Sunday, May 2, 8:30 AM - 10:30 AM Grand B Symposium Program Number Range: 1 - 6
101. Genetic and Epigenetic Regulation of Eye Development and Disease: The Future of Vision Research Contributing Section: RC, BI, PH, RE, VN
1 - 8:30 AM
Introduction
2 - 8:42 AM
Analysis Of Retinal Amacrine Cells At Single Cell Resolution
D. Chen. Ophthalmology/Harvard, Schepens Eye Res Inst, Harvard Med Sch,
Boston, MA.
C.L. Cepko. Genetics, Harvard Medical School/HHMI, Boston, MA.
The vertebrate retina uses diverse neuronal cell types arrayed into complex neural
circuits to extract, process and relay information from the visual scene to the higher
order processing centers of the brain. Amacrine cells, a class of interneurons, are
thought to mediate much of the processing of the visual signal that occurs within
the retina. Although amacrine cells display extensive morphological diversity, the
molecular nature of this diversity is largely unknown. Furthermore, it is not known
how this diversity arises during development. Here, we have combined in vivo genetic
labeling, single cell genome-wide expression profiling and classical birthdating to:
1) Identify specific molecular types of amacrine cells; 2) Demonstrate the molecular
diversity of the amacrine cell class, and; 3) Show that amacrine cell diversity arises
at least in part from the temporal patterning.
CR: D. Chen , None.
Support: None
CR: C.L. Cepko, None.
Support: NIH EY08064
3 - 9:04 AM
Alterations in Cell Cycle and Cell Fate Signaling Increase Retinal Progenitor
Cell Proliferation and Late-Born Neurogenesis
4 - 9:26 AM
Regulation of Alternative Splicing in the Retina
D.J. Zack. Ophthalmology, Wilmer Eye Inst, Johns Hopkins Univ., Baltimore, MD.
D.A. Fox. University of Houston, Houston, TX.
There is increasing evidence that alternative RNA splicing plays an important role in
generating genetic and functional diversity. RNA splicing demonstrates both tissue and
cell type specificity. Errors in splicing can cause disease, and in fact mutations in both
cis-acting elements and trans-acting splicing factors have been found to be associated
with retinal degeneration. We have been using a combination of bioinformatic and
laboratory-based approaches to define the pattern of alternative splicing in the retina,
and also have begun to analyze the mechanisms regulating retina-specific splicing.
A review of the current status of the field as well as a progress report on our ongoing
studies will be provided.
CR: D.J. Zack, None.
Support: NIH, FFB, RPB, Guerrieri Family Foundation
The developing brain and retina are especially vulnerable to drug, environmental,
nutritional and toxicant insult. Millions of young children are or were exposed to lowmoderate levels of lead during early development, which places them at permanent
risk for adverse health effects. Recently, we reported that low-level gestational lead
exposure (GLE) increased the scotopic ERG a- and b-wave amplitudes (supernormality)
in children and adult rats. This talk will describe the retinal anatomical/histological,
cellular and molecular changes in rat and mice models of human GLE. GLE produced
a novel retinal phenotype in adult rodents characterized by an increased number
of late-born retinal neurons (rods and bipolar cells), but not Müller glial cells.
Confocal, BrdU and TUNEL assays showed that during GLE retinal progenitor cell
(RPC) proliferation was increased and prolonged, neurogenesis was increased, and
apoptosis was unchanged. Further, microarray and RT-qPCR findings showed that
GLE upregulated several cell cycle genes as well as certain pro-neuronal bHLH and
homeodomain genes. Western blots and confocal studies confirmed and extended these
results by showing age-dependent upregulation of Cyclin D1 and pRb, downregulation
of the cyclin kinase inhibitor p19[INK4D], and upregulation of the Notch1-Hes1-Mash1
signaling pathway with no change in STAT3 signaling. Together, these findings show
that GLE increased G1-/S-phase transition and produced selective neurogenesis of
late-born retinal neurons. The increased number of rods and bipolar cells in adult
GLE rodents likely occurs in GLE children and underlies the supernormal scotopic
ERG responses in rodents and children. The long-term consequences of this increased
retinal cellularity are unknown, but our recent aging studies with GLE mice reveal
enhanced retinal degeneration.
CR: D.A. Fox, None.
Support: NIH Grants ES012482, EY07551, EY07024, EY11115, EY06671 and US EPA
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1-4
Sunday, May 2, 8:30 AM - 10:30 AM Grand B Symposium Program Number Range: 1 - 6
101. Genetic and Epigenetic Regulation of Eye Development and Disease: The Future of Vision Research Contributing Section: RC, BI, PH, RE, VN
6 - 10:10 AM
Epigenetic Signature In Retinal Development
5 - 9:48 AM
CRX-associated Transcription Factor Network Regulates Target Chromatin
Configurations During Photoreceptor Development and Disease
A. Swaroop. N-NRL, NEI/NIH, Bethesda, MD.
S. Chen. Ophthalmology and Visual Sciences, Washington University School of
Medicine, St Louis, MO.
CR: A. Swaroop, None.
Support: NIH intramural program
A network of transcription factors (TF) centered on CRX regulates the spatial and
temporal expression of retinal photoreceptor genes. Using transcriptional regulation
of opsin genes as a model, we found that during photoreceptor development, CRX
recruits co-activators with histone acetyltransferase (HAT) activity to target gene
chromatin, triggering histone acetylation and chromatin “opening”, allowing access of
the basal transcriptional machinery. Active transcription correlates with the presence
of intrachromosomal loops between enhancer/promoter and coding exons of each
target gene. Two rod-specific TFs, NRL and NR2E3, cooperate with CRX to differentially
regulate additional histone modifications and chromatin configurations of rod vs.
cone genes. These TF-mediated epigenetic modulations are essential for precisely
regulating the expression of rod vs cone genes in their respective photoreceptor
subtypes. Mutations in CRX, NRL, NR2E3, or HAT co-activators can cause epigenetic
regulation errors leading to transcriptional dysregulation and defects in photoreceptor
subtype development and maintenance.
CR: S. Chen, None.
Support: NIH grants R01 EY012543 (to SC) and P30 EY02687 (to WU-DOVS), Research
to Prevent Blindess (Lew R. Wasserman Merit Award to SC and unresticted grant to
WU-DOVS), Foundation Fighting Blindness (to SC)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
5-6
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
25 - A72
Lack of Opticin in Mice Results in Delayed Hyaloid Vascular Regression and
Retinal Vascular Development
26 - A73
HIF1α is Essential for Proper Development of the Retinal Vasculature
C. Caprara, M. Thiersch, C. Grimm. Dept. of Ophthalmology, Lab for Retinal Cell
Biology, Zurich, Switzerland.
M.M. Le Goff 1A,1B, P.N. Bishop1A,1B, R. Mayne2, M. Ugarte1B. AWellcome Trust Centre
for Cell-Matrix Research, Faculty of Life Sciences, BThe Medical School, Faculty
of Medical and Human Sciences, 1University of Manchester, Manchester, United
Kingdom; 2Department of Cell Biology, University of Alabama, Birmingham, AL.
Purpose: Nutrients and oxygen necessary for early retinal development are provided
by a vascular network of the vitreous called the hyaloid system. As the hyaloid system
regresses, the retinal vasculature develops radially from the optic disk. Opticin is a
vitreous glycoprotein that has anti-angiogenic properties. In this study, we investigate
if a lack of opticin perturbs hyaloid regression and retinal vascular development.
Methods: Initially, immunohistochemistry was performed on 5 µm eye sections from
wild-type and Optc-/- mice at P4, P7 and P17 using an anti-mouse opticin antibody.
Subsequently, the hyaloid vessels and retinal vasculature were fluorescently labelled
with isolectin B4 in wild-type and Optc-/- eye cups and retinal flat-mounts prepared
at P4, P7, P17 and P35 to observe vascular development.
Results: Opticin was localized to the non-pigmented ciliary epithelium, vitreous,
hyaloid vasculature and trabecular meshwork. Fluorescent microscopy revealed a
more extensive hyaloid vascularization in the Optc-/- mice at P4 and P7 compared
to their wild-type counterparts. While the hyaloid network had totally regressed in
the wild-type animals at P35, residual vessels were observed in a proportion of the
Optc-/- mice. Analyses of retinal flat-mounts at P4 and P7 revealed a delay in retinal
vascularization in Optc-/- compared to wild-type mice; however, no differences were
observed in the vascular pattern by P17.
Conclusions: Opticin is involved in hyaloid regression and indirectly influences the
rate of retinal vascularization.
CR: M.M. Le Goff, None; P.N. Bishop, None; R. Mayne, None; M. Ugarte, None.
Support: Support for the NIHR Manchester Biomedical Research Centre
27 - A74
Differential Localization of VEGF165 and VEGF165b Associated With
Developing Vasculatures in the Embryonic and Fetal Human Eye
Purpose: Hypoxia-inducible factors (HIFs) are central members of the molecular
oxygen sensing pathway, and their role in response to hypoxia is well known. The retina
of the newborn mouse is virtually avascular lacking especially the two inner vascular
plexi that only form within the first two weeks after birth. This suggests reduced tissue
oxygenation at this time requiring an appropriate molecular response. Indeed, HIF1α levels are high in the young mouse retina but decrease as the retinal vasculature
develops. Here, we tested the involvement of HIF-1α in vascular development in the
mouse retina.
Methods: Conditional HIF-1α knockdowns were generated by breeding HIF1αflox/flox
mice to mice expressing cre recombinase under control of the alpha element of the
Pax6 promoter. This leads to a deletion of HIF-1α genomic DNA and thus to HIF-1α
knockdown in cells of the peripheral but not the central retina. Retinal morphology
was observed in mice by light microscopy. Development of retinal blood vessels
was analyzed using IHC on retinal flat mounts. Gene expression was assessed by
semiquantitative real-time PCR and Western blotting.
Results: HIF-1α knockdown mice had a 50% reduced global retinal expression of HIF1α at post natal day (PND) 21. The peripheral retinal vasculature of HIF1α knockdown
mice was generally underdeveloped and less organized and the development of the
inner vascular plexi was disturbed as compared to control mice. The phenotype
was most pronounced at PND10 and persisted into adulthood. In contrast, retinal
morphology was largely unaffected. Increased levels of GFAP mRNA suggested
retinal stress at PND21. Surprisingly, levels of pan-VEGF mRNA were increased by
50% at PND21 despite the lower expression of HIF-1α. Expression of Egln1 and Glut1,
other HIF-1α target genes, was largely unaffected
Conclusions: The underdeveloped retinal vasculature in the HIF-1α knockdown mice
strongly suggests a prominent role of HIF-1α in retinal angiogenesis and especially in
the formation of the deep retinal vessels. In an attempt to form retinal blood vessels,
retinal cells may increase VEGF expression in a HIF-1α independent manner or in
cells not affected in by the HIF-1α gene knockdown.
CR: C. Caprara, None; M. Thiersch, None; C. Grimm, None.
Support: None
28 - A75
The Apelin/APJ Pathway in Retinal Angiogenesis
F.M. Recchia1, L. Xu2, J.S. Penn2. 1Ophthalmology and Visual Sciences, Retina
Division,Vanderbilt Eye Institute, Nashville, TN; 2Ophthalmology and Visual
Sciences, Vanderbilt Eye Institute, Nashville, TN.
G.A. Lutty, T. Baba, C. Merges, T. Hasegawa, D.S. McLeod. Wilmer Eye Inst, Johns
Hopkins Univ Sch of Med, Baltimore, MD.
Purpose:VEGF-A is a key regulatory component in vascular development. The
VEGF165 isoform of A is thought to be the predominant form for stimulating migration
and proliferation of endothelial cells and angioblasts. An anti-angiogenic form of
VEGF-A has been isolated, VEGF165b, which is generated by alternative splicing. This
study investigates the localization of VEGF165 and VEGF165b during development
of vasculatures in embryonic and fetal human eye.
Methods:Immunohistochemistry was performed on cryosections from 7 to 21 weeks
gestation (WG) human eyes using a rabbit antibody against VEGF165 (Thermofisher)
and a monoclonal antibody against VEGF165b (Abcam) and antibodies against CD31
and CXCR4 to label endothelial cells and progenitors respectively.
Results:The tunica vasculosa lentis (TVL) had moderate VEGF165 immunoreactivity
at 7 WG, and very little VEGF165b. Both forms were elevated at 12 WG but VEGF165
was cytoplasmic and VEGF165b was nuclear in endothelial cells (EC). Both forms of
VEGF-A were present diffusely throughout the retinal neuroblastic layer (NBL) at 7
WG. At 12 WG, VEGF165 was present diffusely through inner retina and inner NBL
and VEGF165 and VEGF165b were present in most CXCR4+ progenitors in the inner
retina, which includes angioblasts. By 21 WG, VEGF165 was present in nerve fibers
and VEGF165b in inner Muller cell processes in central retina. In choroid, VEGF165
was present in forming choriocapillaris (CC) and RPE at 7 WG while VEGF165b was
present in CC and mesenchymal progenitors in choroid and sclera. By 12 WG, both
were present predominantly in developing CC but VEGF165 was present in the basal
portion of RPE as well. At 21 WG, both forms were lower in choroidal blood vessels
than previously but VEGF165b was also present in the nuclei of RPE.
Conclusions:In TVL at 12 WG, VEGF165 was cytoplasmic and VEGF165b was nuclear
in endothelial cells. VEGF165 and VEGF165b had distinctly different localizations
in retina at 12 and 21 WG. Most striking was the succinct nuclear association of
VEGF165b within CXCR4+ progenitors in retina, some of which are angioblasts, and
mesenchymal precursors in choroid. The localization of VEGF165 in nerve fibers and
VEGF165b in Muller cells at 21 WG, suggests that both neurons and glia contribute to
the control of retinal vasculogenesis.
CR: G.A. Lutty, None; T. Baba, None; C. Merges, None; T. Hasegawa, None; D.S.
McLeod, None.
Support: NIH Grants EY09357 (GL), EY016151 (GL), and EY01765 (Wilmer)
Background/Purpose: Our laboratory has recently demonstrated increased retinal
gene expression of apelin (apln), an angiogenic cytokine involved in cardiovascular
development and fluid homeostasis, in two rodent models of oxygen-induced
retinopathy (OIR). The present studies were undertaken to elucidate the role of apelin
and its receptor, APJ, in retinal angiogenesis.
Methods: Primary cultures of human retinal vascular endothelial cells (HRVECs)
were used for in vitro experiments, and the rat model of OIR was used for in vivo
experiments. Near-confluent HRVECs were treated for 24 hrs with either 10% FBS
or serum-free medium, in the presence or absence of 2.5uM VEGF. In each of the
four conditions, expression of the apelin and APJ genes were measured by qRT-PCR
and concentration of apelin in the medium was measured by ELISA. Tube formation
assays were performed in triplicate with HRVECs following addition of varying
concentrations (2.5uM - 20uM) of recombinant apelin-12, and varying concentrations
of two apelin inhibitors (apelin-13[F13A], a competitive antagonist, and siRNA against
apln). Following induction of OIR by oxygen cycling, eyes of P15 rats were treated with
an intravitreal injection of varying concentrations (1uM - 100uM) of apelin-13(F13A)
or vehicle. Six days later, retinal flatmounts were stained with ADPase, and retinal
vascular growth was assessed.
Results: Expression of apelin and APJ mRNA in HRVECs, and secretion of apelin
by HRVECs, were increased ≥ 2-fold following either stress with serum-free media
or stimulation with VEGF. As little as 2.5 uM apelin significantly increased total
tube formation (71.0 mm vs. 25.8 mm, P=0.01), an effect comparable to equimolar
VEGF. Tube formation was reduced by 50% and 80% following treatment with apelin13(F13A) and siRNA, respectively. In vivo, mean retinal avascular area was decreased
(13.8mm 2 vs 29.3mm 2, p=0.001), and mean peripheral retinal neovascularization was
significantly decreased (4.5 clock-hrs vs. 1.2 clock-hrs), in eyes treated with as little
as 10 uM of apelin-13(F13A).
Conclusions: The apelin/APJ pathway appears to be involved in retinal angiogenesis
in vitro. Its effects may result from autocrine actions by retinal endothelial cells
and through both VEGF-dependent and VEGF-independent mechanisms. Apelin
inhibition reduces retinal NV in vivo, possibly by reducing peripheral avascularity.
The apelin/APJ pathway may merit further study as a rational therapeutic target for
anti-angiogenesis.
CR: F.M. Recchia, None; L. Xu, None; J.S. Penn, None.
Support: NIH (EY07533 and P30 EY08126); Unrestricted Departmental Grant from
Research to Prevent Blindness, Inc.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
25-28
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
29 - A76
Microglia and Angiogenesis in the Developing Retina
30 - A77
Implication of Dystrophin Protein 71 (Dp71) in Retinal Angiogenesis
C.J. Breen1, T. Gardiner1, A. Stitt1, T. Cogliati2. 1Centre for Vision Sciences, Queen’s
University Belfast, Belfast, United Kingdom; 2Neurobiology-Neurodegeneration
and Repair Laboratory, National Eye Institute, NIH, Bethesda, MD.
R. Tadayoni1, R. Benard2, B. Dupas1, A. Sene2, A. Gaudric1, F. Sennlaub3, J.-A. Sahel2, A.
Rendon2. 1Ophthalmology, Lariboisiere University Hospital, AP-HP, Paris Diderot
University, Paris, France; 2Département de thérapies, Institut de la Vision, Inserm
UMR-S 968, Université Pierre et Marie Curie, Paris, France; 3Centre de Recherche les
Cordeliers, Inserm U872, Paris, France.
Purpose: Microglia (MG) invade the immature retina prior to angiogenesis. MG are
capable of producing a battery of inflammatory mediators, as well as angiogenic
cytokines, but in the developing retina they assume a benign phenotype that is not
associated with inflammation. During retinal vascularisation MG can be observed
in close proximity to endothelial tip cells of the growing vascular tree. Furthermore,
they phagocytose surplus cells undergoing apoptosis, potentially eliciting an antiinflammatory, yet pro-angiogenic response. In this study, we examined the potential
of MG to induce angiogenesis using in-vivo and in-vitro approaches.
Methods: Eyes from C57BL/6 mice between postnatal day-0 (P0) and P12 were collected
and RNA extracted to examine expression patterns of angiogenic and inflammatory
cytokines. Sectioned and flat mounted eyes were also double stained with Tomato
Lectin and Cleaved-Caspase 3 to study the uptake of MG to apoptotic cells. BV2
MG cells in vitro were fed apoptotic bodies generated from cultured WERI-Rb1
retinoblastoma cells and changes in angiogenic and inflammatory cytokine expression
were analyzed. Finally, conditioned media from BV2 cells either fed apoptotic bodies
or treated with LPS were compared in a Matrigel angiogenesis assay.
Results: During retinal vascular development Caspase-3 / Lectin staining revealed
activated MG actively phagocytosing apoptotic cells, often in close apposition to
endothelial tip cells. In-vivo, expression of VEGF, SDF1, IL10, TNFalpha and MIP1alpha
were maximal at P6 and coincident with waves of apoptosis. Upon ingestion of apoptotic
cells, BV2 cell expression of VEGF and IL10 increased two-fold while TNFalpha and
MIPalpha were unchanged; conditioned media increased angiogenic sprouts in the
Matrigel assay compared to controls (p<0.01).
Conclusions: Ingestion of apoptotic bodies by MG in-vitro causes up-regulation of
angiogenic and anti-inflammatory cytokines and induces angiogenesis. In-vivo, MG
phagocytosis of apoptotic cells in the developing retina may be anti-inflammatory
and promote angiogenesis.
CR: C.J. Breen, None; T. Gardiner, None; A. Stitt, None; T. Cogliati, None.
Support: None
31 - A78
Laminins of the Inner Limiting Membrane Regulate Retinal Angiogenesis
Purpose: To investigate whether dystrophin protein 71 (Dp71), the smallest product
of Duchenne muscular dystrophy (DMD) gene, and key component of the membrane
associated cytoskeleton has an implication in postnatal retinal angiogenesis.
Methods: Flat mounted retinas of wild type and Dp71-null newborn mice at different
ages were studied to quantify retinal vessels angiogenesis and coverage of the retina by
GFAP+ astrocytes. Aortic ring assay was used to evaluate a direct effect of absence of
Dp71 on angiogenesis from vessels. The localization of Dp71 in retinal was investigated
by immunostaining with an anti-dystrophin antibody (H4). Relative expression of
proteins was determined by Western blot and Real Time-PCR.
Results: In absence of Dp71 postnatal angiogenesis was found inhibited until P6. A
delay in the spreading of GFAP+ astrocytes was also observed and Dp71was found
to be expressed in these astrocytes glial cells known to drive angiogenesis. These
observations suggested that the absence of Dp71 delayed the deployment of GFAP+
astrocytes on the retina, which could act as a limiting factor for angiogenesis. After
P6, astrocytes covered completely the retina, and angiogenesis activity was higher
in Dp71-null mice allowing a catch-up of wild type mice vascularization at P12. This
is likely related to pericytes in which we found also Dp71. Indeed, absence of Dp71
from pericytes of microvessels in aortic rings essays increased their angiogenesis at 7
days. Exploration of several angiogenesis regulation pathways (VEGF, NOS, microglial
cells…) suggested that the Dp71 might act independently.
Conclusions: Dp71 is implicated in the regulation of angiogenesis. This original effect
is through cells in which Dp71 is present and then could be pro or anti-angiogenic,
depending on time and localization.
CR: R. Tadayoni, None; R. Benard, None; B. Dupas, None; A. Sene, None; A. Gaudric,
None; F. Sennlaub, None; J.-A. Sahel, None; A. Rendon, None.
Support: Fondation Voir et Entendre, Association Française contre les Myopathies
32 - A79
Core Circadian Regulatory Genes Regulate Endothelial Differentiation of
CD34+ Cells
G. Gnanaguru1A, G. Bachay1A, W.J. Brunken1B. ACell Biology, BCell Biology &
Ophthalmology, 1SUNY Downstate Med Ctr, Brooklyn, NY.
Purpose: Cell migration and spatial patterning are regulated by basement membrane
(BM) derived cues. Here, we test the hypothesis that laminins guide astrocyte migration
and vascular patterning.
Methods: Expression of laminin β2 and γ3 was analyzed using standard
immunostaining methods. Astrocyte migration was studied using optic nerve (ON)
explants. Whole mount preparations of wild type, β2-/-, γ3 -/-, and β2-/-γ3 -/- retinae were
used to study vascular development.
Results: We have previously shown that the β2 and γ3 laminin chains are deposited
in the ILM and differentially in vascular BM, and that the deletion of laminin β2 and
γ3 genes disrupts astrocyte migration and angiogenesis, suggesting a haptotactic
role for laminins. We assayed whether astrocyte migration is laminin-dependent in
vitro. ON explants were treated with exogenous laminins. EHS laminin promoted
astrocyte migration while laminin α3β3γ2 did not. Astrocyte migration/patterning in
β2-/- and β2-/-γ3 -/- retinae were dysmorphic while the γ3 -/- retina was normal; in contrast,
endothelial migration was slowed in all the mutants. This latter result was surprising
given the lack of an effect on astrocyte patterning in γ3 -/- mice. Developmental studies
demonstrate that retinal angiogenesis in γ3 -/- mice was delayed up to 2 days and the
capillary network branched excessively. As the γ3 chain is spatially restricted to the
branch points of sprouting vessels, we posit that γ3 regulates branching. Thus, we
measured vessel density and showed it was significantly increased in the γ3 -/- retina.
We also tested if laminin deletion affected VEGF expression: VEGF isoforms 188/164
regulate branching while 120 regulates tip cell guidance. VEGF188/164 expression was
increased and VEGF120 was decreased in γ3 -/-, whereas all the isoform levels were
reduced in β2-/- and β2-/-γ3 -/- mice.
Conclusions: These data indicate that laminin β2 and γ3 play independent roles in
regulating retinal angiogenesis; moreover, they suggest that laminins are upstream
of VEGF regulation and that the β2 chain is important for astrocyte patterning and
migration, whereas γ3 regulates endothelial migration rate and branching behavior.
CR: G. Gnanaguru, None; G. Bachay, None; W.J. Brunken, Patent Awarded, P.
Support: NIH Grant EY12676; SUNY Eye Institute
A.D. Bhatwadekar1A, Y. Xu1B, V. Stepps2, S. Bartelmez2, J.V. Busik 3, M.B. Grant1A.
Pharmacology and Therapeutics, BImmunology and Microbiology, 1University of
Florida, Gainesville, FL; 2Beta Stem Therapeutics, San Francisco, CA; 3Physiology,
Michigan State University, East Lansing, MI.
A
Purpose:Endothelial progenitor cell (EPC) differentiation is critical for vascular
repair function. We reported that in diabetes there is a loss of circadian rhythmicity
of EPC release due to bone marrow neuropathy which is accompanied by a loss of
circadian regulatory proteins (CCRP). Clock genes encoding CCRP were shown to
control differentiation in neuronal stem cells, we asked whether clock genes similarly
control the differentiation of EPCs into endothelial cells (EC).
Methods:CD34+ cells isolated from healthy volunteers (n=6) were maintained in
either EPC differentiating or control medium. At 12 hr intervals for 4 days, CD34+
cells were collected and stained for CD34, CD45, CD133, CD144, CD146 to study their
hematopoietic and endothelial characteristics using multicolor flow cytometry and
parallel studies were performed to examine clock gene expression (Clock, Bmal, Per1,
Per2, Per3, Cry1, Cry2) every 4 hrs using real time RT-PCR.
Results:Endothelial differentiation of CD34+ cells resulted in a decrease in the
CD133+CD34+CD45+ population, while control medium maintained stem cell
characteristics with 94.1% of cells expressing the stem cell marker CD133. Differentiation
of CD34+ cells into ECs was associated with robust alteration in clock gene expression.
The positive arm of the clock genes involving clock and Bmal1 showed suppression
(p<0.001) in their expression with endothelial differentiation. In contrast, control
media maintained the oscillatory pattern of the clock genes over the 96 hrs examined.
The clock genes in negative loop (Per1, Cry1, Cry2) of clock gene assembly started
oscillating in rhythmic pattern (p<0.001), in particular Cry1 (15 fold; p<0.001) and
Cry2 (5 fold; p<0.001) exhibited a robust response when the cells were placed in EC
differentiating medium.
Conclusions: These novel findings support that CCRP are involved in endothelial
differentiation of CD34+ cells, a step that is reduced in diabetic CD34+ and may be
responsible for their dysfunction. Modifying the period and amplitude of circadian
oscillations in diabetic CD34+ cells may directly impact there reparative potential.
CR: A.D. Bhatwadekar, None; Y. Xu, None; V. Stepps, None; S. Bartelmez, None; J.V.
Busik, None; M.B. Grant, None.
Support: AHA Post Doctoral Grant, NIH grants: 2RO1 EY012601-08 , 2RO1 EY00773917, R01 EY018358
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
29-32
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
33 - A80
α-Crystallin Knockout Mice Have Normal Vascular Development in Advance of
a Delayed Astrocyte Template
T. Baba1, D.S. McLeod1, C. Merges1, I.A. Bhutto1, R. Grebe1, M. Edwards1, E.F.
Wawrousek 2, G.A. Lutty1. 1Wilmer Ophthalmological Inst, Johns Hopkins University,
Baltimore, MD; 2Lab of Molecular & Dev Bio, National Eye Inst/NIH, Rockville, MD.
Purpose: During primary retinal vascular development in rodents, an astrocyte
template has been reported to be critical for guiding endothelial tip cells. Recently,
a small heat shock protein, αB-crystallin (CryαB) was reported to regulate glial
fibrillary acidic protein (GFAP) assembly in astrocytes [Hageman TL et al. Hum Mol
Genet 2009; 18: 1190-9]. We used CryαA/CryαB/heat shock protein-b2 knockout mice
(α-cry -/-) to investigate the role of α-cry’s in retinal astrocyte development, and the
relationship between astrocytes and primary blood vessels during early stages of
retinal vasculogenesis.
Methods: α-cry -/- mice (n= 8, from P3 to P35) and wild-type (WT) controls (129S6/
SvEvTac, n= 4) were used under the ARVO statement for the use of animals in
ophthalmic and vision research. Flatmount retinas labeled with Isolectin B4, as a
vascular marker, and GFAP, as an astrocyte marker, were examined by confocal
microscopy. Morphometric analysis was performed to measure the distance from optic
nerve head to the peripheral edge of Isolectin-labeled vasculature and GFAP-positive
astrocytes and the number of vascular branches and filopodia of endothelial tip cells.
Results: The GFAP-labeled astrocytes were observed in advance of endothelial cells
in 98% of the measurements in WT retinas and 56% of those in α-cry -/- retina. There
was a significant difference in the distance from the optic nerve head to vascular or
astrocyte front in α-cry -/- mice compared to WT (P< 0.0001). In 44% of measurements
in α-cry -/- retina, blood vessels were 74.8 +/- 45.1 μm in advance of GFAP-labeled
astrocytes. The number of vascular branches and endothelial cell filopodia were not
different between WT and α-cry -/- (P= 0.358, P= 0.683). In α-cry -/- retina, the vascular
network had reached the ora serrata by around P7 and the primary vascular pattern
was normal at P7, P14 and P35.
Conclusions: There was a delay in formation of a GFAP-positive astrocyte template
in α-cry -/- mice. However, the development of peripheral vasculature was similar
to WT, suggesting there are processes other than an astrocyte template which guide
vasculogenesis in developing retina.
CR: T. Baba, None; D.S. McLeod, None; C. Merges, None; I.A. Bhutto, None; R. Grebe,
None; M. Edwards, None; E.F. Wawrousek, None; G.A. Lutty, None.
Support: NIH/NEI EY09357 (GL), NIH/NEI EY01765 (Wilmer), JSPS Postdoctoral
Fellowships for Research Abroad (TB)
34 - A81
Vla-1 is Directly Involved in Lymphangiogenesis
S. Grimaldo, D. Yuen, L. Chen. Optometry, University of California, Berkeley,
Berkeley, CA.
Purpose: In the inflamed and lymphatic-rich corneas, the transplantation rejection
rate can be as high as 90%. Unfortunately, many patients who are blind from corneal
diseases fall in this category after inflammatory, traumatic, or chemical insults. To
date, there is no effective treatment for these patients. This study is to investigate the
specific roles of VLA-1 (very late antigen 1; also known as alpha1beta1 integrin) in the
processes of lymphangiogenesis both in vitro and in vivo.
Methods: In vitro human lymphatic endothelial cell (LEC) culture model was used to
study the expression of VLA-1 in LECs by both RT-PCR and immunocytochemistry
assays. The effect of VLA-1 depletion in LECs by small interference RNAs (siRNA) on
LEC proliferation and tube formation were also analyzed. Additionally, murine in vivo
suture-induced corneal inflammatory lymphangiogenesis model was used to study
the effect of VLA-1 neutralizing antibody (provided by Covella Pharmaceuticals, Inc.)
on the development of new lymphatic vessels in the inflamed corneas.
Results: VLA-1 was expressed in human LECs at both mRNA and protein levels. Its
down-regulation by siRNAs suppressed LEC proliferation as well as tube formation.
This effect was also confirmed in the inflamed corneas after the antibody treatment.
Conclusions: VLA-1 plays a direct role in the processes of lymphangiogenesis, holding
the promise of uncovering new therapeutic targets for corneal inflammation and
transplant rejection. It is hopeful that beyond its contributions to eye diseases, this
study also will shed some light on the development of new therapeutic strategies for
other lymphatic-related disorders in the body.
CR: S. Grimaldo, None; D. Yuen, None; L. Chen, None.
Support: This work is supported in part by research grants from NIH, DoD, and
University of California at Berkeley (LC).
35 - A82
Knockdown of Multidrug Resistance-Associated Protein 4 by Sirna Enhances
Cell Migration and Tube Formation in Human Retinal Endothelial Cells
36 - A83
IGFBP-3 Attenuates Vascular Injury by Reducing Activation of Acid
Sphingomyelinase and Retinal Vascular Permeability
M. Tagami, S. Kusuhara, H. Imai, S. Honda, Y. Tsukahara, A. Negi. Department of
Ophthalmology, Kobe Univ. Graduate Sch. of Medicine, Kobe-City, Japan.
J.L. Kielczewski1A, T. Gardiner2, L. Wu1B, S. LiCalzi1A, S. Firth 3, R. Baxter3, R.N. Mames4,
M.E. Boulton1B, J.V. Busik 5, M.B. Grant1A. APharmacology and Therapeutics, BAnatomy
and Cell Biology, 1University of Florida, Gainesville, FL; 2Centre for Vision Science,
Institute of Clinical Science, Belfast, Ireland; 3Molecular Medicine, University of
Sydney, St. Leonards, NSW, Australia; 4Affiliate UF, Retina Center, Gainesville, FL;
5
Physiology, Michigan State University, East Lansing, MI.
Purpose: To explore the angiogenic function of multidrug resistance-associated protein
4 (Mrp4), an efflux ATP-binding cassette transporter , in human retinal endothelial
cell (HREC) culture.
Methods: Mrp4 gene and protein expressions in HRECs were examined by RT-PCR
analysis and immunohistochemical analysis, respectively. Semiconfluent HREC
cultures were transfected with Mrp4 siRNA or nonsilencing siRNA and used in the
following assays 24 hours after transfection. HREC proliferation was evaluated using
DNA synthesis measurements by 5-bromo-2’-deoxyuridine (BrdU) incorporation and
HREC migration was assessed with a modified Boyden chamber assay. The effect
of Mrp4 knockdown on the ability of capillary-like formation was tested by tube
formation assay in Matrigel.
Results: HRECs expressed Mrp4 mRNA and showed immunoreactivity for Mrp4.
Knockdown of Mrp4 by siRNA enhanced the migratory capacity of HRECs (P = 0.002).
Similarly, Mrp4-knockdown HRECs showed a marked increase in tube formation (P
= 0.001). On the other hand, there was no significant difference in cell proliferation
between Mrp4-knockdown and control HRECs (P = 0.134).
Conclusion: Transfection of Mrp4 siRNA promotes cell migration and tube formation
in HRECs. These results suggest that Mrp4 is negatively associated with retinal
angiogenesis.
CR: M. Tagami, None; S. Kusuhara, None; H. Imai, None; S. Honda, None; Y.
Tsukahara, None; A. Negi, None.
Support: None
Purpose: Insulin-like growth factor binding protein-3 (IGFBP-3) has vascular protective
effects. However, the molecular mechanisms which mediate this effect have yet to
be elucidated. Sphingomyelin is a main lipid component of the plasma membrane.
Lysosomal acid hydrolases play an important role in the turnover of membrane lipids
of cells. Among them, the key inflammatory enzyme acid sphingomyelinase (ASM),
hydrolyzes sphingomyelin to ceramide and phosphocoline. Since, retinal injury
activates acid sphingomyelinase (ASMase), we asked (1) if IGFBP-3 reduced acid
sphingomyelinase (ASM) and (2) if IGFBP-3 protected from injury-induced increases
in retinal vascular permeability.
Methods: In the first set of studies, healthy mice received an intravitreal injection of
VEGF (100 ng/μl) to increase retinal vascular permeability, followed by intravitreal
injection of IGFBP-3 (100 ng/μl) at 6 hours (n=6) and 24 hours (n=6) post VEGF injection.
As a control, vehicle was injected either 6 or 24 hours after VEGF injection (n=6). Mice
then received a tail injection of FITC- albumin 2 hours prior to sacrifice and their
retinas were harvested 48 hours following initial VEGF injection. In a second group,
mice underwent laser photocoagulation of retinal vessels and intravitreal injection
of IGFBP-3 expressing plasmid or control plasmid. Mice were sacrificed 4 days post
IGFBP-3 injection, the time of peak IGFBP-3 expression and retinal ASMase changes
were determined by qRT-PCR.
Results: At 24 hours, the combination treatment of VEGF and IGFBP-3 in uninjured
mouse eyes resulted in a 1.5 fold reduction in retinal vascular permeability (p<0.05)
compared to VEGF alone. In mice receiving laser and control plasmid, ASMase mRNA
levels were elevated 4-fold compared to controls (p<0.05). However, in mice receiving
laser photocoagulation and IGFBP-3 plasmid, ASMase levels approached baseline
(p<0.05) and this was accompanied by a reduction in vascular permeability.
Conclusion: Our studies support that the beneficial effects of IGFBP-3 on retinal
vascular permeability may be due to reduction of the pro-inflammatory enzyme
ASMase.
CR: J.L. Kielczewski, None; T. Gardiner, None; L. Wu, None; S. LiCalzi, None; S.
Firth, None; R. Baxter, None; R.N. Mames, None; M.E. Boulton, None; J.V. Busik,
None; M.B. Grant, None.
Support: NIHIR01 EY07739 and NIHR01 EY12601
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
33-36
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
37 - A84
Functional Organization of the Retinal Microvasculature: Differential Actions
of Angiotensin II Within the Feeder Vessel/Capillary Unit
38 - A85
A Dominant Negative Mutant of p38 MAP Kinase Blocks VEGF-Induced uPAR
Expression and Barrier Breakdown in Retinal Microvascular Endothelial Cells
T. Zhang1,2, D.G. Puro1. 1Department of Ophthalmology & Visual Sciences,
University of Michigan, Ann Arbor, MI; 2Department of Ophthalmology, Eye &
ENT Hospital, Fudan University, Shanghai, China.
J. Yang1A, R.B. Caldwell1A, A.M. Behzadian1B. AVascular Biology Center, BVascular
Biology Cnt, Department of Pharmacology & Toxicology, 1Medical College of
Georgia, Augusta, GA.
Purpose: Although decentralization is an important operational feature of the retina’s
circulatory system, much remains to be learned about how its microvasculature is
functionally organized. Here, we characterized the actions of angiotensin II within
the feeder vessel/capillary unit.
Methods: Using a tissue print procedure, we isolated rat retina microvascular
complexes that included a capillary network plus the feeder vessel linking it with a
myocyte-encircled arteriole. A micromanipulator-controlled micropipette was used
to transect isolated microvessels at the capillary/feeder vessel junction. Ionic currents
and membrane capacitances were monitored via perforated-patch pipettes sealed
onto abluminal cells of feeder vessels and capillaries. Calcium-imaging was used to
monitor intracellular calcium in cells loaded with fura-2. Angiotensin-induced cell
death was assessed by trypan blue dye exclusion.
Results: Angiotensin II activated a larger non-specific cation conductance in capillaries
than in feeder vessels. Using transected retinal microvessels, we found that angiotensin
(500 nM) activated a conductance of 1690 ± 460 pS (n = 8) in capillaries and 140 ± 50 pS
(n = 6, P = 0.01) in feeder vessels. In addition, angiotensin evoked an increase in pericyte
calcium of 325 ± 20 nM (n = 81), which was markedly greater than the 55 ± 3 nM (n =
149, P < 0.0001) increase detected in proximal mural cells. Exposure to angiotensin
also resulted in calcium-activated chloride currents that were larger in the capillaries
(610 ± 130 pS, n = 9) than in the feeder vessels (170 ± 60 pS, n = 6, P = 0.01). Suggestive
that angiotensin inhibits gap junction pathways in capillaries, but not feeder vessels,
this vasoactive signal decreased the membrane capacitance only within the capillary
network. In other experiments, we found that a 1-day exposure to 500 nM angiotensin
triggered cell death in the capillary network, but not in the feeder vessels.
Conclusions: In the retinal microvasculature, angiotensin-induced changes in ionic
currents, abluminal cell calcium, cell-to-cell transmission and cell viability are greater
in the capillaries than in the feeder vessels. Thus, although a capillary network and
its feeder vessel constitute a highly interactive operational unit (Microcirculation
14:1-10, 2007), there is functional sub-specialization within this decentralized portion
of the retina’s circulatory system. The capillary network is the predominant site for
mediating the effects of angiotensin II.
CR: T. Zhang, None; D.G. Puro, None.
Support: NIH Grant EY12505, EY07003 and RPB
Purpose: To define the role of MAP kinases in VEGF-induced signaling pathway
that lead to uPAR expression and vascular permeability increase. Activation of MAP
kinases has been shown to correlate with increased permeability in endothelial cells
treated with VEGF or high glucose and in retinas of diabetic animal models. We
have shown previously that p38 MAP kinase inhibitors block VEGF-induced GSK
/ beta-catenin signaling and uPAR expression. Here, we present data showing that
adenoviral vector delivery of dominant negative p38 mutants into retinal endothelial
cells blocks VEGF-induced activation of the GSK / beta-catenin signaling pathway,
prevents uPAR expression and preserves paracellular barrier function.
Methods: Two dominant negative mutants of p38 which were originally obtained
from Dr. J. Han’s lab, one was mutated on the Thr180 and Tyr182 phosphorylation sites;
the other on its ATP-binding site, thus loosing its kinase activity. The mutants of p38
gene were cloned into pShuttle-IRES-hrGFP and then into pAdEasy vector. Bovine
retinal endothelial (BRE) cells were infected with recombinant adenovirus of those p38
dominant negative mutants or without insertions. Fluorescence microscopy was used
to monitor expression of recombinant genes in infected BRE cells and Western blotting
to analyze phosphorylation of GSK, p38 and the p38 substrates. uPAR expression
was measured by quantitative real-time PCR and barrier function was monitored by
measuring transcellular electrical resistance.
Results: Both p38 mutants blocked VEGF-induced phosphorylation of p38 substrates
MAPKAP-2 and ATF2, suggesting that VEGF-induced p38 activation was blocked.
Moreover, VEGF-induced GSK phosphorylation was also reduced by both mutants.
However, the VEGF-induced increase in uPAR expression was more potently inhibited
by the kinase mutant of p38 as compared with either the phosphorylation mutant
or empty vector. This same kinase mutant also strongly prevented VEGF-induced
barrier breakdown.
Conclusions: Expression of a dominant negative p38 Map kinase mutant blocks the
activation of p38 by VEGF, prevents phosphorylation of GSK, expression of uPAR
and preserves endothelial cell barrier function. This indicates that VEGF-induced
permeability depends on p38 kinase activity and offers a new strategy for developing
potent and specific therapies for treatment of retinal diseases associated with vascular
barrier dysfunction.
CR: J. Yang, None; R.B. Caldwell, None; A.M. Behzadian, None.
Support: NIH RO1EY04618; NIH RO1EY11766, AHA predoctoral fellowship, Veterans
Administration
39 - A86
The Effect of Penetrating Ocular Injury on the Regulation of Angiogenesis by
Proteolysis
40 - A87
Expression of Claudins in the Normal Adult Human Retinas
W. Xiao, Y. Luo, X. Chen, J. Li, X. Ding, M. Zeng, S. Tang. Sun Yat-sen University,
Zhongshan Ophthalmic Center, Guangzhou, China.
M.E. Capozzi, W.Y. Boadi, G.W. McCollum, J.S. Penn. Ophthalmology, Vanderbilt
University, Nashville, TN.
Purpose: Penetrating ocular needle injury (POI) induces angiostatic activity in
the vitreous humor and alters the retinal and vitreous humor levels of several
proteins known to regulate angiogenesis. Previous studies suggest that matrix
metalloproteinases (MMP) facilitate neovascularization by remodeling retinal tissue
to allow extension of neovascular structures from the retinal plane into the vitreous
cavity. The purpose of this study was to investigate biochemical assays of MMP
activities in the presence of soluble POI and non-POI rat vitreous.
Methods: To generate POI, a dry 30-gauge needle was inserted twice posterior to the
temporal ora of the left eyes of Sprague-Dawley rats. The right eyes served as nonPOI controls. Vitreous from both eyes was harvested 24 hours after needle injury and
pooled separately. MMP-1, -2, -9, -13, and -14 proteolytic activities were assayed in the
presence of vehicle, 50μg/ml soluble POI or 50μg/ml non-POI vitreous protein in a
biochemical assay that uses an exogenous MMP substrate tagged with a fluorescent
reporter. A potent inhibitor of all these enzymes was used as a negative control. All
measurements were made when the change in fluorescent units was linear with time.
Results: Soluble POI and non-POI vitreous protein had no effect on MMP-1 and -13
activities. The MMP-2 and -14 positive control activities were significantly different
from the MMP activities in the presence of soluble POI and non-POI vitreous protein.
The data suggest that soluble POI vitreous protein decreases MMP-2 and -9 activity
when compared with non-POI vitreous protein.
Conclusions: Soluble vitreous protein from both POI and non-POI eyes showed
inhibition in MMP-2 and -14 activities. Vitreous protein from needle-injured eyes
showed a decrease in MMP-2 and -9 activity, relative to that from non-injured eyes.
Complimentary studies will be designed to further assess effect of needle injury on
MMP activity.
CR: M.E. Capozzi, None; W.Y. Boadi, None; G.W. McCollum, None; J.S. Penn, None.
Support: NIH Grant EY07533
Purpose: Tight junctions (TJs) are important components of the blood-retinal barrier.
However, the evidence for the presence of tight junction-associated proteins in
human retina is lacking. To understand the contribution of each claudin subtype
to TJ formation in the inner blood-retinal barrier of human eye, the expression and
distribution of claudin subtypes in adult human retina were investigated in this study.
Methods: Eye balls of healthy adult individuals were obtained from the Eye Bank of
Zhongshan Ophthalmic Center. Whole retinas were dissected carefully. RT-PCR was
used to assess mRNA expression of claudins in normal adult human retinas. Western
blot analysis and immunofluorescence staining were performed to determine the
protein expression and tissue distribution of selected claudins respectively.
Results: RT-PCR showed that claudin-12 was the most abundant subtype, followed
by claudin-1, -2, -3, -4, -5 and -22. Claudin-6 to -11, -14 to -19 and -23 were not detected
at mRNA level. Expression of claudin-1, -2, -3, -4 and -5 were identified in the retinas
by western blot. By immunofluorescence staining, claudin-1, -2, -3 and -5 were found
to be localized on retinal microvessels.
Conclusions:This study indicates that claudin-1, -2, -3, -4 and -5 are expressed on the
retinal vessels in the normal adult human retinas. These claudins may be responsible
for maintaining the normal function of inner blood-retinal barrier.
CR: W. Xiao, None; Y. Luo, None; X. Chen, None; J. Li, None; X. Ding, None; M.
Zeng, None; S. Tang, None.
Support: The National Natural Science Foundation of China(30872819)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
37-40
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
41 - A88
A Novel Profiling System of Endothelial Gene Expression in Angiogenic Retinal
Vessels
42 - A89
Astrocyte Hypoxic Response is Essential for Pathological but Not
Developmental Angiogenesis of the Retina
S. Kusuhara1,2, A. Uemura1,2, L.M. Jakt2, Y. Shimizu2, A. Negi1, S.-I. Nishikawa2.
1
Ophthalmology, Kobe Univ Grad Sch of Med, Kobe, Japan; 2Laboratory for
Stem Cell Biology, RIKEN Center for Developmental Biology, 2-3-3 Minatojima
Minamimachi, Chuo-ku, Kobe, Japan.
T.U. Krohne1, A. Weidemann2, E. Aguilar1, T. Kurihara2, N. Takeda2, M.I. Dorrell1,
M.C. Simon3, V.H. Haase4, R.S. Johnson2, M. Friedlander1. 1Cell Biology, The Scripps
Research Institute, La Jolla, CA; 2Biology, University of California, San Diego, La
Jolla, CA; 3Cell and Developmental Biology, Howard Hughes Medical Institute,
The Abramson Family Cancer Research Institute, Philadelphia, PA; 4Medicine,
Vanderbilt University Medical Center, Nashville, TN.
Purpose: Deregulated retinal angiogenesis directly cause vision loss in many ocular
diseases, such as diabetic retinopathy and retinopathy of prematurity. To identify
endothelial-specific genes expressed in angiogenic retinal vessels, we conducted global
transcriptional profiling in endothelial cells (ECs) sorted from living retinal vessels.
Methods: Utilizing fluorescence-activated cell sorting (FACS), we isolated ECs from
angiogenic and quiescent retinal vessels in neonatal and adult Tie2-GFP transgenic
mice, respectively. Total RNAs extracted from purified retinal ECs were amplified and
used for microarray analyses. Differentially expressed genes in distinct cell fractions
were statistically analyzed with Significance Analysis of Microarrays and validated
with the eXintegrator software. Bioinformatic analyses for functional annotations
were performed with NIH-DAVID. Whole-mount in situ hybridization was performed
with digoxigenin (DIG)-labeled RNA probes and anti-DIG antibodies conjugated with
alkaline-phosphatase.
Results: We successfully sorted ECs representing 0.1% of entire cell populations in
living mouse retinas. In neonatal retinas, 1623 genes were statistically upregulated in
EC fractions compared to non-endothelial fractions. The Gene Ontology annotations
correlated the functions of a considerable number of the endothelial-specific gene
products with cell motility and cytoskeletal arrangements. Exploiting the eXintegrator’s
pattern-matching function, we further determined angiogenic endothelial genes whose
expression levels were downregulated in quiescent vessels of adult retinas. These
genes displayed spatially unique distribution in ECs of arteries, veins, and capillaries
of the developing retinal vasculature.
Conclusions: Our FACS-array transcriptional profiling system will contribute not only
to the understanding of the pathogenesis of retinal diseases, but also to the discovery
of novel drug targets to treat deregulated retinal angiogenesis.
CR: S. Kusuhara, None; A. Uemura, None; L.M. Jakt, None; Y. Shimizu, None; A.
Negi, None; S.-I. Nishikawa, None.
Support: Grants-in-Aid for Scientific Research on Priority Areas (12219209) and the
Leading Project grant for Realization of Regenerative Medicine in Japan
43 - A90
Investigation of Barrier Characteristics of Zebrafish Retinal Vessels
Purpose: Astrocytes perform crucial functions in both developmental and pathological
angiogenesis of the retina. Vascular endothelial growth factor (VEGF), regulated by the
hypoxic response signaling pathway, is essential for these processes. We have analyzed
the role of the astrocyte hypoxic response in developmental and pathological retinal
angiogenesis using conditional knockout mice and the oxygen-induced retinopathy
(OIR) model.
Methods: Astrocyte-specific (GFAP-cre) conditional knockout mice for VEGF and
its upstream regulators von Hippel-Lindau protein (VHL), hypoxia-inducible factor
(HIF)-1α, and HIF-2α were created. Retinal vascular morphology in these animals
was evaluated by immunohistochemistry at postnatal day 7 (P7) and in adult mice.
OIR was induced by hyperoxia treatment from P7 to P12, and retinal vaso-obliteration
and pre-retinal neovascularization were quantified at P17. Retinal expression of VEGF,
VEGF isoforms, and EPO was assessed by RT-PCR.
Results: Conditional knockout of VEGF, HIF-1α, or HIF-2α in astrocytes did not affect
retinal vascular development and resulted in a normal retinal vasculature in adult
animals. In contrast, knockout of VHL caused a grossly abnormal, hypervascular
retinal phenotype associated with increased retinal VEGF expression. This phenotype
was rescued by additional knockout of HIF-2α or VEGF, but not HIF-1α. Similarly, in
the OIR model, knockout of HIF-2α or VEGF, but not HIF-1α, significantly reduced
hypoxia-induced pre-retinal neovascularization.
