynamics of retinal degeneration in dark-reared VHL mutants.
Methods: Doxycycline injections in inducible VMD2-Cre mice activate Cre
recombinase activity specifically in RPE cells in a mosaic pattern. VMD2-Cre mice
were crossed with VHLflox/flox mice to inactivate VHL expression in RPE cells. We
inactivated VHL only in adult mice to serve as a model of AMD. The RPE and
retinal phenotypes were examined using immunohistochemistry on cryosections,
and confocal and electron microscopy. TUNEL assay was performed to confirm
apoptosis in specific retinal cell types.
Results: Light reared VHL-/- mice exhibit significant photoreceptor degeneration
28 days after VHL gene deletion in the RPE, and long term VHL deletion (after 8
months) shows catastrophic retinal degeneration. In contrast, dark reared VHL-/mice showed significant degeneration only 3 days after VHL inactivation, and
severe degeneration 14 days post inactivation. TUNEL-positive photoreceptor cells
were detected in both light and dark reared mice.
Conclusions: Mice reared in the dark prior to RPE specific VHL gene deletion
display dramatically accelerated and pronounced retinal degeneration when
compared to VHL mutant mice maintained in conventional light-dark cycles. We
will discuss potential mechanisms underlying these observations and their potential
application to better understand human AMD pathology.
Commercial Relationships: Stephen Bravo, None; Toshihide Kurihara,
None; Peter D. Westenskow, None; Edith Aguilar, None; Martin Friedlander,
Support: NEI Grant EY11254
Program Number: 4764 Poster Board Number: A439
Presentation Time: 1:45 PM - 3:30 PM
Protective Role of Superoxide Dismutase 1 in the retina against NMDAinduced neurotoxicity
Kenya Yuki1, Seiji Miyake2, Tetsu Yoshida3, Kazuo Tsubota4, Yoko Ozawa5.
Ophthalmology, Keio Univ School of Medicine, Shinjyuku-ku, Japan; 2Laboratory
of Retinal Cell Biology, Keio University School of Medicine, Sinjyuku, Japan;
Keio University, Shinjuku, Japan; 4Ophthalmology, Keio Univ School of
Medicine, Shinjuku-ku, Japan; 5Department of Ophthalmology, Keio University,
School of Medicine, Shinjuku-ku, Japan.
Purpose: Superoxide dismutase 1 (SOD1) is an enzyme that metabolizes
superoxide anion, one of reactive oxygen species. It binds the metal cofactor,
copper and zinc, and acts in the cytosol. N-methly-D-aspartic acid (NMDA) is an
amino acid which mimics the action of glutamate and is a well-known excitotoxin.
Its toxicity is reported to be associated with ischemia-reperfusion injury, Alzheimer
disease, and glaucoma. In this study, we analyzed the role of SOD1 on NMDA
induced-neurotoxicity in the retina.
Methods: Eight-week old SOD-1 knock-out (SOD1KO) mice (C57BL/6J
background) and the control wild type (SOD1WT) mice were prepared, and
NMDA was administered as follows. After anesthetized with diethyl ether, 2 µl of
either 10mM NMDA or phosphate-buffered saline (PBS) was injected intravitreally
using a Hamilton syringe with a 32-G needle. First, the levels of SOD1 in the retina
in SOD1WT mice were measured by immunoblot analysis at 24 hours after the
injection (n=8,9). Then, the apoptotic cells were counted using TdT-dUTP terminal
nick-end labeling (TUNEL) in the retinal cryosections cut through the vertical
meridian including the optic nerve head, and compared between SOD1KO and
SOD1WT mice (n=5).
Results: The level of SOD1 in the retina significantly increased at 24 hours after
10 mM NMDA administration compared to PBS administration in the SOD1WT
mice (SOD1WT-PBS 1.0±0.1, SOD1WT-10mM NMDA 1.8±0.4, p<0.01 t-test).