Conclusions: Our findings demonstrate that (i) astrocyte-derived VEGF is essential
for pathological, but not developmental, retinal angiogenesis, and that (ii) the hypoxic
response in retinal astrocytes is mediated predominantly by HIF-2α, not HIF-1α.
CR: T.U. Krohne, None; A. Weidemann, None; E. Aguilar, None; T. Kurihara,
None; N. Takeda, None; M.I. Dorrell, None; M.C. Simon, None; V.H. Haase, None; R.S.
Johnson, None; M. Friedlander, None.
Support: Grants from the National Eye Institute (EY11254) and the MacTel Foundation
to MF; fellowships from the German Research Foundation (DFG) to TUK (KR 2863/61) and AW (WE 4275/1-1)
44 - A91
Limited Pericyte Ensheathment Predisposes Human Choroidal Vessels to
Vascular Instability & Poor Blood Flow Regulation
E. Yang1, J.H. Kim1, K.-W. Kim2, Y.S. Yu1, J.H. Kim1. 1Ophthalmology, Seoul National
University Hospital, Seoul, Republic of Korea; 2College of Pharmacy and Research
Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of
Korea.
T. Chan-Ling1, M. Koina2, J.R. McColm1, J. Dahlstrom2, E. Bean2, L. Baxter1. 1Department
of Anatomy, University of Sydney, Sydney, Australia; 2Department of Pathology,
Australian National University, Canberra, Australia.
Esther Yang1, Jin Hyoung Kim1, Kyu-Won Kim 2, Young Suk Yu1, Jeong Hun Kim1
Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Department of
Ophthalmology, College of Medicine, Seoul National University & Seoul Artificial
Eye Center Clinical Research Institute, Seoul National University Hospital, Seoul,
Korea; 2NeuroVascular Coordination Research Center, College of Pharmacy and
Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul,
Korea
1
Purpose: To investigate barrier characteristics of zebrafish retinal vessels.
Materials and Methods: Tg(fli1:EGFP) zebrafish, which expresses enhanced green
fluorescent protein (EGFP) in all blood vessels throughout embryogenesis, were
sacrificed at 5 and 7 days post-fertilization (dpf). Immunofluorescence staining of
GFAP (astrocyte marker), NG2 (pericyte marker), and tight junction proteins (ZO-1,
Occludin, and Claudin-5) was done for evaluation of barrier characteristics of retinal
vessels. Vascular permeability was assessed in the retina and brain after injection
of FITC-dextran (2000kDa), Rhodamin-dextran (10kDa), and DAPI (350Da) tracers.
Results: With staining of GFAP and NG2, only NG2 was found to be co-localized with
EGFP- expressing endothelial cells. ZO-1, Occludin, and Claudin-5 were all expressed
in the zebrafish retina, but only Claudin-5 was expressed in the EGFP-expressing
endothelial cells. In the assessment of vascular permeability with FITC-dextran,
Rhodamin-dextran, and DAPI, retinal vessels were found to be permeable to all
tracers, while brain vessels were impermeable to even DAPI.
Conclusion: Our results show that the zebrafish retinal vessel at 5 to 7 dpf was found
to be lack of barrier characteristics, different from the brain vessel.
CR: E. Yang, None; J.H. Kim, None; K.-W. Kim, None; Y.S. Yu, None; J.H. Kim, None.
Support: None
Purpose:During development, smooth muscle cells (SMCs) and pericytes are thought
to play an important role in regulating endothelial proliferation, vascular remodelling,
vessel stabilisation and synthesis of the extracellular matrix. We sought to examine
normal mural cell differentiation and its relationship to vascular formation and related
our findings to the different ability of the retina and choroid to autoregulate.
Methods:Mural cells were investigated following triple-label immunohistochemistry
applied to human retinal and choroidal wholemounts and histological cross sections, aged
8 to 40 weeks gestation (WG). Antibodies to αSMA, Desmin, NG2, Calponin, Caldesmon,
CD34 and CD39 were used to visualise the relationship between smooth muscle cells,
pericytes and the forming vasculature. Transmission Electron Microscopy (TEM) was
also undertaken.
Results:SMA+ mural precursor cells were found scattered and isolated over the primordial
vascular tree at 12WG. αSMA+ smooth muscle cells were restricted to major arteries and
veins. At 16WG, SMA aggregated into concentric filaments, forming a continuous sheath
around large choroidal arteries, whilst in the veins there were gaps in SMA expression.
At 18WG the choriocapillaris had an extensive CD34+ vascular bed, but only a small
portion of these vessels were ensheathed by SMA+ and NG2+ mural cells. Calponin and
Caldesmon (calcium regulating proteins) were expressed only on large arteries, not the
veins. Pericyte ensheathment of human adult capillaries as sampled with TEM was 11%
in the choroid versus 94.5% in the retina. Desmin is seen on ultrastructural examination
as an intermediate filament. In adult choroidal pericytes no intermediate filaments
were observed. Remarkably, pericytes, as indicated by Desmin and NG2 labeling and
visualisation of intermediate filaments with TEM, were absent on human choroidal
capillaries.
Conclusions:We provide evidence in support of the role of CD44+ stem cells in the
formation of mural cells of the human choroidal vasculature. Vast amounts of nonvascular associated SMA filaments were observed throughout the stroma, suggestive
of a possible role in rapid changes in choroidal volume in emmetroprisation. Moreover,
we have provided evidence of a marked difference in pericyte ensheathment between
choroidal and retinal vessels, underlying the marked difference in vessel stability and
autoregulatory ability reported between the two vascular plexii.
CR: T. Chan-Ling, None; M. Koina, None; J.R. McColm, None; J. Dahlstrom, None; E.
Bean, None; L. Baxter, None.
Support: NHMRC Project Grants & Fellowship (#464859, #57100), Baxter Charitable
Foundation, Macular Vision Loss Support Society, Rebecca L. Cooper Medical Research
Foundation
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
41-44
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
45 - A92
Lack of Doxycycline Angioinhibitory Activity in Retinal and Choroidal
Neovascularization
46 - A93
Effects of Triamcinolone Acetonide on Vessels of the Posterior Segment of the
Eye
N. Sheibani, S. Wang, S. Park, E.A. Scheef, C.M. Sorenson. Ophthalmology and Visual
Sciences, Univ of Wisconsin-Madison, Madison, WI.
F. Valamanesh1A,2, M. Berdugo1, F. Sennlaub1B, M. Savoldelli3, C. Goumeaux1A, M.
Houssier1A, J.-C. Jeanny1A, A. Torriglia1A, F. Behar-Cohen1A,3. APhysiopatho of Ocular
Diseases, BCentre de Recherche les Cordeliers, 1INSERM UMRS 872, Paris, France;
2
Centre de Recherche les Cordeliers, Rothschild Ophthalmic Foundation, Paris,
France; 3Hôtel-Dieu Hospital, Paris, France.
Purpose: Doxycycline-mediated gene regulation has been extensively utilized for
evaluation of various gene functions in a tissue specific manner. The purpose of
the current study was to determine the effect of doxycycline administration on
postnatal retinal vascular development and neovascularization, as well as choroidal
neovascularization (CNV) in vivo and retinal endothelial cell (EC) function in vitro.
Methods: The potential antiangiogenic activity of doxycycline was evaluated
during postnatal development of mouse retinal vasculature using established
immunohistological methods. The impact of doxycycline on retinal neovascularization
was assessed during oxygen-induced ischemic retinopathy (OIR). We also evaluated
the activity of doxycycline in the mouse laser-induced CNV model and ex vivo aortic
sprouting assay. In addition, we determined the effects of doxycycline on mouse
retinal EC function in culture using various cell biological and biochemical methods.
Results: We show that administration of doxycycline minimally impacted normal
postnatal retinal vascularization and retinal neovascularization during OIR. We
observed no significant effect on CNV or aortic sprouting following administration of
doxycycline. Doxycycline also had minimal effect on retinal EC proliferation, apoptosis,
capillary morphogenesis, and permeability. These observations were consistent with
minimal effect of doxycycline on expression of cell adhesion molecules PECAM-1 and
VE-cadherin on the surface of retinal EC and their junctional localization.
Conclusions: The impact of doxycycline administration on retinal vascular
development and neovascularization in vivo, and retinal EC function in vitro, was
minimal. Doxycycline also showed no significant effect on CNV or aortic sprouting.
CR: N. Sheibani, None; S. Wang, None; S. Park, None; E.A. Scheef, None; C.M.
Sorenson, None.
Support: NIH grants EY016995, EY018179, and P30 EY016665; Retina Research
Foundation
47 - A94
Time-Lapse Imaging of Retinal Angiogenesis Demonstrates That Anecortave
Desacetate Attenuates Both the Development and Progression of Neovascular
Sprouting
Purpose:To investigates the effects of Triamcinolone Acetonide (TA) on bovine retinal
endothelial cells (BRECs) in vitro and explores the potential vascular toxic effect of TA
injected into the vitreous cavity of rats.
Methods: Subconfluent BRECs were treated with either 0.1 or 1 mg/mL TA in 1% ethanol.
Cell viability was evaluated at 24 h,72 h and 5D using MTT and lactate dehydrogenase
(LDH) assays. Cell proliferation was evaluated by BrdU test. Apoptosis was evaluated
by TUNEL assay, annexin-binding and caspase 3 activation. Caspase-independent cell
deaths were investigated by immunohistochemistry using antibodies against apoptosis
inducing factor (AIF), Microtubule-associated protein-light chain 3 (MAP-LC3) and
LEI/L-DNase II. In vivo, semi-thin and ultrathin structure analysis and vascular casts
were performed to examine TA induced changes of the choroidal vasculature. In addition,
outer segments phagocytosis assay on primary RPE cells was performed to assess COX-2
and VEGF mRNAs up-regulations with or without TA.
Results: The inhibitory effect of TA on cell proliferation could not explain the significant
reduction in cell viability. Indeed, TA induces a time-dependent reduction of BRECs
viability. Annexin-binding positive cells were observed. L-DNase II was found
translocated to the nucleus, meaning that LEI was changed into L-DNase II. AIF was
found nuclearized in some cells. Positive LC3-labelled cells were not observed. No
autophagy or caspase dependent apoptosis was identified. Thereof, while 1mg/mL TA
induces mostly necrosis, exposure to lower concentration for 3 to 5 D induces caspase
independent apoptosis involving AIF and LEI/L-DNase II. Semi-thin and ultrathin
structure analysis and vascular casts revealed that TA mostly affected the choroidal
vasculature with a reduction of choroidal thickness and increased the avascular areas
of the choriocapillaries. Experiments performed on primary RPE cells showed that TA
down-regulates the basal expression of COX-2 and VEGF and inhibits the OS-dependent
COX-2 induction but not the OS-dependant VEGF induction.
Conclusions: This study demonstrates that TA exert direct toxic effect on BRECs through
caspase-independent cell death mechanisms. The choroidal changes observed after TA
intravitreous injection may have important implications regarding the safety profile of
TA use in human eyes.
CR: F. Valamanesh, None; M. Berdugo, None; F. Sennlaub, None; M. Savoldelli, None; C.
Goumeaux, None; M. Houssier, None; J.-C. Jeanny, None; A. Torriglia, None; F. BeharCohen, None.
Support: None
48 - A95
Influence of Netrin-4 and Its Receptors on the Neovascularization in the Mouse
Retina
M. Nukada1, N. Unoki2, K. Ogino2, T. Murakami2, N. Yoshimura2. 1University of Kyoto,
54 kawaramachi shogoin sakyoku kyoto city, Japan; 2University of Kyoto, 54
Kawaramachi Shogoin Sakyoku Kyoto City, Japan.
Purpose: Neovascularization is a dynamic phenomenon, whereas the kinetics of retinal
angiogenesis remains ill-defined. Here we evaluated the dynamics of neovascular
sprouting and its molecular mechanisms in the retinal angiogenesis treated with an
angiostatic steroid, anecortave desacetate (AD).
Methods: Retinas were isolated from 7- to 8- week old C57BL6/J mice and cultured for 96
hours in the presence or absence of AD, followed with each additional experiment. For
in vivo experiments, AD was injected intraperitoneally into C57BL6/J mice on postnatal
day 2 (P2), and the retinas were isolated on P4. After the fixation and permeation,
immunohistochemistry was performed with fluorescent 2nd antibodies. Real time
PCR was performed for the measurements of mRNA levels. Time-sequential images
were obtained at every 15 minute using confocal microscopy, and the movements of
neovascular sprouts were quantified
Results: AD treatment significantly reduced the number of neovascular sprouts in
retinal explants in a dose dependent manner. Time-lapse imaging demonstrated that
AD suppressed both the development and the elongation of sprouts, and the motility of
the leading edges in tip cells. AD treatment further disturbed the filopodial extension
and significantly decreased the transcriptional levels of KDR and platelet-derived
growth factor-B (PDGFB), which are highly expressed in tip cells, suggesting the
tip cell dysfunction. The transcriptional level of stromal derived factor-1 (SDF-1),
which regulates tip cell motility, was also reduced by AD, whereas AD did not alter
the expression of its receptor, CXCR4. Additionally, immunostaining and electron
microscopy demonstrated the immature basement membrane in neovascular
sprouts treated with AD. Further in vivo experiments showed that AD inhibited the
neovascularization and filopodial extension in tip cells in retinal vascular development.
These data suggest that AD suppresses both development and progression of sprouting
angiogenesis.
Conclusions:AD attenuated VEGF-induced retinal angiogenesis, mediated via the
suppression of the development and progression of neovascular sprouts and tip cell
motility at least partially.
CR: M. Nukada, None; N. Unoki, None; K. Ogino, None; T. Murakami, None; N.
Yoshimura, None.
Support: Alcone
S.V. Klein1, Y. Liang2, E. Abari1, K. Schmidt1, I. Semkova1, K.L. Meyer3, W.J. Brunken4,
N. Kociok1, M. Koch 5, A.M. Joussen1. 1Ophthalmology, Heinrich-Heine-University
Duesseldorf, Duesseldorf, Germany; 2Ophthalmology, Peking University, Peking,
China; 3Functional Genomics of Microorganisms, Heinrich-Heine-University
Düsseldorf, Duesseldorf, Germany; 4Department of Cell Biology, SUNY Downstate
Medical Center, New York, NY; 5Institute for Biochemistry II, University of
Cologne, Cologne, Germany.
Purpose: Vascular diseases of the retina are the most common reasons for blindness.
The pathological proliferation of vessels is mostly accompanied by degeneration of the
neuronal tissue. Netrins are path finding proteins, known to play a role in establishment
of the nervous system and in normal and pathological neoangiogenesis. Especially
macrophages are involved in the development of neovascularisations and neutrophil
granulocytes play a main role in the regulation of VEGF expression in endothelial
cells. The aim of this study was to examine if and how frequently netrin-4 and its
receptors are expressed on those cells.
Methods: Red blood cells were lysed from C57/Bl6 whole blood and leukocytes were
marked with cell specific antibodies. To determine receptors, cells were additionally
stained with antibodies against netrin receptors and analysed using flow cytometry.
Leukocytes were separated to obtain homogeneous populations of neutrophil
granulocytes and monocytes. Activated peritoneal macrophages were obtained 3
days after intraperitoneal injection of 0.1ml thioglycolate.Total RNA was isolated from
these cells and Real Time RT-PCR was carried out, verified and quantified.
Results: FACS Analysis showed that leukocytes were positive for netrin-4 and its six
receptors DCC, Neogenin and Unc5H1 to 4. The corresponding Real Time RT-PCR
confirmed these results and showed that the expression profiles of the tested netrin
receptors differ between the cell types.
Conclusions: The combination of Real Time RT-PCR and flow cytometry allows
analysing the receptors on RNA and protein level. The fact that netrin-4 and its
receptors are expressed in the majority of leukocytes suggests its importance as
path finding molecule. Furthermore the different expression profiles of the netrin-4
receptors on the particular cell types indicate the importance of netrin-4’s functions.
CR: S.V. Klein, None; Y. Liang, None; E. Abari, None; K. Schmidt, None; I. Semkova,
None; K.L. Meyer, None; W.J. Brunken, None; N. Kociok, None; M. Koch, None; A.M.
Joussen, None.
Support: DFG - SFB612
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
45-48
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
49 - A96
Regulatory Effect of Vascular Endothelial Cell PD-L1 on Angiogenesis
50 - A97
Stromal Cell-Derived Factor-1 (SDF-1)/CXCR4 Promotes the Activation of Tip
Cells and Microglia in Retinal Angiogenesis
Y. Jin1, S.K. Chauhan1, P. Sage2, A. Sharpe2, R. Dana1. 1Schepens Eye Research, Boston,
MA; 2Department of Pathology, Harvard Medical School, Boston, MA.
Purpose: To investigate the role of program death ligand-1 (PD-L1) expression on
vascular endothelial cell (VEC) proliferation and corneal angiogenesis.
Methods: Expression levels of PD-L1, PD-1 and CD80 (B7.1) in MS1 cells (mouse VEC
cell line) and primary VEC from murine lung and heart were analyzed using real-time
PCR and flow cytometry. PD-L1 and CD80 expression levels in the MS1 cells were
inhibited using respective siRNA, and the cell proliferation was then analyzed by
BrdU incorporation assay. Neovascularization in the PD-L1 knockout (KO) mice and
wild-type (WT) mice were scored during 2 weeks after suture-placement in the cornea
using slit-lamp biomicroscopy, and the percentage of CD31hiLYVE-1- blood vessels
in the corneas were measured on Day 14 using confocal microscopy. The expression
levels of VEGF-A and VEGFR2 on the MS1 cells or the suture-placed corneas were
analyzed by real-time PCR.
Results: Both primary murine VEC and MS1 cells expressed PD-L1 and CD80, but
not PD-1, at mRNA and protein levels. After inhibition of PD-L1 or CD80 expression
in the MS1 cells by siRNA transfection, the mRNA expression level of VEGFR2 and
the cell proliferation level were significantly enhanced, compared to the control
group (p<0.01, t-test). After suture placement in the cornea, the expression level of
VEGFR2 was significantly higher in the PD-L1 KO mice vs. WT mice (p<0.05, t-test).
The CD31hiLYVE-1- blood vessel density was significantly promoted in the PDL-1KO
group relative to the WT group (22+2% vs.11+1%, n=6 per group, p<0.001, t-test).
Conclusions: The expression of PD-L1 and its receptor CD80 on VEC has an inhibitory
function on VEC proliferation and angiogenesis.
CR: Y. Jin, None; S.K. Chauhan, None; P. Sage, None; A. Sharpe, None; R. Dana, None.
Support: NEI-12963
51 - A98
Girdin Phosphorylation at Serine-1416 by Akt/PKB Promotes Postneonatal
Angiogenesis and Ischemic Neovascularization in the Retina
N. Unoki1, T. Murakami2, K. Nishijima3, N. Yoshimura4. 1Ophthalmology, Kyoto Univ
Grad Sch Med, Kyoto, Japan; 2Ophtahlmology, Kyoto University Graduate School of
Medicine, Kyoto, Japan; 3Department of Ophthalmology, Kyoto Univ Grad School of
Med, Kyoto, Japan; 4Ophthalmology, Kyoto University, Sakyo-ku, Japan.
Purpose: Although stromal cell-derived factor-1 (SDF-1) contributes to angiogenesis,
its effects upon sprouting angiogenesis remain ill-defined. We investigated how SDF-1
and its receptor, CXCR4, regulate the tip cells and microglia in neovascular sprouting.
Methods: The retinas isolated from 7- to 8- week old C57BL6/J mice were cultured
for 96 hours with or without inhibitors, and applied to each experiment. For in vivo
experiments, inhibitors were intraperitoneally administrated into C57BL6/J mice on
postnatal day 2 (P2), and the eyes were isolated on P4. After the fixation and permeation,
immunostaining with fluorescent 2 nd antibody were performed. To quantify the
transcriptional levels, mRNA was applied to reverse transcription and following
real time PCR. Time-sequential images were obtained at every 15 minute intervals,
using confocal microscopy. The movements of tip cells and microglia were quantified.
Results: Neutralizing antibodies against SDF-1 or an antagonist of CXCR4, AMD3100,
decreased the radius of the vascularized area in retinal vascular development. These
inhibitions disturbed the filopodial extensions in tip cells and decreased the mRNA
levels of KDR/Flk-1, UNC5B, and PDGFB, which are highly expressed in tip cells. In
ex vivo experiments, VEGF increased SDF-1 mRNA expression, and the inhibition
of SDF-1/CXCR4 decreased the number of VEGF-induced neovascular sprouts. We
investigated the kinetics of sprouts using time-lapse imaging, and found that SDF-1/
CXCR4 contributes to the elongation of neovascular sprouts and also to the motility
of leading edges in tip cells. These data deomonstrate that SDF-1/CXCR4 promotes
the sprouting angiogenesis per se. In addition, the number of microglia in in vivo
neonatal retinas was reduced by SDF-1/CXCR4 inhibitons. The movements of resident
microglia by VEGF treatment were also reduced by these inhibitions, suggesting this
axis contributes to the activation of microglia partially.
Conclusions: These data suggests that SDF-1/CXCR4 promotes sprouting angiogenesis
in retinal neovascularization, mediated via the activation of both tip cells and microglia
at least partially.
CR: N. Unoki, None; T. Murakami, None; K. Nishijima, None; N. Yoshimura, None.
Support: a Grant-in-Aid for Scientific Research from the Ministry of Education, Science
and Culture of the Japanese Government and Ono Pharmaceutical Co.
52 - A99
Tetraiodothyroacetic Acid (tetrac) Inhibits Pathological Retinal Angiogenesis
T. Yoshida, J. Gong, Z. Xu, E.J. Duh. Wilmer Eye Inst, Johns Hopkins Univ Sch of
Med, Baltimore, MD.
T. ITO1A, K. Komeima1A, T. Yasuma1A, A. Enomoto1B, N. Asai1B, M. Takahashi1B, H.
Terasaki1A. ADepartment of Ophthalmology, BDepartment of Pathology, 1Nagoya
University Graduate School of Medicine, Nagoya, Japan.
Purpose: The serine/threonine kinase Akt/PKB substrate Girdin regulates vascular
endothelial growth factor (VEGF)-mediated angiogenesis (Kitamura T et al. (2008) Nat
Cell Biol.). In this study, we sought to determine if Girdin phosphorylation of serine at
position 1416 by Akt/PKB is associated with postneonatal angiogenesis or pathological
neovascularization induced by ischemia in the retina.
Methods: To test the role of Girdin phosphorylation at serine-1416 in vivo, we used
knockin mice in which serine-1416 is mutated to alanine. Postneonatal retinal
angiogenesis in knockin mice or wild-type (WT) mice was evaluated at postnatal day
7 (P7) and P10 using confocal microscopy of whole-mounted retinas. Ischemic retinal
neovascularization was induced using well-described mouse model of oxygen-induced
retinopathy (OIR). Briefly, P7 mice and their nursing dam were exposed to hyperoxia
(80% O2) for 5 days and returned to room air at P12. Retinal neovascularization of
knockin mice or WT mice was quantified at P17 using confocal microscopy of retinal
flatmounts.
Results: Postneonatal vascular development in the retina of knockin mice was
significantly slower than that of WT mice. The ratios of vascular area to whole retinal
area in knockin mice and WT mice at P7 were 66.7% and 88.4% respectively (p<1.0x10 -6),
and those at P10 were 97.7% and 99.8% respectively (p<1.0x10 -6). Pathological retinal
neovascularization by OIR in knockin mice was significantly reduced by 62.6%
comparing to WT mice (p<1.0x10 -16).
Conclusions: These data demonstrate that phosphorylation of serine at position
1416 in Girdin by Akt/PkB promote postneonatal angiogenesis and pathological
neovascularization in the retina, and that inhibition of Girdin phosphorylation by
Akt/PKB may reduce pathological retinal neovascularization in patients with ischemic
retinal diseases such as retinal vein occlusion, retinopathy of prematurity and diabetic
retinopathy.
CR: T. Ito, None; K. Komeima, None; T. Yasuma, None; A. Enomoto, None; N. Asai,
None; M. Takahashi, None; H. Terasaki, None.
Support: None
Purpose:Tetrac (tetraiodothyroacetic acid) is a deaminated analogue of L-thyroxine
(T4)) that blocks the pro-angiogenesis actions of (T4) and 3, 5, 3’-triiodo-L-thyronine
as well as VEGF and FGF-2 by a mechanism involving integrin alphaVbeta3. We
investigated the potential anti-angiogenic activity of tetrac on retinal angiogenesis
in vitro and vivo.
Methods:Human retinal endothelial cells (HREC) were treated with or without tetrac
and stimulatory factors, and ERK1/2 phosphorylation assessed by western blotting.
The oxygen-induced retinopathy model (OIR) was used, in which newborn mice are
exposed to hyperoxia from postnatal day 7 (P7) to P12 and then returned to room
air. Tetrac was administered by intravitreal injection at P12 and P15, and vehicle was
used as control. Retinal neovascularization was assessed at P18.
Results:Tetrac significantly inhibited the activation of ERK1/2by both VEGF and T4
treatmentin HREC. In the mouse OIR model, intravitreal injection of tetrac significantly
reduced pathological retinal neovascularization at P18 as compared to vehicle. Tetrac
did not have a significant effect on avascular retinal area.
Conclusions:Our study demonstrates the antiangiogenic effects of Tetrac in retinal
neovascularization in vitro and in vivo. Tetrac could be used as a therapeutic agent
for retinal neovascularization.
CR: T. Yoshida, None; J. Gong, None; Z. Xu, None; E.J. Duh, None.
Support: NIH 1R01EY018138, Research to Prevent Blindness, CLF-MTAP
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
49-52
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
53 - A100
Tissue Factor Regulates Fibroblast Growth Factor-2-Induced Angiogenesis in
Retinoblastoma
54 - A101
Development of Capillary Network Despite Persistent Hyperoxia in the Mouse
Retina
M.S. Jeong1, J.H. Kim2, K.-W. Kim 3, Y.S. Yu2, J.H. Kim2. 1Opthalomology, Seoul
National University hospital, Seoul, Republic of Korea; 2Opthalomology, Seoul
National University Hospital, Seoul, Republic of Korea; 3College of Pharmacy and
Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul,
Republic of Korea.
Y. Feng, S. Gross, H.-P. Hammes. 5th Medical Department, Medical Faculty
Mannheim, University of Heidelberg, Mannheim, Germany.
Purpose: To investigate whether tissue factor (TF) regulates fibroblast growth factor
(FGF)-2-induced angiogenesis in retinoblastoma.
Methods: In an experimental model of retinoblastoma, immunofluorescence staining
for TF and CD31 as an endothelial cell maker was performed. With treatment of FGF2 (10 ng/ml), TF expression in human umbilical vein endothelial cells (HUVECs)
was measured by Western blotting. To confirm the role of TF in tumor angiogenesis
in retinoblastoma, anti-angiogenic activity of TF pathway inhibitor (TFPI) was
evaluated by FGF-2-induced proliferation, migration and in vitro tube formation
assay of HUVECs. In addition, inhibition of ERK-1/2 phosphorylation by TFPI was
measured by Western blot analysis.
Results: TF was highly expressed on vascular endothelial cells of retinoblastoma,
co-localized with CD31. With FGF-2-induced proliferation of HUVECs, TF expression
was significantly up-regulated. Interestingly, TFPI effectively blocked FGF-2induced proliferation, migration and in vitro tube formation of HUVECs, which was
accompanied by inhibition of ERK-1/2 phosphorylation.
Conclusions: Our results suggest that TF on tumor vessels of retinoblastoma could
be involved in regulation of FGF-2 induced angiogenesis, which was mediated by
ERK pathway.
CR: M.S. Jeong, None; J.H. Kim, None; K.-W. Kim, None; Y.S. Yu, None; J.H. Kim,
None.
Support: None
55 - A102
The Effects of Amphotericin B and Voriconazole on in vivo Angiogenesis in
Chick Chorioallantoic Membrane
Purpose: Oxygen regulates vessel growth in the retina both in physiological and
pathological angiogenesis. Hyperoxia and hypoxia contribute to retinal vasoregression
and neovascularisation, respectively. However, we occasionally observed an outgrowth
of deep capillary layer during hyperoxia in a mouse model of oxygen-induced
retinopathy (OIR). The aim of the current study was to investigate this escape effect
by using long-term hyperoxia and its outcome on intraretinal angiogenesis in the
developing mouse retina.
Methods: Mice in the conventional OIR model at p17 (5 days of hyperoxia plus 5 days
of hypoxia) and in the modified OIR mouse model at p17 (10 days of hyperoxia) were
used for the study. PAS staining on paraffin sections was performed to determine the
location of neovascularization in prolonged hyperoxia. Outgrowth of deep capillary
layers, avascular zone and neovascular tufts were quantitated in whole mount retinas
stained with collagen IV.
Results: Mice in the modified OIR model with prolonged hyperoxia did not show
any preretinal neovascularization, whereas mice in the conventional OIR model
demonstrated numerous preretinal neovascular tufts at p17. In retinal whole mounts,
retinas of the modified OIR model showed substantial outgrowth of capillaries in the
deep layers despite of persistent hyperoxia and similar avascular zones compared with
the conventional OIR model. Moreover, neovascular tufts in the modified OIR model
were located at the border of avascular zones in the retina, while neovascularizations in
the conventional OIR model were predominantly preretinal at the border of avascular
zone. The capillaries in the modified OIR model were more regularly formed than
those in the conventional OIR model.
Conclusion: Our study showed the outgrowth of the capillary network and intraretinal neovascularization during persistent hyperoxia in a modified OIR model. The
data may indicate a new concept of retinal angiogenesis under VEGF depression. The
underlying mechanisms need to be further investigated.
CR: Y. Feng, None; S. Gross, None; H.-P. Hammes, None.
Support: None
56 - A103
810 nm Micropulse Laser Irradiation Selectively Regulates VEGF 165 Isoforms
Expression Acting on RNA Binding Splice Factor Activation in Indocyanine
Green Loaded ARPE19 and Caco2 Cultured Cells
G. Gokce1A, T. Ozgurtas1B, G. Sobaci1A. AOphthalmology, BBiochemistry, 1Gulhane
Military Medical Academy, Ankara, Turkey.
Purpose: To evaluate the in-vivo anti-angiogenic effect of Amphotericin B and
Voriconazole which are commonly used in the treatment of fungal endophthalmitis
either systemically or intravitreally in chick chorioallantoic membrane (CAM) model.
Methods: Atak-S type fertilized eggs obtained from Poultry Institution (Ankara,
TURKIYE) were used. The eggs were kept under 37o C at 85-90 % relative humidity
until fifth day when the application would be performed. Amphotericin B and
Voriconazole were applied to eggs at different concentrations (Amphotericin B 125ng,
125μg; Voriconazole 100ng, 100μg). Thirteen eggs were used for each group. The eggs
were kept under appropriate condition throughout the experiment. The results were
evaluated at the 48th hour of application of the drugs and recorded by digital camera
(Konica Minolta, Tokyo, JAPAN).
Results: No anti-angiogenic effect was observed in the eggs treated with all
concentrations of voriconazole and all the eggs treated with 125 ng Amphotericin B.
A significant anti-angiogenic effect was determined in 125 μg Amphotericin B group
(11 of 13 eggs, 84.6 %).
Conclusions: Anti-angiogenic effect seen in therapeutic doses of Amphotericin B
in this model may indicate potential use of this drug as an anti-angiogenic agent.
CR: G. Gokce, None; T. Ozgurtas, None; G. Sobaci, None.
Support: None
F.U. Ricci1A, P. Mazzarelli1B, M.J. Zonetti1B, F. Missiroli, Jr.1A, M. Cesareo, Sr.1A, S. Pucci1B.
A
Ophthalmology, BBiopathology, 1University of Rome Tor Vergata, Rome, Italy.
Purpose: In order to characterize the biological effects and molecular mechanism
underlying indocyanine green (ICG)-mediated photodynamic therapy , ICG loaded
cultured retinal pigmented epithelium cells (ARPE19) and colon cancer epithelial cells
(Caco2) were exposed to 810-nm laser radiation. Cell viability and death induction
were examined, as well as the modulation of proteins involved in cell death and DNA
repair and the effect on the expression of VEGF165 antagonistic isoforms .
Methods: ICG preloaded ARPE19 cells and Caco2 cells were exposed to micropulse 810
nm laser radiation. VEGF165a and b isoforms, HIF1α and the shift of the antagonistic
isoforms of Clusterin (nCLU and sCLU) expression were evaluated. Bax release from
Ku70 protein was also investigated. In addition,Caco2 cells were underskin injected
in nude mice. ICG-preloaded mice were laser irradiated and molecular mechanisms
inducing the specific VEGF165a inhibition and the preferential VEGF165b expression,
growth arrest and apoptosis induction were studied.
Results: A selective induction of VEGF165b isoform was found in irradiated ARPE19
and Caco2 cells, as compared to un-irradiated controls. In colon cancer xenografts a
strong induction of the anti-proliferative and anti-angiogenic VEGF 165b isoform was
observed, as compared to untreated tumors. The VEGF165b expression correlated to
an increased RNA binding splice factor activation.
Conclusions: VEGF is a key component involved in physiological and pathological
angiogenesis. Inhibition of VEGF has been shown to be effective in cancer
and eye diseases. Two different splicing isoforms of VEGFA165 (a and b) have
been recently identified, exerting pro- or anti-angiogenic actions and whose
formation is strictly controlled by micro-environmental factors. The production
of these forms of VEGF depends upon splice site selection of RNA, controlled
by binding splice factors previously shown to regulate VEGF splice site choice.
Our data show that ICG mediated photodynamic therapy could stimulate
the selective and specific expression of antiangiogenic isoform of VEGF,
influencing its splice variant formation (VEGF165b) in cultured ARPE19
and Caco2 cells as well as in experimental models of colon cancer.
Uncovering the molecular mechanisms involved in the differential expression of the
2 VEGF isoforms may yield novel therapeutic strategy in the therapy of neovascular
eye diseases and cancer.
CR: F.U. Ricci, None; P. Mazzarelli, None; M.J. Zonetti, None; F. Missiroli, Jr.,
None; M. Cesareo, Sr., None; S. Pucci, None.
Support: Iridex Corp. Free Research Grant
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
53-56
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
57 - A104
Hypoxia-Induecd Insulin-Like Growth Factor-2 Regulates Expression of
Vascular Endothelial Growth Factor in Retinal Endothelial Cells
58 - A105
17β Estradiol-Induced Proliferation of Monkey Retinal Endothelial Cells is
Mediated by PEDF and Modulated by Tamoxifen and Raloxifene
S. Park1,2, J. Kim1,2, K.-W. Kim 3, Y. Yu1,2, J. Kim1,2. 1Fight against Angiogenesis-Related
Blindness (FARB) Laboratory, Department of Ophthalmology, College of Medicine,
Seoul National University, Seoul, Republic of Korea; 2Seoul Artificial Eye Center
Clinical Research Institute, Seoul National University Hospital, Seoul, Republic of
Korea; 3NeuroVascular Coordination Research Center, College of Pharmacy and
Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul,
Republic of Korea.
K. Parvathaneni1, J.G. Grigsby1, D.A. Allen2, E. Kotchan Vidro1, S. Kalhor1, B. Yendluri1,
A.T.C. Tsin1. 1Biology, University of Texas at San Antonio, San Antonio, TX; 2Biology,
The University of Texas Permian Basin, Odessa, TX.
Purpose: To investigate the role of insulin-like growth factor (IGF)-2 in vascular
endothelial growth factor (VEGF) expression in retinal endothelial cells under hypoxia.
Methods: In human retinal microvascular endothelial cells, IGF-2 and VEGF expression
were measured by reverse transcriptase-polymerase chain reaction (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-2 (1 to 1000 ng/ml)
In addition, VEGF expression in HRMEC was evaluated under hypoxia with treatment
of a blocking antibody to IGF-2 by RT-PCR and Western blot analysis.
Results: Under hypoxic condition, IGF-2 and VEGF were up-regulated in HRMEC.
VEGF expression increased in dose-dependent manner with treatment of IGF-2, which
was accompanied by the significant increase of ERK as well as Akt phosphorylation.
Moreover, hypoxia-induced VEGF expression was effectively inhibited by blocking
of IGF-2.
Conclusions: Our results suggest that hypoxia-induced IGF-2 could directly regulate
VEGF expression in HRMEC via ERK as well as PI3K/Akt pathway. Therefore, IGF-2
might be involved in regulation of developing retinal vessels which is mediated by
hypoxia-induced VEGF expression.
CR: S. Park, None; J. Kim, None; K.-W. Kim, None; Y. Yu, None; J. Kim, None.
Support: None
Purpose: Proliferative diabetic retinopathy (PDR) results from the proliferation of
retinal capillary endothelial cells. In many tissues, estrogen induced proliferation of
cells is well documented. We hypothesized that 17β estradiol (E2) induces proliferation
in retinal capillary endothelial cells and that this estrogen effect is mediated by PEDF.
Methods: Monkey retinal capillary endothelial cells (mREC) in culture were exposed
to 1nM E2 for 1, 3, 5 and 7 days to study the effect of E2 on the change in the total
number of mREC in culture. mREC were also exposed to 1 nM E2, 1 nM E2 + 1nM
tamoxifen (TM) and 1 nM E2 + 1nM raloxifene (RA) for 6 days. The numbers of viable
mREC were counted using a hemocytometer and trypan blue method. Conditioned
media were collected and assayed for PEDF levels by ELISA.
Results: The total number of mREC in culture increased from 17k (day 0) to 70k (day
7) in a control media of MEMα-1X with 10% charcoal-stripped FBS. When exposed
to E2, the number of mREC in culture increased at all sampling points (90k cells on
day 7). In a subsequent experiment, E2 treatment of mREC increased the viable cell
number from 130k (control) to 260k in 6d. However, the addition of TM and RA reduced
cell proliferation counts to 120k and 133k, respectively. E2 treatment also reduced the
level of PEDF in the conditioned media from 0.2 pg/10k viable cells in control media
to 0.1 pg/10k viable cells. The addition of TM and RA returned the PEDF level in the
conditioned media to 0.2 pg/10k viable cells.
Conclusions: E2 (1 nM) induced a significant increase in the number of mREC in
culture, suggesting a possible role of E2 in the development of PDR. Selective estrogen
receptor modulators TM and RA were effective modulators of this estrogen effect on
mREC proliferation. E2 also induced a significant decrease in PEDF in the conditioned
media and this effect of estrogen was modulated by TM and RA. Since PEDF is
known to inhibit the proliferation of endothelial cells, the estrogen-induced mREC
proliferation we observed may well be mediated by PEDF.
CR: K. Parvathaneni, None; J.G. Grigsby, None; D.A. Allen, None; E. Kotchan Vidro,
None; S. Kalhor, None; B. Yendluri, None; A.T.C. Tsin, None.
Support: The University of Texas at San Antonio Center for Research and Training in
the Sciences (CRTS), The UTSA MBRS/MARC/RISE Program
59 - A106
Bim-Deficient Mice Exhibit Reduced Retinal Vaso-Obliteration and Subsequent
Neovascularization in the Mouse Model of Oxygen-Induced Retinopathy
60 - A107
Acharan Sulfate: A Heparin-Like Compound With Anti-Angiogenic Effect. A
Potential Drug to Treat Eye Neovascular Diseases
M.R. Powers1A, M.H. Davies1B, A.J. Stempel1A. APediatrics & Ophthalmology,
B
Pediatrics and Ophthalmology, 1Casey Eye Institute-OHSU, Portland, OR.
J.L. Dreyfuss1A, T.F. Gesteira1A, G.L.A. Cunha1A, V.J. Coulson-Thomas1A, C.V. Regatieri1B,
H.B. Nader1A. AMolecular Biology, BOphthalmology, 1Federal University of Sao
Paulo, Sao Paulo, Brazil.
Purpose: Previously our laboratory has demonstrated that the extrinsic apoptotic
pathway plays an important role in retinal neovascularization (NV) during oxygeninduced retinopathy (OIR) via death ligands. We used Bim deficient (-/-) mice in the
model of OIR to determine if this proapoptotic Bcl-2 family member (BH3-only)
contributes to the control of retinal NV via the intrinsic apoptotic pathway.
Methods: Bim expression was evaluated in room air and hyperoxia exposed C57BL/
B6 (B6) retinas by RT-PCR and Western blot. Vascular development was assessed
at postnatal day 5 (P5) and P7 in B6 and Bim-/- mice in lectin stained retinal whole
mounts. B6 and Bim-/- mice were exposed to 75% oxygen from P7 to P12. Retinal
vaso-obliteration was quantified from lectin-labeled retinas collected at P8 and P12.
NV was quantified from H&E stained cross-sections by counting pre-retinal vascular
tuft nuclei from P17 and P21 retinas. Qualitative analysis of retinal vascularization
was performed in lectin-labeled retinal whole mounts in B6 and Bim-/- mice on P17.
Results: Bim expression was confirmed at both the RNA and protein level in room air
and hyperoxia exposed B6 retinas on P8, P12, P17 and P21. There was no significant
difference in retinal vascular development in Bim-/- mice compared to controls. At P8,
after hyperoxia exposure, 0.7±0.05% of the retina was avascular in Bim-/- mice compared
to 39.3±2.6% avascular in B6 mice (p<0.0001; n=6). At P12 following hyperoxia exposure,
9.7±0.4% of the retina was avascular in Bim-/- mice compared to 30.3±1.7% in B6 mice
(p<0.0001; n=5-6). Subsequently, at P17, Bim-/- mice exhibited a significant reduction in
NV (9.2±1.2 neovascular nuclei per section) compared to B6 mice (25.1±1.2; p<0.0001;
n=8-10 eyes). At P21, Bim-/- mice had 5.1±0.4 neovascular nuclei per section compared to
9.8±2.5 in B6 mice (p=0.179; n=6-10 eyes). These findings were qualitatively confirmed
in lectin-labeled retinal whole mounts.
Conclusions: The immature retinal vasculature was dramatically protected from vasoobliteration in Bim-/- mice during hyperoxia exposure. Bim is known to be strongly
induced by growth factor withdrawal. We speculate that Bim contributes to retinal
endothelial cell death and vaso-obliteration as a result of VEGF down-regulation from
hyperoxia. The reduced NV observed during room air recovery in the Bim-/- mice is
likely related to a reduction in retinal ischemia as compared to controls. Modulation of
the intrinsic apoptotic pathway offers unique therapeutic opportunities for retinal NV.
CR: M.R. Powers, None; M.H. Davies, None; A.J. Stempel, None.
Support: NIH Grant EY011548 (MRP) and an unrestricted grant from Research to
Prevent Blindness
Purpose: The heparin-like glycosaminoglycan obtained from the giant African snail
Achatina fulica is capable to modulate inflammatory responses without interfering
on hemostasis. Thus the objective of this study was to evaluate the anti-angiogenic
effects of this new compound on “in vitro” models.
Methods: Acharan sulfate was purified and analyzed by Nuclear Magnetic Ressonance
and electrospray ionization mass spectrometry (ESI-MS). The ability of endothelial
cells form capillary-like structures “in vitro” when plated on top of a reconstituted
basement membrane extracellular matrix (Matrigel) was investigated in cells treated
with acharan sulfate (0.01, 0.1 and 1 mg/mL). The cytotoxicity of this compound was
evaluated by MTT test in retinal pigmented epithelial cells (ARPE-19) and endothelial
cell cultures.
Results: Electrospray ionization mass spectrometry (ESI-MS) analysis was used to
determine the molecular weight of acharan sulfate-derived oligosaccharide. A doubly
charged negative ion at m/z 481.1 was also observed for the tetrasaccharide. 1H NMR
spectra spectra were obtained and the signal at 3.27 ppm indicates the presence of
glucosamine N-sulfated which is an unusual variation of previously purifed acharan
sulfate. Only the 1 mg/mL of acharan sulfate induced a significant decrease (p<0.05)
in total length of tubes formed by endothelial cells in Matrigel. No cytotoxic effect
was detected in ARPE-19 or endothelial cell culture in all tested doses.
Conclusions: The acharan sulfate was resolved and the in vitro studies showed
that the optimal dosage to reduce angiogenesis in vitro was 1 mg/mL and it was no
cytotoxic effect in ARPE-19 cells or endothelial cells. These findings suggest that the
inhibitory effect of acharan sulphate on angiogenesis and the non-cytotoxic effect on
ARPE-19 cells indicates acharan sulfate as a potential compound that can be used in
the future as a therapy to control neovascularization
CR: J.L. Dreyfuss, None; T.F. Gesteira, None; G.L.A. Cunha, None; V.J. CoulsonThomas, None; C.V. Regatieri, None; H.B. Nader, None.
Support: FAPESP, CNPq, CAPES
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
57-60
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
61 - A108
Cell Signaling Alterations During Cell Shape Changes Induced by Protein
Kinase CK2 Inhibition in Cultured Human Vascular Endothelial Cells
62 - A109
The Effect of Acute Insulin Exposure on P38 Kinase Production in Human
Retinal Epithelial Cells
A.A. Kramerov, A.V. Ljubimov. Ophthalmology Research, Cedars-Sinai Medical
Center, Los Angeles, CA.
M.D. Cooke, P. Kothary, M.A. Del Monte. University of Michigan Medical School,
Ann Arbor, MI.
Purpose: Protein kinase CK2 (CK2) is an important regulator of cell migration,
proliferation, and tumor growth. CK2 is abundant in retinal vascular endothelial
cells and astrocytes, and its inhibition blocks retinal neovascularization in a mouse
model. In human cultured astrocytic and vascular endothelial cells, CK2 inhibition
caused dramatic cell shape change leading to cell rounding. The purpose was to study
the effects of CK2 inhibition on activation of some signaling mediators during cell
shape changes in human cultured vascular endothelial cells.
Methods: Cultured human brain microvascular endothelial cells (HBMVEC) were
used. Specific CK2 inhibitor TBB, and p38 activity inhibitor SB 202190 (both from
Calbiochem) were added at 0.02-0.10 mM in medium with 0.5% serum. Western
analysis was used to examine phosphorylated forms of signaling molecules ERK and
p38MAPK after the inhibitor treatment.
Results: After TBB treatment of HBMVEC, an increase in phospho-p38 (p-p38) was
observed, whereas no such effect was found for SB 202190. When the cells were
treated with both TBB and SB 202190, we observed a decrease in p38 phosphorylation
suggesting an important role of autophosphorylation of p38 that may be downregulated by CK2. Surprisingly, SB 202190 (that did not induce cell shape changes)
enhanced the rounding effect caused by suboptimal concentration of TBB on the
cells cultured with serum that suggests p38 may be involved in serum-mediated
cell spreading. We observed also that TBB and SB 202190 each increased pERK1/2
levels, whereas their combined action had an additive effect leading to a higher
phosphorylation of ERK1/2. Thus, there is a correlation between the Western blot and
morphological data on combined action of TBB and SB 202190: p38 inhibitor enhanced
both TBB-induced cell rounding and phosphorylation of ERK1/2.
Conclusions: Our data suggest possible important roles of signaling molecules in
mediating the CK2 inhibitor-induced cell shape changes that may underlie the antiangiogenic effect of CK2 inhibition in vivo, and may allow for the development of
novel anti-angiogenic therapeutic approaches.
CR: A.A. Kramerov, None; A.V. Ljubimov, None.
Support: Eye Defects Research Foundation; OneSight Research Foundation;
Department of Surgery, Cedars-Sinai Medical Center
Purpose: Metaplastic human retinal pigment epithelial (hRPE) cells have been
implicated in the pathogenesis of proliferative eye diseases and proliferative diabetic
retinopathy (PDR). Acute insulin therapy given to diabetic patients has been linked
to a transient increase in PDR by a mechanism involving up-regulation of Vascular
Endothelial Growth Factor (VEGF). Since VEGF causes abnormal proliferation of
vascular endothelial and hRPE cells via a mechanism that may involve regulation by
the P38 mitogen-activated protein kinase (P38), we investigated the effect of insulin
and of SB203580 (SB), an inhibitor of P38 activity, on P38 production in in vitro cultured
hRPE cells.
Methods: Primary hRPE cell cultures were prepared from donor human eyes
obtained from the Michigan Eye Bank. 3H-thymidine incorporation and direct cell
counting by the trypan blue exclusion method (T) were performed to quantitate cell
proliferation. Synthesis of P38 was measured quantitatively by immunoprecipitation of
14
C-methionine labeled P38 and qualitatively by immunocytochemistry. Cell numbers
are expressed as cells/μL. A Student “t” test was used to compare groups of data.
Results: Insulin (0-5 μg/mL) increased hRPE cell proliferation in a dose-dependent
manner. In addition, insulin (0-5 μg/mL) stimulated 14C-methionine-P38 synthesis
in hRPE cells also in a dose-dependent manner. SB inhibited the insulin-stimulated
14
C-methionine-P38 synthesis (774±110 vs. 1039±156, CPM±SEM, n=6, p≤0.05) in hRPE
cells. SB also decreased proliferation as determined by T (42±6 vs. 75±14, n=3, p≤0.05).
Immunocytochemical studies confirmed increased immuno-reactivity of P38 in hRPE
cells exposed to insulin alone compared to the immuno-reactivity in cells exposed
to insulin and SB.
Conclusion: Insulin is a mitogen for hRPE cells. It stimulates P38 production in hRPE
cells. SB, an inhibitor of P38, inhibited insulin-stimulated P38 production. Thus, a P38
inhibitor given concurrently with acute insulin treatment may be effective in reducing
the exacerbation in retinopathy seen in some diabetic patients.
CR: M.D. Cooke, None; P. Kothary, None; M.A. Del Monte, None.
Support: Skillman Foundation, Summer Biomedical Research Program (University
of Michigan Medical School program)
63 - A110
Identification of a γ-Secretase-Catalyzed Transmembrane Cleavage Site for
VEGFR1 in Retinal Vascular Endothelial Cells
64 - A111
Dystrophin 71 a Membrane Cytoskeleton Protein Involved in Corneal
Neovascularization
J. Cai1A, S. Han1B, Q. Ruan1A, L. Wu1A, M.B. Grant1C, M.E. Boulton1A. AAnatomy and
Cell Biology, BSurgery, CPharmacology and Therapeutics, 1University of Florida,
Gainesville, FL.
G. Ortiz1, R. Bernard1, E. Gabison2, A. Rendon1, J.-A. Sahel1, R. Tadayoni3. 1Département
de thérapies, Institut de la Vision, Inserm UMR-S 968, Université Pierre et Marie
Curie, Paris, France; 2CNRS UMR 7149, Université Paris 12, Créteil, France;
3
Ophthalmology, Lariboisiere University Hospital, AP-HP, Paris Diderot University,
Paris, France.
Purpose: The antiangiogenic activity of PEDF is associated with the γ-secretase-catalysed
cleavage of VEGF receptors. The aim of this study was to identify the VEGFR1 cleavage
site and determine how amino acid substitution affects VEGFR1 binding to γ-secretase
and the intracellular translocation of VEGFR-1.
Methods: The GFP-tagged human VEGFR1 expression vector (pVEGFR1-EGFP) underwent
site-directed mutagenesis to substitute valine767 (a putative cleavage site) with alanine
(pVEGFR1-EGFP-A167). Porcine aortic endothelial cells (PAECs) lacking VEGF receptors
were transfected with either pVEGFR1-EGFP, pVEGFR1-EGFP-A167 or empty vector. Stably
transfected cells were selected using kanamycin resistance and exposed to VEGF (100ng/
ml) in the presence or absence of PEDF (100ng/ml) for up to 24 hours. VEGFR1 binding
to γ-secretase complex was determined by immunoprecipitation of VEGFR1 followed by
Western Blot analysis for the components of the γ-secretase complex (presenilin [PS1],
nicastrin [NTC], APH-1 and PEN-2). Peptide analysis by LC-MS/MS confirmed the identity
of the bound proteins. Intracellular translocation of VEGFR1 was analyzed using confocal
microscopy and Western blot following subcellular fractionation.
Results: Neither VEGF nor PEDF affected the overall expression levels of VEGFR1 in
PAECs. Both Western blot analysis and LC-MS/MS demonstrated that PEDF+VEGF
resulted in NTC binding to VEGFR1 by 5 minutes followed by APH-1 and PS1 at 30min
and finally, PEN-2 at 1hr for both wild type and mutant-VEGFR1. Neither VEGF nor
PEDF alone promoted binding of VEGFR1 to the γ-secretase complex. Intracellular
translocation of VEGFR1-EGFP was followed by confocal microscopy. VEGF alone induced
nuclear translocation of VEGFR1 in cells transfected with both wild type and mutantVEGFR1, whereas addition of PEDF alone did not induce VEGFR1 translocation. However,
VEGF+PEDF in combination blocked the VEGF-induced nuclear translocation of VEGFR1
in the cells transfected with wild type-VEGFR1 vector but not mutant, confirming that
V767 plays a critical role in translocation. Western blot for the intracellular fragment of
VEGFR1 confirmed PEDF-induced cleavage of wild type-VEGFR1 in the presence of
VEGF resulting in the appearance of a cytosolic VEGFR1 fragment which was absent in
cells with mutant VEGFR1.
Conclusions: V767 is a γ-secretase-catalyzed cleavage site on VEGFR-1 and may offer
a novel therapeutic strategy to regulate VEGF activity in conditions associated with
pathological angiogensis.
CR: J. Cai, None; S. Han, None; Q. Ruan, None; L. Wu, None; M.B. Grant, None; M.E.
Boulton, None.
Support: NIH Grant EY018358
Purpose: The objective of this study is to investigate whether Dp71 the smallest product
of Duchenne muscular dystrophy (DMD) gene, and key component of the membrane
associated cytoskeleton plays a modulator role in corneal neovascularization (CNV).
Methods: The study of CNV was performed on Wild-Type C57BL6 (WT) and Dp71-null
mice. CNV was induced by alkali injury in both strains and the mice were sacrified
7 days after. Neovascularization was observed in corneal flatmounts using vessel
immunostaining with the CD31 antibody. The degree of angiogenesis was analyzed
using Photoshop Cs. The influence of Dp71 on the expression of VEGF was quantified
using an Elisa Kit. Expression of Dp71 in the cornea was analyzed by Western blot
with a panspecific antibody against dystrophins (H4).
Results: An increase of mean percentage corneal surface covered by CNV was observed
in Dp71-null mice compared to WT mice, respectively 40.72% vs. 26.33%. (P<0.005).
In WT mice, no increase of Dp71 during cornea neovascularization compared to a
healthy cornea was found. It was observed also in WT that Dystrophin Dp116, normally
expressed in the peripheral nervous system was present in the cornea. Moreover in
the absence of Dp71 the Dp116 was up-regulated when compared with the WT mice
strain. The concentrations of VEGF, a pro-angiogenic factor were higher in vascularized
corneas of Dp71-null compared to WT mice.
Conclusions: The results suggest that dystrophin Dp71 could play a role in the
modulation of corneal neovascularization
CR: G. Ortiz, None; R. Bernard, None; E. Gabison, None; A. Rendon, None; J.-A.
Sahel, None; R. Tadayoni, None.
Support: Fondation Voir et Entendre, Association Française contre les Myopathies
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
61-64
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
65 - A112
A Protective Role for Microglial Cells in TNFα -/- Mice in Ischemic Retinopathy
D.M. McDonald, L. Stevenson, N. Matesanz, K. Edgar, L. Colhoun, A. Devine, T.A.
Gardiner, D.M. McDonald. Vision Science, Queens University Belfast, Belfast, United
Kingdom.
Purpose: Neovascularisation occurs in response to tissue ischemia and growth factor
stimulation. In ischemic retinopathies however, new vessels fail to recover the hypoxic
tissue; instead, they infiltrate the transparent vitreous for reasons that are poorly
defined. In a model of oxygen-induced retinopathy (OIR) TNFα and iNOS, upregulated
in retinal Müller and microglial cells in response to tissue ischemia, are cytotoxic and
inhibitory to vascular repair. Here we investigated the mechanism for this effect.
Methods: Wild type C57WT and TNFα -/- mice were subject to OIR by exposure to 75%
oxygen (postnatal days 7-12). Retinas were subsequently removed during the hypoxic
phase of the model (P12-P13). Retinal cell death was determined by TUNEL staining
and microglial cells quantified following Z series capture with a confocal microscope.
In situ peroxynitrite and superoxide were measured using the fluorescent dyes DCF
and DHE. iNOS, nitrotyrosine, arginase and VEGF were analysed by real-time PCR,
western blotting or the conversion of radiolabeled arginine.
Results: In the TNFα -/- animals there was a significant reduction in TUNEL positive
apoptotic cells in the inner nuclear layer of the avascular retina compared to the WT
group. This coincided with an increase in the number of microglial cells which were
actively engaged in phagocytosing apopotic debris and displayed low ROS/RNS/NO
production and high arginase activity.
Conclusions: Collectively, our results demonstrate that enhanced vascular recovery
in the absence of TNFα is associated with preservation of a microglial cell population,
which display an anti-inflammatory phenotype during the early ischemic phase of OIR.
CR: D.M. McDonald, None; L. Stevenson, None; N. Matesanz, None; K. Edgar,
None; L. Colhoun, None; A. Devine, None; T.A. Gardiner, None; D.M. McDonald,
None.
Support: Wellcome Trust UK, Fight for Sight UK
66 - A113
Retinal Pericyte Contractile Phenotype: Regulation by Myosin PhosphataseRhoA Interacting Protein (MRIP) and the β-Actin Specific Capping Protein,
βcap73
I.M. Herman, J.T. Durham, H.K. Surks. Physiology, Tufts University School of
Medicine, Boston, MA.
Purpose: Pericyte-endothelial interactions are likely to modulate microvascular
morphogenesis during diabetes and aging. Indeed, regulation of endothelial cell
growth and capillary tonus have been linked to pericyte contractile phenotype
(Kutcher and Herman, 2009). Through opposing functions of myosin light chain
phosphatase (MLCP) and myosin light chain kinase (MLCK), pericytes sustain a
relaxed or contractile state. While it has been postulated that the RhoA/Rho kinase
(ROCK) pathway may regulate retinal pericyte contractile phenotype, the mechanisms
by which the key signaling components orchestrate these events remain largely
equivocal. Similarly, pericyte RhoA-GTP status has recently been shown to modulate
retinal endothelial growth in co-culture studies.
Methods: Yeast-two hybrid analyses followed by co-immunoprecipitation and
co-immunolocalization studies were employed to characterize protein-protein
interactions. Additionally, real-time and immunofluorescence-based assays were
performed in MRIP-silenced vs. control-treated pericytes so that cell shape, spreading
and cytoskeletal dynamics could be assessed.
Results: Here, we report that a novel interaction exists between MRIP and βcap73
as well as βcap73 and ROCK. Interestingly, upon MRIP silencing, we observe that
βcap73 is displaced from the cell periphery without perturbing β-actin localization.
Further, MRIP-silenced pericytes exhibit a 2-fold increase in cellular spreading rates.
Conclusion: Pericyte MRIP regulates MLCP- and RhoA-ROCK interactions by binding
βcap73, therein creating a molecular scaffold through which cell shape and spreading
are likely to be controlled. Further, βcap73 binding to MRIP may coordinate RhoA,
ROCK and MLCP function by targeting these components to actomyosin containing
stress fibers. In turn, perturbing MRIP-βcap73 association could disrupt pericyteendothelial interactions and contribute to the pathologic angiogenesis and vascular
complications, which accompany diabetic retinopathy or wet age-related macular
degeneration.
CR: I.M. Herman, US Patent #6,780,987, P; J.T. Durham, None; H.K. Surks, None.
Support: NIH EY15125, EY19533 (IMH), T32-DK07542 (JTD)
67 - A114
A Regulatory Role for γ-Secretase in VEGF-Induced Retinal Vascular
Permeability via Phosphorylation of the VE-Cadherin Complex
68 - A115
Angiogenesis Induced by Proliferative Diabetic Retinopathy and Eales’ Disease
Vitreous is Mediated by a Common Pro-Inflammatory Mechanism
L. Wu1A, J. Cai1A, M.B. Grant1B, M.E. Boulton1A. AAnatomy and Cell Biology,
B
Pharmacology and Therapeutics, 1University of Florida, Gainesville, FL.
M. Ponnalagu1, R. Kim2, D. Shukla2, P. Namperumalsamy2, V.R. Muthukkaruppan3,
A.W. Stitt4. 1Dr.G.Venkatasamy Eye Research Institute;Queens’ University Belfast,
Madurai, India; 2Vitreous and Retina Service, Aravind Eye Care Systems, Madurai,
India; 3Dr.G.Venkatasamy Eye Research Institute, Madurai, India; 4Centre for Vision
and Vascular Sciences, Queens University, Belfast, United Kingdom.
Purpose: We have previously shown that γ-secretase controls vascular permeability,
in part, via regulating the binding of VEGFR-1 to the VE-cadherin complex. The aim
of this study was to determine if γ-secretase can regulate the phosphorylation state
of the VE-cadherin complex.
Methods: Bovine retinal microvascular endothelial cells (BRMECs) were treated
with VEGF, and/or PEDF (each at 100ng/ml) in the presence or absence of γ-secretase
inhibitor for up to 12 hours. Protein samples were extracted from the different
subcellular fractions and immunoprecipitated with an antibody against tyrosine
phosphorylation (PY20). Total tyrosine phosphorylation of VE-cadherin and β-catenin
was analyzed by Western blot. The proteins fractions were also subjected to Western
blot using antibodies against VE-cadherin tyrosine phosphorylation sites (pY658 or
pY731). In order to knockdown γ-secretase, BRMECs were transfected with siRNA
against presenilin 1(PS1) or nicastrin (NTC) and the change in endothelial permeability
after the treatment with VEGF and/or PEDF was determined.
Results: VEGF-induced a rapid increase in tyrosine phosphorylation of VE-cadherin
in the membrane fraction which could be prevented by PEDF. The effect of PEDF
was reversed by addition of a γ-secretase inhibitor. This was confirmed by Western
blot using antibodies against VE-cadherin pY658 and pY731. However, cytosolic
and nuclear VE-cadherin was not phosphorylated at pY658 and pY731 even though
immunoprecitation with PY20 and subsequent Western blot for VE-cadherin showed
strong tyrosine phosphorylation of VE-cadherin within both the fractions. The total
tyrosine phosphorylation of β-catenin showed a very similar pattern to that of
VE-cadherin upon treatment with VEGF, PEDF and γ-secretase inhibitor. siRNA
knockdown of PS1 and NTC was able to reduce the inhibitory effect of PEDF on
VEGF-induced permeability of endothelial cells.
Conclusions: This study demonstrates that PEDF can block VEGF-induced permeability
via γ-secretase modulating the tyrosine phosphoryation of the membrane bound VEcadherin complex further supporting the regulatory role of γ-secretase in angiogenesis.
CR: L. Wu, None; J. Cai, None; M.B. Grant, None; M.E. Boulton, None.
Support: NIH Grant EY018358
Purpose: There is an increasing emphasis on inflammatory processes in the aetiology
of diabetic retinopathy (DR). Eales’ Disease (ED) is an overt inflammatory eye disease
that can result in pre-retinal neovascuarisation, however, the connection between
this disease and the proliferative stage of DR (PDR) remains uncertain. In patients
with PDR or ED, this study has compared pro-inflammatory cytokines occurring in
vitreous from both groups and explored the role that they play in angiogenesis ex vivo.
Methods: PDR (n=9), ED (n=5) vitreous samples were quantified for various cytokines
by Cytokine Biochip Array (Randox, UK) and ELISA. Vitreous from patients with
Macular Hole (MH) was used as a control (n=5). Tubulogenesis assay was performed
using Human Dermal Microvascular Endothelial cells (HDMECs) and tube length
was quantified using Nikon-NIS-Elements software
Results: PDR and ED vitreous had elevated levels of many cytokines, but especially
IL6, MCP-1 and VEGF. PDR and ED vitreous showed greater ability to induce tube
formation compared with the untreated controls (p<0.05; p<0.01) and there was a
significant correlation between angiogenic capacity and the quantified levels of
cytokines. Angiogenic capacity of vitreous samples, when mixed with Lucentis (0.5mg/
ml) and/or anti-IL6 (0.1ug/ml) were neutralized, (mean fold decrease ranging from
0.2 to 1.2).
Conclusions: In addition to VEGF, inflammatory cytokines (IL6 and MCP-1) also
play a central role in inducing retinal neovascularization in PDR and ED. The present
result also indicates the possibility of interaction between cytokines and vascular
growth factor.
CR: M. Ponnalagu, None; R. Kim, None; D. Shukla, None; P. Namperumalsamy,
None; V.R. Muthukkaruppan, None; A.W. Stitt, None.
Support: Commonwealth and Fellowship Plan, Department of Science and Technology.
TIFAC-CORE
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
65-68
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 25 - 70 / A72 - A117
104. Retinal Angiogenesis: Development and Diseases Organizing Section: RC
69 - A116
MT1-MMP Modulates the Expression of FGF Receptor and VEGF in Mouse
Corneal Fibroblast Cell Lines
70 - A117
Additive Effect of Nicotine and Hyperglycemia on Upregulation of Vascular
Endothelial Growth Factor and Inflammation in Human Retinal Pigment
Epithelial Cells and Retinal Endothelial Cells
K. Han, J.-H. Chang, D. Azar. Ophthalmology and Visual Sciences, University of
Illinois at Chicago, Chicago, IL.
Purpose: To evaluate the pro-angiogenic role of stromal fibroblast-derived MT1-MMP
in mouse corneal fibroblasts.
Methods: Immortalized MT1-MMP knockout and knock-in corneal fibroblast cell
lines were generated. Levels of ERK, phospho-ERK proteins and activated Ras were
examined by western blot analysis in WT, MT1-MMP KO and MT1-MMP KI corneal
fibroblast cells. Expression levels of the tyrosine kinase receptors FGFR-1 and -2,
VEGFR-1 and EGFR were quantitated by real-time PCR. The VEGF protein expression
level was determined by western blotting and immunohistochemistry in the corneal
fibroblast cell lines. The VEGF mRNA expression levels were measured in the absence
and presence of ERK and Ras inhibitors in bFGF-stimulated corneal fibroblast cell
lines by real-time PCR.
Results: VEGF expression and ERK activation were significantly diminished in
bFGF stimulated MT1-MMP knockout mouse corneal fibroblasts when compared to
that of wildtype fibroblasts. Diminished FGFR-1 and EGF receptor expression was
demonstrated in MT1-MMP knockout cells (KO) cells. MT1-MMP- and bFGF-mediated
VEGF expression is prevented by ERK and Ras inhibitors in WT corneal fibroblasts.
The transcription factor HIF-1α, was activated by bFGF on MT1-MMP wildtype (WT)
and MT-MMP knock-in (KI), but not in MT1-MMP knockout (KO) fibroblasts.
Conclusion: MT1-MMP modulates the activity of bFGF-induced signaling molecules
ERK and Ras in corneal fibroblasts. Transcription factor HIF-1α may play a role in
MT1-MMP- and bFGF-induced VEGF expression in corneal fibroblasts. In this study,
we demonstrated that MT1-MMP potentiates bFGF-induced VEGF expression, likely
by modulating the bFGF signal transduction pathway.
CR: K. Han, None; J.-H. Chang, None; D. Azar, None.
Support: EY10101, EY01792
N. Tirgan, P. Gupta, G. Kulp, A. Boretsky, E. van Kuijk, B.F. Godley, R.G. Tilton, M.
Motamedi. Department of Ophthalmology, University of Texas Medical Branch,
Galveston, TX.
Purpose: Previous studies have reported that hyperglycemia and nicotine exposure
individually can cause alterations in retinal cell function and proliferation. Much less
is known about the combined effect of nicotine exposure with hyperglycemia in the
retina. Here, we studied the expression of vascular endothelial growth factor (VEGF)
and inflammatory responses of two cell types, ARPE-19 (human retinal pigment
epithelial cells) and ACBRI-181 (human retinal microvascular endothelial cells), after
simultaneous exposure to both nicotine and hyperglycemia.
Methods: ARPE-19 cells and ACBRI-181 cells were exposed to four experimental
conditions, including: i) 5 mM glucose, ii) 30 mM glucose, iii) nicotine at concentrations
ranging from 10^-4 to 10^-10 M, and iv) a combination of 30 mM glucose and nicotine.
Cells were exposed to physiological glucose (5 mM) or hyperglycemia (30 mM) for two
days, followed by treatment with nicotine or combination of nicotine and hyperglycemia
for an additional 48 hours. Cell proliferation was assessed by MTT assay after five
days. The extent of NF-kB activation was assessed using immunocytochemistry
to measure RelA nuclear localization after five days. VEGF, MCP-1, and IL-8 were
measured from cell culture supernatant using Millipore’s human cytokine kit at
baseline, 8, 24, and 48 hours.
Results: ACBRI-181 cell proliferation occurred in the hyperglycemia, nicotine, and
combination treatment. The hyperglycemia-treated cells had the greatest proliferation
as compared to control (176 %). Cells treated with nicotine at doses of 10^-6 M had
exhibited increased proliferation (161% with 5 mM glucose, 159% with 30 mM glucose as
compared to control). In addition, increased nuclear localization of RelA accompanied
combinational treatment compared to normal glucose-exposed cells. Also, increases
in VEGF, MCP-1, and IL-8 were observed when ARPE-19 and ACBRI-181 cells were
exposed to a combination of nicotine and hyperglycemia.
Conclusions: The combination of nicotine and hyperglycemia promoted upregulation
of VEGF and inflammatory responses in both cell lines. In addition, RelA activation was
stronger for the combination treatment, suggesting an additive effect on inflammation.
These results suggest that in diabetic patients who smoke, the retinal tissue may
experience increased production of VEGF and NF-kB-linked cytokines due to the
potential additive effects of chronic exposure to hyperglycemia and nicotine.
CR: N. Tirgan, None; P. Gupta, None; G. Kulp, None; A. Boretsky, None; E. van
Kuijk, None; B.F. Godley, None; R.G. Tilton, None; M. Motamedi, None.
Support: NIH ES007254
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
69-70
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
459 - A23
7-Ketocholesterol Efflux is Mediated by HDL and Independent of ABCG1 in
RPE-Derived D407 Cells
460 - A24
Bruch’s Membrane (BM) Aging Induces Down Regulation of CD46 in Retinal
Pigment Epithelium: Implications for AMD
J.W. Lee, I.R. Rodriguez. Mechanisms of Retinal Disease Section, LRCMB, National
Eye Institute/NIH, Bethesda, MD.
H. Cai1, Y. Roh1, C. Cai2, L.V. Del Priore3. 1Ophthalmology, Columbia University
Medical Center, New York, NY; 2Bryn Mawr College, Bryn Mawr, PA;
3
Ophthalmology, Columbia University, New York, NY.
Purpose: Recent studies have shown that 7-ketocholesterol (7KCh) efflux was
dependent on the expression of ABCG1 and the presence of HDL in media in Abcg1+/+
macrophages. The purpose of this study is to test whether 7KCh efflux is dependent
on ABCA1 or ABCG1 expression in D407 cultured RPE cells.
Methods: 7KCh-enriched human LDL (7KLDL) was used to deliver 7KCh to the cells
without interference from other oxidized lipids. 7KCh and cholesterol were quantified
by HPLC-UV 24 h post-treatment. Efflux studies were performed by treating the cells
with 7KLDL (50 μg/mL) in serum-free media for 24 h. Cells were then washed with
PBS and incubated for 20 h in fresh serum-free media in the absence and presence of
apoA-1 (10 μg/mL), HDL (100 μg/mL) or LDL (100 μg/mL). Cell pellets and conditioned
media were collected and analyzed for 7KCh and cholesterol. ABCA1 and ABCG1
expression were suppressed using specific siRNAs before 7KLDL treatment.
Results: In D407 cells, 7KCh uptake had increased in a dose-dependent manner after
7KLDL treatment. 7KCh was not detectable in the conditioned media without acceptors.
By adding 100 µg/mL of HDL or LDL to the media, 7KCh efflux from D407 cells was
increased 40% and 25%, respectively. ApoA1 did not promote 7KCh efflux. ABCA1
and ABCG1 siRNAs suppressed ABCA1 and ABCG1 mRNA levels 65% and 85%,
respectively. A double ABCA1 and ABCG1 knockdown decreased cholesterol efflux
by 10% when compared to the negative siRNA control. 7KCh was not detectable in the
media from any of siRNA knockdown samples. Interestingly, the knockdown of ABCA1
and ABCG1 did not alter the 7KCh efflux in the presence of HDL or LDL in the media.
Conclusions: Our data suggests that 7KCh efflux is dependent on lipoprotein acceptors
in the media but independent of ABCA1 and ABCG1. This indicates that 7KCh efflux is
occurring either via other transporters or by mass-action exchange with lipoproteins.
CR: J.W. Lee, None; I.R. Rodriguez, None.
Support: NEI intramural research program (IRR)
461 - A25
The 5HT 1A Agonist 8OH DPAT Decreases Oxidative Stress-Induced
Mitochondrial Damage and Reduces Accumulation of Lipofuscin Granules in
ARPE19 Cells
Purpose: CD46, a complement regulatory protein located on the basal surface of
the retinal pigment epithelium, is in the correct anatomic location to connect the
complement system to Bruch’s membrane changes, and thus may play a role in the
pathogenesis of age-related macular degeneration (AMD).Herein we determine the
effects of BM aging on CD46 gene expression and post-translational modifications,
and the role of these changes in regulating RPE function.
Methods: Human ARPE19 cells were cultured to confluence either on regular Petri
dish or human Bruch’s membrane explants harvested from young (age < 50) or older
(age > 70) human eye bank eyes. Confluent ARPE monolayers were harvested and
protein lysates were prepared using standard techniques. Expression levels of CD46
protein were determined by densitometry of western blotting images. To study posttranslation modifications, immuno-precipitates of ARPE19 cell (cultured on young
or older BM) were prepared using anti-CD46 monoclonal antibodies. The immunoprecipitated samples were subjected to western blot analysis using 8-12% SDS-PAGE
and transferred to a nitrocellulose membrane. Sulfo- or phospho-tyrosine specific
antibodies were used to detect sulfo-or phospho-tyrosine modified CD46. Phosphatase
inhibitor sodium orthovanadate (Na3VO4) were added during the tissue culture.
Results: The levels of CD46 protein expression in ARPE19 were 2.0 -fold higher in RPE
cultured on young compared to older human Bruch’s membrane. Tyrosine-sulfated
CD46 was present in RPE cultured on both young and older BM. However the levels
are 1.5 fold higher in RPE cells cultured on young BM. Tyrosine-phosphorylated CD46
was undetectable in RPE cultured on a Petri dish or human BM. When phosphatase
inhibitor sodium orthovanadate was added into RPE culture trace amounts of tyrosinephosphorylated CD46 could be observed.
Conclusions: The results suggest that BM aging affect CD46 expression, with a down
regulation of CD46 as a function of increasing BM age. In addition, preliminary
evidence suggests that post translation modification of tyrosine residues within CD46
may also be affected by BM age. Since CD46 plays a role in inhibition of complementmediated damage, down regulation of CD46 would be expected to render the RPE
more susceptible to complement mediated damage.
CR: H. Cai, None; Y. Roh, None; C. Cai, None; L.V. Del Priore, None.
Support: Research to Prevent Blindness, Robert L. Burch III Fund, Retina Society, and
the Foundation Fighting Blindness
462 - A26
Stimulation of the P2X7 Receptor on RPE Cells Triggers a Rapid Release of IL-6
L.-A. Tu1, S. Guha2A, J.C. Lim1, T. Eysteinsson3, A.M. Laties2B, C.H. Mitchell1,2A.
1
Anatomy and Cell Biology, Sch of Dental Med University of Pennsylvania,
Philadelphia, PA; APhysiology, BOpthalmology, 2Sch of Med University of
Pennsylvania, Philadelphia, PA; 3Physiology, University of Iceland, Reykjavik,
Iceland.
P. Thampi1, H. Vittal Rao1, S. Jarrett2, J. Cai1, C. Romano3, M.E. Boulton1. 1Anatomy &
Cell Biology, University of Florida, Gainesville, FL; 2Molecular and Biomedical
Pharmacology, University of Kentucky, Lexington, KY; 3Retina Discovery Rsch,
Alcon Laboratories, Inc, Fort Worth, TX.
Purpose: To investigate whether the 5HT1A agonist, 8OH DPAT, decreases oxidative stressinduced mitochondrial damage and reduces lipofuscin accumulation in cultured human
retinal pigment epithelial (RPE) cells.
Methods: ARPE19 cells in basal media were 1) maintained for 4 weeks to generate autophagy
derived-lipofuscin, 2) fed photoreceptor outer segments (POS) or peroxidized POS (oxPOS)
every two days for 14 days to induce phagocytosis-derived lipofuscin, 3) fed mature
lipofuscin granules and 4) treated with oxidative stressors 200µM H2O2 (1 hr) or 40µg/ml
7-ketocholesterol (7-kCh) (8 hr). In all conditions, 8OH DPAT was replenished every two days
while controls received vehicle only. Lipofuscin accumulation and superoxide (O2.) anion
generation were quantified by FACS and fluorescence microscopy, lipofuscin phototoxicity
was assessed by the MTT assay, REDOX activity was measured by ELISA and mitochondrial
DNA damage assessed by long chain QPCR.
Results: 8OH DPAT induced a dose-dependent decrease in both autophagy- and POSderived lipofuscin granules in RPE cells. Maximal inhibition (>50%) of lipofuscin formation
was observed at 10 µM 8OH DPAT and this was used as the concentration of choice for
all subsequent experiments. 8OH DPAT decreased autophagy-derived lipofuscin by 30%
at 3 weeks and 67% at 4 weeks. 8OH DPAT induced a 28% and 20% decrease at 14 days in
phagocytosis-derived lipofuscin accumulation in POS and oxPOS fed cells respectively
compared to untreated controls fed POS or oxPOS. Similar changes were observed by
fluorescence microscopy. However, 8OH DPAT failed to reduce the existing mature lipofuscin
granules. In cells fed oxPOS for 14 days phototoxicity was decreased by 56% in 8OH DPAT
treated cells compared to untreated controls. H2O2 and 7-kCh induced a 102% and 37% increase
in O2. generation respectively and this increase was blocked by 67% and 50% respectively
following treatment with 8OH DPAT. In addition, 8OH DPAT protected cells from loss of
REDOX potential caused by exposure to H2O2 and 7-kCh. H2O2-induced mitochondrial DNA
damaged was significantly reduced by >70% in the presence of 8OH DPAT.
Conclusions: 5HT1A agonists have the capacity to reduce the generation of both autophagyand phagocytosis-derived lipofuscin in RPE cells. These agonists can also protected against
oxidative damage to mitochondria thus reducing the autophagic load. 5HT1a agonists may
prove useful in the treatment of AMD.
CR: P. Thampi, Alcon Laboratories, F; H. Vittal Rao, Alcon Laboratories, F; S. Jarrett,
None; J. Cai, None; C. Romano, Alcon Laboratories, E; M.E. Boulton, Alcon Laboratories, F.
Support: Alcon Laboratories, Inc.
Purpose: The cytokine IL-6 may stimulate choroidal neovascularization (CNV),
and inhibition of IL-6 reduces the vessel growth and inflammation in CNV models.
While expression of the IL-6 gene is increased in RPE cells from diseased eyes, it is
not currently known how IL-6 release is triggered. In other tissues, stimulation of the
P2X7 receptor for ATP activates a pannexin hemichannel, which in turn activates the
inflammasome and triggers cytokine release. In this study we asked whether a similar
pathway could trigger IL-6 release from RPE cells.
Methods: Calcium levels were measured in ARPE-19 cells grown on 96 well plates
loaded with fura-2 and excited at 340 and 380 nm. IL-6 release was determined with
a chemiluminescent Elisa assay.
Results: Stimulation of RPE cells with the P2X7 receptor agonist BzATP led to a
large, sustained rise in calcium. This elevation was dependent upon the presence of
extracellular calcium, was increased by removal of magnesium, and was inhibited by
antagonists Brilliant Blue G, A438079 and KN-62. Together this strongly implicates the
P2X7 receptor. The level of IL-6 in the bath was increased several fold after exposure
of RPE cells to BzATP. Release was rapid, with levels raised 1, 15 and 60 min after
exposure to BzATP began. The release of IL-6 was inhibited by pannexin channel
blocker carbenoxolone (10 µM), suggesting pannexins may be involved in the release.
Conclusions: RPE cells possess functional P2X7 receptors. Their stimulation triggers
a release of IL-6 that requires pannexin channels. It remains to be determined whether
IL-6 release following stimulation of the P2X7 receptor enhances CNV.
CR: L.-A. Tu, None; S. Guha, None; J.C. Lim, None; T. Eysteinsson, None; A.M.
Laties, None; C.H. Mitchell, None.
Support: EY-013434, EY-015537, EY-001583 (CHM); Research to Prevent Blindness, the
Paul and Evanina Bell Mackall Foundation Trust (AML)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
459-462
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
463 - A27
Rewiring Pathologic Cellular Responses Using the Tetraspan Web Alters
Collagen Gel Contraction by ARPE-19 Cells
464 - A28
Pyruvate Enhances the Differentiation Profile of the Human Retinal Pigment
Epithelium Cell Line ARPE-19
L.K. Gordon1A, S.A. Morales1B, M. Wadehra1C, D.G. Telander2, J. Braun1C. AJules Stein Eye
Inst, BOphthalmology, CPathology and Laboratory Medicine, 1Univ of CaliforniaLos Angeles, Los Angeles, CA; 2Ophthalmology, University of California, Davis,
Sacramento, CA.
A. Ahmado1,2, A.-J.F. Carr1, A.A. Vugler1, M. Semo1, J.M. Lawrence1, L. da Cruz2, P.J.
Coffey1. 1Ocular Biology and Therapeutics - Institute of Ophthalmology, University
College London, London, United Kingdom; 2Vitreoretinal Surgery, Moorfields Eye
Hospital, London, United Kingdom.
Purpose: Collagen gel contraction by ARPE-19 is an in vitro model for proliferative
vitreoretinopathy (PVR), an aberrant wound healing response. Expression of the
tetraspan protein epithelial membrane protein 2 (EMP2) controls gel contraction
through FAK activation. Peripheral myelin protein 22 (PMP22), another member of the
tetraspan web, is closely related to EMP2. The purpose of this study was to determine
if PMP22 also controls the contractile phase associated with PVR.
Methods: PMP22 levels in ARPE-19 cells were increased through stable transfection
(ARPE-19/PMP22). A PMP22 specific siRNA was used to decrease PMP22 expression
in the cells. AKT activation was blocked using a small molecule inhibitor Ly294002.
Integrin expression, adhesion, and protein expression were assessed respectively
through flow cytometry, binding to collagen type I and IV, and Western Blot analysis.
Collagen gel contraction was assessed using an in vitro assay.
Results: In comparison to the ARPE-19 cells, ARPE-19/PMP22 cells exhibited an
increased collagen adhesion. Gel contraction, however, was reduced by greater than
50% in the PMP22 overexpressing cells (P<0.001). In contrast to the FAK activation
observed by increasing EMP2 expression, PMP22 overexpression led to increased
AKT activation and reduced FAK activation. The decrease in gel contraction by the
ARPE-19/PMP22 cells was partially reversed through either PMP22 siRNA or by
blockade of AKT.
Conclusions: Relative expression of EMP2 or PMP22 within the tetraspan web drives
a cellular response towards a FAK or AKT dependent pathway, respectively. EMP2
and PMP22 differentially regulate collagen gel contraction in the ARPE-19 cell line,
an in vitro model for PVR. The implication of this finding adds a new dimension to
the concept of the tetraspan web that can drive the downstream response depending
on both the presence and abundance of specific proteins.
CR: L.K. Gordon, patent application pending, P; S.A. Morales, Patent application
pending, P; M. Wadehra, Patent application pending, P; D.G. Telander, Patent
application pending, P; J. Braun, Patent application pending, P.
Support: VA Merit Grant, A.P Giannini Foundation
Purpose: Transplantation of the retinal pigment epithelium (RPE) requires cells that
are phenotypically close to native RPE. Cultured RPE may become an option for
transplantation in RPE degenerative disease such as age-related macular degeneration.
However RPE undergo de-pigmentation and lose markers of differentiation when
grown in culture. Pyruvate is a potent intracellular buffer and a scavenger of hydrogen
peroxide. Many studies suggest pyruvate is protective against oxidative stress and
other stressors in the retina. We are proposing a simple culture protocol that promotes
differentiation markers such as CRALBP, RPE65 and dense pigmentation in higher
passages of ARPE-19 cells when pyruvate is added to culture medium. This is the
first study to demonstrate significant RPE65 expression by western blot analysis in
the human RPE cell line ARPE-19.
Methods: Passages p22 - p28 of the human RPE cell line ARPE-19 were grown on
polyester filters in either DMEM/F12, DMEM with or without pyruvate and foetal
calf serum concentration of 1% for up to 3 months. Cultures were fed twice a week.
Pigmentation, immunocytochemistry, apical-basal polarity, VEGF secretion and
western blots were used to assess differentiation.
Results: Pyruvate accelerated pigmentation in combination with DMEM both at
low (1g/L) and high (4.5g/L) glucose concentration. CRALBP and RPE65 expression
is significantly increased by pyruvate as shown by western blot analysis. Pyruvate
supplementation induced both quicker onset of circumferential actin distribution
as well as suppression of cytokeratin 8 (CK 8) irrespective of glucose concentration.
Immune positivity of CK 8 was found to be inversely related to pigmentation and
pmel17 expression as shown by confocal microscopy. VEGF secretion was not affected
by pyruvate.
Conclusions: Pyruvate is a valuable growth media component whose beneficial effect
on ARPE-19 is demonstrated by promoting multiple differentiation characteristics. The
exact mechanism of pyruvate action on RPE differentiation is unknown but maybe
due to its potent buffering capacity and anti-oxidative properties. It is unlikely that
pyruvate acts simply as an additional energy source since favourable effects occurred
both at low and high concentrations of glucose.
CR: A. Ahmado, None; A.-J.F. Carr, None; A.A. Vugler, None; M. Semo, None; J.M.
Lawrence, None; L. da Cruz, None; P.J. Coffey, None.
Support: The Lincey Foundation
465 - A29
Uptake and Storage of Bevacizumab in RPE Cells Affect Its Physiological
Function
466 - A30
The COOH-Terminal Tails of Mct3 and Mct4 Contain Novel Motifs That Target
the Heteromeric Transport to the Basolateral Membrane
A.K. Klettner1A, T. Meyer1B, F. Möhle1A, M.-L. Kruse1C, D. Wesch1B, D. Kabelitz1B, J.
Roider1A. AOphthalmology, BImmunology, CMolecular Gastroenterology, 1University
of Kiel, Kiel Medical Center, Kiel, Germany.
N.J. Philp1, J.J. Castorino1, E.J. Rodriguez-Boulan2. 1Path/Anat/Cell Biology, Thomas
Jefferson University, Philadelphia, PA; 2Ophthalmology, Weil Med Coll-Cornell
Univ, New York, NY.
Purpose: The VEGF-antagonists Bevacizumab and Ranibizumab are widely used
in anti-VEGF therapy to treat wet age-related macular degeneration. Both products
have been developed from the same monoclonal murine anti-VEGF antibody and are
often considered equally effective and interchangeable. In our studie, we investigate
potential differences between these two agents with regard to their effects on retinal
pigment epithelial cells (RPE).
Methods: In order to investigate the effect of Bevacizumab and Ranibizumab on the
RPE, we used porcine primary RPE cells to analyse the uptake of either substance
by flowcytometry and fluorescence microscopy, compared with the chimeric IGg1
antibody Rituximab. Additionally, we investigated the effect of the VEGF antagonists
on cell proliferation by determining the cell number at day 0, day 3 and day 7 with
a trypan blue exclusion assay, their effect on wound healing using a wound healing
scratch assay and their effect on phagocytosis using photoreceptor outer segment
(POS) opsonized FITC-labeled beads and fluorescence microscopy.
Results: We found that Bevacizumab, but not Ranibizumab or Rituximab, is taken up
and stored in RPE cells for at least 7 d (latest investigated time point). Both Bevacizumab
and Ranibizumab slow down RPE cell proliferation, with a more profound effect of
Bevacizumab compared to Ranibizumab. Neither substance impairs RPE wound
healing ability. Most importantly, Bevacizumab, but not Ranibizumab or Rituximab,
seem to impair the ability of the RPE to ingest POS-opsonized latex-beads, indicating
a negative effect of Bevacizumab on the ability of RPE cells to phagocytose POS.
Conclusions: Bevacizumab and Ranibizumab are not identical in their effects on RPE
cells. Bevacizumab is taken up and stored in RPE cells, which has a negative effect on
the RPE cells ability to phagocytose photoreceptor outer segments.
CR: A.K. Klettner, Novartis, R; T. Meyer, None; F. Möhle, None; M.-L. Kruse,
None; D. Wesch, None; D. Kabelitz, None; J. Roider, None.
Support: DOG Forschungsförderung
Purpose: Epithelial cells depend on the polarized distribution of metabolic transporters
for vectorial transport of ions, fluid and metabolites into and out of tissue compartments
to maintain metabolic homeostasis. The transport of lactate into and out of cells is
facilitated by proton-coupled monocarboxylate transporters (MCT) 1, 3, and 4, which
are expressed as heteromeric complexes comprised of a catalytic subunit (MCT) and
an accessory subunit, CD147. Trafficking of MCT-CD147 heterocomplexes in epithelial
cells presents a novel sorting paradigm because MCT-CD147 complexes are found in
both apical and basolateral membranes depending on the epithelium. The basolateral
sorting sequence of CD147 was identified as a single leucine residue in the C-terminal
cytoplasmic tail. In the present studies, we identified the basolateral sorting sequences
of MCT3 and MCT4.
Methods: Co-expression of MCT3 and MCT4 truncation mutants in cells stably
expressing CD147-L252A provided us with a model system to identify the general
location of their basolateral sorting sequences (BLSS). p75-MCT chimeric constructs
transfected into MDCK cells were used to identify specific residues required
for basolateral sorting. Polarity of all constructs was assessed using confocal
immunofluorescence microscopy.
Results: BLSS were found within the C-terminal cytoplasmic tails of MCT3 and MCT4,
while the C-terminal tail of MCT1 did not harbor basolateral sorting activity. Two BLSS
were present in the C-terminal tail of MCT3 which both contained acidic clusters. A
single bipartite sorting sequence comprised of an upstream acidic cluster and a down
stream di-proline was identified in the C-terminal tail of MCT4.
Conclusions: Trafficking of most heteromeric transporters to the plasma membrane
is regulated by the catalytic subunit. In contrast, we have shown that trafficking of
MCT/CD147 lactate transporters to the plasma membrane can be regulated by either
the catalytic subunit or the accessory subunit depending on the MCT isoform. Our
studies pave the way to a molecular understanding of the trafficking of heteromeric
solute transporters.
CR: N.J. Philp, None; J.J. Castorino, None; E.J. Rodriguez-Boulan, None.
Support: EY-012042 (NP), EY08538 (ERB) GM 34107 (ERB)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
463-466
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
467 - A31
Modulation of Cytokine Expression by Protein Tyrosine Phosphatase 1b
Treatment in Retinal Pigment Epithelial Cells
468 - A32
Role of Lipofuscin in Differential Sensitivity of RPE Cells to Sub-Lethal and
Lethal Photic Stress
D.A. Maerker, R. Foeckler, V. Milenkovic, O. Strauss, H. Helbig, T. Dietrich.
Ophthalmology, University of Regensburg, Regensburg, Germany.
M. Zareba1, T.J. Sarna2, J.M. Burke1. 1Ophthalmology, Medical College of Wisconsin,
Milwaukee, WI; 2Biophysics, Jagiellonian University, Krakow, Poland.
Purpose: The retinal pigment epithelium (RPE) plays an important role in degenerative
and neovascular diseases of the retina. Protein tyrosin phosphatases (PTP) regulate
signaling pathways of essential cellular processes; their role in diseases of retina and
RPE has not been determined yet. The PTP interacting protein 51 (PTPIP51) has been
identified in human embryonic and adult RPE tissue. In order to analyze PTPs and
their functional role in RPE, immunohistochemistry and cell culture experiments
using specific PTP inhibition were performed.
Methods: Immunohistochemical analysis of human and mouse RPE in situ was
performed using specific PTP1B antibodies (Abcam, Calbiochem). Human retinal
pigment epithelium cells (ARPE19) were cultured and incubated for 24 hours
and 48 hours with the specific protein tyrosine phosphatase inhibitor bpV(phen)
(peroxovanadium 1,10-phenanthroline) to analyze the impact on cytokine expression.
Cell lysates were used for quantitative real time RT-PCR. The expression of mRNA
of the cytokines vascular endothelial growth factor A (VEGF-A), thrombospondin-1
(TSP-1), transforming growth factor beta 1 and 2 (TGFß-1/-2), connective tissue growth
factor (CTGF), and pigment epithelium-derived factor (PEDF) was analyzed.
Results: Immunohistochemistry of human and mouse adult tissue revealed PTP1B
positive staining in retinal pigment epithelium in situ. A focal staining pattern of
PTP1B was observed in human tissue. PTP inhibition by incubation for 24h/48h with
bpV(phen) resulted in a 9 fold/6 fold higher expression of VEGF-A in ARPE19 cells
compared to control, detected by RT-PCR. TSP-1 expression was 2 fold higher after
24h while the expression level of TSP-1 was not significantly different from control
after 48h incubation. TGFß-1 expression was increased 3 fold after 24h and remained
on that level after 48h. TGFß-2 and CTGF expression doubled the control after 24h,
while there was no detectable difference after 48h incubation. PEDF mRNA levels
remained unchanged after 24h, whereas 48h incubation resulted in a 2 fold increase.
Conclusions: PTP1B is expressed in human and mouse RPE. PTPs seem to be of
functional importance for cellular processes as cytokine expression, because treatment
with PTP inhibitor bpV(phen) resulted in modulation of cytokine expression, e.g. in
increased VEGF-A, TSP-1, CTGF, TGFß-1 and -2, and PEDF levels. This finding might
offer new insights and therapeutic options for neovascular degenerative retinal
diseases such as age related macular degeneration.
CR: D.A. Maerker, None; R. Foeckler, None; V. Milenkovic, None; O. Strauss,
None; H. Helbig, None; T. Dietrich, None.
Support: REFORM program (project 3005302), University of Regensburg
Purpose: Photic stress induced by sub-lethal visible light slows organelle movement in
cultured RPE cells, especially for organelles made experimentally photoreactive. Here
we asked whether endogenous human RPE lipofuscin granules and melanosomes, both
of which are photoreactive (though lipofuscin is more so), are differentially sensitive
to light-induced motility slowing. We also asked whether variable lipofuscin content
predicts the susceptibility of cells to killing by higher light doses.
Methods: Confluent primary cultures of human RPE containing both granule types
were treated with blue light (405 nm) delivered by the light source of a microscope
equipped for live cell imaging. Movement of the same granules was tracked before
and after sub-lethal light treatment using MetaMorph software and bright field images
captured at 1 s intervals over 3 min. Lipofuscin granules were discriminated from
melanosomes in the same cells by fluorescence imaging. Cell death on higher light
doses was determined by adding propidium iodide (PI) to the medium and recording
nuclear PI fluorescence in real time using images captured at 30 s intervals over 6 hrs.
Results: Sub-lethal blue light stress slowed the movement of both melanosomes and
lipofuscin granules, with greater slowing for lipofuscin granules within the same cells.
Cell death induced by higher doses of 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.
Conclusions: RPE cells are exposed throughout life to blue light. The results here
suggest that photic stress impairs the trafficking and placement of both melanosomes
and lipofuscin granules, and especially the latter. With aging as lipofuscin accumulates
and melanin declines, not only may granule movement become more sluggish, but
individual RPE cells may differ in their vulnerability to photic stress depending on
their variable lipofuscin content. Since oxidative stress, including photic stress, is
believed to diminish RPE support for the retina, those photoreceptors adjacent to RPE
cells with high lipofuscin may be preferentially at risk for degeneration.
CR: M. Zareba, None; T.J. Sarna, None; J.M. Burke, None.
Support: NEI grants R01 EY013722 and P30 EY01931; RPB
469 - A33
Vegf and Pedf Secretions Over Time Following Various Laser Irradiations on an
Rpe Organ Culture
470 - A34
Neurotrophic Factors Affect RPE Physiology
Y. Miura1,2, F. Treumer2, A. Klettner2, J. Hillenkamp2, R. Brinkmann1, R. Birngruber1, J.
Roider2. 1Institute of Biomedical Optics, University of Luebeck, Luebeck, Germany;
2
Department of Ophthalmology, University of Kiel, Kiel, Germany.
Purpose: To investigate the influences of different laser power settings for retinal
photocoagulation (from sub- to over-threshold) on the secretion of growth factors
from RPE.
Methods: RPE-choroid sheets (9 mm diameter) were isolated from freshly enucleated
porcine eyes and cultivated in perfusion culture system. Laser irradiation was
performed on the first day of cultivation (wavelength: 532nm, spot diameter: 300μm,
irradiation time: 0.1ms, power: 20-120 mW, 120 spots per explant). The threshold laser
power was determined using calcein-AM cell viability test directly after irradiation.
From the next day on, the culture medium was collected daily until day 6 and the
concentrations of vascular endothelial growth factor (VEGF) and pigment-epithelium
derived factor (PEDF) were measured with Elisa assay. The wound healing was
assessed using FITC-phalloidin staining to visualize the actin of RPE cells.
Results: Under the condition described above, the threshold laser power for immediate
RPE cell death was around 50 mW. Most of the RPE-defects by up to 120 mW were closed
until day 4. The irradiation with 40 mW did not induce any immediate cell death, but
the wound healing analysis disclosed that some small wound healing occurred after
this laser power too, which suggests the late cell death. VEGF expression on day 1 was
up-regulated in the cultures irradiated with over 40 mW in a dose-dependent manner.
After that, the VEGF secretion declined gradually and reached the control level around
day 3. The amount further decreased and showed a lower level than control on day
4 and 5. On day 6, the level came back to the control level. PEDF secretion was also
up-regulated by over-threshold laser power on day 1 and then decreased on day 2, but
again started to increase from day 3 and kept high level until day 6. With 40 mW, the
PEDF secretion increased on day 2 significantly and then decreased to the control level.
Conclusions: The secretions of VEGF and PEDF from RPE are up-regulated by laser
irradiation, even by sub-threshold laser power for immediate cell death. VEGF is
up-regulated directly following the irradiation, while PEDF secretion increases over
time. Considering these secretion patterns, it is assumed that VEGF is secreted by
the cells heated by laser irradiation, while PEDF is secreted by the cells covering the
wound or by the surrounding RPE cells secondarily-stimulated during wound healing.
CR: Y. Miura, None; F. Treumer, None; A. Klettner, None; J. Hillenkamp, None; R.
Brinkmann, None; R. Birngruber, None; J. Roider, None.
Support: German Ministry of Education and Science (BMBF) Grant 01 EZ0734
R. Li1, R. Wen2, T. Banzon1, A. Maminishkis1, S.S. Miller1. 1National Eye Institute,
National Institutes of Health, Bethesda, MD; 2Bascom Palmer Eye Institute, Miller
School of Medicine, University of Miami, Miami, FL.
Purpose: Ciliary neurotrophic factor (CNTF) has been shown to protect photoreceptors
in several models of retinal degeneration. In this work, we examined the effects of
CNTF, cardiotrophin 1 (CT1), oncostatin M (OsM), and leukemia inhibitory factor
(LIF) on RPE signaling and physiology.
Methods: Gene expression of CNTF, CT1, OsM, LIF and their receptor subunit
expression and localization were examined by quantitative RT-PCR, immunoblot,
and immunofluorescence analysis using confluent monolayers of human fetal retinal
pigment epithelium (hfRPE) primary cultures. Signal transduction was studied by
measuring the phosphorylation of STAT3 and ERK in hfRPE as well as in adult RPE
(ARPE-19). Cell proliferation was assessed by BrdU incorporation. Fluid absorption
(JV) across RPE (retina to choroidal side) was determined by a capacitance probe.
Results: The expression of CNTF, CT1, OsM and LIF, as well as receptor subunits,
including CNTFRα, LIFRβ, gp130 and OsMRβ were all detected in hfRPE by RT-PCR
and immunoblotting. The amounts of LIF and CT1 mRNA are more than CNTF and
OsM, and CNTFRα protein is below the detection level of immunoblotting. All of these
receptor subunits, including CNTFRα, LIFRβ, gp130, and OSMRβ are mainly localized
at the apical membrane. Treatments of CNTF, CT1 and OsM induced phosporylation
of STAT3 in hfRPE and ARPE-19. In addition, OsM significantly activated P44/P42
(ERK) MAP kinase pathway, but not CNTF and CT1. CT1 shows a stimulatory effect
(25%) on RPE proliferation at 50 - 100 ng/ml whereas OsM significantly inhibited
hfRPE proliferation between 10 and 100 ng/ml. CNTF had no significant effect on
hfRPE proliferation. Furthermore, CNTF significantly increased fluid transport (JV)
from 8.7 ± 0.7 to 20.7 ± 3.3 μl·cm-2· hr-1 (n= 3; P < 0.05).
Conclusions: These studies demonstrate that CNTF, CT1, and OsM can activate the
JAK/STAT3 signaling pathway in human RPE (Li et al., AJP cell physiology, 2009),
raising the possibility that the neuroprotective effects of these factors on photoreceptors
are partially mediated by RPE activity.
CR: R. Li, None; R. Wen, None; T. Banzon, None; A. Maminishkis, None; S.S.
Miller, None.