After injection of 10 mM NMDA, the number of TUNEL positive cells in the inner
nuclear layer (INL) of SOD1KO mice was significantly larger than in the INL of
SOD1WT mice (SOD1WT-PBS; 0.0±0.0 cells, SOD1WT-10mMNMDA;
163.8±8.4 cells, SOD1KO-PBS; 0.0±0.0 cells, SOD1KO-10mMNMDA;
208.0±11.0 cells, ANOVA p<0.001, Scheffe p<0.05, SOD1WT 10 mM NMDA vs
Conclusions: SOD1 has a protective role in the retina against NMDA-induced
Commercial Relationships: Kenya Yuki, None; Seiji Miyake, None; Tetsu
Yoshida, None; Kazuo Tsubota, None; Yoko Ozawa, None
Support: None
Program Number: 4765 Poster Board Number: A440
Presentation Time: 1:45 PM - 3:30 PM
Oxidative Stress-Induced p62/SQSTM1 Upregulation in the RPE
Chunjuan Song, Sayak K. Mitter, Haripriya V. Rao, Jun Cai, Xiaoping Qi, Michael
E. Boulton. Anatomy and Cell Biology, University of Florida, Gainesville, FL.
Purpose: p62/SQSTM1 is a multifunctional protein implicated in oxidative stress
responsive pathways, the ubiquitin-proteasome system (UPS) and selective
macroautophagy. The aim of this study was to determine the mechanism and role
of p62 upregulation under oxidative stress.
Methods: Confluent ARPE19 cells in serum-free medium (Ham’s F-10) were
treated with oxidative stressor (200, 400 or 800 µM H2O2) for 6, 24 or 48 hours. In
some cases, to better mimic chronic oxidative stress, cells received sustained
exposure to H2O2 (400 µM) for up to 14 days. Western blot was used to a) assess
the LC3II:LC3I ratio as a marker of autophagy, b) to characterize the level of p62
protein and c) to determine polyubiquitinated protein accumulation and d) caspase3 proteolytic cleavage. Caspase-3 activation was also examined by caspase-3
enzyme activity using its fluorogenic substrate Ac-DEVD-AFC. Apoptotic cell
death was determined by an ELISA sandwich assay for DNA fragmentation. p62
mRNA expression was assessed by real time qRT-PCR. The proteasomal peptidase
assay was performed to examine proteasome activity. 75nM bafilomycin A1, which
blocked the fusion of autophagosome and lysosome, was used together with H2O2.
Results: Exposure of cells to H2O2 resulted in an increase in autophagy. p62
expression increased at both the protein and mRNA level after H2O2 exposure
compared to control. Co-treatment with bafilomycin A1 further enhanced the
protein level of p62 in ARPE19 cells. In addition, we also found H2O2 inhibit
proteasomal activity and caused the aggregation of polyubiquitinated proteins. As
expected, proteasome inhibitor also increased p62 expression in ARPE19 cells.
Sustained treatment with H2O2 dysregulated p62, decreased autophagy and
activated caspases leading to apoptotic cell death.
Conclusions: Oxidative stress inhibits proteasomal activity and aggregates
polyubiquitinated proteins, which in turn upregulates p62 and triggers the
autophagy-lysosome pathway to remove the protein aggregates. Furthermore, acute
and chronic oxidative stress activate different pathways.
Commercial Relationships: Chunjuan Song, None; Sayak K. Mitter,
None; Haripriya V. Rao, None; Jun Cai, None; Xiaoping Qi, None; Michael E.
Boulton, None
Support: NIH grants EY019688 and EY021626
Program Number: 4766 Poster Board Number: A441
Presentation Time: 1:45 PM - 3:30 PM
Effects of Memantine on the mitochondrial membrane potential (∆Ψm) and
Lactate Dehydrogenase (LDH) production on ARPE-19 and Müller cells
(MIO-M1) exposed to Hydroquinone (HQ) In Vitro
Claudio A. Ramirez1, Mohamed Tarek1, Khoa D. Pham1, Marilyn Chwa1, Astrid
Limb2, Baruch D. Kuppermann1, M C. Kenney1. 1Gavin Herbert Eye Institute
Ophthalmology, University of California, Irvine, CA; 2Department of Pathology
and Cell Biology, University College, London, United Kingdom.