Support: NIH Intramural Research Program
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
467-470
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
472 - A36
Lipofuscin Distribution in Retinal Pigment Epithelium of Rhesus Monkeys:
Effects of Age, n-3 Fatty Acids, and Xanthophylls
471 - A35
The Critical Role of Bcl-xL in Mouse Retinal Pigment Epithelial Cells
S.A. Medearis, I. Han, P. Yang, N. Zhang, J.J. Peairs, G.J. Jaffe. Duke Eye Center, Duke
University Medical Center, Durham, NC.
Purpose: We have previously demonstrated a crucial role for Bcl-xL as a human RPE
(hRPE) cell survival protein. Mouse cells and tissues are frequently used as a model
for human disease. Herein, we determined the role of Bcl-xL as a survival protein in
cultured mouse RPE (mRPE) cells.
Methods: Cultured mRPE cells were treated with media alone, or with human IL1-β, human TNF-α or mouse TNF-α for 24 hours. Real-time reverse transcription
polymerase chain reaction (qRT-PCR) was used to determine survival factor gene
expression. Cultured human and mouse cells were transfected with modified,
2’-O-methoxyethoxy Bcl-xL-mismatched control antisense oligonucleotides (ASOs)
and Bcl-xL-specific ASOs, and protein and RNA were isolated. Relative gene expression
of anti- and pro-apoptotic survival genes was determined by qRT-PCR, and western
blot-analyzed proteins. Cell count quantified cell viability post-transfection.
Results: In mRPE cells, Bcl-xL was the most highly expressed of all survival factors.
Expression of the anti-apoptotic genes Traf-1, c-IAP1, and c-IAP2 was upregulated
in mRPE cells treated with TNF-α, while Bcl-xL expression was not significantly
affected, similar to what we have found in hRPE cells. Upon treatment with Bcl-xLspecific ASOs, Bcl-xL gene expression and protein levels were markedly decreased
after 3 days post-transfection. Following this same treatment, Traf-1 expression was
increased, while expression of all other anti-apoptotic genes was either unchanged,
or modestly decreased, relative to controls. The number of human and mouse cells
treated with Bcl-xL-specific ASO was significantly decreased when compared to the
number of cells treated with control ASOs (p < 0.05).
Conclusions: Cultured mRPE cells are highly similar to cultured hRPE cells with
respect to the role and function of the survival factor Bcl-xL. Bcl-xL is a critical protein
present in mRPE cells that promotes the cell’s survival. Based on the similarity between
survival factors within hRPE in situ and in vitro to that of mRPE in vitro, cultured
mRPE may serve as a useful model for hRPE cell studies and human pathology,
particularly for macular diseases in which RPE cell survival is critical.
CR: S.A. Medearis, None; I. Han, None; P. Yang, None; N. Zhang, None; J.J. Peairs,
None; G.J. Jaffe, None.
Support: Core Grant 930EY05722
473 - A37
Increased RPE Microvillus Density in Mice Lacking MFRP
M. Snodderly1, I.Y. Leung2, M. Neuringer3,4. 1Nutritional Sci, University of Texas at
Austin, Austin, TX; 2Wilmer Eye Institute, Johns Hopkins University, MD; 3ONPRC,
Beaverton, OR; 4Casey Eye Institute, Oregon Health and Science University, OR.
Purpose. To study the effects of n-3 fatty acids, lutein and zeaxanthin on the distribution
of lipofuscin in the retinal pigment epithelium (RPE).
Methods. Seventeen rhesus monkeys, 7-18 years of age, were fed xanthophyll-free
semipurified diets with either low or adequate amount of n-3 fatty acids from birth and
had no macular pigment. Five were supplemented with lutein and six with zeaxanthin
for 6 to 24 months while six remained on the xanthophyll-free diet. Following sacrifice,
the central retina was sectioned at 2 µm along the vertical meridian through the fovea.
Fluorescent images of the central retinal sections were taken with Texas Red and/or
FITC filter sets. The areas occupied by the autofluorescent lipofuscin granules of the
RPE were measured along the vertical meridian from zero to 2.64 mm eccentricity
and compared with data from age-matched control monkeys (n = 7) fed a standard
laboratory diet.
Results. In animals fed the xanthophyll-free diet low in n-3 fatty acids, the RPE
accumulated more lipofuscin and at a faster rate with advancing age than control
animals (p 0.1).
Conclusions. Long-term dietary deprivation of n-3 fatty acids, in the absence of lutein
and zeaxanthin, increased the accumulation of lipofuscin in the parafoveal RPE.
Shorter-term supplementation with zeaxanthin or lutein did not reverse this effect.
Reduction in lipofuscin levels is one mechanism by which n-3 fatty acids may confer
protection from age-related macular disease.
CR: M. Snodderly, DSM Nutritional Products Ltd, F; I.Y. Leung, DSM Nutritional
Products Ltd, F; M. Neuringer, DSM Nutritional Products Ltd, F.
Support: DSM Nutritional Products Ltd., NIH P30 EY03790, NIH DK-29930, NIH RR00163, The Foundation Fighting Blindness, John Linn Foundation, Dennis Gierhart
Foundation
474 - A38
ARPE-19 Cell Death by the Alternative Complement Cascaded. Role of Cell
Surface Regulatory Proteins, Calcium, PKC and Oxidative Stress
J. Fogerty, J.C. Besharse. Cell Biology, Neurobiology, and Anatomy, Medical College
of Wisconsin, Milwaukee, WI.
P. Baciu, M. Etheridge, B. Parker, E. Sung. Biology, Allergan, Inc, Irvine, CA.
Purpose: MFRP is a type-II transmembrane protein that is expressed on the apical
membrane of the RPE. Rd6 mice have a splicing mutation in mfrp, rendering it
undetectable at the protein level. Previously we identified a novel mutation in mouse
mfrp, called rdx, which is likewise a null mutation at the protein level. The phenotype
of both of these mutants includes progressive photoreceptor degeneration and white
fundus flecks corresponding with pigmented cells in the subretinal space. Furthermore,
we have previously shown evidence of geographic atrophy of the RPE in 21-month
old rdx mice. We have now further characterized these mutants by examining the
ultrastructure of the RPE of mutant animals before onset of RPE atrophy, in an effort
to determine a key deficiency that may lead to photoreceptor degeneration. We also
have evaluated mRNA and protein expression of MFRP and its binding partner,
CTRP5. Finally, since CTRP5 has been shown to activate AMPK in L6 myoblasts, we
tested for the presence of a similar pathway in the RPE, and examined the possibility
that MFRP might mediate this signaling mechanism.
Methods: For ultrastructural studies of RPE, eyecups were treated with 1%
hyaluronidase in HBSS for 40 minutes, and the neural retina was gently removed.
Tissues were then processed for standard TEM. We used quantitative PCR and western
blotting to analyze expression of MFRP and CTRP5. Activation of AMPK was measured
using antibodies specific to total AMPKα and phospho-AMPKα (Thr 172).
Results: We found that the density of RPE microvilli is increased in both rd6 and
rdx animals, while their length is unaffected. Furthermore, we noted large, lipid-rich
cytoplasmic inclusions in rdx mice that were not observed in controls. MFRP protein
is undetectable in mutants, but the corresponding mRNA is upregulated. CTRP5
message is likewise upregulated, and CTRP5 protein is increased as well. We also
found AMPK to be activated in mutants compared to controls.
Conclusion: Mice with mutations in mfrp have increased RPE microvilli. Cytoplasmic
inclusions in rdx mice were suggestive of lipoprotein accumulation. Mfrp mRNA
is upregulated in mutant animals, suggesting that the gene may be somewhat
autoregulatory. Interestingly, our evidence suggests that mfrp mRNA in rdx mice
is not subject to nonsense-mediated decay. The upregulation of CTRP5 in mutant
RPE, which clearly has an abnormal phenotype, is consistent with previous reports
of CTRP5 upregulation and subsequent AMPK activation in L6 myoblasts during
stress response. Finally, we have also shown that MFRP is not required for AMPK
activation in RPE in vivo.
CR: J. Fogerty, None; J.C. Besharse, None.
Support: NIH Grants EY02414, EY014537
Purpose: To investigate mechanisms regulating complement mediated cell death
of RPE cells.
Methods: ARPE-19 cells were primed with a complement fixing antibody followed
by challenge with human serum deficient in C1q. Effects of complement attack on
cell lysis were monitored by measuring uptake of cell impermeant nuclear dyes.
Development of membrane attack complexes (MAC) were monitored by cell swelling,
C5b-9 FACS, Ca++ influx and ATP release. SiRNA’s were used to knock down either
CD46, CD55 or CD59. Contribution of calcium and PKC to protect ARPE-19 cells
was determined by depletion of extracellular calcium or pretreatment with PKC
inhibitors chelerythrine and G06976. The effects of oxidative stress on complement
mediated death was examined by pretreatment of cells with H2O2 or t-BH followed
by complement challenge.
Results: Complement driven cell death of primed ARPE-19 cells in C1q deficient
serum was dependent on the alternative cascade and correlated with the formation of
functional MAC on the cell surface. Down regulation of either CD46 or CD59 modestly
enhanced cell death while inhibition of PKC double that observed in CD46 and CD59
knockdown studies. The greatest increase in cell death was observed after calcium
depletion or pretreatment with H2O2.
Conclusions: We have established an in vitro assay of complement driven RPE cell
death mediated by the alternative cascade. While complement regulatory proteins
provided protection, cellular pathways regulated by calcium and PKC appear to play
a more significant role in prevention of cell death. The most significant finding is the
observed synergy between complement and oxidative stress. This synergy appears
to reflect the impact of oxidative stress on cellular pathways involved in protection
against the MAC and not modulation of cell surface regulatory proteins.
CR: P. Baciu, Allergan inc., E; M. Etheridge, Allergan Inc, E; B. Parker, Allergan Inc,
E; E. Sung, Allergan Inc, E.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
471-474
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
475 - A39
Effect of VEGF and Anti-VEGF Compounds on Retinal Pigment Epithelium
Permeability: An in vitro Study
476 - A40
Reduction of Lipofuscin-Like Autofluorescence in RPE Cells by Sustained D1/
D5 Receptor Stimulation
C. Campa1,2, V. Kearns1, C. Sheridan1, R. Williams1, I. Grierson1, S.P. Harding1,2.
1
Ophthalmology Research Unit, School of Clinical Sciences, University of
Liverpool, Liverpool, United Kingdom; 2St. Paul’s Eye Unit, Royal Liverpool
University Hospital, Liverpool, United Kingdom.
S. Guha1A, L.-A. Tu1B, G. Baltazar1B, A. Argalla1A, A.M. Laties1C, C.H. Mitchell1A,1B.
A
Physiology, School of Medicine, BAnatomy and Cell Biology, School of Dental
Medicine, COpthalmology, School of Medicine, 1University of Pennsylvania,
Philadelphia, PA.
Purpose: The purpose of this study was to evaluate the effect of two VEGF isoforms
(121 and 165) and two anti-VEGF compounds (ranibizumab and pegaptanib sodium)
on the permeability of retinal pigment epithelium (RPE) cells in vitro.
Methods: RPE permeability was assessed on ARPE19 cells grown onto inserts of
polytetrafluoroethylene previously treated with ammonia gas plasma. Paracellular
permeability to ions was measured by means of transepithelial electrical resistance
(TEER). Permeability to non-ionic molecules was gathered by the amount of 4kDa
or 70 kDa fluorescein dextran (FD) passing across the monolayer within 2 hours.
VEGF isoforms, ranibizumab and pegaptanib sodium were added at the culture
media singularly or as a combination (VEGF + anti-VEGF compound) before each
permeability assay. Doses were: for recombinant human VEGF121 and VEGF165 10ng/
ml, for ranibizumab 0.125mg/ml and pegaptanib sodium 0.08 mg/ml. Control wells
contained standard culture media with 1% fetal bovine serum and IgG from human
serum 0.1mg/ml, respectively. All the experiments were performed in triplicate with
the final value being averaged.
Results: Only VEGF165 applied at the apical side of the monolayer induced a statistically
significant decrease of TEER (p=0.001). No changes in TEER were observed when
pegaptanib sodium or ranibizumab were apically administered together with VEGF165.
Both VEGF isoforms significantly increased permeability to 4kDa dextran (p<0.01).
Apical administration of ranibizumab or pegaptanib sodium per se as well as
co-administration with VEGF121 induced a statistically significant increase of
permeability to 4 kDa FD (p=0.001, p=0.009, p=0.0003, p=0.006, respectively). Little
or no effect on permeability was noted after co-administration of VEGF165 with
pegaptanib (p=0.02) or ranibizumab (p=0.07).
Conclusions: Both VEGF isoforms and anti-VEGF compounds seem to have an effect
on human RPE permeability in vitro. The extent and the characteristics of this effect
is however complex and requires further investigations.
CR: C. Campa, None; V. Kearns, None; C. Sheridan, None; R. Williams, None; I.
Grierson, None; S.P. Harding, None.
Support: Foundation for the Prevention of Blindness
Purpose: Spent photoreceptor outer segments are engulfed and degraded by RPE
cells. Incomplete or faulty degradation may lead to the accumulation of amorphous
material including autofluorescent lipofuscin. Degradative lysosomal enzymes are
pH dependent and function optimally in an acidic environment. We have previously
demonstrated that elevation of lysosomal pH can reduce the ability of RPE cells to
clear labeled outer segments, that elevation of cAMP can restore an acidic pH, and
that receptors coupled to the Gs protein can restore lysosomal pH in compromised
cells. In this regard, the joint D1/D5 dopamine receptor agonist SKF81297 reacidifies
lysosomes of RPE cells. In this study, we asked whether SKF81297 can induce a
prolonged reacidification of RPE cells, whether the agonist can actually reduce outer
segment autofluorescence, and which receptor subtype mediates the reacidification.
Methods: Lysosomal pH was determined from ARPE-19 cells grown in 96 well plates
using Lysosensor Yellow/Blue dye. Cells were treated with chloroquine to raise the
lysosomal pH and the ability of SKF81297 to restore this pH was tracked over time.
Cells were transfected with RNAi against the D1 or D5 dopamine receptors and
lysosomal pH measured 2-3 days later to determine which receptor was mediating
the effects of SKF81297. Cells were exposed to bovine photoreceptor outer segments,
then SKF8197, 3x over a week before autofluorescence at 488 nm was determined
using a flow cytometer.
Results: A single exposure to 10 µM SKF81297 lowered lysosomal pH in chloroquinetreated RPE cells for 7 days. The restoration of acidity was cumulative, with the pH
fully recovered after 7 days. SKF81297 acidified lysosomal pH in control cells, but had
no effect in cells transfected with RNAi for the D5 receptor. Transfection with RNAi
against the D1 receptor did not interfere with the acidifying actions of SKF81297.
The autofluorescence excited at 488 nM was substantially increased in cells fed outer
segments, but treatment with SKF81297 decreased this autofluorescence by 54 ±4%.
Conclusions: SKF81297 induced a sustained restoration of lysosomal pH in compromised
RPE cells and a reduction of lipofuscin-like autofluorescence. These actions are likely
mediated by stimulation of the D5 receptor. It remains to be determined whether
stimulation of the D5 receptor can reduce lipofuscin accumulation in vivo.
CR: S. Guha, None; L.-A. Tu, None; G. Baltazar, None; A. Argalla, None; A.M. Laties,
0247483, P; C.H. Mitchell, 0247483, P.
Support: EY-013434, EY-015537, EY-001583 (CHM);Research to Prevent Blindness,Paul
and Evanina Bell Mackall Foundation Trust (AML)
477 - A41
Age-Related Changes in the Cytoskeleton of the Retinal Pigment Epithelium
478 - A42
MicroRNA-204/211 Regulates Human Retinal Pigment Epithelial Physiology
K.G. Shadrach, J.G. Hollyfield, V.L. Bonilha. Ophthalmology, Cole Eye Institute /
Cleveland Clinic Lerner College of Medicine, Cleveland, OH.
C. Zhang1, F. Wang1, A. Maminishkis1, L. Dong1, C. Zhi1, J. Zhao1, V. Majerciak 2, S. Chen1,
S.S. Miller1. 1National Eye Institute, National Institutes of Health, Bethesda, MD;
2
National Cancer Institute, National Institutes of Health, Bethesda, MD.
Purpose: An intimate interaction between the apical microvilli of the retinal pigment
epithelium (RPE) and the outer segments of the retinal photoreceptors is essential
for vision. Age-related changes in the retina accompany visual impairment in the
elderly. As the RPE ages it displays a number of key changes that are notable at the
ultrastructural level. The precise molecular mechanisms underlying these changes are
not well understood. In the present study the age-related changes in ezrin and other
cytoskeletal proteins present in the RPE apical microvilli were investigated in rats.
Methods: The eyes of F344BN 3-4 month old (young adult), 18 month-old (intermediate)
and 24-25 month old (aged) rats were analyzed. For transmission and scanning electron
microscopy eyes were fixed in 2.5% glutaraldehyde. For tissue immunohistology eyes
were fixed in 4% paraformaldehyde and processed for cryosectioning. Cryosections
were probed with several antibodies specific to proteins previously localized to the
RPE apical surface such as ezrin, EBP50, and neuroglycan C.
Results: Electron microscopy revealed a decrease in RPE apical microvilli density of in
aged rats. The labeling in cryosections of the RPE apical surface with ezrin and EBP50
was reduced while neuroglycan C staining did not significantly change during aging.
In addition, overall ezrin content in RPE lysates were similar in young and old animals.
Conclusions: RPE apical microvilli decreased in density in aging rats. However,
total levels of ezrin in RPE lysates did not significantly decrease in aged RPE cells.
Additional experiments will analyze the role of posttranslational modifications and
protein associations of ezrin during RPE aging.
CR: K.G. Shadrach, None; J.G. Hollyfield, None; V.L. Bonilha, None.
Support: Supported by NIH grants EY017153 and EY15638, a Research to Prevent
Blindness Unrestricted Grant and funds from the Cleveland Clinic Foundation.
Purpose: To determine a set of microRNAs that are enriched in human RPE compared
to retina and choroid. Using a human fetal retinal pigment epithelial (hfRPE) culture
model, we studied the regulation of miR-204/211 on RPE physiology.
Methods: Primary hfRPE cultures were obtained as described previously. miRNA
in human RPE, retina and choroid were profiled using the TaqMan® MicroRNA
Assays Human Panel Early Access Kit. Q-RT-PCR was carried out with TaqMan®
microRNA assay. miRNA Northern blots were done with the LNATM probes and
in situ hybridization was performed with digoxigenin (Dig)-labeled probes. Direct
miRNA targets were determined by a dual luciferase reporter assay using pEZXMT01- target constructs. RPE physiology on intact monolayers of hfRPE was studied
as previously described.
Results: We determined eight miRNAs that are enriched (>10 fold) in RPE (miRs184,187, 200a/200b, 204/211, 221/222) compared to neuroretina or choroid (p<0.05).
Five of these miRNAs are enriched in RPE compared to 20 tissues throughout the
body and are >10,000 fold more highly expressed (p<0.005) and miR-204/211 are the
most highly expressed. We determined that TGFβR2 and Snail2 are direct targets of
miR-204. Addition of anti-miR-204 significantly decreased: (1) transepithelial resistance
(TER); (2) cell membrane potentials; (3) claudin expression (RNA and protein); (4) Kir
7.1 (K channels) (protein).
Conclusions: miR-204 regulation of RPE membrane physiology is mediated through
a TGFβ /SNAI2 signaling pathway that controls the expression of claudins in the tight
junctions and Kir 7.1 in the plasma membranes. The present experiments provide the
basis for understanding how miR-204/211 regulates RPE tight junction integrity and
maintain the blood/retina barrier in a quiescent state, which is a hallmark of epithelia.
CR: C. Zhang, None; F. Wang, None; A. Maminishkis, None; L. Dong, None; C.
Zhi, None; J. Zhao, None; V. Majerciak, None; S. Chen, None; S.S. Miller, None.
Support: NIH intramural research
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
475-478
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
479 - A43
Lactic Acid Activates Kir7.1 and ClC-2 in Human Retinal Pigment Epithelium
J. Adijanto1,2, Q. Wan1, N.J. Philp3, R. Li1, S.S. Miller1. 1National Eye Institute, Bethesda,
MD; 2Chemical and Biomolecular Engineering, The University of Maryland,
College Park, MD; 3Path/Anat/Cell Biology, Thomas Jefferson University,
Philadelphia, PA.
480 - A44
Extracellular ATP Induces Ca 2+ Signaling and Apoptosis in Human Retinal
Pigment Epithelium
D. Yang1, S.G. Elner1, A.J. Clark1, H.R. Petty1,2A, V.M. Elner1,2B. 1Ophthal & Vis Sci, Univ
of Michigan-Kellogg Eye Ctr, Ann Arbor, MI; AMicrobiology and Immunology,
B
Pathology, 2University of Michigan, Ann Arbor, MI.
Purpose: In the dark, the photoreceptors produce lactic acid that is released to the
subretinal space (SRS) and crosses the RPE apical membrane via monocarboxylate
transporters (MCT1). In this study, we describe the ensuing physiological changes
that take place within the RPE, which could in turn affect the chemical composition
of the SRS.
Methods: All experiments were performed with confluent monolayers of cultured
human fetal RPE (hfRPE) grown on transwells (Maminishkis et al., IOVS, 2006). A pHsensitive fluorescence dye (BCECF) was used to monitor intracellular pH (pHi) while
simultaneously recording transepithelial potential (TEP) and total epithelial resistance
(RT). Intracellular microelectrodes measured apical and basolateral membrane voltages
and resistances (VA, VB & R A/RB), TEP and RT.
Results: Microelectrode experiments showed that adding lactate to the apical bath
acidified the cells & produced a 2-phased electrical response: V B depolarization
followed by VA hyperpolarization. VB depolarization is consistent with activation of
Cl-channels at the basolateral membrane, possibly ClC-2. Immunofluorescence show
that ClC-2 is localized at the basolateral membrane, and physiological experiments
with ClC-2 inhibitor (Zn 2+) indicate that ClC-2 was activated by apical lactate. In
addition, microelectrode experiments show that apical lactate activated a Ba2+-sensitive
K-channel at the apical membrane, probably Kir7.1. Intracellular acidification also
activated ClC-2 and Kir7.1.
Conclusions: Our data show that apical H/Lac entry via MCT1 caused intracellular
acidification that subsequently activated Kir7.1 at the apical membrane and ClC-2 at
the basolateral membrane. Lactic acid transport has been shown to cause cell swelling,
and by stimulating KCl efflux via Kir7.1 and ClC-2, the RPE prevents swell-induced
osmotic stress. In addition, lactate induced activation of Kir7.1 allows the RPE to
regulate K-level in the subretinal space following transitions between light and dark.
CR: J. Adijanto, None; Q. Wan, None; N.J. Philp, None; R. Li, None; S.S. Miller, None.
Support: NIH intramural research
Purpose: Studies have indicated that a variety of cell systems express P2 purinoceptors
and undergo functional changes in response to extracellular adenosine triphosphate
(ATP). The purpose of this study was to determine whether extracellular ATP induces
Ca2+ signaling and apoptosis in primary cultures of human retinal pigment epithelial
(RPE) cells.
Methods: Intracellular calcium levels were determined using calcium probe, indo1-AM. P2X7 mRNA and protein expression were detected by reverse transcriptionpolymerase chain reaction (RT-PCR) and immunofluorescence microscopy, respectively.
RPE apoptosis was evaluated by caspase-3 detection, Hoechst staining and cell death
detection ELISA.
Results: Both ATP and the P2X7 agonist benzoylbenzoyl ATP (BzATP) increased
intracellular Ca2+ in RPE cells. The Ca2+ induced by ATP was delayed and significantly
inhibited by oxidized ATP (oATP), a P2X7 antagonist. Suramin only slightly inhibited
the Ca2+ increase. ATP treatment increased the number of caspase-3 positive and
apoptotic RPE cells, and DNA fragmentation. All these increases were significantly
reduced by oATP. RT-PCR and immunofluorescence microscopy revealed the presence
of P2X7 receptor mRNA and protein in RPE cells.
Conclusions: Extracellular ATP induces RPE Ca2+ signaling and apoptosis. Activation
of P2 purinoceptors including P2X7 might contribute to ATP-induced Ca2+ signaling
and apoptosis in the RPE.
CR: D. Yang, None; S.G. Elner, None; A.J. Clark, None; H.R. Petty, None; V.M.
Elner, None.
Support: NIH grants EY09441 (V.M. Elner), CA74120 (H.R. Petty) and P30EY07003
(core). VME is a recipient of Lew R. Wasserman Merit Award from Research to Prevent
Blindness.
481 - A45
Lipofuscin Can Be Eliminated From Retinal Pigment Epithelium After Drug
Treatment
482 - A46
Effect of Terminal Complement Membrane Attack Complex/C5b-9 on RPE Cell
Viability
S. Julien, A. Biesemeier, P. Heiduschka, M. Rittgarn, S. Schultheiss, E. Winkler, S.
Hofmeister, U. Schraermeyer. Section of Exp Vitreoretinal Surgery, Institute for
Ophthalmic Research, Tuebingen, Germany.
P. Yang, S.A. Medearis, G.J. Jaffe. Ophthalmology, Duke University Eye Center,
Durham, NC.
Purpose: Lipofuscin is a pigment that is formed in tissues with high oxidative stress. It
is generally believed that the gradual accumulation of this pigment is the constant sign
of ageing and that the retinal pigment epithelial (RPE) cells could not eliminate their
lipofuscin during life. In a pre-clinical study involving 66 monkeys and focused on a
new treatment for acid-related diseases, we discovered that the oral administration
of tetrahydropyridoethers (THPE) leads to a significant removal of lipofuscin from
RPE cells. The aim of this study was to confirm this observation in cultures of human
aged RPE cells.
Methods: The human RPE cells were obtained from seven organ donors aged between
36 and 75 years. The cells were incubated with different concentrations of a THPE
compound or vehicle. Changes in pigmentation were regularly observed by light,
fluorescence and electron microscopy till 36 days. Cell viability and phagocytic
activity were tested as well.
Results: We previously showed that after exposure to THPE, RPE cells
of monkeys lose lipofuscin. These in vivo experiments indicated that
lipofuscin granules, melanosomes as well as melanolipofuscin granules were
released to macrophages. These were frequently present and were located
between the Bruch’s membrane and the RPE or in the subretinal space.
The cultured RPE cells showed 100% survival at THPE concentrations up
to 0.25 mM, whereas most of the cells died within days at 0.5 mM. Instead
of the grainy yellow-golden lipofuscin autofluoerescence, a diffuse whitebluish glow appeared in the RPE cells indicating degradation of lipofuscin
under the influence of the THPE compound. Pigment granules containing
lipofuscin disappeared completely in an increasing portion of the cultured cells.
The RPE cells regained their ability to phagocytose after THPE-induced removal of
lipofuscin. Depending on the degree of lipofuscin loss, phagocytic activity increased
by 10 to 20 times compared to controls.
Conclusion: The present study demonstrates that the dogma that lipofuscin can not be
removed from RPE cells has not be perpetuated any more. Our finding that removal
of lipofuscin is possible has great implication for the treatment of lipofuscin-related
diseases, in particular the dry age-related macular degeneration.
CR: S. Julien, None; A. Biesemeier, None; P. Heiduschka, None; M. Rittgarn, None; S.
Schultheiss, None; E. Winkler, None; S. Hofmeister, None; U. Schraermeyer, None.
Support: None
Purpose: Complement activation has been implicated increasingly in the pathogenesis
of age-related macular degeneration (AMD). Complement membrane attack complex
(MAC)/C5b-9, the end product of complement cascade, is present in drusen and in
experimental CNV mouse models. Interestingly, sublytic MAC/C5b-9 induces cell
proliferation by survival pathways such as PI3K/Akt or MAPK pathways in some
non-ocular nucleated cells. Herein, we investigate the effect of MAC/C5b-9 deposition
on RPE viability and signal transduction pathways.
Methods: MAC/C5b-9 was assembled on cultured human RPE cells with either purified
complement components (C5b6-C9) or emmprin antibody and 10% normal human
serum (NHS, a complement source). MAC/C5b-9 complement components minus C7
(a MAC/C5b-9 complement component) and heat-inactived NHS (HiNHS) were used
as negative controls. MAC/C5b-9 formation was determined by immunofluorescent
stain using C5b-9 antibody. The lytic activity of MAC/C5b-9 was examined in sheep
erythrocytes. The effect of MAC/C5b-9 deposition on RPE cell viability was assessed
by tetrazolium salt (WST-1) assay and a lactate dehydrogenase (LDH) release assay.
MAC/C5b-9 deposition-induced phosphorylation of Erk, Akt, P38, and JNK was
examined by Western blot. MAC/C5b-9 deposition-induced NF-κB translocation was
evaluated by immunofluorescent double stain.
Results: There was specific immunofluorescent C5b-9 membrane staining in the
emmprin-NHS and C5b6-C9 groups, but not in the emmprin-HiNHS or minus C7
groups 15 minutes, 30 minutes, and 1 hour after complement incubation. MAC/
C5b-9 deposition-mediated cell lysis was confirmed in sheep erythrocytes 30 minute
following complement treatment. However, there was no significant LDH release or
decreased cell viability in the emmprin-NHS and C5b6-C9 groups 1 hour and 24
hours, respectively, after complement addition. Phosphorylation of Erk, Akt, P38,
and JNK, measured 15 minutes and 30 minutes following complement treatment
was not detected in the C5b6-C9 group. NF-κB translocation was not detected in the
C5b6-C9 or emmprin-NHS groups 1 hour and 2 hours after complement treatment.
Conclusions: RPE cells are resistant to MAC/C5b-9 deposition-mediated cell lysis.
This resistance may help to protect RPE cells from complement-mediated injury
normally, and in diseases such as AMD.
CR: P. Yang, None; S.A. Medearis, None; G.J. Jaffe, None.
Support: P30EY05722 (Core Grant)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
479-482
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
483 - A47
High Expression of Epithelial Membrane Protein 2 (EMP2) Decreases
Phagocytosis by ARPE-19
484 - A48
The Role of C3a Receptor and C5a Receptor in Maintenance of Retinal Function
and Structure
A.M. Chan1A, S.A. Morales1B, A. Nagy2, J. Braun1C, L.K. Gordon1A. AJules Stein Eye
Institute, BPathology & Laboratory, CPathology and Laboratory Medicine,
1
University of California, Los Angeles, Los Angeles, CA; 2Surgery & Research,
Greater Los Angeles VA Healthcare System, Los Angeles, CA.
J. Liu1A, M. Yu1B, N.S. Peachey1B, T.M. McIntyre1A. ACell Biology, BOphthalmic
Research, 1Cleveland Clinic Foundation, Cleveland, OH.
Purpose: Circadian cycle-dependent phagocytosis of photoreceptor outer segments
(POS) by retinal pigment epithelial (RPE) cells is essential for the mammalian visual
system. Integrin αvβ5 and activated focal adhesion kinase (FAK) are crucial for POS
phagocytosis. EMP2 regulates formation of α/β integrin heterodimers and activation
of integrin-FAK signaling complexes. The goal of this study was to determine whether
changes in EMP2 expression levels influence POS phagocytosis.
Methods: Wild type, high EMP2-expressing (ARPE-19/EMP2) and EMP2-knock
down (ARPE-19/EMP2siRNA) cells were assayed for phagocytosis using labeled
bovine POS. Bound and internalized POS were quantitated by immunfluorescence
image capture (Ariol SL-50). Levels of αvβ5 integrin were detected by flow cytometry,
immunofluorescence and immunoprecipitation followed by Western Blot analysis.
Results: In comparison to the ARPE-19 cells, ARPE-19/EMP2 cells exhibited decreased
POS phagocytosis (P<0.01). Concordantly, ARPE-19/EMP2siRNA cells showed
increased POS phagocytosis (P<0.05). Analysis of the percent distribution of bound
and internalized POS identified a significant reduction in binding of POS to the
surface of high EMP2-expressing cells (P<0.01), but enhanced internalization (P<0.05)
consistent with the enhanced FAK activation by EMP2 overexpression. β5 integrin
and αvβ5 integrin complex expression is significantly decreased in the high EMP2
expressing cells (P<0.05).
Conclusions: Elevated EMP2 level significantly reduces the overall POS phagocytosis
in cultured ARPE-19 cells by down-regulating αvβ5 integrin. The increased POS
engulfment rate resulting from the enhanced activation of the downstream signal pFAK
cannot compensate for this diminished binding capacity. Changes in EMP2 expression
levels in RPE cells in vivo could potentially lead to alterations in phagocytosis of POS.
CR: A.M. Chan, None; S.A. Morales, None; A. Nagy, None; J. Braun, None; L.K.
Gordon, None.
Support: VA MERIT Grant (LKG) and AI52031 (SAM)
485 - A49
The R162W Mutation in Kir7.1 Causes Dominant-Negative Dysfunction of WildType Kir7.1 Channels
X.-M. Zhang1, H. Wang1, A.K. Sharma2, A.O. Edwards3, B.A. Hughes1. 1Ophthalmology
& Visual Sciences, University of Michigan, Ann Arbor, MI; 2Experimental
Pathology, Mayo Clinic, Rochester, MN; 3Institute for Molecular Biology, University
of Oregon, Eugene, OR.
Purpose: Kir7.1 channels, which are tetramers formed by subunits encoded by KCNJ13,
comprise the apical membrane K+ conductance of the RPE. A mutation in KCNJ13
resulting in a R162W change in the Kir7.1 amino acid sequence was recently found to
be associated with Snowflake vitreoretinal degeneration (SVD), an inherited autosomal
dominant disease with vitreous degeneration, increased risk of retinal detachment,
Fuchs corneal dystrophy, and mild retinal degeneration. The objective of this study
was to investigate the mechanism by which the R162W mutation causes Kir7.1 channel
dysfunction.
Methods: cRNA encoding wild-type (WT) or R162W mutant human Kir7.1 was injected
alone or together into Xenopus oocytes. Protein expression was confirmed by Western
blot analysis using Kir7.1 antibodies. The two-electrode voltage clamp technique was
used to assess channel function.
Results: Western blots of membrane proteins from oocytes injected with
WT or mutant Kir7.1 cRNA revealed appropriately sized bands, confirming
the expression of both WT and mutant proteins. Oocytes injected with WT
Kir7.1 and bathed in 98 mM Rb+ exhibited large inwardly rectifying currents
(-22.1 ± 4.4 µA at -150 mV, n = 6), consistent with Kir7.1 channels. In oocytes injected
with mutant cRNA, Rb+ currents were small and likely mediated by endogenous
channels (-3.0 ± 0.9 µA at -150 mV, n = 9). Co-injection of WT and mutant Kir7.1 cRNAs
resulted in Rb+ currents that were of intermediate amplitude (-7.6 ± 1.9 µA at -150 mV,
n = 8), indicating that mutant subunits are capable of forming heteromeric channels
with WT Kir7.1 subunits and suppressing channel activity.
Conclusions: We conclude that the Kir7.1 R162W mutation results in non-functional
subunits that have a dominant negative effect on WT Kir7.1 channels. Our results
provide new mechanistic insight into the R162W mutation and may lead to a better
understanding of the multiple ocular phenotypes seen in SVD.
CR: X.-M. Zhang, None; H. Wang, None; A.K. Sharma, None; A.O. Edwards,
None; B.A. Hughes, None.
Support: NIH R01EY08850 (BAH), NIH R01EY014467 (AOE), NIH P30EY07003 (BAH),
RPB (BAH), and FFB (AOE).
Purpose: The complement activation products C3a and C5a, interacting with C3a
receptor (C3aR) and C5a receptor (C5aR), a family of G-protein couple receptors,
play an important role in preventing cell apoptosis. This study investigates their role
in retinal function.
Methods: C3aR-/-C5aR-/- (n=20), C3aR-/- (n=7), C3 -/- (n=10) mice and their WT littermates
from the age of 6 weeks up to 11 months old were studied. ERGs were used to analyze
the function of the outer retina and retinal pigment epithelium (RPE). The histological
changes of the retina were analyzed with H&E staining/light microscopy and electron
microscopy. Tissue culture studies conducted using an RPE cell line (ARPE-19)
examined the potential for complement proteins to modify apoptosis.
Results: In comparison to WT, ERG a- and b- waves were significantly decreased
in C3aR-/-C5aR-/-, C3aR-/-and C3 -/-mice, as early as 6 weeks of age. RPE function was
also decreased in C3aR-/-C5aR-/- and C3 -/-animals. Electron microscopy showed the
nuclei of RPE cells shrank in C3 -/- and C3aR-/-C5aR-/- mice. Photoreceptor cells of both
knockouts lost the normal structure of the outer segment discs. Complement activation
fragment C5a prevented ARPE-19 mitochondrial apoptosis while C5aR antagonist
induced ARPE-19 cell mitochondrial apoptotic pathway.
Conclusions: This study discovered that C3aR/C5aR deficiency impaired retinal
function and structure, and suggests that these proteins play an important role in
congenital or age-related retinal disease.
CR: J. Liu, None; M. Yu, None; N.S. Peachey, None; T.M. McIntyre, None.
Support: American Health Assistance Foundation Award M2008-063, Foundation
Fighting Blindness Center Grant, and an unrestricted grant from Research to Prevent
Blindness.
486 - A50
Phagocytic Activity of ARPE-19 Cells on Biological and Artificial Substrata
A. Dobias1A, A.K. Salz1B, P. Walter1A, G. Thumann1A,1B. ADepartment of Ophthalmology,
B
IZKF “Biomat.”, 1RWTH Aachen University, Aachen, Germany.
Purpose: Vision recovery in retinal degeneration first requires the replacement of
degenerated RPE cells and secondly, the transplanted cells must be able to communicate
with the exposed photoreceptors and fulfill the critical function of phagocytizing
outer segments (OS). Since transplantation of cell suspensions does not lead to
vision recovery, it will be necessary to transplant cells as preformed monolayers on
a biocompatible substrate. To evaluate the influence of the substrate on phagocytosis
we have investigated the phagocytic activity of pigment epithelial cell monolayers
grown on silk, amniotic membranes, and plastic.
Methods: ARPE-19 cells were cultured on biologic human amniotic membranes, silk
membranes, and plastic. When the cells reached 80% confluence they were exposed to
unlabeled or FITC-labeled photoreceptor outer segments for 24 hours at 37°C and at 4°C
to inhibit internalisation. After 24 hours the cells were trypsinized and fluorescence
intensity was determined using flow cytometric analysis. Data are expressed as the
median fluorescent index (MFI) of 5000 cells (n=4). The fluorescence at 4°C was assumed
to be surface-bound ROS and was subtracted from fluorescence at 37°C.
Results: After 24 hours at 4°C fluorescence intensity was similar on all three substrata.
The MFI on plastic was 3.1, on silk 3.7 and on amniotic membranes was 5.2. After 24
hours at 37°C fluorescence intensity was significantly higher on amniotic membranes
and on silk than on plastic. The MFI on plastic was 152.4, on amniotic membranes
270.6, a 78% increase, and on silk 261.6, a 70% increase.
Conclusions: The results show that OS phagocytosis is influenced by the substratum
and that natural biodegradable substrata enhance the phagocytic activity of retinal
pigment epithelial cells. Cells on biomaterials adhere and develop the right polarity
to accomplish the essential RPE functions.
CR: A. Dobias, None; A.K. Salz, None; P. Walter, None; G. Thumann, Spintec
Engineering GmbH, Aachen, F.
Support: IZKF “Biomat.”
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
483-486
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
487 - A51
Inhibition of Calcium-Independent Phospholipase A 2 Prevents Retinal Pigment
Epithelium Cell Death
M. Kolko, E.C. Andersen, A. Kehler, B.S. Westlund, M.H. Nissen. Eye Research Unit,
University of Copenhagen, Copehnhagen, Denmark.
488 - A52
Regulation of the Casein Kinase -2 in Retinal Pigment Epithelial Cells
S. Hoffmann1, H.S. Walter1, B. Seitz1, M. Montenarh2. 1Ophthalmology, University
Eye Hospital Saarland, Homburg, Germany; 2Department of Biochemistry,
Biochemistry, Homburg/ Saar, Germany.
Purpose: The causal mechanisms of retinal pigment epithelium (RPE) cell death
remain intriguing. We have previously identified calcium-independent phospholipase
A2 (iPLA2-VIA) in the RPE cells and shown a role of iPLA2-VIA in RPE proliferation.
Since the proliferative phenotype of RPE is only seen during pathological conditions
in which both proliferation and cell death takes place, the present study elucidates a
potential role of iPLA2-VIA in RPE cell death.
Methods: ARPE-19 cells and primary mouse-RPE cultures were treated with sodiumiodate (SI) to induce cell death. Cells were transfected with an iPLA 2-VIA promoterluciferase construct to evaluate the regulation of iPLA2-VIA after exposure to SI.
Activity assays and western blot analysis were performed to evaluate the protein level
and activity levels of iPLA2-VIA after SI-exposure. Inhibitors of iPLA2-VIA were used
to explore a potential protective role in cells exposed to SI. Primary RPE cell cultures
were grown from iPLA2-VIA knockout mice and wild type mice, respectively. The
cultures were exposed to SI to investigate a possible increased protection against SI
in iPLA2-VIA KO mice compared to wild type mice.
Results: The present study revealed an upregulation of iPLA2-VIA promoter activity,
iPLA2-VIA protein as well as iPLA2-VIA protein activity in ARPE-19 cells exposed o
SI. SI-induced cell death could be ameliorated by iPLA 2-VIA specific inhibitors in
both APRE-19 cells and in primary mice RPE cultures. RPE cultures from iPLA2-VIA
knockout mice were less vulnerable to SI-induced cell death compared to RPE cultures
from wild type mice.
Conclusions: SI -induced RPE cell death involves iPLA 2-VIA upregulation and
activation and inhibition of SI-induced RPE cell death can be ameliorated by inhibitors
of iPLA2-VIA. Hence, the present study reveals a role of iPLA2-VIA in RPE cell death,
and it is tempting to suggest iPLA2-VIA as a possible pharmaceutical target in retinal
diseases.
CR: M. Kolko, None; E.C. Andersen, None; A. Kehler, None; B.S. Westlund,
None; M.H. Nissen, None.
Support: The Danish Eye Research Foundation, The Danish Eye Health Society
Purpose: Proliferative vitreoretinopathy (PVR) is a common complication of failed
retinal reattachment surgery. The disease is considered as a form of an exaggerated
wound healing stimulated by growth factors. Retinal pigment epithelial cells (RPEs)
are regarded as the main cell type responsible for vitreoretinal scars in PVR. Their
proliferation, migration and contractions leads to retinal detachment and blindness.
Growth factors and cytokines are determinants of the disease process. Caseinkinase-2
(CK-2) is a cell signalling molecule involved in epithelial- mesenchymal transition.
Until now, the regulation, function and expression of CK-2 in RPE cells has not been
enlightened. Therefore the regulation of CK-2 by the PVR associated growth factors
PDGF and bFGF was investigated.
Methods: Bovine RPE cells were stimulated with the growth factors bFGF (10 ng/ml) and
PDGF (10 ng/ml) for 24 hours and 48 hours in DMEM with 1% fetal growth serum (FBS).
Unstimulated bovine RPE cells in DMEM with 1% FBS were used as a control. After this
time course the activity of the CK-2 was evaluated with a Western Blot. Densitometry of the
Western Blot Gel was done for quantification purposes. Furthermore, the modulation of
CK-2 in RPE cells by the specific inhibitors TBB and Quin was investigated. In addition,
the effect of simultaneous bFGF and PDGF-BB stimulation and CK-2 inhibition on RPE
cell proliferation was investigated by an MTT-assay.
Results: The PVR associated growth factors bFGF and PDGF-BB have no impact on
the expression of CK-2 in RPE cells. Furthermore, these growth factors can not abolish
the inhibitory effects of CK-2 blockage on RPE cell proliferation.
Conclusions: CK-2 expression in RPE cells is not modulated by the PVR associated
growth factors bFGF and PDGF-BB. CK-2 seems to have impact on the inhibition of
RPE cell proliferation most likely by modulating cellular apoptosis.
CR: S. Hoffmann, None; H.S. Walter, None; B. Seitz, None; M. Montenarh, None.
Support: None
489 - A53
IInduction of the Cystine/Glutamate Transporter xc- by Selenomethionine in
ARPE-19 Cells
490 - A54
Clathrin Adaptors and SNARE Proteins Direct Lysosome Exocytosis in
Polarized Retinal Pigment Epithelial Cells
P.M. Martin1A,1B, S. Ananth1A, J.P. Gnana Prakasam1A, S.B. Smith1C,1B, V. Ganapathy1A.
A
Biochemistry/Molecular Biology, BOphthalmology, CCellular Biology/Anatomy,
1
Medical College of Georgia, Augusta, GA.
A. Lakkaraju1, F. Diaz2, R. Schreiner2, E.J. Rodriguez-Boulan2. 1Ophthalmology & Visual
Sciences, University of Wisconsin-Madison, Madison, WI; 2Ophthalmology, Cornell
University Weill Medical College, New York, NY.
Purpose: Oxidative stress is believed to contribute significantly to the pathogenesis
of many diseases including age-related macular degeneration, diabetic retinopathy,
and glaucoma. Hence, development of methods for antioxidant protection is of vital
importance and may be broadly relevant in disease prevention. Selenomethionine
(SeMet) is the main dietary form of selenium, an essential trace mineral whose
antioxidant properties are well recognized. It is postulated also that dietary
supplementation of SeMet may be of benefit in preventing degenerative retinal diseases.
However, the mechanism(s) of SeMet action is not totally clear. In mammalian cells,
the cystine/glutamate exchanger xc- is the major supplier of cysteine, the limiting
amino acid in the synthesis of glutathione (GSH) and thus is an important determinant
of GSH status. The retinal pigment epithelium (RPE) normally contains high levels
of GSH. The purpose of this study was to investigate the influence of SeMet on the
activity of xc- in ARPE-19 cells, a human RPE cell line.
Methods: ARPE-19 cells were cultured in the presence or absence of SeMet. Xcactivity was monitored by the uptake of glutamate in the absence of Na+. RT-PCR,
Northern blot, immunohistochemistry and Western blot techniques were used to
analyze changes in mRNA and protein levels of xCT and of 4f2hc, the light and heavy
chains of xc-, respectively. Similar techniques were used to analyze also expression of
Nrf2, a key regulator of the antioxidant response. GSH levels were measured using
a commercial kit.
Results: Xc- activity was 3- to 4-fold higher in SeMet-treated ARPE-19 cells than in
control cells. Na+-dependent uptake of glutamate was not affected by SeMet treatment,
demonstrating specificity of the SeMet effect on xc-. Nrf2 mRNA and protein expression
was upregulated in SeMet treated cells. GSH levels were also significantly increased.
Conclusions: SeMet upregulates the cystine/glutamate transporter xc-, increases Nrf2
expression and raises intracellular GSH levels in ARPE-19 cells. This influence of SeMet
on xc- may be beneficial in protecting RPE cells against oxidant-induced damage.
CR: P.M. Martin, None; S. Ananth, None; J.P. Gnana Prakasam, None; S.B. Smith,
None; V. Ganapathy, None.
Support: None
Purpose: To identify the molecular machinery involved in the polarized exocytosis
of lysosomes in epithelial cells. Lysosomes can be induced to fuse with the plasma
membrane in response to a localized increase in intracellular calcium. This phenomenon
is essential for generating an immune response, for repairing torn plasma membranes
and can also be exploited as a mechanism to remove cellular debris.
Methods: Lysosome exocytosis in response to a calcium ionophore (ionomycin,
5-10 µM, 10 min, 37°C) was studied in polarized filter-grown ARPE-19 cells and in
model epithelia, Madin-Darby canine kidney (MDCK) cells. Polarity of lysosome
fusion was assessed by (a) the appearance of the lysosome-associated membrane
protein Lamp-2 on the cell surface by confocal microscopy and (b) the release of
lysosomal hydrolases into the apical or basal compartments by a fluorimetric assay. We
investigated the involvement of actin, microtubules and cholesterol by treating cells
with pharmacological agents. Roles of the clathrin adaptor AP-1B and the t-SNARE
syntaxin 4 in lysosome exocytosis were studied by microscopic and biochemical
analyses of stable cell lines either lacking the µ1B subunit of the AP-1 complex and/
or exogenously expressing myc-tagged syntaxin 4.
Results: In MDCK cells, lysosomes predominantly fused with the basolateral
membrane after ionomycin treatment. Actin depolymerization and cholesterol
depletion both induced exocytosis at the apical surface, whereas cholesterol overload
inhibited lysosome fusion. In µ1B-knockdown cells, lysosomes fused mainly with the
apical membrane. The polarity of syntaxin 4 correlated with the polarity of lysosome
exocytosis - basolateral in wild-type cells and non-polar in µ1B-knockdown cells.
RPE cells do not express µ1B and exhibit a non-polar distribution of syntaxin 4.
In keeping with this, although lysosomes in ARPE-19 cells mainly fused with the
basolateral membrane, there was a significant proportion (~40-45%) that fused with
the apical surface.
Conclusions: Lysosomes in RPE cells are the sites of lipofuscin accumulation, which
contributes to age-related macular degeneration. Our results show that clathrin
adaptors, t-SNAREs, membrane cholesterol content and the actin cytoskeleton specify
the polarity and extent of lysosome fusion. Identification of the molecular pathway of
lysosome exocytosis will help to assess whether this can be a viable strategy to help
RPE cells decrease their lipofuscin burden.
CR: A. Lakkaraju, None; F. Diaz, None; R. Schreiner, None; E.J. Rodriguez-Boulan, None.
Support: NIH EY08538, AHAF M2006-081, AHAF M2009093
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
487-490
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
491 - A55
Molecular Mapping of Toll-Like Receptor Mediated Retinal Pigment Epithelial
Cell Responses With Functional Genomics
M.E. Kleinman, W. Cho, B. Fowler, H. Kaneko, S. Dridi, J.Z. Baffi, J. Ambati.
Ophthalmology & Visual Sciences, University of Kentucky, Lexington, KY.
492 - A56
Copper Homeostasis in the Retinal Pigment Epithelium
F. Mazzoni1, J. Camakaris2, E. Rodriguez-Boulan1. 1Ophthalmology, Cornell University
Weill Medical College, New York, NY; 2Genetics, University of Melbourne,
Melbourne, Australia.
Purpose: Toll-like receptors (TLRs) are ubiquitously expressed components of the
innate immune system that mediate host cell responses to pathogenic invasion.
TLR3 is a membrane receptor found both on the cell surface and in endosomes that
recognizes viral double-stranded RNA (dsRNA) and small-interfering RNA (siRNA).
We recently showed that TLR3 activation induces death of human choroidal endothelial
and retinal pigmented epithelial (RPE) cells (Kleinman et al. Nature 2008; Yang et al.
NEJM 2008). In this study, we utilized a functional genomics approach to map TLR3
induced immune and pro-apoptotic pathways in RPE.
Methods: Primary isolates of human RPE (hRPE) were cultured and genotyped for
TLR3. Protein analyses confirmed functional membrane surface receptor. Cultures
were treated with TLR3 agonists with appropriate controls followed by RNA harvest
at 8 hours. Wild-type C57BL/6 mice received intravitreous injections of the same
TLR3 agonists and controls followed by RNA harvest from RPE/choroid at 16 hours.
Relative quantitative PCR was performed for 84 genes related to pro-apoptotic and
TLR induced pathways (n=3 for each group). Data were analyzed using hierarchical
clustering and principle component analyses (PCA) to determine significant changes
(p<0.05) in gene regulation within specific molecular pathways.
Results: In treated primary hRPE isolates, critical molecular components involved
in tumor necrosis factor/stress, caspase, p53, and FADD pathways were upregulated.
In vivo in mice, there were significant changes in interleukin and interferon profiles
coupled with increases in TLR3 expression and various downstream signaling
cascades. PCA and heat-map visualization demonstrated a strong pattern of gene
expression related to apoptosis and TLR signaling.
Conclusions: The expression and activation of TLR3 on RPE is a potent immune signal
that shifts gene expression to pro-apoptotic and TLR mediated molecular pathways.
Through further investigations, critical signaling events will be identified in order to
target and prevent TLR3 mediated RPE cell death in the presence of specific agonists.
CR: M.E. Kleinman, None; W. Cho, None; B. Fowler, None; H. Kaneko, None; S. Dridi,
None; J.Z. Baffi, None; J. Ambati, Quark, C; Genentech, C; Allergan, C; University
of Kentucky, P.
Support: NIH/NEI, Doris Duke Charitable Fund, Burroughs Wellcome Fund, Research
to Prevent Blindness
Purpose: Recent work suggests that Cu2+ deficiency may play a role in Age Related
Macular Degeneration (AMD). We examined the polarized expression in Retinal
Pigment Epithelium (RPE) of three Cu2+ transporters, Menkes, Wilson and CRT1,
which are poorly characterized in RPE.
Methods: Polarized filter-grown ARPE-19 cells were exposed to a Cu2+ chelator, BCS
(300 µM) or to excess Cu2+ (100 µM CuCl2) for 2 h. Localization of endogenous and
overexpressed transporters was analyzed by quantitative microscopy. Experiments
in young and aged mice were conducted in compliance with the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. Enucleated eyes were fixed
in 4% PFA and analyzed identically to cultured cells.
Results: RPE expresses both Menkes and Wilson proteins and in low Cu2+ these
transporters are stored in the Golgi compartment and move to the plasma membrane
(PM) in high Cu2+. We found that in high Cu2+ both transporters were expressed at
the basolateral PM of ARPE19 cells (i.e., facing the choroid circulation). In contrast,
CTR1, the channel mediating the entrance of Cu2+ displayed a non-polar punctate
pattern in the cytoplasm, in agreement with the concept that this transporter cycles
between surface and intracellular pools. In aged mouse eyes the localization of the 3
endogenous transporters was comparable to that observed in cultures.
Conclusions: The basolateral localization of Menkes and Wilson in RPE differs from
that reported in other tissues, e.g. placental trophoblasts, in which they are polarized
to opposite PM domains. Our observations suggest that Menkes and Wilson play
overlapping roles in RPE. The presence of CTR1 suggests an important role of this
transporter in Cu2+ entry. We speculate that age may decrease the ability of these 3
transporters to maintain Cu2+ homeostasis, resulting in the Cu2+ deficiency recently
reported in AMD donor eyes. We are currently characterizing the sorting of these
three transporters in RPE and the participation of clathrin and clathrin adaptors in
their polarized localization and function.
CR: F. Mazzoni, None; J. Camakaris, None; E. Rodriguez-Boulan, None.
Support: NIH EY08538
493 - A57
Retinal Pigment Epithelial Signature Gene Function and Retinal Disease
494 - A58
Kinetics of Oxidized Rod Outer Segment Phagocytosis Under Chronic
Oxidative Stress in an in-vitro Model of Age-Related Macular Degeneration
N.V. Strunnikova1, W. Chen2, A. Maminishkis3A, C. Zhi3A, D. Stambolian4, A.O. Edwards5,
A. Swaroop3B, G. Abecasis2, P. Munson6, S.S. Miller3A. 1OGVFB, National Eye Institute,
Bethesda, MD; 2Biostatistics, School of Public Health, University of Michigan,
Ann Arbor, MI; ASERPD, BNeurobiology-Neurodegeneration & Repair Laboratory,
3
NIH, Bethesda, MD; 4Opthalmology, University of Pennsylvania, Philadelphia,
PA; 5Ophthalmology, Mayo Clinic, Rochester, MN; 6Mathematical and Statistical
Computing Laboratory, Center for Information Technology/NIH, Bethesda, MD.
Purpose: The gene signature of the RPE distinguishes it from other cell types and we
seek to understand the role of these genes in RPE physiology and pathophysiology.
Methods: A set of 154 RPE “signature” genes was determined using expression profiles
of native human adult and fetal RPE, and fetal RPE cell cultures. The association
of RPE signature genes with AMD was tested in a genome wide association study
(GWAS), which included 2.5 million SNPs collected in 1150 controls and 2157 AMD
cases. Proteins of interest were downregulated using shRNA lentiviral particles
(Sigma). Expression and localization of signature proteins was determined by
immunoblotting and immunofluorescence in primary cultures of human fetal retinal
pigment epithelium.
Results: A cross-sectional analysis of the RPE signature genes against an AMD GWAS
dataset identified novel candidate genes potentially relevant to AMD. The TIMP3 gene
(rs5754221, p = 5×10 -5), GRAMD3 (rs4836255, p = 3×10 -4), PITPNA (rs17821234, p = 4×10 -4)
and CHRNA3 (rs11072791, p = 6×10 -4) were significantly associated. SNPs near 44 other
signature genes show some association to AMD at p <0.01. Functional analysis of the
48 genes by DAVID revealed that 18 genes have a signal sequence at the N-terminus
that targets these proteins to the ER. Mutations in signal sequences can cause protein
misfolding, ER stress, and cell degeneration. In functional experiments, signature
RPE gene products such as DCT were modified by shRNA in confluent monolayers.
Down regulation of DCT significantly altered transepithelial resistance (TER) and
the expression levels of proteins critical for RPE function (bestrophin, Kir7.1, NaKATPase, claudin 10, ezrin, ZO-1).
Conclusions: Our identification of RPE signature genes has uncovered highly
expressed genes whose functions in RPE physiology are still unknown. This data
set has also revealed novel candidates and pathways that could underlie retinal
degenerative disease.
CR: N.V. Strunnikova, None; W. Chen, None; A. Maminishkis, None; C. Zhi, None; D.
Stambolian, None; A.O. Edwards, None; A. Swaroop, None; G. Abecasis, None; P.
Munson, None; S.S. Miller, None.
Support: None
P. Gupta, N. Tirgan, N.M. Kalariya, B.F. Godley, M. Motamedi. Ophtha & Visual Sci,
Univ of Texas Medical Branch, Galveston, TX.
Purpose: There is a constant rebuilding of photoreceptor cells, mainly through the
shedding of the photoreceptor outer segment tips, followed by their phagocytosis in
the retinal pigment epithelial (RPE) cells. During this process, there is a continuous
protein turn over and replacement by RPE cells for maintaining homeostatic regulation.
This study was undertaken to determine how the rate of phagocytosis of oxidizedrod outer segments (ox-POS) behave under daily sub-lethal oxidative stress and/or in
combination with a lysosomal inhibitor in retinal pigment epithelial cells in culture.
Methods: Duplicate ARPE-19 cell cultures were seeded into 96 well plate and were left
untreated or treated daily with a sub-lethal dose of 40uM H2O2 and/or in combination
with 40uM leupeptin for 3 and 7 days following standard incubation protocols. Purified
bovine POS were oxidized under UV irradiation (302 nm light source) for 16 hours
and labeled using Rhodamine conjugation kit (Invitrogen). Rhodamine-labeled oxPOS (1x106/ml) were fed to the cultures (both treated and untreated) for a period of 5
hours and continuous phagocytosis was assessed every 20 min using live cell confocal
imaging system (BD pathway 855). Also, fluorescence was measured at time 0 and time
5 hrs in a fluorometer plate reader at the excitation filter of 530/25 nm and emission
filter of 590/35 nm after careful washing to read only the phagocytosed ox-POS.
Results: We found that there was a decrease in the rate of phagocytosis at the end of
3 days of stress both in H2O2 group and in combination group that received H2O2 and
leupeptin when compared to the untreated groups. However, at the end of 7 days of
oxidative stress treatment we found that in both the treatment groups there was an
increase in the rate of phagocytosis with time following the order of H2O2+ leupeptin
> H2O2 > control. The linear slope for the rate of phagocytosis per cell was 12.11 for
combination group, 10.43 for H2O2 and 5.48 for the control group (mean average from
3 experiments). Additionally, we noted that there was a delay in the ingestion of oxPOS in the control group where as the process was faster in both the oxidant-induced
groups after 7 days of exposure to oxidative stress.
Conclusion: Our studies conclude that RPE cells respond differentially to a single
stress or a combination of stressors with respect to phagocytosis. This synergistical
effect of oxidative stress may contribute to the formation of lipofuscin in the RPE cells
which may have direct implications in ageing and age-related macular degeneration.
CR: P. Gupta, None; N. Tirgan, None; N.M. Kalariya, None; B.F. Godley, None; M.
Motamedi, None.
Support: RPB
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
491-494
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
495 - A59
Activation of M-Type Current in Mouse RPE Cells by Zinc Pyrithione
496 - A60
Effect of HDAC Inhibitors on Oxidative Apoptosis of RPE Cells
H. Wang1, B.R. Pattnaik 2, B.A. Hughes1. 1Ophthalmology and Visual Sciences,
University of Michigan, Ann Arbor, MI; 2Pediatrics, Univ of Wisconsin, Madison,
WI.
S.J. Upadhyay1, J.A. Hossain1, M.P. Krebs1, P. Baciu2, S. Kaushal3. 1Department of
Ophthalmology, University of Florida, Gainesville, FL; 2Allergan, Inc, Irvine, CA;
3
Department of Ophthalmology, University of Massachusetts Medical School,
Worcester, MA.
Purpose: We have shown previously that freshly isolated human, monkey, and bovine
RPE cells exhibit an M-type K+ current, which recent evidence suggests is mediated
by KCNQ/Kv7 channels. The purpose of this study was to determine whether M-type
currents are also expressed in mouse RPE.
Methods: RPE cells from C57BL/6 mice were isolated enzymatically with papain.
Whole-cell currents were recorded using the patch-clamp technique. M-type current
was identified by its characteristic tail currents and kinetics. The pipette solution
contained (in mM) 100 K gluconate, 30 KCl, 5 HEPES, 5.5 EGTA-KOH, 0.5 CaCl2, 4
Mg-ATP (pH 7.2) and the control bath solution consisted of (in mM) 5 KCl, 135 NaCl,
10 HEPES, 10 glucose, 1.8 CaCl2, and 1 MgCl2 (pH 7.4).
Results: Mouse RPE cells had an average membrane potential of -55.2 ± 3.3 mV (mean
± SE, n = 9) and exhibited a prominent inwardly rectifying K+ current, but no M-type
K+ current. Exposure of cells to 10 µM zinc pyrithione (ZnPy), a potent KCNQ channel
opener, activated M-type current in every cell tested, with a maximal conductance
of 5.1 ± 1.0 nS (n = 8) and a half-maximal voltage for activation of -58.1 ± 5.5 mV (n =
8). Erbstatin (20 µM), a Src tyrosine kinase inhibitor, also activated M-type current in
resting mouse RPE cells, albeit to a lesser extent than did ZnPy.
Conclusions: Our findings that M-type current is absent in freshly isolated mouse
RPE cells and that it can be activated by ZnPy suggest that KCNQ channels are present
but inactivated at rest. Tyrosine phosphorylation of these channels might contribute
to this inactivation, but it is likely that other mechanisms are also involved.
CR: H. Wang, None; B.R. Pattnaik, None; B.A. Hughes, None.
Support: NIH Grants R01EY08850 and P30EY07003, and RPB
Purpose: To determine the effect of the HDAC inhibitors valproic acid (VPA),
4-phenylbutyrate (PBA) and the VPA analog valpromide (VPD) on the survival and
function of RPE cells in response to oxidative stress and complement attack both in
vitro and in vivo.
Methods: ARPE-19 cells were cultured by standard methods. For hydroquinone
(HQ)-induced death, HQ was used at concentrations previously established to induce
RPE oxidative damage (250-450 µM). ARPE-19 cells were then pretreated for 24 hrs
with various concentrations of VPA, VPD, or PBA, followed by re-treatment and
challenged with HQ for 48 hrs. Apoptosis and necrosis were assayed by dual Annexin
V and propidum iodide staining, as well as cleaved caspase-3 analysis via flow
cytometry. For complement-induced death, pretreated ARPE-19 cells were primed with
a complement-fixing antibody, followed by treatment with C1q-deficient serum in the
presence or absence of conditioned media, and level of cell death was monitored by
accumulation of a cell membrane impermeant nuclear stain. VPA effects in vivo were
also investigated. Oxidative stress was induced by intravitreal paraquat injection in
SOD1+/- mice, and VPA protection was determined by ERGs.
Results: Dose-dependent ARPE-19 cell death was observed at all concentrations of
HQ, with more than 80% of cells dead at concentrations of 350 μM and above. Both
VPA and VPD significantly increased cell viability in response to HQ challenge, and
VPA reduced apoptosis and downstream caspase-3 activation. VPA and VPD also
protected against complement-mediated cell death, however conditioned media from
pretreated cells was required for protection. VPA also protected against paraquatinduced oxidative stress in vivo.
Conclusions: VPA can reduce RPE cell death in in vitro models of oxidative injury
and complement attack and in vivo against paraquat-induced oxidative stress. This
protective and anti-apoptotic activity of VPA on the RPE and photoreceptors supports
the use of VPA in clinical ARMD studies.
CR: S.J. Upadhyay, None; J.A. Hossain, None; M.P. Krebs, None; P. Baciu, None; S.
Kaushal, None.
Support: None
497 - A61
Metabolism of 7-Ketocholesterol in Cultured RPE Cells
498 - A62
Pathophysiology of Outer Blood-Retina Barrier Breakdown
J.-D. Huang, J.W. Lee, I.R. Rodriguez. Mechanisms of Retinal Disease Section,
LRCMB, National Eye Institute/NIH, Bethesda, MD.
Y.-Z. Le1A,2, J. Wang1B,3, H. Xu1B,3. AMedicine, Cell Biology, and Harold Hamm
Oklahoma Diabetes Center, BMedicine and Harold Hamm Oklahoma Diabetes
Center, 1University of Oklahoma Health Sciences Center, Oklahoma City, OK; 2Dean
A. McGee Eye Institute, Oklahoma City, OK; 3Ophthalmology, Xiangya Hospital,
Central South University, Changsha, China.
Purpose: In atheromatous plaques 7-ketocholesterol (7KCh) is the major oxysterol
that induces cytotoxicity. The cytochrome P450’s, CYP27A1 and CYP46A1 as well
as the sulfotransferase SULT2B1b are known to hydroxylate and sulfate 7KCh. The
purpose of this study is to quantify the expression of these genes in the retina and to
determine whether overexpression of these enzymes will protect cultured RPE cells
from 7KCh cytotoxicity.
Methods: The mRNA expression of CYP27A1, CYP46A1 and SULT2B1b in human
tissues and cultured RPE cells were determined by qRT-PCR. Full-length ORFs of
CYP46A1, SULT2B1b, and PAPSS1 were respectively cloned into pcDNA4/HisMax
TOPO expression vector. Cultured D407 cells were either transfected with CYP46A1
construct or co-transfected by SULT2B1b construct and PAPSS1 construct. Expression
of these enzymes was confirmed by immunoblot. The transfected cells were treated
with various concentrations of 7KCh in serum-free medium for 24 hours and the cell
viability was measured using the Dojindo Cell Counting Kit.
Results: In the retina, the per cell gene copy number for CYP27A1 (2548) is much higher
than CYP46A1 (625) and SULT2B1b (3). CYP27A1 mRNA level in the retina was 1/3 of
that found in the liver. CYP46A1 mRNA level in the retina was approximately 1/5 of
that found in the brain. Overexpression significantly increased the levels of CYP46A1
(105-fold), SULT2B1b (100-fold), and PAPSS1 (10-fold) over the mock-transfected cells.
In four independent experiments overexpression of CYP46A1 and SULT2B1b did not
provide statistically significant protection from 7KCh cytotoxicity. Experiments with
CYP27A1 are pending. Analyses for 7KCh metabolites, 24-hydroxy-7-ketocholesterol
and 7-ketocholesterol-3-sulfate are also in progress.
Conclusions: Our results indicate that the retina contains significant levels of
CYP27A1 but considerably lower levels of CYP46A1 mRNA. The levels of SULT2B1b
are essentially nil. Overexpression of CYP46A1 and SULT2B1b do not convey any
protection from 7KCh cytotoxicity. Our data suggests that these enzymes are unlikely
to play a significant role in the detoxification of 7KCh in the RPE.
CR: J.-D. Huang, None; J.W. Lee, None; I.R. Rodriguez, None.
Support: NEI Intramural Research Program
Purpose: Although outer blood-retina (BRB) barrier is responsible for approximately
80% of blood circulation in the retina, the pathophysiology of outer BRB is not wellstudied. To determine the significance of outer BRB breakdown in retinal vascular
diseases, we investigated outer BRB-specific leakage in mice with altered RPE barrier
in ischemia and uveitis models.
Methods: Mice with altered outer BRB were generated by disrupting vascular
endothelial growth factor (VEGF) or VEGF-receptor 2 (R2) in the RPE. Ischemia
was induced with an oxygen-induced retinopathy (OIR) and uveitis was generated
with endotoxin-induced uveitis (EIU). The alteration of outer BRB was assessed
by measuring tight-junction protein occludin in the RPE with Western blot and
immunohistochemistry. The outer BRB-specific leakage of macromolecules were
visualized and measured by a fluorescent microscopic assay.
Results: For the first time, we were able to visualize and measure outer BRB-specific
leakage in ischemic mice. Outer BRB-specific breakpoints and macromolecule leakage
were significantly reduced in the OIR-treated conditional VEGF or VEGF-R2 KO mice.
EIU-treated conditional VEGF KO mice demonstrated a significant reduction in the
size of retinal detachments and the amount of outer BRB-specific leakage. These
pathological changes were associated with an attenuation of occludin depletion in
the conditional VEGF or VEGF-R2 KO mice.
Conclusions: Our newly established fluorescent microscopic assay permits the
visualization and measurement of outer BRB breakdown in ischemic mice for the first
time. Our results suggest that the breakdown of outer BRB contributes significantly
to overall blood-content leakage under ischemic condition and exudative retinal
detachment under uveitic condition, through an autocrine VEGF signaling mechanism.
Therefore, our study may have significant implications to the mechanism, diagnosis,
and therapeutics of retinopathy of prematurity, uveitis, and macular edema.
CR: Y.-Z. Le, None; J. Wang, None; H. Xu, None.
Support: NIH grants P20RR17703, P20RR024215, P30EY12190, ADA grants 1-06-RA76, 1-10-BS-94, AHAF grant M2008-059, FFB grant BR-CMM-0808-0453-UOK, OCAST
grant HR09-058
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
495-498
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
499 - A63
Altered Outer Segment Phagocytosis Causes RPE Mitochondrial Dysfunction:
Molecular Mechanisms
500 - A64
Mertk as a Common Phagocytic Receptor for Tubby and Tulp1 to Facilitate RPE
Phagocytosis
S.C. Finnemann, C.-C. Yu, Y. Dun. Biological Sciences, Fordham University, Bronx,
NY.
N.B. Caberoy, G. Alvarado, W. Li. Ophthalmology, Bascom Palmer Eye Institute Univ
of Miami, Miami, FL.
Purpose:In mice lacking the integrin recognition receptor αvβ5, oxidative processes
secondary to loss of the diurnal rhythm of photoreceptor outer segment phagocytosis
cause age-related loss of photoreceptor function (but not viability) and RPE lipofuscin
accumulation. We seek to explore this experimental model to elucidate how changes
in phagocytic capacity or kinetics of the RPE (1) specifically cause oxidative stress,
(2) how such chronic, sublethal oxidative stress alters RPE functionality causing agerelated retinal disease, and (3) how we can prevent secondary oxidative damage to
retina/RPE preserving photoreceptor function in vivo.
Methods:We explored primary cultures of RPE derived from mice with complete or
defective phagocytic machineries that were exposed to different sources of oxidative
stress in assays quantifying experimental outer segment uptake, reactive oxygen
species, or mitochondrial activity. We determined levels, phosphorylation, oxidative
modification and subcellular localization of phagocytic signaling proteins in these
cells using immunoblotting and laser scanning confocal microscopy. Furthermore, we
tested in vivo oxidative burden, phagocytic signaling, and mitochondrial functionality
in RPE/retina of wild-type and β5 null mice fed or not diet supplemented with grapes
rich in antioxidants.
Results:Our experiments demonstrate that RPE cells lacking αvβ5 integrin are more
sensitive to accumulation of the lipofuscin component A2E or to sublethal levels of
hydrogen peroxide than RPE cells that possess a complete phagocytic machinery.
Phagocytosis of photoreceptor outer segments further enhances this discrepancy
by increasing intracellular reactive oxygen species. Our data also suggest that this
is due to altered phagocytic signaling pathways and that a major target of reactive
oxygen species produced by β5 null RPE in vivo and in vitro is the mitochondrial
respiratory chain.
Conclusions:Cumulative oxidative damage of the RPE leads to formation of prooxidant lipofuscin and contributes to RPE and retinal atrophy in dry AMD. Our
work pinpoints molecular changes due to delayed phagocytosis of outer segments
shed daily by photoreceptor cells as possible causative factor in RPE mitochondrial
dysfunction that can be directly responsible for such outcome.
CR: S.C. Finnemann, None; C.-C. Yu, None; Y. Dun, None.
Support: NIH Grant EY13295, The California Table Grape Commission
Purpose: Mutations in tubby or tubby-like protein 1 (Tulp1) cause retinal degeneration
with undefined molecular mechanisms. Both proteins have been recently identified as
novel eat-me signals to facilitate retinal pigment epithelium (RPE) phagocytosis by a
new strategy of phagocytosis-based functional cloning. The purpose of this study is
to further characterize tubby and Tulp1 as novel eat-me signals by elucidating their
phagocytic receptors and signaling cascades.
Methods: Binding of tubby and Tulp1 to phagocytic receptor MerTK was analyzed
by co-immunoprecipitation. MerTK activation was investigated by receptor
phosphorylation. MerTK-dependent signaling of non-muscle myosin II activation
was characterized by immunohistochemistry. Receptor-binding domains of Tulp1
were delineated by mutation and deletion analyses.
Results: Tubby and Tulp1 bound to MerTK, while Tulp1 additionally interacted with
Axl and Tyro3 in the same receptor tyrosine kinase subfamily. Tubby and Tulp1 induced
MerTK phosphorylation, MerTK-dependent myosin II redistribution and colocalization
with phagosomes. Mutation analyses revealed five minimal phagocytosis determinants
(MPDs) with a consensus motif in the N-terminus of Tulp1, which were essential for
Tulp1-mediated MerTK binding, receptor activation and RPE phagocytosis. Tubby and
Tulp1 bound to both MerTK and phagocytosis preys to facilitate phagocytosis that
was blocked by excessive amount of soluble MerTK extracellular domain (Mer-Fc),
tubby N-terminal domain (Tubby-N) or Tulp1-N.
Conclusions: These data demonstrated that tubby and Tulp1 are novel MerTK ligands
that function as bridging molecules to facilitate RPE phagocytosis. These results
provide in-depth understanding of RPE phagocyte biology and its regulation by
tubby and Tulp1.
CR: N.B. Caberoy, None; G. Alvarado, None; W. Li, None.
Support: NIH grant EY016211and P30EY014801
501 - A65
Expression of Succinate Receptor Gpr91 in Retinal Pigment Epithelium (RPE) in
Control and Hfe Knockout Mice
502 - A66
Differentiation Potential of Mesenchymal Stem Cells to RPE Cells
J. Gnana Prakasam1A,1B, S. Ananth1A,1B, P.M. Martin1A,1B, S.B. Smith1B,1C, V. Ganapathy1A,1B.
A
Biochemistry and Molecular Biology, BVision Discovery Institute, CCellular
Biology and Anatomy, 1Medical College of Georgia, Augusta, GA.
Purpose: GPR91 is a G-protein-coupled receptor for succinate. It plays an important
role in the production of vascular endothelial growth factor (VEGF); it is also a
potent mediator of vascular growth in normal retina and in diseases associated
with proliferative retinopathy. RPE-derived VEGF is important for maintenance of
choriocapillaris and plays a pathogenic role in age-related macular degeneration
(AMD). However, nothing is known on GPR91 expression in RPE. Excessive iron
accumulation is a hallmark of AMD retinas and is also seen in patients with
hemochromatosis. Here we examined the expression GPR91 in RPE in normal mice
and in a mouse model of hemochromatosis.
Methods: Expression and distribution of GPR91 in mouse retina was determined
by RT-PCR and confocal immunofluorescence. Bioluminescence Resonance Energy
Transfer (BRET) assay was done to confirm that succinate is a ligand for GPR91. Hfe-/mice were used as an animal model for hemochromatosis. The influence of excessive
iron on expression of GPR91 in RPE was analyzed in vitro by exposing RPE cell lines
and primary cells to ferric ammonium citrate (FAC).
Results: GPR91 mRNA and protein are expressed in RPE as well as in neural retina.
The expression is restricted to the apical membrane in RPE. Succinate functions as a
ligand for GPR91 based on BRET assay. Expression of GPR91 is markedly higher in
Hfe-/- mice throughout the retina including RPE. Primary RPE cells from Hfe-/- mice
also show increased expression of GPR91 compared to control RPE cells. RPE cell
lines and primary RPE cells treated with FAC show increased expression of GPR91
similar to Hfe-/- mice.
Conclusions: RPE expresses GPR91. Localization of GPR91 in the apical membrane
of RPE suggests that succinate in the subretinal space plays a role in the activation
of GPR91. Increased GPR91 expression in RPE and in retina during iron overload
indicates that excessive iron accumulation in retina as seen in patients with AMD
and hemochromatosis might play a pathologic role in neovasculatization through
increased GPR91-mediated VEGF secretion.
CR: J. Gnana Prakasam, None; S. Ananth, None; P.M. Martin, None; S.B. Smith,
None; V. Ganapathy, None.
Support: None
C.M. Sheridan1, D. Pattwell1, S. Thompson1, S. Mason1, D.L. Kent2, I. Grierson1. 1School
of Clinical Sciences, University of Liverpool, Liverpool, United Kingdom; 2The
Vision Clinic, Kilkenny, Ireland.
Purpose: To investigate whether human Mesenchymal Stem Cells (hMSC) could
differentiate to human Retinal Pigmented Epithelium (hRPE)-like cells and therefore serve
as a potential source of RPE cells for translational studies.
Methods: Female primary hRPE cells were cultured for up to 5 days in 4 well lab-teks in
HAMS F10 medium + 20% FCS @ 37°C, 5% CO2. Male hMSC, were sorted using specific
positive markers; CD73, CD105, and CD166 were negative for haematopoetic markers CD34,
CD45 and CD14. hMSC cells were cultured for up to 5 days in MSC basal medium + 20%
FCS in 4 well lab-teks @ 37°C, 5% CO2. immunocytochemistry (ICC) for primary antibodies
against STRO-1, cytokeratin, RPE-65, MITF, Chx,10 Nestin and Pax-6 was compared in both
cells types. For co-culture experiments, hMSC cells were grown in 25cm 2 flasks for 5 days
until confluent, trypsinised and loaded with 10mM Cytotracker Green (Molecular Probes).
hMSC cells were added to monolayers of hRPE cells in 4 well lab-teks and co-cultured for
one week in a 50:50 mixture of HAMS F10, MSC basal medium + 2% FCS. As a control group,
hMSC cells were seeded in 4 well lab-teks and grown in conditioned medium from hRPE
cells (50:50 mixture of HAMS F10, MSC basal medium + 2% FCS ) for the same period of
time. Cells were examined for cytokeratin expression.
Results: Under standard culture conditions hMSC cells were shown by ICC to
express STRO-1 hMSC cells in culture whether in standard medium or in a 50:50
mix of HAMS F10, MSC basal medium + 2% FCS retained a monolayer appearance.
hRPE cells were also found to express STRO-1 and only hRPE cells expressed cytokeratin.
hRPE cells also retained a normal monolayer formation during culture under standard or
co-culture conditions. Both cell types were positive for MITF expression, but negative for
Chx10 expression. RPE-65 expression was only found in hRPE cell. hRPE cells and hMSC
cells were both negative for the expression of neuroepithelium markers, nestin and Pax6.
hMSC cells grown in conditioned medium expressed STRO-1 and MITF but did not express
cytokeratin, RPE-65, Chx10, Nestin or Pax6. Results under co-culture conditions showed that
3 populations of cells existed, hRPE (positive for cytokeratin alone), hMSC (positive for the
expression of Cytotracker green alone) and a population of cells that was positive for the
cytotracker green and cytokeratin.
Conclusions: Co-culture conditions demonstrated a population of green labelled hMSC
cells also positive for cytokeratin expression, whilst this was not seen in conditioned media
experiments indicating cell-cell contact is required.
CR: C.M. Sheridan, None; D. Pattwell, None; S. Thompson, None; S. Mason, None; D.L.
Kent, None; I. Grierson, None.
Support: Foundation for the prevention of Blindness; Dunhill Medical Trust
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
499-502
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
503 - A67
The Effects of Knock Down of Mecp2 on TGFβ Signaling Involved in RPE
Transdifferentiation
504 - A68
Caspase-8, Deficient in Retinal Pigment Epithelial Cells, is Necessary for
Degradation of IRF-3
S. He, P. Zhou, N. Chan, J. Xu, S. Ryan, D. Hinton. Ophthalmology-USC, Doheny Eye
Institute, Los Angeles, CA.
N.C. Sears, S. Chattopadhyay, G. Sen, G. Stark. Molecular Genetics, Cleveland Clinic
Foundation, Cleveland, OH.
Purpose: Retinal pigment epithelial (RPE) cell transdifferentiation is a critical step in
the wound healing response in proliferative vitreoretinopathy (PVR). Our previous
studies have shown that human PVR membranes are rich in methyl CpG binding
protein 2 (MeCP2). In current experiments, we investigated the effects of knocking
down MeCP2 by siRNA on TGFβ signaling involved in RPE transdifferentiation.
Methods: Early passage human fetal RPE cells were used in the study. MeCP2 expression
in scratch wounded RPE monolayer was studied by immunocytochemistry. The effect
of methylation inhibitor on the expression of proteins MeCP2, TGFβ II receptor, and
PPAR-γ was evaluated by western blot after 48 hours 5-aza-2’-deoxycytidine (1, 2 and
6 uM) treatment. For the analysis of the role of MeCP2 in TGFβ mediated fibrosis, RPE
were pretreated with siRNA MeCP2 (0.1 or 10nM) and then with or without TGFβ (10ng/
ml) for 48 hours ,the response of MeCP2 knockdown on the expression of Smad/2/3,
MeCP2, alpha Smooth muscle actin (a-SMA), and fibronectin (FN) induced by TGFβ
were determined by western blot.
Results: MeCP2 was prominently expressed at the wounded edge of the scratched
RPE monolayer, however MeCP2 expression was low away from wounded area. In
the presence of 5-AZA (2-6 uM), the expression of MeCP2 and TGFβ receptor II were
significantly inhibited. A strong increase of FN and α-SMA stimulated by TGFβ is seen
when RPE were treated with control (scrambled) siRNA, while up regulation of FN,
α-SMA and activation of Smad2/3 by TGFβ was much attenuated by pretreatment with
MeCP2 specific siRNA. Inhibition of MeCP2 also up-regulated Smad 7 and PPAR-γ.
Conclusions: These results support the idea that RPE transdifferentiation may be
under epigenetic control and that MeCP2 may be a critical factor in the regulation of
the PVR pathogenesis.
CR: S. He, None; P. Zhou, None; N. Chan, None; J. Xu, None; S. Ryan, None; D.
Hinton, None.
Support: NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel
Beckman foundation
Purpose: Intracellular dsRNA is a chief sign of replication for many viruses. dsRNA
binds endosomal toll-like receptor (TLR)-3 or cytoplasmic helicase RIG-I, to activate
interferon response factor 3 (IRF3), leading to the induction of many antiviral genes,
including interferon-beta. Within 12 hours of activation, IRF3 is proteasomally
degraded to attenuate the inflammatory gene induction program. The purpose of this
investigation is to determine the molecular pathways that underlie IRF3 degradation.
Methods: RIG-I dependent N terminal IRF-3 degradation was analyzed by western
blot in P2.1 cells, a mutagenized HT1080 derived cell line low in IRF3, using lentivral
directed expression of IRF3-Flag. We measured the time dependent degradation
of IRF-3 in HT1800 cells after RIG-I or TLR3 activation with and without selective
caspase inhibition. We next compared IRF3 degradation in ARPE-19 cells after stable
transfection of caspase-8. Cells were cultured in DMEM with 5% FBS and treated with
transfected dsRNA (4 ug/ml) to selectively activate the RIG-I pathway.
Results: A 40 kD C-terminal fragment of IRF-3 was observed after RIG-I activation.
Broad inhibition of caspases by Z-VAD-FMK as well as specific inhibition of caspase 8
by Z-IETD-FMK prevented IRF3 degradation. ARPE-19 cells, which express 100x less
caspase-8 than HT1080 cells, showed no signal dependent IRF3 degradation. Ectopic
expression of caspase-8 in ARPE-19 cells allowed IRF-3 degradation.
Conclusions: We have defined a necessary role for caspase-8 in the degradation
of IRF-3. We think it possible that viral dsRNA can initiate two different antiviral
responses, depending on the level of caspase 8 expression. First, if caspase-8 is present,
an immediate type-I interferon response is initiated which is then attenuated by IRF3
degradation, followed by programmed cell death. However, if caspase-8 is not present,
cells initiate a sustained type-I interferon response, not followed by IRF3 degradation
and cell death. This new finding has significant implications in the response of the
RPE to inflammatory stimulus from viral dsRNA.
CR: N.C. Sears, None; S. Chattopadhyay, None; G. Sen, None; G. Stark, None.
Support: NIH Grant CA95851
505 - A69
Evaluation of in vitro Effects of Trivaris (Triamcinolone Acetonide) on Human
Retinal Pigment Epithelial Cells and Müller Cells
506 - A70
Effects of Arginase II on Retinal Pigment Epithelial Cells in Gyrate Atrophy
N.K. Gupta1, S. Mansoor2, A.U. Sapkal1, A.G. Limb3, M.C. Kenney1, B.D. Kuppermann1.
1
Ophthalmology, Univ of California, Irvine, Anaheim, CA; 2Department of
Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta,
GA; 3Division of Pathology and Cell Biology, University College London, Institute
of Ophthalmology, London, United Kingdom.
Purpose: To study the in vitro effects of Trivaris (Allergan, Inc.) on human retinal
pigment epithelial (ARPE-19) cells and Müller cells (MIO-M1).
Methods: ARPE-19 cells and MIO-M1 cells were treated for 24 hours with 1000, 500,
200 or 100 μg/mL of Trivaris or hydrogel control. Cell viability (CV) was measured
using trypan blue dye exclusion assay, mitochondrial membrane potential (ΔΨm)
was measured using JC-1 assay, and caspase-3/7 activity was measured to determine
apoptosis.
Results: There was a significant dose-dependent decrease in cell viability of ARPE-19
and MIO-M1 cells when treated with 1000 μg/mL (clinical dose), 500 μg/mL or 200
μg/mL Trivaris compared to hydrogel controls (ARPE-19 cells 32.9±0.05, 39.5±1.05, and
51.3±0.6 respectively versus 87.2±0.7, 87.4±0.5, and 91.2±1.05 respectively; MIO-M1 cells
13.3±0.7, 27.9±0.7, 48.9±1.75 versus 89.3±0.4, 86.2±1.3, 84.5±0.5). ΔΨm was decreased and
caspase 3/7 activity increased in ARPE-19 and MIO-M1 cells treated with Trivaris 1000
μg/mL, 500 μg/mL and 200 μg/mL compared to hydrogel controls (ΔΨm ARPE-19 cells
1.3±0.01, 2.2±0.2, 3.8± 0.3 versus 5.7±0.09, 7.8±0.42, 8±0.4; ΔΨm MIO-M1 cells 0.7±0.05,
0.6±0.1, 1.0±0.06 versus 1.8±0.03, 2.3±0.03, 3.6±0.06; caspase 3/7 activity ARPE-19 cells
18800±893, 16260±66.9, 11800±425.2 versus 10440±282.4, 6956±140.9, 4519±34.65; caspase
3/7 MIO-M1 cells 26850±4794, 18090±3348, 13530±1029 versus 9157±293, 7216±59.3,
6927±363.8). Cells treated with 100 μg/mL Trivaris were not significantly affected:
CV 71.3±0.6 for ARPE-19 cells and 74.9±3.4 for MIO-M1 cells versus 81.2±1.05 and
82.5±2.5 respectively; p>0.05.
Conclusions: Trivaris is a preservative-free triamcinolone acetonide formulation in a
hydrogel vehicle approved by FDA for intraocular use. Our in vitro study shows that
Trivaris exhibits cytotoxicity at doses comparable to other triamcinolone acetonide
preparations such as Kenalog, Triesence, and preservative free Triamcinolone
Acetonide.
CR: N.K. Gupta, None; S. Mansoor, None; A.U. Sapkal, None; A.G. Limb, None; M.C.
Kenney, None; B.D. Kuppermann, Consultant for Allergan Inc. and Novagali Pharma, C.
Support: Supported by Discovery Eye Foundation, the Henry L. Guenther Foundation,
the Iris and B. Gerald Cantor Foundation, Gilbert Foundation, Ko Family Foundation
and Research to Prevent Blindness Foundation
M. Ohnaka1A, E. Okuda-Ashitaka1B, S. Kaneko1A, A. Ando1A, K. Takahashi1A, S. Ito1B.
A
Ophthalmology, BMedical Chemistry, 1Kansai Medical University, Moriguchi,
Japan.
Purpose: Gyrate atrophy (GA) is a type of progressive chorioretinal atrophy
characterized by hyperornithinemia related to deficiency of ornithine-δaminotransferase (OAT). Experiments using genetically engineered mice lacking OAT
have revealed that retinal pigment epithelial (RPE) cells are the initial sites of insult in
GA, while several clinical studies have demonstrated that reduction of ornithine by an
arginine-restricted diet restrains the progression of chorioretinal atrophy. However,
the mechanisms of RPE cell damage and protection remain unclear. We established
an in vitro model of GA by addition of ornithine to OAT-deficient human RPE cells
using a specific irreversible inhibitor (5-fluoromethylornitine; 5-FMO) that induced
cell death. We found upregulation of arginase (ARG) II (a mitochondrial enzyme)
in OAT-deficient RPE cells by ornithine using microarray analysis and attempted to
clarify the role of ARGII with our in vitro GA model.
Methods: The expression of ARGII mRNA in cultured human RPE cells (TERT-RPE)
was examined using real-time RT-PCR. The effects of ARGII gene silencing using
siRNA on RPE cell viability were investigated by morphologic observations and MTT
colorimetric assays. Since arginine is a precursor for synthesis of NO, the production
of NO was quantified using the Griess method.
Results: (1) The expression of ARGII mRNA was increased by ornithine in both timeand dose-dependent manners. (2) ARGII silencing resulted in increased RPE cell
death. (3) NO production was increased in our in vitro GA model and ARGII silencing
resulted in greater production. (4) NO donors induced RPE cell death.
Conclusions: These results suggest that upregulation of ARGII by ornithine plays
a critical role in cytoprotection against RPE cell damage in GA. Furthermore, insult
to RPE cells caused by downregulation of ARGII might be the result of increased
NO production.
CR: M. Ohnaka, None; E. Okuda-Ashitaka, None; S. Kaneko, None; A. Ando,
None; K. Takahashi, None; S. Ito, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
503-506
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 459 - 507 / A23 - A71
120. RPE: Cell Biology and Disease Organizing Section: RC
507 - A71
Suppression of TGF-β-Induced Upregulation of Connective Tissue Growth
Factor by Ceramide in Human RPE Cells
S. Sonoda1,2, M. Kitamura2, C. Spee2, S.J. Ryan2,3A, T. Sakamoto1, R. Kannan2,3B, D.R.
Hinton2,3B. 1Department of Ophthalmology, Kagoshima University, Kagoshima,
Japan; 2Arnold and Mabel Beckman Macular Research Center at the Doheny
Eye Institute, Los Angeles, CA; ADepartment of Ophthalmology, BDepartment of
Pathology, 3Keck School of Medicine of the University of Southern California, Los
Angeles, CA.
Purpose: Connective tissue growth factor (CTGF) is a matricellular protein found
in the extracellular matrix and is reported to be induced in response to TGF-β in
fibroblasts and RPE. CTGF plays an important role in the pathogenesis of proliferative
vitreoretinopathy. The aim of the present study was to investigate the effect of oxidative
stress from the lipid messenger ceramide on CTGF secretion from human RPE cells.
Methods: Early passage fetal human RPE cells were cultured in DMEM containing
10% fetal bovine serum (FBS). After overnight incubation with DMEM containing 0.5%
FBS, cells were treated with 8 ng/ml recombinant human (rh) TGF-β for 24 h. The effect
of shortchain ceramide was studied by pretreatment for 1 hour with or without 25
μM C2-ceramide prior to rh-TGF-β exposure. Incubations with C2-dihydroceramide
served as negative controls. CTGF protein secreted in the culture medium and its gene
expression were analyzed by ELISA and real-time PCR respectively.
Results: TGF-β increased CTGF levels in the medium significantly from 185±32 pg/
ml in non-treated controls to 592±82 pg/ml with TGF-β (p<0.01). Pretreatment with
C2 ceramide inhibited this response significantly by decreasing CTGF (285±63 pg/
ml vs 592±82 pg/ml in TGF-β alone group; p<0.01). Real-time PCR analysis revealed
that CTGF mRNA expression in untreated and TGF-β-treated conditions was lower
than that in the presence of C2 ceramide. The doses of TGF-β and C2 ceramide used in
the present study had no appreciable effect on the viability of RPE cells by MTTassay.
Conclusions: Our results show that C2 ceramide suppresses the action of TGF-β
and downregulates CTGF secretion in human RPE. Investigating the mechanism of
this phenomenon would be valuable in identifying drug targets for ocular disease.
CR: S. Sonoda, None; M. Kitamura, None; C. Spee, None; S.J. Ryan, None; T.
Sakamoto, None; R. Kannan, None; D.R. Hinton, None.
Support: NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel
Beckman foundation
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
507
Sunday, May 2, 2:45 PM - 4:30 PM Hall B/C Poster Session Program Number/Board # Range: 887 - 898 / A11 - A22
144. Retinal Neurons Organizing Section: RC Contributing Section: VN
887 - A11
Expression of Specific Ganglion Cell Proteins by Brn3-Positive Retinal
Ganglion Cells in Mouse
888 - A12
Distribution and Topography of the Ganglion Cells and Displaced Amacrine
Cells in the Howler Monkey Retina (Alouatta Caraya)
V. Jain1, D. Poria1A, O. Saha1A, N.K. Dhingra1A. ASystems Neuroscience, 1National Brain
Research Centre, Gurgaon, India.
L.C.L. Silveira1A, J.P.C. Muniz1B,2, L.M. Athaide1A, B.D. Gomes1B, B.L. Finlay3. ANucleo
de Medicina Tropical, BInstituto de Ciencias Biologicas, 1Universidade Federal do
Para, Belem, Brazil; 2Centro Nacional de Primatas, Belem, Brazil; 3Department of
Psychology, Cornell University, Ithaca, NY.
Purpose: Brn3 family of POU domain transcription factors are required for the
development and survival of retinal ganglion cells (RGCs). Interestingly, these proteins
are expressed by ~70% of RGCs even in adult retina, raising a possibility that these
cells have a defined physiological role. To explore this, we studied the expression by
Brn3-positive RGCs of specific ganglion cell proteins, including nonphosphorylated
neurofilaments, parvalbumin and melanopsin.
Methods: Retinal wholemounts and radial sections from adult C57BL6 mouse
were subjected to fluorescent double immunostaining for Brn3 or Brn3a and SMI32, parvalbumin or melanopsin. Digital images were taken under epifluorescence
microscope using two different emissions, and the data analyzed for co-expression
of Brn3/Brn3a with the other proteins.
Results: A small proportion of Brn3-positive RGCs expressed SMI-32 or parvalbumin.
Approximately 3% of Brn3-positive cells were also positive for SMI-32. However, more
than 40% of SMI32-positive RGCs expressed Brn3. Similarly, a subset of parvalbuminpositive cells expressed Brn3. With melanopsin C-terminus antibody that labels
predominantly M1 type of intrinsically photosensitive RGCs (ipRGCs), we found
that none of the Brn3a-positive RGCs, and a 0.2% of Brn3-positive RGCs expressed
melanopsin. However, with melanopsin N-terminus antibody that labels both M1 and
M2 type of ipRGCs, we found that none of the Brn3a-positive RGCs, but a relatively
larger proportion of Brn3-positive cells expressed melanopsin.
Conclusions: Our study on co-localization of Brn3 and SMI-32 or parvalbumin
is consistent with the hypothesis that Brn3-positive RGCs comprise multiple
morphological and physiological subtypes, and therfore may not have a single
physiological role. The results on coexpression of Brn3/Brn3a and melanopsin suggest
that a small subset of Brn3b- and/or Brn3c-, but not Brn3a-positive RGCs coexpress
melanopsin, and that the M2, but not the M1 type of ipRGCs preferentially express
these Brn3 isoforms.
CR: V. Jain, None; D. Poria, None; O. Saha, None; N.K. Dhingra, None.
Support: None
Purpose: Differently from all other Platyrrhini, males and females howler monkeys
are regular trichromats. Previous studies have shown that the howler monkey has
a well developed fovea and a very high cone density in the foveola (Franco et al.,
2000; Finlay et al., 2008). In the present work, the density distributions of ganglion
cells (GC) and displaced amacrine cells (DAC) were determined in four retinas from
different Alouatta caraya.
Methods: The animals were deeply anesthetized and perfused transcardially. The eyes
were removed and the retinas were prepared as whole, flat-mounts, and stained with
cresyl violet using the method of Nissl. The criteria to distinguish GC from DAC were
refined by inspecting a collection of capuchin monkey retinas retrogradely labeled
after horseradish peroxidase or biocytin deposits in the optic nerve and couterstained
with cresyl violet (Silveira et al., 1989; Yamada et al., 1996).
Results: GC density peaks at 0.5 mm from the fovea, reaching 50,000 / mm2. In
comparison with the capuchin monkey retina, the howler monkey retina has a lower
peripheral GC density which compensates for an increased foveal packing. The
increased central GC number means that the increased cone density in this primate
could be available for increased acuity measured behaviorally. The GC density
decreases towards the retinal periphery at approximately the same rate along all
meridians, but is 1.2-1.8 times higher in the nasal periphery when compared to the
temporal region at the same eccentricities. The DAC have a shallow density gradient,
their peak density in the central region was about 1,500-2,000 / mm2. The means and
standard deviations for the retinal area and total numbers of cells (n = 4) were: 661 ±
62 mm2; 1,147,975 ± 83,507 GC and 544,909 ± 68,305 DAC.
Conclusions: The GC density distribution in the howler monkey retina, in general
topography is consistent with that of other diurnal Anthropoidea, reflects the increased
central and decreased peripheral cone density reported previously in the howler
monkey.
CR: L.C.L. Silveira, None; J.P.C. Muniz, None; L.M. Athaide, None; B.D. Gomes,
None; B.L. Finlay, None.
Support: CNPq, CAPES, NSF, and FINEP IBN-Net
889 - A13
Morphology, Mosaics and Targets of Diverse Ganglion Cell Populations in
Macaque Monkey Retina: Approaching a Complete Account
890 - A14
Morphological Classification of Retinal Ganglion Cell Types in the Pigeon
Retina
D.M. Dacey, H.R. Joo, B.B. Peterson, T.J. Haun. Biological Structure, University of
Washington, Seattle, WA.
A.M. Querubin1A, B.J. O’Brien1B, K.M. Bumsted O’Brien1A. AARC Centre for Excellence
in Vision Science, BDepartment of Psychology, 1The Australian National University,
Canberra, Australia.
Purpose: Characterization of diverse ganglion cell structure and function in primate is
ongoing (Szmajda, et al. J Comp Neurol 510; 251 2008; Yamada, et al. Vis Neurosci 22; 383
2005; Rodieck, & Watanabe, J.Comp.Neurol. 338, 289 1993; Dacey, D M, in The Cognitive
Neurosciences, MIT Press, 2004; Crook, et al., J Neurosci 28, 12654 2008) but an overall
synthesis has not yet been achieved. Our goal is a complete description of primate
visual pathway origins, providing an anatomical basis for targeted physiological
analysis, dissection of underlying circuitry and for making trans-species comparisons
most notably with the mouse, for which transgenic technology offers increasing
access to retinal pathways.
Methods: To observe ganglion cell dendritic morphology we used retrograde
photostaining (Dacey et al., 2003) from tracer injections made into either the lateral
geniculate nucleus (LGN; n = 16) or the superior colliculus-pretectum (SC; n = 8). This
method had the advantage of providing complete staining of the dendritic tree for large
numbers of retrogradely labeled ganglion cells, permitting measurements of relative
density and inner retina stratification depth derived from the mosaic organization
of overlapping dendritic trees.
Results: In addition to previously well-described midget, parasol, smooth
monostratified and melanopsin-expressing inner-outer cell pairs, and the single
small bistratified-blue-ON cell type, 6 additional low-density populations have been
clearly established. These include a large-field bistratified blue-ON cell that precisely
costratifies with the small bistratified blue-ON cell, an inner-outer pair of narrowly
stratified (30/75% IPL depth), highly branched ‘thorny’ cells and possible primate
correlates of the mammalian local edge detector (ON-OFF; broad thorny) and the ONand ON-OFF-direction selective cells (‘recursive’ mono- and bistratified types).
Conclusions: Density estimates derived from mosaic coverage suggest that the 15 types
quantified thus far account for only 85% of all macaque ganglion cells and a number
of additional very low density populations remain to be clearly identified.
CR: D.M. Dacey, None; H.R. Joo, None; B.B. Peterson, None; T.J. Haun, None.
Support: NIH Grants EY06678, RR00166, EY01730
Purpose: The pigeon has two areas of high acuity vision, the fovea and the area dorsalis.