Purpose: To study the in vitro effects of memantine, a neuroptotectant used in
patients with Alzheimer’s disease and glaucoma, on human ARPE-19 and MIO-M1
cells exposed to hydroquinone (HQ), one of the toxic components of cigarette
Methods: ARPE-19 and MIO-M1 cells were pretreated for 6 hours with 30µM of
memantine and then exposed to 200µM, 100µM, 50µM and 25µM of HQ
(dissolved in DMSO), while the memantine was still present. JC1 assay was
performed to evaluate the mitochondrial membrane potential (∆Ψm). LDH
production was measured by the LDH-Cytotoxicity Assay Kit II.
Results: The ∆Ψm for ARPE-19 cells exposed to 200µM, 100µM, 50µM and
25µM of HQ was 3770±43.08, 4015±66.51, 5139±40.76 and 7475±94.06,
respectively compared to the highest (200 µM) DMSO equivalent-treated cells
(9097±76.06). Pretreatment with 30µM memantine resulted in an increased ∆Ψm
only at the 50 µM HQ dose, at the level of 6997±75.45 (p<0.0001). The values for
memantine pretreated cultures plus 200µM, 100µM and 25µM HQ were
3777±23.23(p=0.8787), 4181±39.52(p =0.0573) and 7637±41.71(p =0.1461),
respectively. ∆Ψm for MIO-M1 treated cultures plus 200µM, 100µM, 50µM and
25µM HQ were 3275±81.50, 4527±61.49, 5625±57.80 and 7763±34.77,
respectively compared to the highest (200 µM) DMSO equivalent -treated cells
(9118±45.9). In the 50µM HQ dose, after memantine pretreatment the ∆Ψm
increased to 6695±34.03(p <0.0001 ). There were no statistically significant
differences for the other HQ doses. LDH levels for ARPE-19 cells treated with
200µM, 100µM, 50µM and 25µM HQ were 5.50±0.25, 4.8±0.20, 4.16±0.06 and
2.0±0.10 . The LDH levels were decreased significantly after pretreated with
memantine at 50 µM and 25 µM HQ doses to 2.233±0.08(p <0.0001) and
1.567±0.12(p< 0.0314). MIO-M1 cells treated with the same concentrations of HQ
alone showed LDH levels of 6.20±0.11, 5.66±0.08, 5.35±0.13 and 2.06±0.03. The
level of LDH decreased significantly after pretreatment with memantine only at the
25 µM HQ to 1.250±0.08 (p< 0.0007).
Conclusions: Memantine showed an in vitro protective effect against HQ induced
toxicity by increasing the ∆Ψm at 50µM HQ dose on both cell lines and decreasing
LDH levels at 50µM and 25µM HQ doses in ARPE-19 cells and at the 25µM HQ
dose on MIO-M1 cells.
Copyright 2012 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any abstract, contact the ARVO Office at [email protected]
ARVO 2012 Annual Meeting Abstracts by Scientific Section/Group – Retinal Cell Biology (RC)
Commercial Relationships: Claudio A. Ramirez, None; Mohamed Tarek,
None; Khoa D. Pham, None; Marilyn Chwa, None; Astrid Limb, None; Baruch
D. Kuppermann, None; M. C. Kenney, None
Support: Supported by Discovery Eye Foundation, Polly & Michael Smith
Foundation, Beckman Initiative for Macular Research, Lincy Foundation, Iris & B.
Gerald Cantor Foundation, Research to Prevent Blindness
Program Number: 4767 Poster Board Number: A442
Presentation Time: 1:45 PM - 3:30 PM
The Wnt/beta-catenin Pathway Cross-talks with STAT3 Signaling to Regulate
Survival of Retinal Pigment Epithelium Cells
Karen Alvarez-Delfin1, Miryam A. Fragoso1, Amit K. Patel1, Rei E. Nakamura2,
Hyun Yi1, Abigail S. Hackam1. 1Ophthalmology, University of Miami, Miami, FL;
Ophthalmology and Visual Sciences, Washington University in St. Louis, St.
Louis, MO.