There is some evidence of a midget-like retinal ganglion cell (RGC) in these regions;
however, a large scale morphological classification of pigeon RGC types using modern
techniques is not yet available. The aim of this study was to characterize RGC types
to determine the array of RGCs and the prevalence of midget-like ganglion cells
in all areas of the pigeon retina (the yellow field, the red field containing the area
dorsalis, and the fovea).
Methods: RGCs (n = 376) were labeled with DiI or DiO in pigeon retinal wholemount
preparations (n=20) using a Di-olistics approach with coated tungsten beads (PDS1000/He System). Labeled RGCs were imaged using a confocal microscope. Soma size,
dendritic field size and branching pattern, inner plexiform layer (IPL) stratification
and eccentricity were measured and then RGCs were classified based on these
parameters.
Results: Our data demonstrate that the pigeon retina contains 17 identifiable types
of RGC. The dendritic field diameter varied from the smallest (14 μm or 7 min of arc)
found in the area dorsalis in the red field, to the largest (394 μm or ~3 deg) found in the
yellow field. Seven RGC types were similar to previously identified mammalian RGC
types with horizontally oriented dendrites. Four pigeon RGC types were completely
different from any reported mammalian RGC types in that the cells projected only
vertically oriented dendrites. Stratification varied from clearly monostratified (6 RGC
types) to quadruple stratified (1 RGC type). Within these classes, stratification occurred
at different IPL depths. Some RGC types with mainly horizontally oriented dendrites
also had vertically protruding dendrites lacking further horizontal branching. RGC
dendrites varied from very “loosely” organized to very densely packed and were
classified as “radiate” or “recursive”. Near the fovea, four RGC types were identified
that had a small dendritic field diameter (35 μm or 17.5 min of arc).
Conclusions: Of the 17 different types of pigeon RGCs, the smallest dendritic field
diameter observed in our sample (14 μm or 7 min of arc in the area dorsalis) was
approximately twice the size required to resolve 12 cpd (the behaviorally measured
acuity for this region, Rounsley & McFadden, 2005). None of the RGC types
corresponded exactly to the primate midget type, though one pigeon RGC type in
the area dorsalis shared similarities with the Golgi-impregnated midget-like RGC
previously reported by Lockhart (1979).
CR: A.M. Querubin, None; B.J. O’Brien, None; K.M. Bumsted O’Brien, None.
Support: ARC Centre of Excellence in Vision Science
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
887-890
Sunday, May 2, 2:45 PM - 4:30 PM Hall B/C Poster Session Program Number/Board # Range: 887 - 898 / A11 - A22
144. Retinal Neurons Organizing Section: RC Contributing Section: VN
891 - A15
Morphological and Functional Characterization of the Octodon degus Retina
892 - A16
Role of Melatonin in Photoreceptor Phagocytosis in the Rodent Retina
N. Cuenca, L. Fernández-Sánchez, G. Esquiva, J. Martín-Nieto, P. Lax. Fisiología,
Genética y Microbiología, Universidad de Alicante, Alicante, Spain.
V. Laurent-Gydé, D. Hicks. Neurobiologie des Rythmes, CNRS-INCI-UPR3212,
Strasbourg, France.
Purpose: Octodon degus is a rodent with a cone-dominant retina also displaying diurnal
crepuscular activity. The purpose of this study was to describe neuronal phenotypes
and visual functions in this diurnal rodent, and compare them with the retinas of
nocturnal mammals commonly studied.
Methods: Octodon degus retinas were singly or doubly immunostained with
antibodies against calcium-binding proteins and classical neurotransmitters and
then visualized by confocal microscopy. Visual function was studied by means of
electroretinogram.
Results: The outer nuclear layer thickness ranged from 2 rows of photoreceptors at the
ora serrata to 4-5 rows in the central retina. The inner nuclear layer contained 3-4 rows
of neurons. Recoverin, ChAT, TH and connexin36 showed cellular immunoreactivity
patterns similar to other mammalian retinas. However, horizontal cells were labeled
with antibodies to parvalbumin, as in the primate retina, but also to calbindin, as in
other rodents. PKCα was found in rod bipolar cells and a subtype of amacrine cells,
as expected, but also in a cone bipolar cell subtype and the outer segments of blue
cones. Finally, a substance P-immunoreactive plexus was found at the stratum S5 of
the IPL, as a difference with other mammals. ERG a- and b-wave thresholds were
higher in dark-adapted Octodon degus (0.8 ± 0.1 and 2.1 ± 0.1 log cds/m 2, respectively)
as compared with rats (-1.7 ± 0.1 and < -3 log cds/m 2, respectively) and mice (-1.0 ±
0.1 and < -3 log cds/m 2, respectively). Moreover, the double-flash protocol showed
percentages of cone contribution to the mixed dark-adapted a- and b-waves over
fivefold higher in Octodon degus than those found in rats and mice. Finally, Octodon
had a significantly higher flicker fusion (> 50 Hz) than that observed in rats (35.2 ±
4.2 Hz) and mice (42.5 ± 2.7 Hz).
Conclusions: The Octodon degus retina exhibits a number of differences compared
with nocturnal rodents regarding visual function, morphological features and cellular
characteristics, but also several similitudes to diurnal animals. Therefore, it constitutes
an interesting experimental subject for retinal research.
CR: N. Cuenca, None; L. Fernández-Sánchez, None; G. Esquiva, None; J. MartínNieto, None; P. Lax, None.
Support: MEC (BFU2006-00957/BFI, BFU2009-07793/BFI),), MSyC (RETICS
RD07/0062/0012), FUNDALUCE and ONCE.
Purpose: Melatonin (mel) a zeitgeber hormone, for which Aryl-alkylamine-N acetyl
transferase (Aa-nat) is the limiting enzyme of synthesis, is thought to play roles in
retinal photoreceptor phagocytosis (PP), critical for photoreceptor survival. In order
to investigate mel impact on mammalian retinal physiology, we first studied PP in rat
retinas treated or not with mel. We then evaluated Aa-nat gene and protein production
in rodent retinas under different lighting conditions.
Methods: Adult rats that had received a melatonin implant were kept in constant
darkness (DD) and examined for retinal immunohistochemistry using an antirhodopsin antibody to assess PP by fluorescence microscopy. In parallel, retinas of
adult Arvicanthis ansorgei a cone-rich diurnal rodent, kept in a 12-hour light-dark cycle
(LD) were processed for non-radioactive in situ hybridization (ISH) using an Aa-nat
riboprobe. Also, circadian retinal Aa-nat gene expression was studied by real time
PCR in animals housed under different lighting conditions (LD, DD, and constant
light (LL) cycles). Finally, retinas of adult Arvicanthis maintained under DD cycles for
72h were processed for western-blotting using a specific anti-AA-NAT antibody in
order to investigate circadian AA-NAT expression.
Results: Rat PP displays a marked circadian expression with a maximum at CT2 in
a DD cycle. Aa-nat gene expression revealed by ISH reached a maximum at night
(ZT19) and a trough during the day (ZT6). Aa-nat expression was restricted to cone
photoreceptors in both species. Arvicanthis retinal Aa-nat showed robust circadian
expression, maximum at ZT19 in a 12h LD cycle and conserved in DD conditions.
36h LL exposure altered the slope and time of maximum Aa-nat mRNA expression.
Finally, AA-NAT protein expression in Arvicanthis retinas was actually maximal at
CT7, in the middle of the subjective day.
Conclusions: Mel implants do not modify PP in DD rat retina. Aa-nat expression
occurs uniquely in cones of Arvicanthis retina. However, LL conditions induce marked
alterations in the expression profile, and protein presence in subjective daytime
under DD conditions appears paradoxical. Use of cone-rich mammalian species
such as Arvicanthis ansorgei may help understand melatonin regulation and action
on retinal physiology.
CR: V. Laurent-Gydé, None; D. Hicks, None.
Support: None
893 - A17
Clock Gene Dependence of Mouse Retinal Circadian Clock
894 - A18
Spatial Regularity in the Arrangement of Excitatory Synapses Within the Inner
Plexiform Layer
D.G. McMahon, G. Ruan. Biological Sciences, Vanderbilt University, Nashville, TN.
Purpose: Mammalian circadian rhythms are generated by an autoregulatory network
of “clock genes” that are expressed in a wide variety of tissues and cells. The effects
of clock gene deletion on clock function have been found to be tissue-specific, with
the highly coupled central neural clock (suprachiasmatic nucleus, SCN) being more
resistant to genetic disruption than peripheral tissue clocks in which there is little
evidence of cellular coupling. Here we tested the effects of genetic deletion of canonical
clock genes on the function of the retinal biological clock in vitro.
Methods: Knockout mice for Per1, Per2, Per3, Cry1, Cry2, and Clock (gifts of D. Weaver,
A. Sancar, and S. Reppert) were crossed with luciferase reporter strains and molecular
circadian rhythms were measured in retinal whole-mounts as in Ruan et al., 2008.
Results: Retinas from Per1-/-, Cry1-/-, and Clock-/- mice were arrhythmic or showed
severely disrupted gene cycling, whereas retinas from Per2-/-, Per3-/- and Cry2-/- mice
were robustly rhythmic. In addition, Cry1 gene dosage on a Cry2 KO background
affected retinal freerunning period with Cry1+/-Cry2-/- exhibiting a lengthened
period of ca. 27 hours compared with ca. 25 hours for Cry1+/+Cry2-/-.
Conclusions: The clock gene dependence of the retinal molecular circadian clock is
unique among tissues tested to date. Whereas Per1, Cry1 and Clock are individually
dispensable for SCN rhythmicity, their deletion disrupts the retinal clock, similar to
peripheral tissue clocks. On the other hand, Per2, which is necessary for peripheral
clock function, is dispensable for retinal clock function. These data suggest that the
retinal circadian clock may lack the compensatory coupling mechanisms of the SCN
neural clock and that it may be more vulnerable to genetic perturbation than the
central clock. Supported by NEI R01 EY15815 to DGM.
CR: D.G. McMahon, None; G. Ruan, None.
Support: NEI R01 EY15815 to DGM
A. Koizumi1, T.C. Jakobs2, R.H. Masland2. 1Cell Physiology, Natl Inst for Physiol Sci,
Okazaki, Japan; 2Massachusetts Eye and Ear Infirmary, Harvard Medical School,
Boston, MA.
Purpose: In the inner plexiform layer (IPL) of the retina, excitatory outputs from
functionally distinct types of bipolar cells make synapses upon the dendrites of
ganglion and amacrine cells. These contacts occur between defined types of cells
and within precisely defined laminae. Here we asked whether the arrangement
of synapses onto ganglion cells within the same lamina manifests any discernible
spatial regularity.
Methods: Pieces of adult rabbit retina were maintained in organotypic tissue culture
for four days. Retinal ganglion cells were transfected with plasmids encoding a
fusion protein of PSD95 with EGFP using a gene gun. Fifty-six ganglion cells were
imaged by confocal microscopy, and 10 were chose for detailed analysis in using
the NeuroLucida program package. Synapse distributions were analyzed for nonrandomness using nearest neighbor analysis and a modified version of the density
recovery profile test.
Results: The absolute density of excitatory synapses throughout the whole sample
of reconstructed cells was 0.19 ± 0.04 puncta/linear μm of dendrite and varied very
little across the different types of cells, or across dendrites of different order. Even
upon casual inspection, the synaptic inputs to a ganglion cell did not appear to be
random. Statistical analysis showed that the synaptic puncta are spaced regularly
along the dendrites with mean inter-synapse interval ranging from 2 to 4 μm. This
was true for ganglion cells of many different functional types, for starburst amacrine
cells, and for the varicosities of bipolar axon terminals.
Conclusions: (1) The linear density of synaptic inputs (PSD95 sites / linear μm) varied
very little between ganglion cells of different functional types. (2) The excitatory
synapses upon the dendrites of retinal ganglion (and amacrine) cells are regularly
spaced along the dendrites: the presence of a PSD95 zone decreases the probability that
another excitatory synapse lies nearby. The mean inter-synapse interval - the exclusion
zone -- ranges from 2 to 4 μm for different cells. (3) The same spacing regularity was
observed for the varicosities of bipolar axon terminals. (4) Thus it appears that the
regular spacing of whole cells across the retinal surface has a correspondence on the
smaller scale of the individual synaptic sites within the IPL.
CR: A. Koizumi, None; T.C. Jakobs, None; R.H. Masland, None.
Support: NIH Grant R01-EY017169
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
891-894
Sunday, May 2, 2:45 PM - 4:30 PM Hall B/C Poster Session Program Number/Board # Range: 887 - 898 / A11 - A22
144. Retinal Neurons Organizing Section: RC Contributing Section: VN
895 - A19
Amacrine and Müller Cells Have Different Immunoreactivity for Aquaporin 4
in Light and Dark Adapted Teleost Retinas
896 - A20
Retinal Photoreceptors in Fossorial Tuco-Tucos (Rodentia, Ctenomys): Types,
Topographies, and UV Sensitivity
J. De Juan1, J.M. Romero del HombreBueno1, N. Martínez-Ruiz1, A. García-Alcaraz2. 1Dept
Biotecnologia, Universidad de Alicante, San Vicente del Raspeig, Spain; 2Centro
Oceanográfico de Murcia, Instituto Español de Oceanografía, Murcia, Spain.
L. Peichl1, A. Vielma2, M. Glösmann3, A.G. Palacios2, C.E. Schleich4. 1Max Planck Inst
for Brain Research, Frankfurt am Main, Germany; 2Centro de Neurociencia de
Valparaíso, Universidad de Valparaíso, Valparaíso, Chile; 3Inst of Physiology and
Pathophysiology, University of Veterinary Medicine, Vienna, Austria; 4Laboratorio
Ecofisiología, FCEyN, Universidad Nacional de Mar del Plata, Mar del Plata,
Argentina.
Purpose: Aquaporins (AQPs) are membrane proteins that facilitate water transport
across biological membranes, being essential for the correct function of the visual
system. Although AQPs have been described for most cellular types in the mammalian
retina, their identity in teleosts fish retinas remain unknown. In turn AQP4, expressed
in mammalian Müller cells, has an important role in the maintenance of extracellular
potassium homeostasis. We investigated the immunoreactivity of AQP4, in teleost
fish retina during light/dark adaptation.
Methods: The study was performed in two species of teleosts: Dicentrarchus labrax
and Sparus aurata. Animals were maintained on a daily 12 h light/dark cycle. Fish
were light- and dark-adapted at least 2 hours prior to sacrifice. Dark-adapted animals
were sacrificed under dim red light. We investigated the expression pattern and the
identity of AQP4 immunoreactive cells by immunocytochemistry
Results: AQP4 immunoreactivity was present in both species for amacrine cells
at the Inner Nuclear Layer and its processes through the inner plexiform layer. In
addition, Müller cells were also immunoreactive, being strongly labelled for S. aurata.
No differences between light/dark adaptation were observed in D. labrax. However,
AQP4 immunoreactivity for Müller cells in S. aurata was stronger in light condition.
Co-localization studies with GAD67 showed the GABAergic type identity of AQP4
immunoreactive amacrine cells in D. labrax.
Conclusion: Amacrine and Müller cells are immunoreactive for AQP4 in teleost
retinas, and show different cell expression patterns specie dependent. In addition,
AQP4 immunoreactivity for S. aurata changes between light- and dark- adapted retinas.
The presence of AQP4 in the teleost retina could be indicative of water redistribution
through different retinal cells and its implication in potassium regulation at the
inner retina.
CR: J. De Juan, None; J.M. Romero del HombreBueno, None; N. Martínez-Ruiz,
None; A. García-Alcaraz, None.
Support: Spanish Grants BFU2004-03727-C02-02
897 - A21
Conservation of Retinal GNB3 Expression in Vertebrate Species
Purpose: Cone and rod populations in two fossorial (facultative subterranean) rodent
species, Ctenomys talarum and C. magellanicus (tuco-tucos), were analyzed to elucidate
whether their retinas are more adapted to their near-lightless burrows or to their
occasional diurnal surface activity.
Methods: Paraformaldehyde-fixed retinas were dissected and prepared as flatmounts.
Overall photoreceptor densities were assessed with differential interference contrast
optics. Middle-to-longwave sensitive (L) cones and shortwave sensitive (S) cones were
immunolabeled by opsin-specific antisera, and their numbers and retinal distributions
determined. Genomic DNA was used to PCR-amplify and sequence the tuning-relevant
part of the S opsin gene.
Results: C. talarum and C. magellanicus had normally developed eyes. Overall
photoreceptor densities were comparatively low at 110,000-170,000/mm 2. 69-85%
of these were rods. However, the retinas showed high cone densities (15-31% of the
photoreceptors). The majority of cones expressed the L opsin, and a 6-16% minority
expressed the S opsin. No coexpression of L and S opsin was seen in any cones. L and S
cones had their peak densities in mid-ventral retina and lowest densities in the retinal
periphery. In C. talarum, L cone densities were 19,000-47,500/mm 2, S cone densities
1,800-7,300/mm 2. In both tuco-tuco species, the tuning-relevant amino acids of the S
opsin indicate sensitivity in the near UV rather than the blue/violet range.
Conclusions: The eyes of Ctenomys have low rod densities, similar to those of other
subterranean rodents (Nemec et al, BRB 75:356, 2008). However, their cone proportions
are higher than those of strongly subterranean species. Avoiding predators and
selecting food during the brief above-ground excursions may have exerted pressure to
retain robust cone-based vision in Ctenomys. The UV tuning of the S pigment is shared
by a number of rodents, while its adaptive advantage remains enigmatic.
CR: L. Peichl, None; A. Vielma, None; M. Glösmann, None; A.G. Palacios, None; C.E.
Schleich, None.
Support: PBCT-CONICYT ACT45 (to A.G.P.), PIP 5670 CONICET (to C.E.S.)
898 - A22
Theoretical and Behavioural Limit of Visual Resolution in Temperate and
Tropical Seahorses
E.R. Ritchey1A, R.E. Bongini1B, K.A. Code1C, C. Zelinka1B, S.M. Petersen-Jones2, A.J.
Fischer1B. ACollege of Optometry, BNeuroscience, CSchool of Allied Medical
Professions, 1Ohio State University, Columbus, OH; 2Small Animal Clinical
Sciences, Michigan State University, East Lansing, MI.
H. Lee, K. Bumsted O’Brien. Research School of Biology, ANU, ARC Centre of
Excellence in Vis Sci, Canberra, Australia.
Purpose: Guanine nucleotide-binding protein β3 (GNB3) is an isoform of the β subunit
of the heterotrimeric G protein second messenger complex. In the retina, GNB3 has
been shown to play a critical role in photoreceptor signal transduction. In Retinopathy,
Globe Enlarged (RGE) chickens, a spontaneous 3 base pair mutation in the GNB3
gene has been shown to cause vision loss with a progressive retinopathy and globe
enlargement. Despite the apparent importance of GNB3 for normal retinal function, the
patterns of expression of GNB3 in the retina remain relatively unknown. We examine
the expression patterns of GNB3 in the retinas of representative vertebrate species and
the presence of GNB3 mRNA and protein in wild-type and RGE chickens.
Methods: Retinal tissue from chickens, mice, guinea pigs, dogs and macaques
were fixed, sectioned and immunolabeled with antibodies for photoreceptors (Red/
Green Opsin, Human Cone Arrestin), bipolar cells (PKC, Islet1) and GNB3. Reversetranscription PCR and Western blotting were performed on postnatal day 7 wild-type
and RGE chickens retinas per standard protocols.
Results: We find that the pattern of expression of GNB3 in the retina is highly
conserved across vertebrate species, including wild-type chickens, mice, guinea pigs,
dogs and macaques. We find that chickens homozygous for the RGE allele maintain
mRNA transcription yet completely lack GNB3 protein in the retina. Regardless of
the species, we find that GNB3 is expressed by Islet1-positive ON-bipolar cells and
by cone photoreceptors.
Conclusions: GNB3 is expressed by cone photoreceptors and ON-bipolar cells across
multiple species. Given that analogous types of retinal neurons express GNB3 in
different species, we propose that the function of GNB3 and the mechanisms that
regulate its expression are highly conserved across species.
CR: E.R. Ritchey, None; R.E. Bongini, None; K.A. Code, None; C. Zelinka, None; S.M.
Petersen-Jones, None; A.J. Fischer, None.
Support: NIH Grant EY016043 (AJF); K12EY015447 (ERR)
Purpose: Seahorses are visually guided feeders that prey upon small, fast moving
crustaceans. Their feeding behaviour strongly suggests a mechanism for high
resolution vision although the presence of a fovea has been debated. The purpose of
this study was to determine whether seahorses have a fovea and to investigate the
limit of their spatial resolution by determining the density of ganglion cells (GC) in
order to calculate their theoretical limit of visual acuity, and to behaviourally measure
their visual resolution.
Methods: Retinas from two seahorse species, a temperate H. abdominalis (Ha, n=8)
and a tropical H. taeniopterus (Ht, n=4), were flat-mounted or processed for frozen
sections. GC densities were determined in both sections and flat-mounts stained
with propidium iodide. The theoretical limit of visual resolution was estimated
based on GC density and a lens diameter based retinal magnification factor. Reactive
distance, a behavioural measure of visual resolution, was tested in mature Ha (n=3)
and Ht (n=10) seahorses.
Results: Both species posses a rod free convexiclivate fovea in the ventro-temporal
retina. GC density was highest along the foveal slope (40,000 cells/mm 2 Ha and 55,000
cells/mm 2 Ht). GC densities of both species declined in the foveal center (25,000 cells/
mm 2). In the far periphery, GC densities gradually decreased to 18,000cells/mm 2. The
calculated theoretical limit of visual resolution on the foveal slope was 2.62 cpd for
Ha and 3.07 cpd for Ht. Therefore, at a distance of 10 cm, the theoretical limit of prey
size resolution was 0.33 mm (Ha) and 0.28 mm (Ht). In the behavioural experiments,
the limit of prey resolution for Ha was 3.28 cm while the limit for Ht was 2.04 cm at
a distance of 10 cm.
Conclusions: Both Ha and Ht retinas possessed a fovea. Ht had a greater density of GC
on the foveal slope and a higher behaviourally measured visual resolution compared
to Ha, although in both species, the calculated theoretical limit of visual resolution
was higher compared to behavioural measurements.
CR: H. Lee, None; K. Bumsted O’Brien, None.
Support: ARC Centre of Excellence in Vision Science
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
895-898
Monday, May 3, 8:30 AM - 10:15 AM Grand A Paper Session Program Number Range: 1230 - 1235
214. Programming and Signaling of iPS Cells and Retinal Progenitors
Organizing Section: RC Contributing Section: VI
1230 - 8:30AM
Isolation, Differentiation and Developmental Modeling of Early Retinal
Progenitor Cell Populations From Human Pluripotent Stem Cells
1231 - 8:45AM
The Lim-Homeodomain Gene Lhx2 Is Required for the Self-Renewal of Retinal
Progenitor Cells (RPCs) During Development
J.S. Meyer1A, E.E. Capowski1A, K.A. Wallace1A, A.D. Verhoeven1A, A.V. Sloman1A, J.M.
Martin1A, L.S. Wright1A, R. Stewart1B, J.A. Thomson1B, D.M. Gamm1A,1C. AWaisman
Center, BMorgridge Institute, COphthalmology and Visual Sciences, Eye Research
Institute, 1University of Wisconsin, Madison, WI.
E.M. Levine, S. Yun. Ophthalmology & Visual Science, Moran Eye Center at
University of Utah, Salt Lake City, UT.
Purpose: Established methods for deriving early retinal progenitors from human
pluripotent stem cells (hPSCs) typically yield a heterogeneous population of cells,
which complicates studies of retinal development. Therefore, we sought to develop a
simple method of isolating a highly enriched population of optic vesicle (OV) stage,
multipotent retinal progenitor cells from human ES and iPS cells.
Methods: hPSCs were differentiated toward a retinal lineage using a previously
described protocol. Highly enriched populations of OV stage retinal progenitors
were manually separated from forebrain progenitor populations and allowed to
differentiate for up to 120 days. Differences in gene expression between retinal and
forebrain progenitor populations were determined via PCR and microarray analyses.
Differentiating OV populations were then examined by qPCR and ICC to ascertain
their ability to generate retinal cell types. Subsequently, selected inducing factors
were added for discrete periods to study effects on cell fate choice.
Results: Upon initial isolation, >90% of all OV stage hPSC populations expressed
the definitive neural retinal progenitor marker Vsx2 (Chx10). Comparison of retinal
and forebrain progenitor populations revealed key differences in the expression
of numerous transcription factors, including Rx, Six6, Dlx1 and Nkx2.1. In vitro
maturation of OV populations produced all major classes of retinal cell types in a
manner reminiscent of normal development. Furthermore, treatment of hPSC-derived,
multipotent early progenitors with factors known to influence retinal development
affected cell fate choice in a predictable fashion.
Conclusions: Results from this study demonstrate that highly enriched populations of
OV stage, multipotent retinal progenitors can be isolated from hPSCs. This capability
will facilitate future studies of mechanisms of human retinogenesis and disease as
well as efforts to develop hPSC-based therapies.
CR: J.S. Meyer, None; E.E. Capowski, None; K.A. Wallace, None; A.D. Verhoeven,
None; A.V. Sloman, None; J.M. Martin, None; L.S. Wright, None; R. Stewart,
None; J.A. Thomson, None; D.M. Gamm, None.
Support: Foundation Fighting Blindness, Lincy Foundation, Research to Prevent
Blindness McCormick Scholar Award, Walsh Foundation, Retina Research Foundation
and Heckrodt Foundation, and NICHD P30 HD03352
1232 - 9:00AM
Differentiation of Rat Hair Follicle Stem Cells Into Photoreceptor Cells
Purpose: Lhx2 is a key regulator of early eye development. Lhx2 knockout mice
have anophthalmia and we recently showed that Lhx2 acts as a molecular node
linking eye field specification with lens formation and the patterning of the optic
neuroepithelium. Lhx2 is also expressed in the retina during the subsequent stages
of eye development, but the anophthalmic phenotype precludes studying its later
requirements. We therefore generated mice carrying an Lhx2 conditional allele
(Lhx2flox) with the Pax6 alpha enhancer cre driver (alpha-cre) and a tamoxifen (TM)
regulated cre driver active in RPCs (Hes1creERT2) to determine the requirements of Lhx2
during retinal histogenesis.
Methods: Lhx2flox; alpha-cre and Lhx2flox; Hes1creERT2 embryos (TM exposure starting
at E10.5) were harvested at several developmental timepoints. Phenotypes were
examined by immunohistochemistry and in-situ hybridization.
Results: Lhx2 is expressed in the majority of RPCs during retinal histogenesis. Loss of
Lhx2 resulted in an extensive depletion of RPCs due to a failure to remain in the cell
cycle regardless of the cre driver utilized or the time of Lhx2 inactivation. Interestingly,
the exited RPCs adopted fates that were characteristic of the stage when Lhx2 was
eliminated and this occurred at the expense of later generated cell types.
Conclusions: Our results suggest that Lhx2 regulates histogenesis by preventing
RPCs from exiting the cell cycle and from acquiring a state that predisposes or biases
them toward the cell fates being generated at that time. By acting in this manner,
Lhx2 maintains the RPC pool by actively promoting the self-renewal of an otherwise
uncommitted state.
CR: E.M. Levine, None; S. Yun, None.
Support: NIH Grant EY013760, RPB Sybil Harrington Scholar
1233 - 9:15AM
Transplantation of Adult Mouse iPS Cell-Derived Photoreceptor Precursors
Restores Retinal Structure and Function in Retinal Degenerative Mice
C. Jomary1, S.E. Jones2, A.J. Lotery1. 1Clinical Neurosciences, University of
Southampton, Southampton, United Kingdom; 2Biochemistry, Kings College
London, School of Biomedical and Health Sciences, United Kingdom.
Purpose: The aim of this study was to evaluate the potential of rat hair follicle stem
cells to transdifferentiate into photoreceptor-like cells, following modulation of the
microenvironment using retinal-specific conditions or after genetic modification
using the Crx transcription factor.
Methods: Epithelial stem cells were isolated from the hair follicle bulge region by
mechanical dissection, enriched by clonal expansion, and either subcultured in
retinal-conditioned media, or genetically modified by electroporation to express
exogenous epitope-tagged murine Crx. Changes in the expression of stem cell markers
(homeodomain transcription factor Pax6, POU transcription factor Oct3/4) and
putative skin stem cell markers (K15, alpha 6 integrin), neuronal markers (nestin,
neuron-specific class III ß-tubulin and neurofilament), and photoreceptor-specific
markers (rhodopsin, cyclic nucleotide-gated cation channel-3, blue-cone opsin, cyclic (c)
GMP phosphodiesterase) were evaluated by immunocytochemistry, Western blotting,
and quantitative reverse transcription-polymerase chain reaction.
Results: Isolated stem cells from the hair follicle bulge were successfully expanded by
clonal growth without a feeder layer. Both media derived from cultured retinal cells
and exogenous expression of Crx by genetic modification were found to be effective
in inducing a photoreceptor -like phenotype. Expression of stem cell markers of
proliferation and pluripotency was decreased. Concomitantly, expression of neuronal
and photoreceptor-specific markers was up-regulated.
Conclusions: The present study suggests that rat hair follicle epithelial stem cells
are capable of differentiation into photoreceptor phenotype cells ex vivo when either
exposed to retinal-specific microenvironment or genetically modified with Crx. The
present study extends our previous findings that exogenous Crx expression can induce
mouse and human retina-derived stem cells into functional photoreceptor cells and
is consistent with the notion that Crx has a broadly-applicable ability to promote
differentiation of such cells into photoreceptor phenotypes.
CR: C. Jomary, None; S.E. Jones, None; A.J. Lotery, None.
Support: Gift of Sight appeal, British Retinitis Pigmentosa Society
B.A. Tucker1, I.-H. Park 2, H.J. Klassen 3, S.M. Redenti1, C. Jiang4, S.D. Qi1, G.Q. Daley2,
M.J. Young1. 1Ophthalmology, Schepens Eye Research Institute, Harvard Medical
School, Boston, MA; 2Pediatric Hematology/Oncology, Children’s Hospital Boston
and Dana Farber Cancer Institute, Harvard Medical School, Boston, MA; 3Univ
of CA-Irvine, Orange, CA; 4Ophthalmology, Chinese Military General Hospital,
Beijing, China.
Purpose: To determine whether iPS cells, generated from adult mouse fibroblast,
could be used to produce retinal precursors and subsequently photoreceptor cells for
retinal transplantation and subsequent restoration of retinal function.
Methods: iPS cells were generated using fibroblasts isolated from the dermas of adult
dsRed mice via infection with retrovirus expressing Oct4, Sox2, KLF4 and c-Myc.
Retinal precursor cells were derived via targeted differentiation of iPS cells with
exogenous delivery of dkk-1, noggin, IGF1, bFGF, aFGF and DAPT. Western blotting,
immunocytochemistry, H&E staining, rt-PCR, microarray, in vitro Ca++ imaging, ERG
and subretinal transplantation were used to determine the capacity for pluripotency,
fate and functionality of undifferentiated and differentiated iPS cells.
Results: To identify pluripotency, adult mouse iPS cells grown under standard
undifferentiating conditions were tested via immunocytochemistry, rt-PCR and
teratoma formation assays. As with normal mouse ES cells, iPS cells expressed
the pluripotency genes SSEA1, Oct4, Sox2, KLF4, c-Myc and Nanog. Following
transplantation into the eye of immune-compromised retinal degenerative mice
these cells proceeded to form teratomas containing tissue comprising all three germ
layers. At 33 days post-differentiation a large proportion of the cells expressed the
retinal progenitor cell marker Pax6 and went on to express the photoreceptor markers,
CRX, recoverin, and rhodopsin. When tested using calcium imaging these cells were
shown to exhibit characteristics of normal retinal physiology, responding to delivery
of neurotransmitters. Following subretinal transplantation into degenerative hosts
these cells took up residence in the retinal outer nuclear layer and gave rise to increased
electro retinal function as determined by ERG.
Conclusion: Adult fibroblast-derived iPS cells provide a viable source for the
production of retinal precursors to be used for transplantation and treatment of
retinal degenerative disease.
CR: B.A. Tucker, None; I.-H. Park, None; H.J. Klassen, None; S.M. Redenti, None; C.
Jiang, None; S.D. Qi, None; G.Q. Daley, None; M.J. Young, None.
Support: Minda de Gunzburg Research Center for Retinal Transplantation, Foundation
Fighting Blindness Canada & USA, Discovery Eye and Lincy Foundations, and
Research to Prevent Blindness.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1230-1233
Monday, May 3, 8:30 AM - 10:15 AM Grand A Paper Session Program Number Range: 1230 - 1235
214. Programming and Signaling of iPS Cells and Retinal Progenitors
Organizing Section: RC Contributing Section: VI
1234 - 9:30AM
In vitro Induction of Retinitis Pigmentosa-Specific Photoreceptor Cells From
Patient-Derived Induced Pluripotent Stem Cells
1235 - 9:45AM
PAX6 and MITF Play a Dose-Dependent Role in Determining Cell Fate of the
Retinal Pigment Epithelium (RPE) and Putative Ocular Stem Cells
Z.-B. Jin, S. Okamoto, M. Takahashi. Laboratory for Retinal Regeneration, RIKEN
Center for Developmental Biology, Kobe, Japan.
K. Bharti1A, M. Gasper1A, M. Brucato1A, A. Maminishkis1B, S.S. Miller1B, H. Arnheiter1A.
A
NINDS, BNEI, 1National Institutes of Health, Bethesda, MD.
Purpose: Retinitis pigmentosa (RP) is a group of inherited retinal degeneration
characterized by night blindness and visual field defects which are caused by rod
photoreceptor death. Most causative genes are involved in phototransduction cascade
or signaling specifically in rod photoreceptor, and still are there some widely expressed
genes but their mutations only cause rod degeneration. The genetic heterogeneity
together with the inter-familiar and intra-familiar phenotypic variances in RP make
the disease research complex. It is thus attempted to investigate the phenotype of
individual photoreceptor cell with distinct genotype and its response to candidate
drugs. In this study, we aimed to generate patient-specific photoreceptor cells by
using an induced pluripotent stem (iPS) cells technology and an in vitro differentiation
strategy.
Methods: This study was approved by local ethical committee with informed consent
from patients. RP patients with different mutations/genes were studied. Fibroblast
cells were cultured from skin sample and were re-confirmed the genotype. The
fibroblasts were infected with retrovirus and/or non-integrating virus harboring four
reprogramming factors (OCT3/4, KLF4, c-MYC and SOX2). Established iPS cell lines
were amplified for in vitro differentiation. iPS cells were cultured under a serum-free
suspension conditions followed by an adherent culture. Immunocytochemistry and
gene expression profiling were performed to monitor the differentiation.
Results: Morphologically embryonic stem (ES) cell-like colonies were appeared after
infection of each type of virus. These cells were positive for pluripotent markers
(Nanog, Oct3/4, Tra-1-60, SSEA3). Furthermore, teratoma formation was confirmed by
all three-derm derivatives. Through in vitro differentiation, neural retinal progenitor
cells, retinal pigment epithelia (RPE) progenitor cells, RPE, and photoreceptor
precursor cells were induced sequentially. And rod photoreceptor cells emerged with
specific markers again rhodopsin and recoverin. Additionally, other types of retinal
cell, including cone, bipolar cells and ganglion cells, were also confirmed.
Conclusions: We successfully generated iPS cells from RP patients. These RP-derived
iPS cells do have differentiation potential into most retinal cells including the rod
photoreceptor cells which were lost in the patients. These induced patient-specific
rod photoreceptor cells may be useful for drug discovery, disease modeling, and
regenerative medicine.
CR: Z.-B. Jin, None; S. Okamoto, None; M. Takahashi, None.
Support: The Project for Realization of Regenerative Medicine from MEXT, Japan and
the RIKEN Foreign Postdoctoral Researcher program
Purpose: Previous observations in mice with mutations in the bHLH-zipper
transcription factor MITF suggested a potential role for increased levels of the paired/
homeodomain transcription factor PAX6 in regulating dorsal-restricted RPE-retina
transdifferentiation. Therefore, we evaluated the role of Pax6 gene dose in the RPE
and the ciliary epithelium (CE).
Methods: Histological and molecular evaluation of RPE-retina transdifferentiation
in mice harboring a combination of different Mitf and Pax6 alleles.
Results: A reduction of Pax6 gene dose in an Mitf mutant background markedly
enhances the hyperproliferation and dorsal-restricted RPE-retina transdifferentiation
observed with Mitf mutations alone. Conversely, an increase in Pax6 gene dose
decreases Mitf-mutation mediated hyperproliferation and transdifferentiation. This
regulation of RPE proliferation by Pax6 and Mitf is concomitant with an increase in
levels of alpha B crystallin which, based on studies in other cell types, decreases Cyclin
D1 protein stability through the ubiquitin ligase SCFFBX4 and so inhibits the cell cycle.
Moreover, PAX6 positively regulates TFEC, an MITF homolog, which, like MITF, is
thought to promote RPE development and inhibit retinogenic gene expression in the
RPE. In postnatal CE, high PAX6 levels expand a pool of putative stem cells while
low PAX6 levels, in conjunction with Mitf mutations, decrease this pool and increase
a pool of cells expressing retinal progenitor markers.
Conclusions: In the Mitf mutant RPE, a decrease in Pax6 gene dose leads to an increase
in retinal gene expression, and an increase in Pax6 gene dose to a decrease in retinal
gene expression. Consistent with this, in the postnatal CE, PAX6 levels regulate the
balance between putative stem cells and retinal progenitor cells. Thus, in both the
developing RPE and the postnatal CE, the coordinate reduction in PAX6 and MITF
activities helps to initiate the transition of cells towards a neuroretinal fate while an
increase in PAX6 and MITF/TFEC activities has antiretinogenic effects.
CR: K. Bharti, None; M. Gasper, None; M. Brucato, None; A. Maminishkis, None; S.S.
Miller, None; H. Arnheiter, None.
Support: NINDS
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1234-1235
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1400 - D614
Redox Proteomic Identification of Molecules Undergoing Disulfide Formation
as a Result of Experimental Retinal Ischemia
1401 - D615
Antioxidant Treatment Prevents Cigarette Smoke-Induced Cell Death and
Attenuates HO-1 Upregulation in ARPE-19 Cells
A.M. Dins1, C.J. Lieven1, J.D. Ribich1, L.A. Levin2,1. 1Ophthalmology and Visual
Sciences, University of Wisconsin, Madison, WI; 2Ophthalmology, University of
Montreal, Montreal, QC, Canada.
K.M. Bertram1A,1B, C.J. Baglole1A, R.P. Phipps1A,1B, R.T. Libby1C,1B. AEnvironmental
Medicine, BFlaum Eye Institute, CDepartment of Ophthalmology and Center for
Visual Sciences, 1University of Rochester, Rochester, NY.
Purpose:Retinal ischemia is a common result of embolic disease, diabetic vasculopathy,
venous disease, and very high elevations of intraocular pressure (IOP). Ischemia
followed by reflow causes production of certain reactive oxygen species (ROS), which
both cause direct cellular injury and may signal apoptosis. To elucidate the latter
signaling pathways, we used a redox proteomic approach to identify molecules
undergoing formation of intermolecular disulfide bonds as a result of acute retinal
ischemia.
Methods:The ischemia model employed raising the IOP to 110 mmHg in one eye of
anesthetized Long Evans rats for 60 min. The contralateral eyes served as controls.
Retinas were removed immediately or 4 hours after ischemia and prepared for gel
electrophoresis. Following a first dimension run in non-reducing conditions, the gel
lanes were excised and incubated in dithiothreitol (DTT) to reduce disulfide bonds,
followed by iodoacetamide to alkylate free sulfhydryls. After this incubation the gel
lanes were rotated 90 degrees and run in the second dimension. Gels were stained
with SYPRO Ruby and individual spots differing between conditions identified for
subsequent mass spectrometry.
Results:The vast majority of the proteins in the second dimension were on a 45 degree
diagonal, indicating that they did not contain disulfide bonds. Several spots appeared
below the diagonal, indicating the presence of disulfides that had been reduced during
the DTT incubation. There were a small number of protein spots that were present in
the 4 hour ischemia model and not in the control or 0 hour ischemia model, indicating
the presence of ischemia-dependent formation of intermolecular disulfide bonds.
Conclusions:Proteins that undergo disulfide formation after ischemia may be
important ROS-dependent signaling molecules for retinal neuronal death. These
proteins may be novel therapeutic targets for intervention in diseases characterized
by retinal ischemia.
CR: A.M. Dins, None; C.J. Lieven, None; J.D. Ribich, None; L.A. Levin, Wisconsin
Alumni Research Foundation, P.
Support: NIH R21EY017970, P30EY016665, Retina Research Foundation, Research
to Prevent Blindness.
Purpose: Cigarette smoking is a major risk factor for developing age-related macular
degeneration (AMD). We recently showed that cigarette smoke extract (CSE) damages
human retinal pigment epithelial cells (ARPE-19) in vitro (Bertram et al. Am J Physiol
Cell 2009). In these studies, heme oxygenase-1 (HO-1), an enzyme induced under
conditions of oxidative stress, was shown to be important for ARPE-19 cell viability
after CSE exposure. Here, we tested the hypothesis that antioxidants would prevent
cell death and cigarette smoke-induced HO-1 expression in human RPE cells.
Methods: ARPE-19 cells were pretreated with 1mM N-acetyl-cysteine (NAC), exposed
to CSE, and HO-1 protein expression determined by western blot. The MTT viability
assay was utilized to determine if antioxidant pretreatment could prevent cell death in
ARPE-19 cells exposed to CSE. All experiments were repeated at least three times.
Results: A non-lethal dose of CSE (0.1% CSE, 104 ± 7.9% cell survival compared to
unexposed cells) caused increased HO-1 protein expression compared with HO-1
levels in media-only treated cells (8.8 ± 0.9 fold increase; P<0.05). NAC treatment did
not prevent HO-1 upregulation (3.2 ± 0.7 fold increase compared to media-only treated
cells; P>0.05); however, the upregulation was significantly less than the increase in
HO-1 without NAC treatment (P<0.001). The viability of ARPE-19 cells was significantly
reduced after exposure to 1% CSE (51.5 ± 3.2% compared to unexposed cells; P<0.05).
Antioxidant pretreatment with NAC prevented the cell loss caused by 1% CSE exposure
(106 ± 12.7% compared to unexposed cells).
Conclusions: Antioxidant treatment prevented CSE induced cytotoxicity, but
was unable to fully prevent HO-1 upregulation. These data highlight the complex
regulation of HO-1 in human RPE cells exposed to cigarette smoke and suggest that
components of CSE besides oxidizing agents might contribute to HO-1 upregulation
after exposure to CSE. Further investigations are warranted to determine how HO-1
is regulated in RPE cells.
CR: K.M. Bertram, None; C.J. Baglole, None; R.P. Phipps, None; R.T. Libby,
None.
Support: Parker B. Francis Fellowship (CJB), Research to Prevent Blindness; ES01247,
Toxicology Training Grant; T32 ES07026
1402 - D616
Combined Effects of Aging and a Pro-Diabetic Diet on Retinal Function in a
Murine Model of Aging of the Human Eye
1403 - D617
Radioprotective Effects of a Cell-Permeable Redox Agent on Retinal
Endothelial Cells
E. Simon1, N. Acar1, A.M. Bron2,1, C.P. Creuzot-Garcher2,1, L. Bretillon1. 1INRA,
University of Burgundy, Eye & Nutrition Research Group, Dijon, France;
2
Ophthalmology, University Hospital, Dijon, France.
A.F. Thompson1, C.J. Lieven1, N. Sheibani1, L.A. Levin1,2. 1Ophthalmology and Visual
Sciences, University of Wisconsin, Madison, WI; 2Ophthalmology, University of
Montreal, Montreal, QC, Canada.
Purpose: Improvement of life expectancy and changes in the dietary behaviour of
developed populations are accompanied with the prevalence of diabetes and agerelated ocular pathologies. Aging of the retina is characterized by accumulation of
lipids at the basement of the retinal pigment epithelium - Bruch’s membrane complex.
Meanwhile increased oxidative stress is one of the features of aging and diabetes. A
fructose-rich diet induces insulin resistance and hypertriglyceridemia, mimicking
diabetes. The goal of our study was to evaluate the effects of a pro-diabetic fructoseenriched diet on the retinal function of the ApoB100,LDLR-/- mice, a murine model of
aging of the human eye.
Methods: Four month-old ApoB100,LDLR-/- and SF2J control mice were bred under either
a standard chow or a fructose-enriched diet (60%) during 6 months (n=6 in each group).
The functionality of the retina was evaluated by single flash electroretinography (ERG)
under scotopic conditions to assess the global response of the retina. The a-wave was
used to reflect the response of photoreceptors, and the b-wave the response of the
inner layers of the retina. Flicker ERG was monitored by increasing flash intensity
at a 10Hz-fixed frequency of the stimulus to specifically assess the sensitivity of
rods and cones.
Results: The fructose-enriched diet significantly decreased a- and b-wave amplitudes
of the retina to single flash ERG in both strains of animals. This fructose-induced
diminution was intensified in ApoB100,LDLR-/- mice compared to controls at a flash
intensity of 2500mcds/m²: -55.5% versus -33.4% for the a-wave (p<0.01), -41% versus
-38% for b-wave (p<0.05), respectively. The fructose-enriched diet impaired the
sensitivity of rods by 0.3 log unit (44 versus 20mcds/m², respectively) in both strains
of mice without affecting that of cones.
Conclusions: A pro-diabetic diet enhanced the effects of aging on the retina by
reducing its functionality and impairing its sensitivity to light stimulus. Oxidative
stress may be one of the mechanisms involved in these effects.
CR: E. Simon, None; N. Acar, None; A.M. Bron, None; C.P. Creuzot-Garcher, None; L.
Bretillon, None.
Support: None
Purpose: Radiation retinopathy and radiation optic neuropathy are common adverse
effects of radiation treatment of ocular tumors. One of the mechanisms for neuroretinal
radiation damage is injury to endothelial cells (Levin LA et al., Ophthalmology 107:370,
2000), possibly mediated by generation of reactive oxygen species. We hypothesized that
a phosphine-borane complex which we had previously shown to be neuroprotective
of retinal ganglion cells following axotomy (Schlieve CR et al., Exp Eye Res 83:1252,
2006) would protect retinal endothelial cells from radiation damage.
Methods: Retinal endothelial cells (Su X et al., Mol Vis 9:171, 2003) were subjected to
ionizing radiation from a 137Cs source, using single doses from 1 to 30 Gy. Experimental
groups of cells were treated with bis(3-propionic acid methyl ester)phenylphosphine
borane complex (PB1) at concentrations ranging from 100 pM to 100 uM 18 hours before,
immediately preceding, or 18 hours after irradiation. Cell viability was assessed by
staining live cells with calcein-AM 72 hours after irradiation. Cells were photographed
and batch-analyzed using ImageJ.
Results: Retinal endothelial cell viability was reduced to 56.1 ± 6.3% and 37.1 ±
3.5% of control following 10 and 30 Gy irradiation, respectively (p < 0.004 for both
comparisons). Treatment with 10 nM or 100 nM PB1 18 hours before, immediately
preceding, or 18 hours after 10 Gy irradiation significantly increased numbers of viable
cells (p < 0.05). Treatment with 10 nM PB1 18 hours after 10 Gy irradiation showed
the strongest protective effect (viable cells increased to 127.8 ± 20.3% of control, p =
0.005). Treatment with all PB1 concentrations tested between 100 pM and 100 µM at
any of the three experimental time points significantly increased numbers of viable
cells following 30 Gy irradiation (p < 0.05). 10 nM PB1 treatment immediately before
irradiation was most protective against this highest 30 Gy radiation dose (viable cells
increased to 101.1 ± 9.6% of control, p < 0.0001).
Conclusions: The redox modulating agent PB1 has a radioprotective (administration
before radiation) and radiomitigative (administration after radiation) effect in
irraditated retinal endothelial cells. The mechanism may relate to interruption of
oxidative signaling pathways for apoptosis induced by radiation.
CR: A.F. Thompson, None; C.J. Lieven, Cytodefense, E; N. Sheibani, Cytodefense, C;
L.A. Levin, Wisconsin Alumni Research Foundation, P; Cytodefense, I; Cytodefense,
E.
Support: NIH R21EY017970 and P30EY016665, an unrestricted departmental grant
from Research to Prevent Blindness, Inc, and a Wisconsin Alumni Research Foundation
Lead Discovery Initiative Grant
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1400-1403
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1404 - D618
Altered Expression of Tight Junctions in Apre19 Cells Under Endoplasmic
Reticulum Stress
1405 - D619
NSAIDs Induce Antioxidant Proteins in Retinal Pigment Epithelial Cells
N. Yoshinaga1A, N. Arimura1A, S. Sonoda1A, K. Takenouchi1B, S. Noma1C, K.-I. Kawahara1B,
T. Hashiguchi1B, I. Maruyama1B, T. Sakamoto1A. AOphthalmology, BLaboratory
and Vascular Medicine Cardiovascular and Respiratory Disorders Advanced
Therapeutics, CDivision of Respiratory Medicine, Respirology and Stress Care
Center, 1Kagoshima University, Kagoshima, Japan.
T. Yoshikawa1, N. Ogata1, I. Hiroshi2, S. Masamitsu2, H. Hideaki2, K. Takahashi 3.
1
Ophthalmology, Kansai Medical University, Moriguchi, Japan; 2Biofunctional
Molecules, Gifu Pharmaceutical University, Gifu city, Japan; 3Dept of
Ophthalmology, Hirakata Hospital, Hirakata, Japan.
Purpose:Endoplasmic reticulum (ER) stress has been linked to the pathogenesis of
the several diseases,e.g.. diabetes mellitus and Parkinson disease. ER stress induces
the expression of inflammatory cytokines, and inflammatory cytokines have been
reported to be the leading cause of diabetic retinopathy (DR) and age-related macular
degeneration (AMD). Because retinal pigment epithelial (RPE) cells are associated with
the development and pathogenesis of DR and AMD, we investigated the expression
of tight junctions, and angiogenic and /anti- angiogenic factors in RPE cells under
ER stress in vitro.
Methods:ER stress was induced in cultured ARPE19 cells , a human retinal pigment
epithelium cell line, by exposure to tunicamycin (TM; 1μg/ml) to inhibit N-linked
glycosylation or thapsigargin (TG; 1μM) to inhibit the sarcoplasmic/endoplasmic
calcium-ATPase. After 6, 12, 24, and 48 hours exposure, RNAs were extracted from
the ARPE cells. The expressions of; GRP78/Bip (Bip) and C/EBP-homologous protein
(CHOP) , markers of ER stress; zonula occluden (ZO)-1, occludin, and claudin1, genes
for tight junctions; and vascular endothelial growth factor (VEGF) and pigment
epithelium derived factor (PEDF) were determined by real time RT-PCR.
Results:The expressions of Bip and CHOP mRNAs were significantly increased in
the ARPE 19 cells time dependently under ER stress induced by both TM and TG.
The expression of CHOP was increased by forty-fold increase at 48 hrs compared to
that of the controls. The expressions of ZO-1, occludin, and claudin1 were increased
by TM and TG. In addition, the expression of VEGF mRNA was increased by both
TM and TG. The expressions of mRNAs of VEGF and occludin increased in a time
dependent way. The correlation between the degree of expression of VEGF and PEDF
was not significant.
Conclusions:The increased expression of tight junctions and VEGF in TM- or TGexposed ARPE19 cells indicate that the ER stress can alter the function of RPE cells
and may be involved in the pathogenesis of DR and AMD.
CR: T. Yoshikawa, None; N. Ogata, None; I. Hiroshi, None; S. Masamitsu, None; H.
Hideaki, None; K. Takahashi, None.
Support: None
Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) are common therapeutic
agents for ocular inflammatory diseases with less adverse effect. We intended to
analyze the effect of NSAIDs on induction of antioxidant proteins in human retinal
pigment epithelial (RPE) cells and in a rat model of choroidal neovascularization
(CNV).
Methods: We treated human RPE cell line, ARPE-19, with indomethacine (0-500
uM), bromfenac (0-200 uM), or vehicle control (dimethylsulfoxide). After treatment,
inductions of transcription factor NF-E2 related factor 2 (Nrf2) in nuclear protein
and its downstream protein heme oxygenase-1 (HO-1) in cytosolic protein were
assessed using western blots. The expressions of Nrf2 and HO-1 also were examined
by immunohistochemistry. In vivo expressions of Nrf2 and HO-1 were analyzed in
a laser-induced CNV model of rat treated with or without bromfenac ophthalmic
solution.
Results: Western blots showed both indomethacine and bromfenac induced the
translocation of Nrf2 into the nucleus and that the robust expression of HO-1 in ARPE19 with a dose- and time- dependent manner. Immunohistochemistry also showed
dose- dependent translocation of Nrf2 into the nucleus accompanied with expression of
Ho-1. Additionally, analysis in vivo showed that treatment with bromfenac ophthalmic
solution potentially led translocation of Nrf2 into the nucleus especially in the part
of laser-induced CNV.
Conclusions: Treatment with NSAIDs led to translocation of Nrf2 into the nucleus
and induction of antioxidant protein HO-1. The results suggest that NSAIDs has
properties as a potential antioxidant agent in ocular retinal diseases.
CR: N. Yoshinaga, None; N. Arimura, None; S. Sonoda, None; K. Takenouchi,
None; S. Noma, None; K.-I. Kawahara, None; T. Hashiguchi, None; I. Maruyama,
None; T. Sakamoto, None.
Support: None
1406 - D620
SS31 Protects Human RPE Cells From Oxidative Damage and Reduce LaserInduced Choroidal Neovascularization
1407 - D621
In-situ Spectroscopic and Morphological Analysis of Chronic Oxidative Stress
Induced ‘Lipofuscin-Like’ Fluorophores in Cultured Retinal Pigment Epithelial
Cells
X. Liang, F. Chen, H. Zhou, C. Yang, G. Sun, Q. Gao, Y. Luo, J. Ge. Key Lab of Ophthal
Ministry of Edu, Zhongshan Ophthalmic Center, Guangzhou, China.
Purpose: To investigate whether a new peptide SS31 may protect human retinal
pigment epithelial(hRPE) cells from oxidative damage and reduce choroidal
neovascularization in mice.
Methods: Cultured hRPE cells were pretreated with SS31 for 4 hours followed by
treatment with tert-butylhydroperoxide(t-BHP). Reactive oxygen species (ROS)
was measured by using H(2)DCF, changes in mitochondrial membrane potential
were detected by JC-1 dye, apoptosis was detected by using Annexin V-FITC Kit,
malondialdehyde(MDA) level was determined by a method based on the reaction with
thiobarbituric acid. Then, five to six-week-old C57BL/6 male mice had laser-induced
rupture of Bruch’s membrane at four locations in each right eye. Daily intraperitoneal
injections of 1mg/Kg(Group1), 9mg/Kg SS31(Group2) or vehicle(Control) were started
the day prior to laser photocoagulation. After one week, the mice were perfused with
FITC-dextran and CNV areas were measured on choroidal flat mounts.
Results: Pretreatment of hRPE cells with 1µM SS31 significantly reduced the t-BHPinduced intracellular ROS and MDA level by 46% and 34% respectively, protected
against t-BHP-induced decreases in mitochondrial membrane potential, and prevented
oxidant-induced cell apoptosis. In vivo study showed the average areas of CNV were
0.013±0.0034 mm 2/eye(Control),0.0068±0.0025mm 2/eye(Group1), 0.0067±0.0026mm 2 /
eye(Group2). Compared with the Control group, the average areas of CNV in Group1
and Group2 decreased 47.4% and 48.1% (p<0.01), but there was no significant difference
between SS31-treated groups.
Conclusions: These results suggest that SS31 could protect against oxidative damage
in hRPE cells and suppress Laser-Induced Choroidal Neovasculari-zation.It could
be effective against age-associated increase in oxidative stress and mitochondrial
dysfunction in RPE cells, and have clinical utility for treatment of Age-related Macular
Degeneration.
CR: X. Liang, None; F. Chen, None; H. Zhou, None; C. Yang, None; G. Sun, None; Q.
Gao, None; Y. Luo, None; J. Ge, None.
Support: Science and Technology Planning Project of Guangdong Province
2006BAI02B05; NSFC30973899; Stealth Peptides International (Shanghai) Inc.
E. van Kuijk, N. Tirgan, P. Gupta, Y. Jiang, B. Godley, M. Motamedi. Ophthalmology &
Visual Sciences, Univ of Texas Medical Branch, Galveston, TX.
Purpose: Lipofuscin is a conglomerate of many molecules with characteristic
autofluorescence and are found to accumulate in pathological conditions in the
retinal pigment epithelial cells (RPE) both in ageing and in various diseases.
Unfortunately, we lack knowledge on the nature of the composition of lipofuscin
and on the mechanistic pathways that lead to such formation. We investigated single
“lipofuscin-like” autofluorescent granule that accumulated at chronic oxidative stress
with the goal of resolving them spectroscopically for insights into its components
and mechanistic pathway.
Methods: Herein, first we present a daily oxidative stress model to induce artificial
lipofuscin material in RPE cells in culture and secondly, we analyzed the morphological
and emission spectroscopic properties of individual lipofuscin granules in-situ using
a Olympus Fluoview FV 1000 Multiphoton Laser Scanning Microscope system. Briefly,
ARPE-19 cells were exposed to 40uM H2O2 and/or leupeptin (lysosome inhibitor) and/
or (+/-) peroxidized rod outer segments (POS) for a chronic 7 day treatment.
Results: We found that there is an increase in the accumulation of autofluorescent
compounds under all the treatment groups when compared to controls after a
treatment of 7 days. Artificial lipofuscin ranged from small granules in the H2O2
group versus a “salt-pepper” dusty appearance in the Leupeptin + H2O2 group and
were coalesced into larger granules in the combination group that received H2O2,
Leupeptin and POS. Spectroscopically, there was a variation in the emission spectra
observed between different treatment groups. In the untreated group there was a
peak at 520nm, and the peak shifted to 540nm with the treatment of Leupeptin and
H2O2. Interestingly, the emission spectrum was broader in the granules from the
RPE cells that were exposed to a combination of H2O2, Leupeptin and UV-POS with
shoulder peaks at 530nm, 560nm and 610nm.
Conclusions: These data suggest that, though, RPE-lipofuscin may look similar
morphologically in various disease states in-vivo, spectroscopically they may be
composed of different fluorophores. These also suggest that the molecular events
that lead to such deposition of lipofuscin are probably different under different
conditions of stress.
CR: E. van Kuijk, None; N. Tirgan, None; P. Gupta, None; Y. Jiang, None; B. Godley,
None; M. Motamedi, None.
Support: Research to Prevent Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1404-1407
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1408 - D622
Expression of the Antioxidant, Heme- and Radical-Scavenger Alpha-1Microglobulin in the Eye
1409 - D623
H 2DCF Oxidation Seems to be Dependent on Free Iron and Cytochrome C as
Studied in ARPE-19 Cells
M. Cederlund1, F.K. Ghosh2A, S. Andreasson2B, B. Åkerström1. 1Infection Medicine,
Lunds University, Lund, Sweden; ADept of Ophthalmology, BOphthalmology,
2
Lund University Hospital, Lund, Sweden.
M. Karlsson1A, T. Kurz1B, U.T. Brunk1B, S.E. Nilsson1A, C.I. Frennesson1A. ADept of
Ophthalmology, BDept of Pharmacology, 1Linkoping University, Linkoping,
Sweden.
Purpose: To messure concentrations of alpha-1-microglobulin (A1M), hemoglobin and
oxidative stress in vitrectomy derived vitreous samples from patients with retinal
detachment, diabetic retinopathy and macular hole, and to investigate the expression
of A1M in the eye. Alpha-1-microglobulin (A1M) is a 26 kDa plasma and tissue protein
that was discovered in human urine 40 years ago. A1M is synthesized predominantly
in the liver, secreted to the blood stream and distributed to all tissues. Recent reports
have shown that A1M has radical-scavenging and reductase properties and it is
suggested that A1M functions as an antioxidant. A1M also binds and degrades heme.
Expression of A1M is induced in the liver and blood cells when the cells are exposed
to hemoglobin and it has also been shown that A1M protects human erythroid cells
against the oxidative damage caused by free heme.
Methods: Measurement of human hemoglobin concentration in the vitreous body
was performed using a competitive ELISA. Measurement of oxidative stress was
performed using a carbonyl group ELISA. A radioimunoassay was used to measure
the concentration of A1M. Real time PCR was performed to investigate the expression
of A1M in rat retina explantats. A1M-forms in the vitreous body was analyzed by
Western blotting.
Results: Hemoglobin, the oxidation product protein carbonyl groups and A1M were
found in various amounts in the vitreous body. Novel forms of A1M, not previously
seen in plasma, were found in the vitreous body. The A1M-gene is expressed in rat
retina explants.
Conclusions: Analysis of vitrectomy derived vitreous samples from patients with
retinal detachment, diabetic retinopathy and macular hole show that hemorrhage
and oxidative stress may be common in patients with these pathological conditions.
The results suggest that A1M plays a protective role against oxidative stress and cell
injury caused by hemorrhage in the eye.
CR: M. Cederlund, None; F.K. Ghosh, None; S. Andreasson, None; B. Åkerström,
None.
Support: None
Purpose: The dihydrodichlorofluorescein diacetate (H2DCF-DA) technique is often
used to assay cellular ”oxidative stress”. H2DCF-DA is a non-fluorescent, lipophilic
ester that is split by unspecific esterases intracellularly. One of the reaction products is
the hydrophilic alcohol H2DCF that is trapped within the cell where it may be oxidized
into fluorescent DCF by a process which is assumed to involve unspecified “reactive
oxygen species (ROS)”. We have previously demonstrated a weak mitochondrial DCFfluorescence in normal human ARPE-19 cells, while strong cytosolic fluorescence was
found only in cells with apoptotic morphology. Here we point out possible underlying
mechanisms for these findings.
Methods: Human immortalized ARPE-19 cells, grown on coverslips, were exposed
to 10 µM H 2DCF-DA for 30 min and then studied by confocal laser scanning
microscopy following mounting in Hank’s buffered salt solution (HBSS) or in
100 µM of the lysosomotropic detergent MSDH, which rapidly induces lysosomal
membrane permeabilization (LMP). In separate experiments, cells were exposed to
the lysosomotropic, metachromatic fluorochrome acridine orange (AO) for 15 min and
then mounted in MSDH as above. The dependence of H2DCF oxidation on free ionic
iron and cytochrome c was investigated in test tube experiments.
Results: Cells that were exposed to H2DCF-DA showed a weak mitochondrial-like
fluorescence pattern, while those exposed to H2DCF-DA + MSDH displayed a timedependent strong increase in cytosolic fluorescence that paralleled a decrease in the
number of intact AO-containing lysosomes. Test tube experiments proved both ionic
iron and cytochrome c to be potent catalysts of H2DCF-oxidation.
Conclusions: It appears that LMP is either an upstream or a potentiating event in
apoptosis that results in release of redox-active iron and lytic enzymes, which attack
mitochondria with ensuing release of cytochrome c. H2DCF-oxidation seems to rely
both on iron-mediated Fenton-type reactions and enzymatic oxidation by cytochrome
c in the presence of hydrogen peroxide and is dependent on initial LMP. The weak
mitochondrial fluorescence pattern of normal ARPE-19 cells seems to originate in
the intermembraneous space of the mitochondria, to which H2DCF has easy access
through the fenestrations of the outer membrane, where it encounters cytochrome
c and hydrogen peroxide.
CR: M. Karlsson, None; T. Kurz, None; U.T. Brunk, None; S.E. Nilsson, None; C.I.
Frennesson, None.
Support: Crown Princess Margareta’s Foundation for the Visually Handicapped and
the Linkoping University Hospital Research Fund
1410 - D624
Structural and Functional Changes in the Mouse Retina Elicited by Blue Light
Exposure
1411 - D625
Computational Modeling of Multiple Concurrent Oxidative Stress Processes in
the Retina
S.M. Hanks, M. Stefanidakis, J. Demirs, I.L. Jones, S. Liao, B.D. Jaffee, C.E. Bigelow.
Ophthalmology, Novartis Institutes for BioMedical Research, Cambridge, MA.
P.K. Fink1A, M.L. Denton2, L. Ibekwe1B, S. Ryan1B, X. Zhu1A, J. Oliver3, G. Pocock 3.
A
Computer Science, BBiology, 1St. Mary’s University, San Antonio, TX; 2Warfighter
Concepts and Applications, Northrup Grumman TASC, San Antonio, TX; 3711
hpw/rhdo, Air Force Research Laboratory, Brooks City-Base, TX.
Purpose: Blue light exposure in rodents is a model for investigating the morphological
and physiological changes that occur in response to oxidative stress. In an attempt
to further characterize this model in mice, we investigated how variation in both
exposure time and animal strain affect the structure, function, and molecular profile
of the retina.
Methods: Nine-week-old C57Bl/6 and Balb/c mice were dark adapted prior to being
treated with between 1 and 50 hours of blue light exposure (420nm, 550 lux). Retinal
function was quantified by electroretinography (ERG) after light exposure. A-wave
and b-wave amplitudes were monitored in response to a series of flashes ranging
from -4 to 1.8 log•scot•cd•s•m-2. Morphological alterations were assessed with optical
coherence tomography (OCT) while molecular changes were quantified by Western
blot and immunofluorescence (IF).
Results: Balb/c mice are highly sensitive to blue light exposure, exhibiting an
exposure-time dependent decrease in retinal function as measured by ERG. A-wave
and b-wave amplitudes were significantly reduced (36% and 42%, respectively) with
1.8 log flashes after as little as 2 hours of light exposure (p<0.001). Balb/c mice also
exhibited significant thinning of the photoreceptor layer as measured by OCT (-64%).
Eye tissue of these blue light exposed Balb/c mice also displayed autophagy specific
marker as measured by both Western blot and IF. In contrast, C57Bl/6 mice were
highly resistant to light exposure. These animals exhibited no retinal abnormalities
by either ERG or OCT.
Conclusions: We have shown that blue light exposure in Balb/c mice inhibits retinal
function as measured by electroretinography. OCT results indicate that the source
of this retinal dysfunction is likely preferential damage to the photoreceptors. Both
IF and Western blot results support these findings and indicate that the damage
involves autophagy.
CR: S.M. Hanks, Novartis Institutes for BioMedical Research, E; M. Stefanidakis,
Novartis Institutes for BioMedical Research, E; J. Demirs, Novartis Institutes for
BioMedical Research, E; I.L. Jones, Novartis Institutes for BioMedical Research,
E; S. Liao, Novartis Institutes for BioMedical Research, E; B.D. Jaffee, Novartis
Institutes for BioMedical Research, E; C.E. Bigelow, Novartis Institutes for BioMedical
Research, E.
Support: None
Purpose: A computer-based model was developed for various oxidative stress
processes on the retina. The model allows for the exploration in silico of questions
concerning the subcellular processes in RPE when exposed to triggers of oxidative
stress, such as lasers and aging.
Methods: The computer-based model was developed using a technique known as
BioFusion, that involves a specific process of collecting, analyzing, documenting, and
organizing knowledge available in the area of interest and then using this knowledge
to build a model. Implementation of the model is in a commercial, off-the-shelf
modeling tool that runs on a standard PC. This modeling technique was developed
specifically for building large, complex models of biological processes and systems.
The modeling approach is hierarchical and modular to deal with the complexity of
the processes. Also, it is capable of modeling the RPE cell in an in vitro or an in vivo
configuration.
Results: We have collected a large repository of information relating RPE cellular
responses to photo-oxidative stresses based on peer-reviewed articles and other
sources. We tested the ability of the model to indicate damage in the RPE upon
simulation of laser exposures of various wavelength and exposure duration. We
followed key intracellular processes in the RPE cell in response to laser irradiation
(metabolism, oxygen consumption, lipid peroxidation, protein damage, GSSH
metabolism, RedOx enzymes, iron and the Fenton reaction, lipofuscin production)
We analyzed the effects of melanin, NO, Vitamin A, and heat shock proteins on the
cellular response to laser exposure.
Conclusions: The model is capable of responding to acute oxidative insults such
as laser exposure, as well as to slower insults such as low-level light exposure over
extended durations. The model allowed for interactive exploration of questions and
theories that would be difficult if not impossible to explore in vitro or in vivo alone.
CR: P.K. Fink, Inventor on Patent, P; M.L. Denton, None; L. Ibekwe, None; S. Ryan,
None; X. Zhu, None; J. Oliver, None; G. Pocock, None.
Support: Contract #FA8650-08-C-6927 with the Air Force Research Labs’ Directed
Energy Division
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1408-1411
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1412 - D626
Complement System Dysregulation and Oxidative Stress in the abca4-/- Mice
R.A. Radu1, J. Hu1, Q. Yuan1, J. Makshanoff 1, D. Welch1, M. Lloyd1, G.H. Travis1, D. Bok 2.
1
Ophthalmology, UCLA/Jules Stein Eye Inst, Los Angeles, CA; 2Jules Stein Eye
Institute, University of California Los Angeles, Los Angeles, CA.
1413 - D627
The Zebrafish Eye: A Model for the Early Detection of Neurodegeneration in
Parkinson’s Disease
J.R. Leheste, B.H. Hallas, G. Torres. Neuroscience, NYCOM of NYIT, Old Westbury,
NY.
Purpose: Chronic inflammation due to activation of the complement system is an
important cause of age-related macular degeneration (AMD). Accumulation of
autofluorescent lipofuscin pigments, such A2E, within RPE cells is seen in several
forms of macular degeneration including AMD. A2E activates complement in
cultured RPE cells following light exposure. Oxidative stress also plays a role in AMD
pathogenesis. Previously, we showed that oxidation products of A2E are increased
in abca4 -/- mice exposed to high intensity light. In the current study, we investigated
the role of lipofuscin pigments and oxidative stress in activating the complement
system in abca4 -/- eyes.
Methods: Retinas and eyecups containing RPE were collected at designated timepoints from age-matched wild-type (WT) and abca4 -/- (KO) mice. Protein extracts
were tested for complement activation components and C-reactive protein by
immunoblotting. The expression levels for complement-regulatory protein (CRP)
and oxidative stress genes were measured by qRT-PCR and immunocytochemistry.
Lipid peroxidation levels in mouse RPE were determined by TBARS (for MDA) and
ELISA (for 4-HNE). Retina sections were analyzed by light and electron microscopy.
Visual retinoids and lipofuscin pigments were quantitated by high-performance
liquid chromatography.
Results: Retinal degeneration was seen in seven-mo-old KO mice. A2E and A2Eprecursors were significantly elevated in the KO vs. WT eyes at all time points.
Consistently, RPE autofluorescence was higher in the KO mice. Bruch’s membrane
was significantly thickened in 11-mo-old KO vs. WT mice. The KO mice also showed
increased complement C3 immunoreactivity predominantly within the basal
infoldings of the RPE. Quantitative Western blotting showed that iC3b was 1.7-fold
higher in KO vs. WT RPE cell homogenates. MDA and 4-HNE levels in KO eyecups
homogenates were 2.1-, and 3.1-fold respectively higher than in WT. By qRT-PCR, all
oxidative-stress genes were upregulated in KO RPE. Surprisingly, expression of all
CRP genes was reduced in the KO eyes.
Conclusions: Our data suggest that loss of ABCA4 causes dysregulation of the
complement system, chronic inflammation, and increased oxidative stress due to A2E
accumulation in the RPE. Moreover, down-regulation of CRP genes, perhaps as a result
of chronic insult, renders the RPE more susceptible to further complement attack.
CR: R.A. Radu, None; J. Hu, None; Q. Yuan, None; J. Makshanoff, None; D. Welch,
None; M. Lloyd, None; G.H. Travis, None; D. Bok, None.
Support: American Health Assistance Foundation M2008-090, Foundation Fighting
Blindness Center Grant for JSEI
Purpose: Key issues associated with age-related disease of the central nervous
system (CNS), such as Parkinson’s Disease (PD), are early detection and concrete
diagnosis. As a result, medical intervention is often delayed leading to a more rapid
aggravation of symptoms. An accessible in vivo system for the identification of early
signs relevant to human neurodegeneration is therefore needed. The larval zebrafish
eye is fully developed after 72h post fertilization (hpf) facilitating easy access to the
eye and its neural retina. We believe that the larval zebrafish eye constitutes the
ideal model system to identify early morphological signs associated with PD that
could be also tested via ophthalmoscopy in humans. The purpose of this study is
therefore to establish a reliable zebrafish model for PD allowing the detection of early
morphological aberrations associated with the disease.
Methods: All zebrafish embryos were treated to prevent pigmentation. Chemically
induced zebrafish PD models were generated with either, 5mM H2O2 or 40μg/ml
1,2,3,6-tetrahydropyridine (MPTP) for 24h. Transient genetic zebrafish PD models
were generated via RNA interference (RNAi) using zebrafish dj1 (park7) and mdm2
small hairpin oligonucleotides in combination. At 72hpf, all embryos were fixed;
dopaminergic (DA) neurons targeted with anti-tyrosine hydroxylase (TH) antibody,
and visualized. DA neurons in the ventral diencephalon of the zebrafish brain were
identified and counted. Embryos with a reduced DA count are now subject for
comparative morphological analysis via confocal microscopy
Results: Exposure to H2O2 specifically ablated approximately 80% of DA neurons
found in the ventral diencephalon of the zebrafish brain, an area that corresponds
with the nigrostriatal pathway in humans. Exposure to MPTP or RNAi reduced the
number of DA neurons in the same area by 10-20% which better mimics the early,
asymptomatic stages of PD.
Conclusion: We successfully ablated DA neurons in an area of the zebrafish brain that
corresponds to the substantia nigra in humans. We thereby recreated the key feature of
human PD in zebrafish. Our chemical and genetic zebrafish PD models mimic different
stages of PD thereby facilitating the search for morphological markers specific for those
stages in the zebrafish retina. Those markers may be useful in the early diagnosis of
PD in humans if they can be accessed via standard ophthalmoscopy.
CR: J.R. Leheste, None; B.H. Hallas, None; G. Torres, None.
Support: None
1414 - D628
Quantification of Expression of αB-Crystallin in Mitochondria in Human
Retinal Pigment Epithelial Cells and Its Secretion Using Enzyme-Linked
Immunosorbent Assay (ELISA)
1415 - D629
Effects of Combined Antioxidant/Cholesterol Supplementation in a Rat Model
of Smith-Lemli-Opitz Syndrome
M. Kitamura1,2A, P.G. Sreekumar1,2A, S. Sonoda1,2A, C. Spee1,2A, S.J. Ryan1,2A, R. Kannan1,2A,
D.R. Hinton1,2B. 1Doheny Eye Institute, Los Angeles, CA; AOphthalmology,
B
Pathology, 2Keck School of Medicine of the University of Southern California, Los
Angeles, CA.
Purpose: The aim of the present study was to a) standardize and validate an enzymelinked immunosorbent assay (ELISA) for quantification of αB-crystallin and b) to
apply the assay for measurement of αB-crystallin in mitochondria and secretion into
extracellular media of human cultured fetal RPE.
Methods: Early passage, confluent, cultured fetal human RPE cells and polarized RPE
cells were used for the study. RPE cells were switched to serum free medium for 16
h and were then exposed to 150 μM tert-butylhydroperoxide (tBH) for 2-4 h. Cytosol
and mitochondria were isolated using a Mitochondria/Cytosol Fractionation Kit for
αB-crystallin determination by ELISA using reagents from Stressgen. In separate
experiments, 24 h secretion of αB crystallin into extracellular medium was measured
in polarized and non-polarized RPE under unstimulated conditions.
Results: The ELISA standard curves were linear in the range 1.25 ng/ml-40 ng/ml
(r2=0.9996). The levels of αB-crystallin (ng/mg protein) in cytosol and mitochondria
of non-polarized RPE were 2522 ± 61 and 162 ± 4.7 respectively. tBH-induced oxidative
stress caused an increase in mitochondrial expression of αB-crystallin while the
cytosolic pool decreased. The 24 h secretion of αB-crystallin in non polarized RPE
was 2.22 ± 0.55 ng/106 cells. αB-crystallin was selectively secreted to the apical side
with negligible secretion to the basolateral side.
Conclusions: Our data show that αB-crystallin is expressed in mitochondria and is
regulated by oxidative stress. Using polarized RPE, we could also obtain quantitative
evidence for preferential secretion of αB-crystallin to the apical surface.
CR: M. Kitamura, None; P.G. Sreekumar, None; S. Sonoda, None; C. Spee, None; S.J.
Ryan, None; R. Kannan, None; D.R. Hinton, None.
Support: NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel
Beckman foundation
L.G. Sheflin1, J.E. Young1, N.S. Peachey2, S.J. Fliesler1. 1Research Serv.-VAWNYHS and
Depts. of Ophthalmol. Biochem., Univ. at Buffalo/SUNY, Buffalo, NY; 2Research
Serv.- Cleveland VAMC and Cole Eye Institute, Cleveland Clinic Foundn,
Cleveland, OH.
Purpose: Previously we showed that feeding a diet containing 2% cholesterol (Chol)
partially prevented retinal function deficits in the AY9944-induced rat model of SmithLemli-Opitz syndrome (SLOS) (Fliesler et al., 2007). Here, we assessed the additional
benefits of dietary antioxidant (AO) supplementation in this model.
Methods: Sprague-Dawley rats were treated with AY9944 (AY) to produce the SLOS rat
model (Fliesler et al., 2004); untreated age-matched rats served as controls. At weaning,
SLOS rats were randomized to three dietary groups: 1) AY (normal (0% Chol) chow);
2) AC (2% (w/w) Chol chow); and 3) AO (chow containing 2% Chol and 5% (w/w)
complex AO mixture). At 3 mo, rats were assessed by electroretinography (ERG); eyes
were harvested for histological analysis; companion tissues (retina, serum, liver) were
harvested for biochemical analyses at 3 mo as well as at earlier time points.
Results: No significant differences in retinal structure or function were observed
comparing AC vs. AO groups. While both showed similar, marked structural and
functional improvements over the AY group, neither were comparable to controls.
AY rats exhibited increased 4-hydroxy-2-nonenal (HNE) modification of three serum
proteins: ca. 120 kDa (120%), 80 kDa (35%), and 37 kDa (55%), relative to controls (p
<0.05). AC rats exhibited decreased HNE modification of these proteins (by 28%, 19%
and 14%, respectively; p<0.05), as did AO rats (by 29%, 32% and 46%, respectively;
p<0.05) compared to AY rats. AY rats also had decreased serum ApoE levels (by 70-90%;
p<0.05), compared to controls; AC and AO rats had serum ApoE levels comparable
to controls.
Conclusions: Under the conditions employed, while AO supplementation provided no
additional beneficial effect over 2% dietary Chol supplementation alone with regard
to retinal structure or function in the SLOS rat model, AO further reduced HNE
modification of some serum biomarker proteins. AO may require further optimization
to achieve desired beneficial effects.
CR: L.G. Sheflin, None; J.E. Young, None; N.S. Peachey, None; S.J. Fliesler, None.
Support: NIH Grant EY007361 (SJF); Veterans Administration (NSP); FFB (NSP);
RPB (SJF, NSP)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1412-1415
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1416 - D630
Chorioretinopathy in LCHAD Deficiency : The Role of Mitochondrial Oxidative
Stress
1417 - D631
Photooxidation Products Activate Complement Attack in Cultured Human
Retinal Pigment Epithelium
D. Stopek1, S. Majzoub2A, M. Le Lez2A, F. Labarthe2B. 1Ophthalmology, C.H.I.P.S.,
POISSY, France; AOphthalmology, BPediatrics, 2C.H.U., Tours, France.
J.G. Hu1, R.A. Radu2, J. Makshanoff 1, G.H. Travis3, D. Bok4. 1Ophthalmology, Jules
Stein Eye Institute, UCLA, Los Angeles, CA; 2Ophthalmology, UCLA/Jules Stein
Eye Inst, Los Angeles, CA; 3Jules Stein Eye Institute, UCLA School of Medicine, Los
Angeles, CA; 4Jules Stein Eye Institute, University of California Los Angeles, Los
Angeles, CA.
Purpose: Long-chain 3-hydroxyacyl CoA dehydrogenase (LCHAD) participates in
the mitochondrial fatty acid oxidation. Genetic fatty acid oxidation defects induce
cellular energetic deficiency, and thus early life threatening manifestations. An
appropriate diet prevents those severe manifestations in organs that consume much
fatty acid for their energetic needs. LCHAD deficiency is the only mitochondrial
fatty acid oxidation deficiency to induce moreover a chorioretinopathy. The attempt
consists in an atrophic degeneration predominating at the posterior pole and begins
histologically at the level of retinal pigment epithelium. What is the pathogenesis of
this specific chorioretinal degeneration ?
Methods: Reviewing of literature and biochemical mechanisms analysis were
combined.
Results: LCHAD deficiency chorioretinopathy appears linked to toxic accumulation
of 3-hydroxyacyl-carnitine rather than mitochondrial energetic defect. 3-hydroxyacyl,
carried by l-carnitine, is the only hydroxylated fatty acid of the pathway. It participates
in mitochondrial oxidative stress. Moreover, 3-hydroxyacyl-carnitine could interfere
with the peroxysomal metabolism of docosahexanoic acid (DHA) which is known
to participate in retinal metabolism. Regarding the prevention of chorioretinopathy,
appropriate energetic diet is not efficient. Administrations of l-carnitine and DHA
have been tested with controversial results.
Conclusion: Chorioretinopathy in LCHAD deficiency points out the chorioretinal
toxicity of 3-hydroxyacyl. Some clinical and histological analogies with age related
macular degeneration (ARMD) lead to hypotheses whether 3-hydroxyacyl is an actor
of mitochondria-based model of ARMD and retinal degeneration, based on retinal
pigment epithelium mitochondrial DNA susceptibility to oxidative stress?
CR: D. Stopek, None; S. Majzoub, None; M. Le Lez, None; F. Labarthe, None.
Support: None
Purpose: Recent studies have suggested that inappropriate complement attack is
involved in the pathogenesis of age-related macular degeneration (AMD). We examined
the response of cultured human retinal pigment epithelium complement activation
by photooxidation products.
Methods: Cultured human fetal RPE cells were grown to confluence in porous culture
wells for 2 months, and were exposed to rod outer segments (ROS) from BALB/C and
Albino abca4-/- mice Thereafter, the expression of complement regulatory proteins and
anti-oxidative stress genes was measured by qRT-PCR, and the localization and content
of the negative regulator, CD46 was imaged by immunohistochemistry. To further test
whether photooxidation products such as A2E activate complement attack on RPE,
we loaded the cells with A2E and irradiated them with blue light in the presence of
10% human serum as a source of complement. We examined the formation of the
C5b-9 membrane attack complex using immunocytochemistry and immunoassay and
measured the formation of C3b and C5b in the medium by western analysis.
Results: qRT-PCR showed that the expression of mRNA for complement regulatory
proteins, CFH, CD46, CD55, and CD59 was increased by 1.67-, 2.85-, 2.28-, and 2.03fold respectively after RPE cells were fed ROS from albino abca4 -/- mice compared
with BALB/c mice. In addition, expression of the anti-oxidative stress genes SOD1,
Metallothionein 1A, Catalase 1,HO and GSTM was increased by 1.9-, 1.35-, 1.22-, 1,29,
and 1.32 respectively. Immunostaining of CD46 was elevated in RPE cells exposed
to ROS of abca4 -/- mice. The membrane attack complex was detected using anti-C5b-9
antibodies after hRPE cells were exposed to A2E and blue light in the presence of
10% human serum. Western analysis of culture medium containing human serum
showed the presence of C3b and C5b.
Conclusions: Our gene expression and morphological data suggest that photooxidation
products such as A2E can activate complement attack of cultured human RPE. These
results support the notion that A2E and complement activation are involved in AMD
pathogenesis.
CR: J.G. Hu, None; R.A. Radu, None; J. Makshanoff, None; G.H. Travis, None; D.
Bok, None.
Support: NIH/NEI R24 EY017404, Dolly Green Prof. Endowment Award
1418 - D632
Clusterin Protects Human Retinal Pigment Epithelial Cells from Oxidative
Stress-Induced Apoptosis
1419 - D633
Protective Effect of 17Beta Estradiol on Benzo(e)pyrene Induced Toxicity in
ARPE-19 Cells
Y.S. Yu1, J. Kim1, J. Kim1, B. Min2, K.-W. Kim 3. 1Ophthalmology/Coll of Med, Seoul
National Univ Hosp, Seoul, Republic of Korea; 2Pharmacology/Coll of Med, Korea
University, Seoul, Republic of Korea; 3Pharmacy, Seoul National Univ, Seoul,
Republic of Korea.
A.U. Sapkal, V.R. Sharma, C.R. Ramirez, R.Z. Migon, N. Gupta, M. Chwa, B.D.
Kuppermann, M.C. Kenney. Gavin Herbert Eye Institute, University of California,
Irvine, CA.
Purpose: The oxidative stress to retinal pigment epithelial (RPE) cell is thought to
play a critical role in the pathogenesis of age-related macular degeneration (AMD).
This study is to investigate whether clusterin protects human RPE cells from ROSinduced apoptosis through PI3K/Akt survival pathway.
Methods: The preventive effect of clusterin on reactive oxygen species (ROS) production
and RPE cell death induced by hydrogen peroxide was determined in ARPE-19 cells.
The ability of clusterin to protect RPE cells against ROS-mediated apoptosis was
assessed by caspase-3 activity and DAPI staining. Furthermore, the protective effect
of clusterin via PI3K/Akt pathway was determined by Western blot analysis.
Results: Clusterin prevented ARPE-19 cells from H2O2-induced cell death and ROS
production. H2O2-induced oxidative stress increased caspase-3 activity, which was
significantly inhibited by clusterin as determined by abrogation of apoptotic bodies.
Interestingly, clusterin induced Akt phosphorylation in human RPE cells under
oxidative stress, which contributed to cellular viability in ARPE-19 cells. This cellular
survival by clusterin was blocked by a PI3K inhibitor.
Conclusion: Clusterin may play a protective role in responding to the local redox
environment of human RPE cells, which contributes to the cellular survival via
PI3K/Akt pathway. Therefore, clusterin couldd be considered for the preventive
approach to AMD.
CR: Y.S. Yu, None; J. Kim, None; J. Kim, None; B. Min, None; K.-W. Kim, None.
Support: None
Purpose: To investigate the in vitro interaction of 17Beta Estradiol (βE2, a hormonal
antioxidant) and Benzo(e)pyrene (B(e)P, a major component of cigarette smoke) on a
human retinal pigment epithelial cell line (ARPE-19).
Methods: ARPE-19 cells were cultured in Dulbecco’s modified Eagle’s medium
containing 10% bovine growth serum. Cells were pretreated with 0 nM (no protective
pretreatment) or 20 nM or 40 nM βE2 for 6 hrs and then 300 µM B(e)P was added to all
cells for an additional 24 hrs. Because B(e)P requires dimethyl sulfoxide (DMSO) for
dissolution, DMSO control was also performed on cells without βE2 pretreatment.
Cell viability was measured using a trypan blue dye-exclusion assay. JC-1 assay was
performed to measure mitochondrial membrane potential (ΔΨm) and caspase-3/7
activity was assayed to measure apoptosis.
Results: The mean cell viability percentage of ARPE-19 cells treated with 300 µM B(e)P
alone decreased to 71.6 ± 1.3 and it increased to 82.4 ± 0.8 with 20 nM βE2 pretreatment
(P<0.05). DMSO equivalent control for B(e)P was 98.40 ± 0.30. ΔΨm after B(e)P treatment
alone decreased to 3.14 ± 0.03 and after 20nM βE2 pretreatment increased to 6.007 ±
0.08 (P < 0.001) and to 5.423 ± 0.75 in cells pretreated with 40 nM βE2 (P < 0.05). DMSO
equivalent control for B(e)P was 6.322 ± 0.7991. Caspase-3/7 activity in ARPE-19 cells
treated with 300 µM B(e)P alone increased to 17260 ± 552.5, and decreased to 6386
± 132.3 with 20 nM βE2 pretreatment (P<0.001) and to 12060 ± 611.2 with 40 nM βE2
pretreatment (P<0.01). DMSO equivalent control for B(e)P was 2817 ± 395.8.
Conclusions: B(e)P is a major component of cigarette smoke known to induce apoptotic
cell death. Estrogen has been previously shown to protect ARPE-19 cells from oxidative
stress. Smoking increases the chances of Age-Related Macular Degeneration (AMD)
while Estrogen may protect against AMD. Our study demonstrates that βE2 plays a
protective role in ARPE-19 cells against B(e)P induced toxicity by reducing apoptosis,
and increasing both cell viability and mitochondrial membrane potential.
CR: A.U. Sapkal, None; V.R. Sharma, None; C.R. Ramirez, None; R.Z. Migon,
None; N. Gupta, None; M. Chwa, None; B.D. Kuppermann, None; M.C. Kenney,
None.
Support: Discovery Eye Foundation, Guenther Foundation, Lincy Foundation, Iris and
B. Gerald Cantor Foundation, Ko Family Foundation, Gilbert Foundation, Research
to Prevent Blindness.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1416-1419
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1420 - D634
Resveratrol Protects Human Retinal Pigment Epithelial Cells From SmokingRelated Oxidative Stress
1421 - D635
Characterization of DJ-1 in the Retina and Retinal Pigment Epithelium
V.L. Bonilha, M.E. Rayborn, Y. Li. Ophthalmology, Cole Eye Inst/ Cleveland Clinic
Lerner College of Medicine, Cleveland, OH.
S.-J. Sheu1,2, N.-C. Liu1. 1Department of Ophthalmology, Kaohsiung Veterans Gen
Hospital, Kaohsiung, Taiwan; 2Medical School, National Yang-Ming University,
Taipei, Taiwan.
Purpose: Although the exact pathogenesis of age-related macular degeneration
(AMD) is not clear, most studies indicate a role for retinal pigment epithelial cell
damage and death caused by oxidative stress. The purpose of this study was to
examine the potential protective effect of lutein, zeaxanthin, meclofenamic acid,
and resveratrol on the acrolein-induced oxidative stress in human retinal pigment
epithelial (RPE) cells.
Methods: Cultured human RPE R-50 cells were treated with acrolein at different
concentrations and treatment times. The protective effects of lutein (100 μM), zeaxanthin
(100 μM), meclofenamic acid (30 μM), and resveratrol (10 μM) were investigated by
pretreatment with the above agents before toxicant exposure in acute toxicity models
and co-treatment with the toxicant in chronic toxicity models. The synergistic effects
of hyperoxide exposure were also studied. Fluorescent latex beads were used to assess
the phagocytic function of the cells.
Results: Acrolein inhibited the phagocytic function of human RPE R-50 cells, and
the inhibitory effects were time dependent. Pretreatment with lutein, zeaxanthin,
meclofenamic acid, or resveratrol alleviated the inhibition of phagocytosis in the acute
acrolein and combined acrolein/hyperoxide toxicity model. Co-treatment with lutein,
zeaxanthin, meclofenamic acid, or resveratrol showed a protective effect against the
damage caused by 7-day acrolein exposure followed by hyperoxide treatment.
Conclusions: Our results indicated an inhibitory effect of compounds found in
cigarette smoke on human RPE phagocytosis, and lutein, zeaxanthin, meclofenamic
acid, and resveratrol each offered protection against this inhibition. Therefore, red
wine polyphenol, resveratrol, might prevent smoking-induced or age-related RPE
degeneration, such as AMD.
CR: S.-J. Sheu, None; N.-C. Liu, None.
Support: The study was supported by grant NSC97-2314-B-075B-011 from the National
Science Council and grants VGHKS 98-063 from Kaohsiung Veterans General Hospital,
Kaohsiung City, Taiwan.
Purpose: DJ-1 was first discovered as a novel oncogene product that transforms mouse
NIH3T3 cells in cooperation with activated ras and was later identified as a causative
gene of familial Parkinson’s disease. DJ-1 is a multifunctional protein involved in
pathways that regulate cell survival, mitochondrial function, modulation of the PTEN/
Akt survival pathway, suppression of Ask1-mediated apoptosis, increased synthesis of
glutathione, expression of Hsp70, tyrosine hydroxylase and antioxidative stress. DJ-1
is highly expressed in the testis and moderately in other tissues. DJ-1 peptides were
detected in rat retinal pigment epithelium (RPE) cell fractions subjected to proteomic
analysis. The present study was conducted to further analyze the localization and
function of DJ-1 in mouse retinal tissues and RPE cells.
Methods: Paraffin sections of mouse retinas were processed for immunofluorescence
with the DJ-1 specific antibodies followed by analysis with confocal microscopy. The
presence of DJ-1 was analyzed in whole cell lysates from retina, RPE, and several RPE
cell lines. To decipher the DJ-1function, RPE cultures were treated with H2O2 (100
to 800μM) and 4-HNE (5 to 100μM) for various times followed by biochemical and
immunohistological analysis.
Results: DJ-1 immunoreactivity is associated with cells in the ganglion cell layer, cells
in the inner nuclear layer, photoreceptors and RPE. Interestingly, DJ-1 was frequently
distributed around the three to five outermost rows of photoreceptor nuclei. In RPE
cells under baseline conditions, DJ-1 displays a diffuse cytoplasmic and nuclear
staining of all the RPE cell lines analyzed. A major band of ~ 25κDa was was observed
in the extracts of mouse retina and RPE and in all the RPE cell lines as compared to
extracts from mouse brain. RPE cells exposed to oxidative stress lead to a significant
increase in DJ-1 expression as analyzed by immunocytochemical and biochemical
assays. Moreover, upon oxidative injury, a large portion of DJ-1 redistributed to
mitochondria. Finally, overexpression of DJ-1 leads to increased viability of cells
exposed to oxidative stress.
Conclusions: DJ-1 is expressed in retinal tissue using both biochemical and
immunohistological studies. Upon exposure to oxidative stress DJ-1 expression
increased. In addition, DJ-1 expression is involved in the oxidative stress response
in RPE cells in vitro.
CR: V.L. Bonilha, None; M.E. Rayborn, None; Y. Li, None.
Support: Supported in part by NIH grants EY017153 and EY15638, a Research to
Prevent Blindness Unrestricted Grant and funds from the Cleveland Clinic.
1422 - D636
Effect of 4-Hydroxynonenal on EGFR Mediated Signaling Pathway in RPE Cells
1423 - D637
Inhibition of Retinal Glycolysis by Oxidative Stress. Prevention by Pyruvate
R. Vatsyayan1, P. Chaudhary2, P. Lelsani2, A. Sharma2, R. Sharma2, S. Awasthi2, Y.C.
Awasthi2. 1Molecular biology and immunology, UNTHSC, FORT WORTH, TX;
2
Molecular biology and immunology, UNTHSC, Forth Worth, TX.
K.R. Hegde1A, S. Kovtun1A, S.D. Varma1B. AOphthalmology & Visual Sciences,
B
Ophthalmology & Visual Sci & Biochem, 1Univ of Maryland Sch of Medicine,
Baltimore, MD.
Purpose: 4-Hydroxynonenal (4-HNE), an stable end-product of oxidative stress
induced lipid peroxidation (LPO) is an important second messenger molecule
involved in the regulation of various cellular processes including proliferation,
transformation and apoptosis. Previous studies have shown that 4-HNE activates EGFR
but the mechanism(s) of activation, its effect on downstream signaling components,
and its biological significance are not understood. Since LPO has been implicated
in the mechanisms of retinopathy we have studied the effects of 4-HNE on the
downstream components of EGFR signaling pathway in RPE cells in order to elucidate
the physiological significance of 4-HNE mediated activation of EGFR.
Methods: SV40-Transformed human fetal male RPE 28 cells cultured in DMEM
containing 10% FBS and antibiotics in a humidified incubator at 37oC in 5% CO2
atmosphere were treated with 4-HNE (0-70 μM) for 12 h. The extent of total cell
death was measured by MTT and apoptosis was determined by TUNNEL assays.
Expression of EGFR and its downstream targets pERK/ERK and pAKT/AKT were
compared in 4-HNE treated RPE cells by immunoblotting and immunofluorescence
studies. These effects of 4-HNE were compared in presence and absence of EGFR,
ERK and AKT inhibitors.
Results: Up to 5 μM final concentration in medium, 4-HNE induced time and
concentration dependent sustained activation of EGFR in RPE cells. 4-HNE also
activated EGFR downstream targets pERK/ERK and pAKT/AKT. Combined treatments
of 4-HNE (1μM and 5μM,) with EGFR inhibitor AG1478 (0.1 μM -10μM), MEK inhibitor
UO126 (0.5 μM -50μM), and PI3K inhibitor LY 294002 (0.5 μM -50μM), in separate
experiments, caused enhanced cell death in RPE cells strongly indicating that the
activation of EGFR mediated by 4-HNE is a protective mechanism against oxidative
stress.
Conclusions: These results suggest that activation of EGFR and its downstream
pathway by 4-HNE is an adaptive response in RPE cells for protection from oxidative
stress.
CR: R. Vatsyayan, None; P. Chaudhary, None; P. Lelsani, None; A. Sharma, None; R.
Sharma, None; S. Awasthi, None; Y.C. Awasthi, None.
Support: NIH Grants-EY04396, ES012171, CA77495
Purpose: We have previously shown that pyruvate prevents retinal oxidative stress
involved in triggering the pathogenesis of senile macular degeneration, diabetic
retinopathy, etc.. Its effect has been previously attributed to its oxyradical scavenging
properties. We hypothesize that its protective effect is also attributable to its ability to
overcome glycolytic inhibition associated with oxidative stress. The present studies
were undertaken to verify this hypothesis.
Methods: The hypothesis has been verified by measuring glycolysis in retinal explants
incubated for 3.5 hrs in medium199 generating ROS with and without pyruvate
(10mM) and determining tritiated water (3H2O) generated from 5-3H glucose. ROS
was generated by addition of xanthine and xanthine oxidase to the medium. The
medium was then analyzed chromatographically for 3H2O. The chromatographic
system consisted of an anion exchange (-OH) mini-column piggybacked on a boronate
column. The radioactivity in the eluate accounted for 3H2O generated by enolase during
glycolysis. Lactate was measured spectrophotometrically by the LDH reaction with
NAD as a co-substrate. GSH was measured in the acid extract of the tissue using
Ellman’s reaction.
Results: Incubation with ROS decreased the yield of 3H2O to about 50% (0.77 µmoles/mg
protein) of the controls incubated without ROS (1.63µmoles/mg). Pyruvate abolished
this decrease. The ROS-induced decrease in lactate as well as GSH were also prevented
by pyruvate.
Conclusion: The results are in conformity with the hypothesis proposed. The
mechanism by which pyruvate overcomes ROS induced inhibition of glycolysis
is attributable to the prevention of oxidative inactivation of key -SH dependent
glycolytic enzymes such as the GAPDH, as well as to its ability to regenerate NAD+
by its conversion to lactate by LDH. Hence the compound protects the tissue against
ROS damage by acting as a metabolic agonist as well as an oxyradical scavenger. The
findings are considered potentially useful from pharmacological point of view.
CR: K.R. Hegde, None; S. Kovtun, None; S.D. Varma, None.
Support: NIH Grant EY01292
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1420-1423
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1424 - D638
Very Low Density Lipoprotein Receptor (VLDLr) Null Mice Exhibit Changes in
Expression of Oxidative Stress Genes Which Are Reversed by Nanoceria
X. Cai1A, L.L. Wong1A, S. Seal2, J.F. McGinnis1A,1B. AOphthalmology, Health Sci Ctr,
B
Cell Biology & OCNS, Health Sci Ctr, 1Univ of Oklahoma, Oklahoma City, OK;
2
3AMPAC, MMAE, Nanosci. and Tech. Ctr., Univ of Central Florida, Orlando, FL.
Purpose: The VLDLr mouse, which develops retinal vascular lesions, choroidal
neovascularization and retinal degeneration, is a model for a form of Age Related
Macular Degeneration (AMD) called Retinal Angiomatous Proliferation (RAP). Because
anti-oxidants have been shown to inhibit the development of the VLDLr phenotype
and Nanoceria (cerium oxide nanoparticles) catalytically scavenge reactive oxygen
species (ROS), we investigated whether the VLDLr null gene affects the expression of
“oxidative stress-related” genes and if the Nanoceria prevent such changes.
Methods: VLDLr-/- mice received a single intravitreal injection of either saline or 344
nanograms of Nanoceria in saline at postnatal day 7 with uninjected VLDLr-/- and
wild type (WT) as controls. Total RNA was isolated from the retinas at postnatal day
28 using TRIzol. cDNA was synthesized by first strand superscript III transcriptase
and oligo-dT primers. Real-time PCR was performed to survey genes involved in
oxidative stress using mouse oxidative stress and antioxidant defense PCR array
(SABiosciences). Data were analyzed using SAB software and shown as fold changes
compared to the controls.
Results: Knocking out the VLDLr gene results in the upregulation, compared to
WT controls, of a number of genes associated with protection from oxidative stress
including Lactoperoxidase, Thioredoxin reductase, Glutathione peroxidase and
Peroxiredoxin. Genes down regulated include Cathepsin, Dual oxigenase and
Nucleoredoxin. Nanoceria application reversed such regulation.
Conclusions: Expression of key anti-oxidative stress genes in the VLDLr-/- retinas is not
sufficient to prevent the retinal lesions. However, the Nanoceria by scavenging ROS,
prevent the development of retinal vascular lesions and result in the downregulation
of the anti-oxidative stress genes.
CR: X. Cai, None; L.L. Wong, 7347987, P; S. Seal, 7347987, P; J.F. McGinnis, 7347987,
P.
Support: NIH:P30-EY12190, COBRE-P20 RR017703, R21EY018306, R01EY018724. FFB
C-NP-0707-0404-UOK08. NSF:CBET-0708172.. Funds from PHF and RPB and anRPB
SSI award to JFM.