Purpose: Wnt/beta-catenin signaling is an essential pathway that regulates
numerous cellular processes, including cell survival. The mechanisms of prosurvival Wnt signaling are mostly unknown. In age related macular degeneration
(AMD), oxidative stress contributes to retinal pigmented epithelium (RPE) cell
death, leading to loss of vision. We previously demonstrated that the Wnt pathway
protects an RPE cell line from oxidative stress. Signal transducer and activator of
transcription proteins (STATs) are a well-described family of transcription factors.
STAT3 induces expression of anti-apoptotic genes in many tissues and is a
downstream mediator of protective growth factors and cytokines. In this study, we
investigated whether pro-survival Wnt signaling is mediated by STAT3 in the RPE.
Methods: Wnt3a was used to activate Wnt/beta-catenin signaling in the human
retinal pigmented epithelium cell line ARPE-19. Oxidative stress was induced with
hydrogen peroxide or paraquat and cell viability was measured using Cell Titer
Blue. Activation of STAT3 was measured using an antibody against the activated
STAT3 protein (Phospho-Tyr705) by immunohistochemistry. STAT3 expression
was reduced using specifically targeted siRNA and confirmed by quantitative PCR
and Western blotting.
Results: The Wnt3a ligand induced Wnt signaling in the ARPE-19 cell line and
significantly increased the viability of cells exposed to oxidative stress by up to
51% (n=4, p<0.05). Wnt3a also increased STAT3 activation and nuclear
translocation by 10-fold (n=3, p<0.001). Furthermore, reducing STAT3 levels,
using siRNA, eliminated Wnt3a-induced protection from oxidative stress.
Conclusions: These data demonstrate a previously unknown link between Wnt3amediated activation of STAT3 and cell survival, indicating a cross-talk between
two important pro-survival signaling pathways in the retina.
Commercial Relationships: Karen Alvarez-Delfin, None; Miryam A. Fragoso,
None; Amit K. Patel, None; Rei E. Nakamura, None; Hyun Yi, None; Abigail S.
Hackam, None
Support: Karl Kirchgessner Foundation, NEI centre Core Grant P30EY014801
Program Number: 4768 Poster Board Number: A443
Presentation Time: 1:45 PM - 3:30 PM
Retinal Degeneration in IRBP(-/-) Mice, and Patterns of Oxidative Stress
Priscilla Gonzalez-Fernandez1,2, Dongjin Sung1,2. 1Medical Research Service,
VAWNYHS, Amherst, NY; 2Ophthalmology and Pathology & Anatomic Sciences,
University at Buffalo/SUNY and SUNY Eye Institute, Buffalo, NY.
Purpose: The mechanism of retinal degeneration in transgenic mice lacking
interphotoreceptor retinoid-binding protein (IRBP -/-), or in humans with an RP
associated IRBP mutation is largely unknown. IRBP, which is the most abundant
soluble protein component of the interphotoreceptor matrix, appears to have a
complex poorly understood role in the translocation of hydrophobic molecules
particularly 11-cis and all-trans retinol, and 11-cis retinal during the visual cycle.
Its role in protecting the oxidative state of retinol in this cycle may be a particularly
important function of the protein. We hypothesized that lack of IRBP in transgenic
mice leads to photoreceptor degeneration by enhancing oxidative stress in the outer
retina compared to the inner retina.
Methods: IRBP (-/-) mice, and C57BL/6 age matched controls were maintained
from birth under cyclic dim light. Eyes were fixed in 4% paraformaldehyde from
p5 to p80. The eyes were sectioned at 5 microns in a vertical orientation. Nuclear
cell layer counts were made at 6 locations evenly distributed on both sides of the
optic nerve. The distribution of 4-hydroxynonenal (HNE) was determined by
avidin-biotin immunohistochemistry using mouse monoclonal HNEJ-2 at a 1:20
dilution. Pre-adsorbed controls utilized HNE-ovalbumin. Successful conjugation of
HNE to the ovalbumin was confirmed by Western blot analysis. Three month-old
albino mice were exposed for 24 hrs to intense green light (1700 lux, 490-580 nm)
as positive controls.
Results: Between p5 and p20 the ONL thickness decreased by 30% and 20% for
IRBP(-/-) and C57BL/6 mice respectively. In