-/-
1426 - D640
Molecular Structure Determination of Several High Molecular Weight
Hydrophobic Components of Human Retinal Lipofuscin
1425 - D639
Detoxicant Actions of Sulfhydryl Compounds in Retina
B.S. Winkler, G. Mane, R. Deda, M. Thomas, M.L. Duenow. Eye Research Institute,
Oakland University, Rochester, MI.
Purpose: A recent publication from our laboratory (Winkler, IOVS, 49:3259, 2008)
proposed that the exceptional vulnerability of photoreceptor cells to metabolic
poisons and oxidants is linked to a specific deficiency in glutathione (GSH) and that
the daily renewal of outer segments serves as a surrogate antioxidant/protectant.
To test this hypothesis, we determined whether an elevation in the level of a GSHmimetic in retinal cells protects against iodoacetate (IAA), a well-known inhibitor
of glyceraldehyde-3-phosphate dehydrogenase (G3PDH) that selectively damages
photoreceptor cells.
Methods: Isolated rat retinas were incubated in HEPES-buffered media of normal
ionic concentration (pH=7.4, ambient air, 37C) for 1-3 hrs in the presence and absence
of 10 mM N-acetylcysteine (NAC), a membrane permeable SH-containing compound
that has antioxidant and detoxicant actions similar to GSH. During the incubations,
samples of the media were withdrawn for estimation of lactate production and the
level of NAC. At the end of the incubations, some control and NAC-treated retinas
were transferred to media containing 0.01 mM iodoacetate (IAA) for 15 min. At the
end of the 15 min exposure to IAA, retinas were rinsed in two successive 30 sec washes
in pre-warmed control media and then homogenized in appropriate solutions for
measurements of soluble SH-content and activity of G3PDH.
Results: The content of soluble SH was similar in retinas incubated in control media
over 3 hrs (20 nmoles/retina), but retinas incubated in media containing 10 mM NAC
showed a time-dependent increase in the level of soluble SH. After 1 hr, SH-content
was increased 3-fold and after 3 hrs SH-content was increased 5-fold. Aerobic lactate
production was increased by 33% in NAC-treated retinas relative to the rate in control
retinas, i.e., 1.35 micromoles lactate/hr/retina. Incubating control retinas in 0.01 mM
IAA for 15 min led to a 60% inhibition of G3PDH, whereas in retinas pre-treated in
NAC-containing media IAA did not inhibit the activity of G3PDH.
Conclusions: These results provide evidence of the beneficial effects of supplementation
of retinas with a membrane-permeant thiol (NAC) against a specific chemical toxin
that is known to selectively target photoreceptor cells. Protection was signalled by the
lack of inhibition of G3PDH in retinas supplemented with NAC and is likely due to
detoxification of IAA by NAC via a carboxymethylation reaction. In contrast, if NAC
or GSH is not present (e.g., in photoreceptor cells), then this reaction will not occur,
resulting in inhibition of G3PDH and death of the cells.
CR: B.S. Winkler, None; G. Mane, None; R. Deda, None; M. Thomas, None; M.L.
Duenow, None.
Support: NIH Grant EY18568
1427 - D641
X-Box Binding Protein 1 is a Novel Regulator of Anti-Oxidant Genes in the RPE
Y. Zhong, J. Li, J. Wang, Y. Le, S. Zhang. Department of Medicine Endocrinology,
Harold Hamm Oklahoma Diabetes Center, The University of Oklahoma Health
Sciences Center, Oklahoma City, OK.
E.R. Gaillard1, L.S. Murdaugh2, J.P. Dillon 3. 1Chemistry & Biochemistry, Northern
Illinois University, DeKalb, IL; 2Chemistry and Biochemistry, Northern Illinois
University, Dekalb, IL; 3Ophthalmology, Columbia University, New York, NY.
Purpose: To determine the structures of the main components of the hydrophobic,
higher molecular weight fraction of human RPE lipofuscin using high pressure liquid
chromatography-mass spectroscopy (LC-MS) with PDA detection. This fraction is ca
10 times more abundant than A2E itself.
Methods: Human RPE lipofuscin granules were isolated as described (Feeney-Burns,
1980) from donor globes (Midwest Eye Banks and Transplantation Centers). The organic
soluble portion was obtained by extraction with equal amounts of CHCl3:CH3OH:H2O,
and the extract was analyzed by LC-MS (Thermo Finnigan, LCQ Advantage, Surveyor;
Surveyor LC with fluorescence and PDA detectors, quadrupole ion trap mass analyzer,
electrospray ion source). 1H NMR spectra were recorded with a Bruker Avance 500
MHz NMR.
Results: A visible absorbing species in RPE lipofuscin has been identified previously
as a bis-retinoid pyridinium compound referred to as A2E. Most of the remainder of
the chromophores in RPE lipofuscin are structurally related to A2E as determined
by their fragmentation pattern with losses of M+- 106, 190, 174 and/or 150 amu and
the formation of fragments of ca. 592 amu. These have discrete molecular weights of
800-900 m/z, 970-1080 m/z and above 1200 m/z regions. The majority of the mixture
consists of relatively hydrophobic components corresponding to derivatized A2E;
most likely formed by the polymerization reaction with aldehydes formed by the
oxidation of other A2E moieties. Detailed mass spectral analysis and NMR structural
analysis of these compounds allowed for the tentative prediction of specific molecular
structures.
Conclusion: Lipofuscin and its reaction products harvested from human RPE are
detected readily by LC-MS and are structurally related to A2E. Aging of RPE lipofuscin
results from the auto and/or photooxidation of A2E to form aldehydes which then
further react with A2E to give a series of polymers that are much more hydrophobic
than A2E. This increases log P (hydrophobicity factor) and induces the sequestering
of these derivatives into granules with a concomitant diminution in reactivity. These
reactions also trap resultant aldehydes which would oxidize cellular components
with concomitant cellular damage.
CR: E.R. Gaillard, None; L.S. Murdaugh, None; J.P. Dillon, None.
Support: None
Purpose: Oxidative damage of the retinal pigment epithelium (RPE) is a significant
pathogenic factor of age-related macular degeneration (AMD). Identifying key
regulators of the anti-oxidant defense system can provide critical information for
developing therapeutic modalities to prevent or halt the development and progression
of AMD. X-box-binding protein (XBP1) is a bZIP transcription factor that induces
genes involved in multiple cellular processes, such as biogenesis of the endoplasmic
reticulum (ER), protein folding and lipogenesis. The purpose of this study is to
investigate the role of XBP1 in the regulation of the anti-oxidant system and oxidative
stress in the RPE.
Methods: Human RPE (ARPE-19) cells were transfected with siRNA specific for XBP1
or control siRNA for 24 h. The mRNA and protein expressions of anti-oxidant genes were
examined by real-time PCR and Western blot analysis, respectively. Intracellular superoxide
(O2-) and reactive oxygen species (ROS) were determined by dihydroethidium (DHE) and
dichlorofluorescein (DCF) fluorescence. Cell viability was determined by 3-[4,5-dimethylthiazol2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay.
Results: Depletion of XBP1 significantly decreased the mRNA expression of antioxidant genes, including nuclear-factor-E2-related factor 2 (Nrf2), superoxide
dismutase (SOD1 and SOD2), catalase, and glutathione synthase, in human ARPE19 cells. Decreased protein levels of Nrf2 and SOD2 in XBP1-deficient cells were
confirmed by Western blot analysis. These changes coincided with increased levels
of intracellular O2- and ROS. In addition, depletion of XBP1 significantly decreased
cell viability in ARPE-19 cells.
Conclusions: Our results suggest XBP1 is a novel transcription factor coordinating
the cellular protective system in the RPE. Insight into the mechanisms of XBP1 on
regulation of anti-oxidant genes and developing XBP1-based therapeutic strategies
may provide novel approaches to prevent and treat AMD.
CR: Y. Zhong, None; J. Li, None; J. Wang, None; Y. Le, None; S. Zhang, None.
Support: NIH grant P20RR024215, JDRF grant 5-2009-475, and a research award
from OCAST
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1424-1427
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1428 - D642
Studies of Zinc and Vitamin C Modulation of AP-1 Gene Regulation in the
Human Retinal Pigment Epithelium Under Oxidative Stress
1429 - D643
Increased Albumin in Cells Exhibiting Oxidative Damage in Retinas of
Glaucomatous Monkey Eyes
E. Chaum1, J. Yin1, W. Huo1, J.C. Lang2. 1Ophthalmology, Univ of Tennessee Health Sci
Ctr, Memphis, TN; 2Alcon Research Ltd., Ft. Worth, TX.
L.D. Carter-Dawson1, R.S. Harwerth2. 1Ophthalmology & Visual Science, Univ
of Texas Houston Med Sch, Houston, TX; 2Optometry, University of Houston,
Houston, TX.
Purpose: We have previously shown that oxidative stress (OS) can quantitatively
regulate AP-1 gene expression changes and that pretreatment with specific doses of
vitamin C alone, in vitro, may play an important protective role in the human retinal
pigment epithelium (RPE) under conditions of OS. Other micronutrients, specifically
dietary zinc, have been shown to reduce the risk of progression of age-related macular
degeneration in the AREDS clinical trials. The purpose of this study was to determine
whether AP-1 biomarker responses to quantified levels of OS are modulated by
pretreatment with zinc alone and if zinc supplementation can synergistically enhance
the protective effects of vitamin C pretreatment in vitro.
Methods: Confluent ARPE-19 cells were cultured for three days in defined media in
the presence of zinc (0 - 60µM) alone, or zinc with vitamin C (100 and 200µM), and
then treated for 1 hour with 500µM H2O2. RNA was isolated using a no-rinse method
at 0-, 1-, 4-, 8-, and 24-hours after OS and compared to untreated controls at each time
point. AP-1 family genes FosB, cFos, JunB, ATF3, and EGR2 transcription factor gene
expression was determined by quantitative PCR.
Results: Zinc pretreatment alone, or in combination with vitamin C, did not alter
AP-1 family or EGR2 gene expression in response to OS compared with the controls.
Alternative transcription factor and zinc-coenzyme pathways that may play a role
in mediating the clinically protective responses to zinc are being evaluated and will
be presented at the meeting.
Conclusions: Unlike vitamin C, the protective effects of zinc supplementation seen
in the AREDS clinical studies do not appear to be mediated directly through AP-1
transcription factors under experimental conditions of acute OS in the human RPE.
CR: E. Chaum, Alcon Research Ltd., F; Alcon Research Ltd., P; J. Yin, Alcon Research
Ltd., F; W. Huo, None; J.C. Lang, Alcon Research Ltd., E; Alcon Research Ltd., P.
Support: Alcon Research Ltd., Research to Prevent Blindness, and the Plough
Foundation
1430 - D644
The Effect of Resveratrol on Oxidative Apoptosis of RPE Cells
A. Kaushal1, S. Upadhyay2, S. Kaushal1. 1Department of Ophthalmology, University
of Massachusetts Medical School, Worcester, MA; 2Department of Ophthalmology,
University of Florida, Gainsville, FL.
Purpose: To determine whether the Phase 2 antioxidant resveratrol can prevent
hydroquinone (HQ)-induced RPE cell death.
Methods: ARPE-19 cells were cultured by standard methods. HQ was added to the cells
to establish a kill curve so that a range of experimentally convenient concentrations
(250-350 μM) could be used in all subsequent studies. These concentrations have been
previously studied and shown to be an accurate model of RPE oxidative damage. ARPE19 cells were then exposed to HQ and co-incubated with various concentrations of
the natural product resveratrol. The number of viable cells was quantified, apoptosis/
necrosis was assayed by Annexin V (Calbiochem), and cleaved caspase-3 assays were
performed via flow cytometry.
Results: A dose-dependent reduction of cell viability in response to HQ exposure
was observed. Significant ARPE-19 cell death occurred at 200 μM HQ, and more than
60% of the cells were dead at concentrations of 250 μM and above. The data showed
a typical cell death curve with increasing concentrations of HQ. In the presence of
increasing concentrations of resveratrol (500nM - 250 μM), there was a significant
increase in viable cells at 48 hrs. Annexin V staining revealed an increase in apoptotic
cells with increasing HQ dosage, and in the presence of 250 μM resveratrol, a significant
increase in unstained cells was observed. Cleaved caspase-3 analysis revealed a
drastic reduction in downstream caspase-3 activation in the presence of 250 μM
resveratrol.
Conclusions: Resveratrol can reduce cell death in an in vitro HQ-induced model of
oxidative injury to the RPE. These in vitro studies warrant further exploration of the
effect of resveratrol in mouse models of ARMD.
CR: A. Kaushal, None; S. Upadhyay, None; S. Kaushal, None.
Support: None
Purpose: We have previously reported an increase of albumin in retinas of
glaucomatous monkey eyes, which was localized to the inner retina. The current
study investigated whether the increased albumin found in glaucomatous monkey
retinas also co-localized with cells exhibiting increased oxidative damage, as indicated
by increased nitrotyrosine immunoreactivity.
Methods: Posterior segments from monkeys with unilateral experimental glaucoma
were fixed in 4 % paraformaldehyde in phosphate buffer. Retina samples, 1mm
in diameter, were taken from the temporal inferior region of each eye, 5mm from
the fovea, and vibratome sections were cut. The sections were immunolabeled
with a monoclonal antibody to nitrotyrosine and a polyclonal antibody to human
serum albumin. Antibody binding was visualized with a Cy3 and a Cy5 conjugated
secondary antibody. Images of retina from the laser treated right eye and the control
left eye of each monkey were taken using identical parameters with a Zeiss confocal
microscope.
Results: Control retinas exhibited light immunoreactivity for nitrotyrosine in the inner
retina. In comparison, many more of the cells in the inner-most part of the inner nuclear
layer and the inner plexiform layer of glaucomatous retinas were heavily labeled for
nitrotyrosine. Albumin immunoreactivity co-localized with nitrotyrosine positive
cells in the inner nuclear layer and with cell processes in the inner plexiform layer.
Co-localization of nitrotyrosine and albumin was also detected in the region of the
ganglion cell/nerve fiber layer in the absence of most ganglion cells.
Conclusions: The co-localization of albumin with nitrotyrosine indicates a link
between accumulation of albumin and oxidative damage. Since albumin is a major
antioxidant, its enhanced presence in nitrotyrosine positive regions suggest that
increased albumin in glaucomatous retinas may be attributed to increased oxidative
stress. Albumin may provide cell protection against oxidizing species in the inner
retina, but the amount of albumin maybe insufficient to protect retinal ganglion
cells.
CR: L.D. Carter-Dawson, None; R.S. Harwerth, None.
Support: NIH/NEI grants R01 EY01139, T32 EY10608 and P30 EY07751 and Research
to Prevent Blindness
1431 - D645
Zeaxanthin Supplementation Reduces Photo-Oxidative Damage and Modulates
the Expression of Inflammation-Related Genes in Retinal Pigment Epithelial
Cells
S. Gao1, Q. Bian1, J. Zhou2, J. Qin1, A. Taylor1, E.J. Johnson1, G. Tang1, J.R. Sparrow2,
D.L. Gierhart3, F. Shang1. 1Human Nutrition Res Ctr on Aging, Tufts University,
Boston, MA; 2Department of Ophthalmology, Columbia University, New York, NY;
3
Zeavision, LLC, Chesterfield, MO.
Purpose: Oxidative damage and excessive inflammatory responses are etiologically related to
the pathogenesis of AMD. Epidemiologic studies suggest that insufficient dietary zeaxanthin
intake increase the risk for AMD. The objective of this work is to test the protective effects
of zeaxanthin against photo-oxidative damage to RPE and oxidation-induced changes in
expression of inflammation-related genes.
Methods: To mimic lipofuscin-mediated photo-oxidation in vivo, cultured ARPE-19 cells were
allowed to accumulate A2E, the major fluorophore and photosensitizer of lipofuscin, for 10
days and then cultured in the presence or absence of 10 μM zeaxanthin for an additional 3
days. The cells were then exposed to blue light for 10 min, followed by 6 h recovery in normal
medium. The levels of mRNA and proteins of IL-6, IL-8, MCP-1 and complement factor H
(CFH) were determined by real-time RT-PCR and ELISA, respectively. Proteasome activity
in the cells was measured using fluorogenic peptides as substrates.
Results: Exposure of RPE to blue light alone had no detectable effect on expression of IL-6,
IL-8, MCP-1 and CFH. Exposure of RPE to A2E alone increased the expression of IL-8 and
MCP-1 by 2-fold. In contrast, exposure of A2E-containing RPE to blue light resulted in 5080% decrease in expression of CFH and MCP-1 and ~ 20-fold increase in expression of IL-8.
Exposure of A2E-containing RPE to blue light also resulted in 40-60% decrease in proteasome
activity. However, pre-incubation of the A2E-containing RPE with zeaxanthin significantly
attenuated the photo-oxidation-induced inactivation of the proteasome and photo-oxidation
induced changes in expression of IL-6, IL-8, MCP-1 and CFH. The photo-oxidation-induced
changes in expression of MCP-1, IL-8 and CFH are similar to those caused by chemical
inhibition of the proteasome, indicating that zeaxanthin modulates photo-oxidation-induced
alterations in expression of MCP-1, IL-8 and CFH partially via protecting the proteasome
from inactivation. Furthermore, supplementation of zeaxanthin to cultured RPE cells also
reduced the expression of IL-6 and IL-8 induced by LPS.
Conclusion: These data indicate that zeaxanthin modulates inflammatory responses in
cultured RPE in proteasome-dependent and proteasome-independent manners. The antiinflammatory effects of zeaxanthin may be a mechanism by which it protects against
AMD.
CR: S. Gao, None; Q. Bian, None; J. Zhou, None; J. Qin, None; A. Taylor, None; E.J. Johnson,
None; G. Tang, None; J.R. Sparrow, None; D.L. Gierhart, Zeavision. LLC, I; Zeavision. LLC,
E; Zeavision, LLC, P; F. Shang, Dennis L. Gierhart charitable gift fund, F.
Support: NIH EY011717 (to FS) USDA CRIS 1950-51000-060-01A; USDA NIFA 2009-3520005014 (to FS); Dennis L. Gierhart charitable gift fund (to FS)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1428-1431
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1432 - D646
Unique Sensitivity of Cone Photoreceptor Precursor Tumor Cells, 661W, to
Cerium Oxide Nanoparticles
1433 - D647
Inhibitory Effect of Cx43 on Oxidative Stress Induced VEGF in Human Retinal
Pigment Epithelial Cells
L.L. Wong1, X. Yu1,2, S.A. Sezate1, S. Seal3, J.F. McGinnis1,4. 1Ophthalmology, Univ of
Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 2Biochemistry, School of Medicine,
Xi’an Jiaotong University, Xi’an, China; 3AMPAC, MMAE, Nanosci. and Tech. Ctr.,
Univ of Central Florida, Orlando, FL; 4Cell Biol. and OCNS, Univ. of Oklahoma
Health Sciences Center, Oklahoma City, OK.
C.M. Hutnik1, S. Khimdas1, H. Liu1, C. Pocrnich1, D. Laird2, C. Shao2. 1Ophthalmology,
Ivey Eye Institute, London, ON, Canada; 2Anatomy and Cell Biology, University of
Western Ontario, London, ON, Canada.
Purpose: Cerium oxide nanoparticles (CNPs) possess regenerative radical scavenging
activities that mimic the catalytic functions of superoxide dismutase and catalase in vitro.
At concentrations as low as 0.000516 µg/ml, CNPs significantly reduced reactive oxygen
species (ROS) generation in primary retinal cell cultures challenged with hydrogen peroxide.
Presently, we investigated whether the CNPs at concentrations up to 33000X higher, had any
cytotoxic effect on five different cell lines.
Methods: We assessed the viability of five cell lines after exposure to CNPs at a number of
concentrations and durations. These were mouse cone photoreceptor precursor tumor cells
(661W, from M.R. Al-Ubaidi), human retinal pigment epithelial cells (ARPE19, from American
Type Culture Collection (ATCC): cat. # CRL-2302), human corneal epithelial cells (THE, from
J. Chodosh), primary human umbilical vein endothelial cells (HUVEC, from M.A. Ihnat), and
human microvascular endothelial cells (HMEC, from M.A. Ihnat). All were adherent cells
with specific culture conditions. Cell viability was determined using either the CellQuantiMTTTMCell Viability Assay Kit (BioAssay Systems) or CellTiter 96® AQueous One Solution Cell
Proliferation Assay Kit (Promega). Apoptosis assessment by flow cytometry was performed
using the Annexin V-PE Apoptosis Detection Kit I (BD Biosciences). Reagents for ROS
detection (5-(and-6)-carboxy-2’,7’-dichlorodihydrofluorescein diacetate, dihydroethidium,
and 3’-p-aminophenyl fluorescein) for flow cytometry were from Invitrogen.
Results: At maximum concentration examined, 17.2 µg/ml of CNPs for 48 hours, there
was no cytotoxicity observed in four of the cell lines. However, 661W cells were uniquely
sensitive to CNPs and showed reduction in viability at 8.6 µg/ml after incubation for 72
hours. This result paralleled the increase in apoptosis and production of ROS observed
under these conditions.
Conclusions: At 1.72 µg/ml, 3300X higher than the concentration observed to be effective for
radical scavenging activity, CNPs are not cytotoxic. However, they do facilitate the production
of ROS, specifically superoxide anions and hydroxyl radicals in 661W cells at concentrations
at or above 8.6 µg/ml after incubation for 72 hours. Under these conditions, CNPs cause an
increase in apoptosis in 661W cells most likely due to oxidative damage.
CR: L.L. Wong, 7347987, P; X. Yu, None; S.A. Sezate, 7347987, P; S. Seal, 7347987, P; J.F.
McGinnis, 7347987, P.
Support: NIH:P30-EY12190, COBRE-P20 RR017703, R21EY018306, R01EY018724. FFB C-NP0707-0404-UOK08. NSF:CBET-0708172. OCAST:HR06-012. unrestricted funds from PHF and
RPB; and an RPB SSI award to JFM.
Purpose: To determine the effect of connexin43 (Cx43) expression and gap junctional
intercellular communication (GJIC) on the expression and secretion of vascular
endothelial growth factor (VEGF) from the retinal pigment epithelium under normal
cell culture and oxidative stress conditions.
Methods: Stable cell lines of ARPE-19 were produced in which Cx43 was either
over-expressed, down-regulated by targeted shRNA, or functionally inhibited by
co-expression of a disease-linked dominant-negative mutant (G21R). Pharmacologic
blockade of GJIC was accomplished with flufenamic acid. Oxidant challenge was
performed with tert-butyl hydroperoxide (tBH). VEGF gene expression and secretion
were assessed by real-time PCR and ELISA, respectively. Serum was collected from
wild-type mice, mice expressing a dominant-negative mutant of Cx43 and Cx43
null mice.
Results: Over-expression of Cx43 in ARPE-19 cells reduced both gene expression and
secretion of VEGF. Down-regulation of Cx43 increased gene expression and secretion
of VEGF. Increased secretion of VEGF was also observed in ARPE-19 cells expressing
a dominant negative mutant of Cx43 or when GJIC was blocked. Over-expression
of Cx43 reduced tBH-induced secretion of VEGF from ARPE-19 cells. Finally, Cx43
mutant mice and Cx43 knock-out mice displayed relatively higher levels of serum
VEGF than wild-type mice.
Conclusions: Expression of Cx43 in ARPE-19 cells reduces VEGF secretion under
normal cell culture conditions and protects against tBH-induced VEGF secretion.
CR: C.M. Hutnik, None; S. Khimdas, None; H. Liu, None; C. Pocrnich, None; D.
Laird, None; C. Shao, None.
Support: None
1434 - D648
Regulation of Oxidative Stress in Light-Induced Retinal Degeneration
1435 - D649
Redox Regulation of Low Molecular Weight PTP in Retinal Endothelial Cells
T. Narimatsu1A, T. Kurihara1B,1A, S. Kubota1B,1A, S. Ishida2,1A, Y. Ozawa1B,1A, K. Tsubota1B,1A.
A
Laboratory of Retinal Cell Biology, BDepartment of Ophthalmology, 1Keio
University School of Medicine, Tokyo, Japan; 2Department of Ophthalmology,
Hokkaido University Graduate School of Medicine, Hokkaido, Japan.
M.A. Abdelsaid, A.B. El-Remessy. Clinical and Experimental Therapeutics,
University of Georgia, Augusta, GA.
Purpose: Excessive visible light induces apoptosis of retinal photoreceptor cells.
Previous reports show that the cell death is caused by oxidative stress generated
through excessive activation of visual cycle. However, our daily light stimuli do
not induce photoreceptor cell death. There should be a physiological self-defending
system. Our final goal is to find a neuroprotective system. In this study, we decided
the threshold stimuli of light exposure which could be overcome by the internal
protective system.
Methods: ICR male mice were dark adapted for 1 night, and then, exposed to 5,
2000, 2500 and 13,000 lux fluorescent light for 1 hour, respectively. The photoreceptor
cell damage was evaluated by analyzing the outer nuclear layer (ONL) thickness
and the number of TUNEL positive cells in the ONL. Then, retinal function was
measured by electroretinogram (ERG). Oxidative stress in the retina was measured
using dihydroethidium.
Results: Decrease in ONL thickness, increase in TUNEL positive cells, and decrease
in a-wave amplitude of ERG were observed in the mice after 2500-or 13,000-lux
light exposure. Intensity of dihydroethidium staining was upregulated under these
conditions. In the 2000 lux-light exposed mice, TUNEL positive cells were slightly
observed, but the other 2 parameters did not change.
Conclusion: The threshold condition which avoided light-induced photoreceptor cell
loss and visual function impairment was 1 hour-exposure of 2000 lux-light. Thus,
further study to find the neuroprotective system which regulates oxidative stress
level should be performed under this condition.
CR: T. Narimatsu, None; T. Kurihara, None; S. Kubota, None; S. Ishida, None; Y.
Ozawa, None; K. Tsubota, None.
Support: None
Purpose: Over-expression of the angiogenic vascular endothelial growth factor
(VEGF) plays a key role in retinal neovascularization. Our previous studies in retinal
endothelial cells have shown that physiological low levels of peroxynitrite (PN)
transduces VEGF’s angiogenic signal. Focal adhesion assembly is regulated by the
balance between activation of focal adhesion kinase (FAK) and its specific phosphatase,
low molecular weight protein tyrosine phosphatase (LMW-PTP). The activity of
LMW-PTP is redox regulated via cysteine oxidation of its active site. The purpose of
this study is to test the hypothesis that PN regulate VEGF-induced FAK activation
via altering cellular redox state of LMW-PTP.
Methods: Human and bovine retinal endothelial cells were treated with VEGF (20 ng/
ml) or PN (1,100 or 500 μM). Cellular redox state was determined by GSH levels using
DTNB-assay. Phosphatase activity was measured using fluorometric assay. Western
blot is used to determine phosphorylation of FAK and LMW-PTP. Thiol oxidation of
LMW-PTP was assessed in 5-IAF labeled cell lysate.
Results: VEGF causes transient oxidation of GSH levels and FAK activation that was
comparable with the low levels of PN (1-100 μM). High levels of PN (500 μM) caused
permanent oxidation of GSH levels. PN caused a dose dependant reduction of the
LMW-PTP phosphatase activity. Decomposing peroxynitrite with FeTPPS (2.5 μM)
prevented LMW-PTP thiol oxidation.
Conclusions: VEGF-induced PN formation transiently alters the redox state of
endothelial cells to inactivate LMW-PTP via thiol oxidation and hence activate FAK.
Treatments that target PN formation and thiol oxidation should be considered for
anti-angiogenic therapy.
CR: M.A. Abdelsaid, None; A.B. El-Remessy, None.
Support: SDG from AHA and CDA from JDRF
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1432-1435
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1436 - D650
Cigarette Smoke-Related Hydroquinone Induces F-Actin Reorganization
and Hsp27 Phosphorylation Through p38 and ERK1/2 in Retinal Pigment
Epithelium
1437 - D651
Filtering Blue Light Reduces Light-Induced Oxidative Stress, Senescence, and
Accumulation of Extracellular Matrix Proteins in Human RPE Cells
M. Kernt1, R.G. Liegl1, C. Hirneiss1, A.S. Neubauer1, M.W. Ulbig1, A. Walch2, A.
Gandorfer1, A. Kampik1. 1Department of Ophthalmology, Ludwig-Maximilians-Univ,
Munich, Gruenwald, Germany; 2Institute of Pathology, GSF-National Research
Center for Environment and Health, Neuherberg, Germany.
M. Pons1, O. Alcazar1, S.W. Cousins2, K.G. Csaky3, M.E. Marin-Castano1.
1
Ophthal-Bascom Palmer, Univ of Miami Miller Sch of Medicine, Miami, FL;
2
Ophthalmology, Duke University, Durham, NC; 3Ophthalmology, Duke
University Eye Center, Durham, NC.
Purpose: Retinal pigment epithelium (RPE)-derived blebs have been observed in eyes with
age-related macular degeneration (AMD). Cumulative oxidative injury caused by cigarette
smoke to the RPE may play a role in AMD. We previously reported that hydroquinone (HQ),
a major pro-oxidant in cigarette smoke, induces actin rearrangement and membrane blebbing
in RPE cells, as well as sub-RPE deposits in mice. Heat-shock protein 25/27 (Hsp25/27) is a
key regulator of actin filaments dynamics regulated by oxidative stress. The objective of the
present study was to further characterize the role of phosphorylated Hsp25/27 in regulating
actin cytoskeleton dynamics and blebbing during non lethal HQ-induced oxidative stress
as well as the involvement of p38 and ERK pathways in vitro and in vivo.
Methods: Human donor eyes with AMD were used for immunohistochemistry. Confluent
ARPE-19 cells were treated with HQ 100μM for various periods of time with or without either
p38 inhibitor SB203580, or okadaic acid or ERK inhibitor PD98059. After treatment, cells were
harvested for RNA and protein extraction. siRNA technology was used to knock-down Hsp27
expression. Western blot, real-time PCR and immunofluorescence staining were performed.
We also used the experimental model for sub-RPE deposits of mice chronically exposed to
oral HQ for 7 months. RPE-choroid complexes were dissected at the time of sacrifice and
protein and RNA were subsequently extracted.
Results: Immunohistochemistry revealed that phosphorylated Hsp27 was upregulated in
RPE from AMD patients. Also, exposure to HQ led to Hsp27mRNA upregulation, dimers
formation and Hsp27 phosphorylation in ARPE-19 cells. A crosstalk between p38 and ERK
pathways mediated HQ-induced Hsp27 phosphorylation and actin aggregates formation.
SB203580 decreased HQ-induced actin rearrangement and blebs formation whereas okadaic
acid increased these effects. Hsp27 siRNA and PD98059 robustly decreased HQ-induced
Hsp27 phosphorylation and actin rearrangement. Also, RPE/choroid from mice chronically
exposed to HQ showed increased Hsp25, p38 and ERK phosphorylation, while Hsp25
expression was dramatically decreased.
Conclusions: Our study suggests that phosphorylated Hsp25/27 may be a major contributor
to RPE-derived blebs formation and HQ-induced actin reorganization which may play
a key role in sub-RPE deposits formation relevant to the pathogenesis of AMD in smokers.
CR: M. Pons, None; O. Alcazar, None; S.W. Cousins, None; K.G. Csaky, None; M.E.
Marin-Castano, None.
Support: NIH Grants RO1-EY015249-01A1 and EY014801
1438 - D652
Deficiency of αB Crystallin Augments ER Stress-Induced Apoptosis by
Accentuating Mitochondrial Dysfunction in RPE Cells
Purpose: Cumulative light-exposure is significantly associated with ageing and
progression of age related macular degeneration (ARMD). In order to prevent the
retina from blue-light damage in pseudophacia, blue-light-absorbing intraocular
lenses (IOL) have recently been developed. This study compares possible protective
effects of a blue-light-absorbing IOL to an untinted, UV-absorbing IOL regarding
light-induced oxidative stress and senescence on human RPE.
Methods: Primary human RPE cells were exposed to white light and either a bluelight-absorbing IOL or a UV-absorbing IOL was placed in the light beam. After 60
minutes of irradiation cellular viability, induction of intracellular reactive oxygen
species (ROS), and senescence-associated β-galactosidase activity (SA β-Gal) was
determined. Expression and secretion of matrix metalloproteinases (MMPs) 1 and 3
and their mRNA were determined by RT-PCR and ELISA.
Results: Light exposure decreased RPE cell viability, and increased ROS, SA β-Gal, and
MMP-1 and 3 expression. These effects were significantly reduced by both, the bluelight-absorbing IOL and the UV-absorbing IOL. In addition, these protective effects
were significantly stronger in presence of the blue-light-absorbing IOL, compared to
the UV-absorbing IOL.
Conclusions: In this study the blue-light-absorbing IOL demonstrated a significant
better protection against light-induced oxidative stress, senescence and extracellularmatrix-protein over-expression than the UV-absorbing IOL. These in vitro findings
support the hypothesis of possibly also preventing retinal damage in clinical use.
CR: M. Kernt, None; R.G. Liegl, None; C. Hirneiss, None; A.S. Neubauer, None; M.W.
Ulbig, None; A. Walch, None; A. Gandorfer, None; A. Kampik, None.
Support: None
1439 - D653
Photooxidation and Cleavage of A2E Releases Methylglyoxal a Dicarbonyl
Known to Form Advanced Glycation End Products. Implications for Drusen
Formation
G. Dou1, P.G. Sreekumar1, C. Spee1, S. He1, S.J. Ryan1, R. Kannan1, D.R. Hinton1,2.
1
Ophthalmology, Doheny Eye Institute, University of Southern California,
Los Angeles, CA; 2Pathology, Keck School of Medicine, University of Southern
California, Los Angeles, CA.
J.R. Sparrow1A, Y. Wu1B, J. Zhou1B, M.M. Siegel2. ADepartments of Ophthalmology and
Pathology and Cell Biology, BDepartment of Ophthalmology, 1Columbia University,
New York, NY; 2Pfizer Research, New York, NY.
Purpose: Disturbance of endoplasmic reticulum (ER) homeostasis is being unveiled
in several pathological conditions including age-related macular degeneration.
Excessive ER stress initiates cell death, which could be orchestrated with mitochondrial
dysfunction. This study aims to examine the cross talk between ER stress and
mitochondrial dysfunction in RPE cells and to identify αB crystallin as a distinct
anti-apoptotic regulator coordinating the interplay.
Methods: Expression levels of αB crystallin, GRP78, CHOP, caspase 12, caspase 3, Bcl-2
and Bax were examined in human fetal RPE (hRPE) cells treated with tunicamycin
(TM, 1-10 µg/ml; 1-24h). Reactive oxygen species (ROS), free glutathione (GSH), release
of cytochrome c and membrane permeability transition (MPT) were measured in TMtreated and untreated hRPE cells. The induction of apoptosis was evaluated by TUNEL
and Annexin V assays in RPE cells from αB crystallin (-/-) mice, αB crystallin siRNA
transfected hRPE, and ARPE-19 cells stably overexpressing αB crystallin.
Results: TM treatment induced activation of caspase 3 and caspase 12 in hRPE. ER
stress was verified by an increase in GRP78 and CHOP. Severe ER stress downregulated
αB crystallin expression in ER and mitochondria. The reduction in αB crystallin
expression occurred prior to caspase 3 activation. TM-treated hRPE exhibited an
increase in ROS (p<0.05), decrease in cellular and mitochondrial GSH (p<0.05), and
50% increase of cytochrome c release from mitochondria into cytosol. Bcl-2 and Bcl-2/
Bax ratio showed slight increases with TM treatment. RPE cells from αB crystallin (-/-)
mice and from αB crystallin siRNA transfection exhibited increased susceptibility
to ER stress-induced apoptosis, while αB crystallin overexpression promoted RPE
cells survival by restoring changes of MPT(p<0.05) or regulating expression of Bax
and caspase 12.
Conclusions: Our data show that αB crystallin plays a crucial role as a protective
mediator in regard to mitochondrial function during ER stress-induced RPE
apoptosis.
CR: G. Dou, None; P.G. Sreekumar, None; C. Spee, None; S. He, None; S.J. Ryan,
None; R. Kannan, None; D.R. Hinton, None.
Support: NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel
Beckman foundation
Purpose:We strove to characterize the structures and behaviours of A2E photocleavage products that are generated subsequent to photooxidation.
Methods: Liquid chromatography (LC) coupled to electrospray ionization mass
spectrometry (ESI-MS) together with tandem mass spectrometry (MS/MS) was
employed to characterize photocleavage products of A2E. ARPE-19 cells that were
grown on a fibronectin substrate were allowed to accumulate A2E and irradiated
at 430 nm. Fibronectin was immunoprecipitated from homogenates of the cells and
substrate and immunoblots were subsequently probed with polyclonal chicken
antibody to advanced glycation end products (AGEs).
Results:A complex mixtures of A2E photo-products of various molecular weights
were detected. Studies in which A2E was incubated with a singlet oxygen generator
yielded results consistent with a mechanism involving bisretinoid photocleavage at
sites of singlet molecular oxygen addition. Structural characterization revealed that
A2E photodegradation releases methylglyoxal, a 72 Da reactive dicarbonyl with the
capacity to form AGE. AGE adducts were detected at sites of blue light irradiation in
association with the fibronectin substrate on which the cells were grown.
Conclusions: Photodegradation of A2E releases methylglyoxal, a low molecular weight
reactive dicarbonyl that initiates AGE formation. Diffusion of A2E photocleavage
products such as methylglyoxal is suggested by the observation that when cultured
RPE cells that have accumulated A2E were irradiated (430 nm), the extracellular
fibronectrin substrate on which the cells are grown becomes AGE-modified.
Methylglyoxal is already known to be generated by carbohydrate and lipid oxidation;
this is the first report of its production via bisretinoid photocleavage. It is significant
that AGE-modified proteins are detected in drusen; drusen have been linked to AMD
pathogenesis. These findings suggest a possible association between RPE bisretinoid
lipofuscin photooxidation and drusen formation.
CR: J.R. Sparrow, None; Y. Wu, None; J. Zhou, None; M.M. Siegel, None.
Support: NIH Grant EY12951
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1436-1439
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1440 - D654
Minocycline Protects Retinal Pigment Epithelial Cells From Chronic Oxidative
Stress
1441 - D655
Alpha Crystallin Derived Peptide Chaperone Protects Human RPE Cells From
Oxidative Injury
J. DaCosta1,2, S. Sivaprasad 3, T.A. Bailey2. 1Moorfields Eye Hospital, London, United
Kingdom; 2Cranfield Health, Cranfield University, Cranfield, Bedfordshire, MK43
OAL, United Kingdom; 3King’s College Hospital, London, United Kingdom.
R. Kannan1, P.G. Sreekumar1, N. Kannan1, S.J. Ryan1,2A, U.B. Kompella 3, K. Sharma4, D.R.
Hinton1,2B. 1Arnold and Mabel Beckman Macular Research Center, Doheny Eye
Institute, Los Angeles, CA; AOphthalmology, BPathology, 2Keck School of Medicine
of the University of Southern California, Los Angeles, CA; 3Pharmaceutical
Sciences, University of Colorado Denver, Aurora, CO; 4Ophthalmology, University
of Missouri-Columbia, Columbia, MO.
Purpose: Multiple pathologic processes are involved in neovascular age related
macular degeneration (AMD). Current therapies focus on the control of active
neovascularisation. There is increasing interest in incorporating therapies that block
other pathophysiologic processes not directly related to neovascularisation such as
inflammation to preserve long-term vision. Minocycline, a semisynthetic tetracycline
has been shown to have immunomodulatory effects on the inflammatory process.
Minocycline is proposed as an anti-inflammatory agent to protect retinal pigment
epithelial cells. This study investigated the effect of Minocycline on oxidative stress
induced RPE cell damage.
Methods: ARPE-19 cells were maintained in Dulbecco’s modified Eagle medium
supplemented with glutamine, penicillin, streptomycin, amphotericin and 10% fetal
calf serum. Cells were exposed to hydrogen peroxide as a source of oxidative stress,
then exposed to varying concentrations of Minocycline. Cell counts for viability
were performed after exposure to acute and chronic oxidative stress conditions and
Minocycline exposure.
Results: Comparison of cell count viability data with the Mann Whitney test showed no
difference between acute oxidative stress exposure alone and with Minocycline (p=0.11)
After chronic oxidative stress exposure cell viability counts increased significantly
after exposure to Minocycline (p=0.04)
Conclusions: The results suggest minocycline protects retinal pigment epithelial cells
from the effects of chronic oxidative stress in culture and supports the potential role
of Minocycline as an adjuvant treatment for neovascular AMD.
CR: J. DaCosta, None; S. Sivaprasad, Allergan, Novartis and Pfizer, C; T.A. Bailey,
None.
Support: None
1442 - D656
PEDF78-121 Limits Oxidative Stress in RPE Cells Through Akt and Erk
Signaling and Reduces Stress Promoted Expression of VEGFR1
A. Gvritishvili1A, Y. Liu1A, J. Tombran-Tink1B. ANeural and Behavioral Science, BNeural
and Behavioural Sciences, 1Penn State College of Medicine, Hershey, PA.
Introduction: PEDF treatment can reduce both retinal cell death and neovascularization.
The neurotrophic activity of the protein is localized to an N terminal fragment
corresponding to amino acid residues 78-121. In this study we examined the effects
of this peptide on oxidative stress in RPE cells.
Methods: PEDF78-121 peptide was cloned into pET32a expressed in E.coli, and peptide
purified using Ni-NTA chromatography. The effects of purified PEDF78-121 peptide
were examined in ARPE19 cultures challenged with 640µM H2O2. Cells were treated
for 2h with H2O2 followed by treatment with various concentration of PEDF78-121 for
24h. The protective effects of PEDF78-121 on H2O2 toxicity were assessed using LDH
assay and PI staining. Modulations in VEGF, VEGF receptors, and various signaling
pathways were also determined.
Results: PEDF78-121 protects RPE cells from hydrogen peroxide toxicity at concentrations
as low as 25 ng/ml. Approximately 30% decrease in cell death is seen in cultures after
treatment with the peptide. This protective effect is mediated through the Akt and
Erk signaling pathways. H202 decreased the phosphorylation of both signal molecules
after 2h, a condition immediately reversed by PEDF78-121. Specificity of PEDF78-121 actions
was shown with the pharmacological inhibitors Akt and MEK1/2 which blocked Akt
and Erk1/2 activation, respectively by PEDF78-121. Inhibition of Akt and Erk signaling
abrogated the protective effects of PEDF78-121 on RPE cells. We also show that oxidative
stress increases the expression of VEGFR1 in RPE cells and that PEDF78-121 reduced
this expression by 3.4 fold, an effect that was abolished when Akt and Erk signaling
pathways were blocked.
Conclusion: Oxidative stress can modulate Akt and Erk signaling and induce
expression of VEGR1 in RPE cells. PEDF78-121 is an effective pharmacological tool that
reduces oxidative stress induced death in RPE cells and may limit the actions of these
cells in angiogenic events in the retina.
CR: A. Gvritishvili, None; Y. Liu, None; J. Tombran-Tink, None.
Support: NIH and the Macular Vision Research Foundation
Purpose: Our previous work established that α-crystallins are anti-apoptotic. Furthermore,
19-20 amino acid sequences were identified from both αA and αB crystallins that exhibited
similar chaperone function as that of full-length proteins (Sharma et al JBC 275, 3767-3771;
Biochemistry 45, 3069-3076). The purpose of the present study is to examine whether these
crystallin-derived peptides have anti-apoptotic functions in RPE cells.
Methods: Protective role of α-crystallins was studied in stable ARPE-19 cell lines
overexpressing full length α-crystallins. Cells were subjected to oxidative stress and
cell protection was assessed by TUNEL analysis and activation of caspase 3. 19-20mer
peptides of αA and αB crystallin having known chaperone function were synthesized
(Neo-Peptide, MA). Early passage human fetal RPE cells were co-treated with varying
doses (50-75µg/ml) of minipeptides of either αA or αB crystallin or scrambled peptides
and tBH or H2O2 to induce oxidative stress. Cell death and caspase activation were
determined. The uptake of mini-peptide was studied using fluorescence labeled αB
crystallin mini-peptide under conditions of oxidative stress. Retinal uptake of the αB
crystallin derived peptide in vivo was studied in a murine model of laser induced
choroidal neovascularization (CNV).
Results: Overexpression of αA and αB crystallins significantly increased protection to RPE
cells from oxidative stress-induced cell death. RPE cells challenged with either H2O2 or
tBH in the presence of either αA or αB crystallin peptides, remained viable and caspase-3
activation was severely inhibited. The uptake of labeled αB crystallin peptide increased
significantly in the presence of oxidative stress vs untreated control RPE cells and most
of the peptide was translocated to the nucleus. In mice, intravenous injection of labeled
αB crystallin-derived peptide showed prominent localization within CNV lesions.
Conclusions: As with full-length α-crystallins, crystallin-derived peptides offer
protection from oxidative stress-induced injury. Stress causes significant translocation
of crystallin peptides to the nucleus which could activate transcription factors involved
in anti-apoptotic pathways.
CR: R. Kannan, None; P.G. Sreekumar, None; N. Kannan, None; S.J. Ryan, None; U.B.
Kompella, None; K. Sharma, None; D.R. Hinton, None.
Support: NIH Grants EY02061, EY03040 & Grants from RPB & the Arnold & Mabel
Beckman Foundation
1443 - D657
Investigating the Ability of Anti-Oxidant Gene Expression in Mesenchymal
Stem Cells to Promote Cell Protection and Recovery in an in vitro Model of
Age-Related Macular Degeneration
A.P. Lynch1, L. McGinley1, C. Sheridan2, C. Coleman1, L. Howard1, D. Kent1, F. Barry1.
1
Regenerative Medicine Institute, Galway, Ireland; 2School of Clinical Sciences,
Opthalmology Research Unit, Liverpool, United Kingdom.
Purpose: The aim of this study was to determine if lentiviral transduced mesenchymal
stem cells (MSCs) expressing anti-oxidant genes could protect and recover the retinal
pigment epithelial cell line, ARPE-19 from oxidative stress with tert-butylhydroperoxide
(tert-BHP) treatment.
Methods: ARPE-19 cells were treated with varying concentrations of tert-BHP, ranging
from 1 - 8 mM, to induce features of age-related macular degeneration (AMD). Control
cells remained untreated. MSCs were transduced with lentiviral vectors expressing
GFP, Catalase, HSP-27, HSP-70, SOD-1 or SOD-3. GFP transduced MSCs were used as a
control. Treated ARPE-19 cells were grown in co-culture with transduced MSCs. Cell
viability was assessed with the MTT and Live / Dead assays, while the production
of ROS was measured with carboxy-H2DCFDA.
Results: Treatment with tert-BHP decreased the viability of ARPE-19 cells.
Morphological changes and cell survival assays revealed that incubation with
transduced MSCs aided ARPE-19 cells to resist the free radicals induced by tertBHP. In addition, activity of SOD-1 and -3 was enhanced in ARPE-19 cells leading to
a reduction in ROS levels.
Conclusions: These results suggest that transduced MSCs have potent antioxidant
activity and protect RPE from oxidative injury. Therefore transduced MSCs are
possible candidates for the modulation of oxidative stress- induced damage of RPE
in the ageing retina.
CR: A.P. Lynch, None; L. McGinley, None; C. Sheridan, None; C. Coleman, None; L.
Howard, None; D. Kent, None; F. Barry, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1440-1443
Monday, May 3, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1400 - 1445 / D614 - D659
229. Oxidative Injury and Protection
Organizing Section: RC
1444 - D658
Regulation of Monocyte Chemoattractant Protein-1 (MCP-1), Growth Factors,
and Pigment Epithelium-Derived Factor in Response to Transient and
Repetitive Non Lethal Oxidative Injury in Human Retinal Pigment Epithelial
Cells
M.E. Marin Castano , O. Alcazar , M. Pons . Ophthalmology, Bascom Palmer Eye
Institute, Miami, FL; 2Ophthal-Bascom Palmer, Univ of Miami Miller Sch of
Medicine, Miami, FL.
1
1
2 1
Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in
the elderly, and tobacco smoking has been shown to be a major risk factor. Cumulative
oxidative injury caused by cigarette smoke to the RPE and inflammation have been
implicated in the pathogenesis of choroidal neovascularization (CNV), the advanced
form of AMD. Although the exact contribution of macrophages remains unknown, they
may play crucial roles both in early AMD scavenging accumulated debris, and wet AMD
stimulating CNV. The study was undertaken to investigate the expression of cytokines
involved in inflammation and angiogenesis (monocyte chemoattractant protein-1 (MCP-1),
vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β) and
pigment epithelium-derived factor (PEDF)) by ARPE-19 cells when exposed to transient
and repetitive cigarette smoke-related hydroquinone (HQ)-induced oxidative stress.
Moreover, since nuclear factor-κB (NF-κB) is known to induce MCP-1 gene, we also
investigated whether HQ-induced MCP-1 expression was mediated by NF-κB.
Methods: Confluent ARPE-19 cells were treated with HQ 100μM for either 24 hours
(transient injury) or 5 weeks (repetitive injury). In some experiments, cells were pretreated
with either proteasome inhibitor MG-132, or IκBα phosphorylation inhibitor Bay 11-7082,
or NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). After treatment, cells were
harvested for RNA extraction and samples were assayed by real-time PCR. Nuclear
translocation of NF-κB p65 was visualized by immunofluorescence staining.
Results: Transient exposure of ARPE-19 cells to HQ increased MCP-1, VEGF and TGF-β
mRNA expression, and decreased PEDF mRNA expression. Induction of MCP-1 by HQ
was mediated by NF-κB because inhibition of this pathway by MG-132, PDTC and Bay117082 resulted in the impairment of MCP-1 transcription activation. Also, exposure to HQ
resulted in nuclear translocation of p65/RelA as well as an increase in p65/RelA and IκBα
mRNA expression. Sustained oxidative injury with HQ decreased MCP-1 expression and
increased VEGF/PEDF ratio.
Conclusions: Taken together, our findings suggest a differential regulation of MCP-1
expression in RPE cells in response to cigarette-smoke related HQ-induced oxidative
injury. HQ might be a key contributor to the progression of late AMD by stimulating
inflammation and angiogenesis leading to CNV.
CR: M.E. Marin Castano, None; O. Alcazar, None; M. Pons, None.
Support: NIH grant EY015249-01A1, NEI P30 Core Grant EY014801,FAMRI ID:072100_CIA,
and an unrestricted grant from Research to Prevent Blindness to the University of
Miami.
1445 - D659
Vulnerability of Retinal Capillaries to Oxidative Stress: Role of Endogenous
Spermine
M. Fukumoto, D.G. Puro. Ophthalmology, Univ of Michigan-Kellogg Eye Ctr, Ann
Arbor, MI.
Purpose: It is unclear why retinal capillaries are particularly vulnerable to diabetes.
We postulate that the oxidized state of cells in the capillaries makes them particularly
sensitive to oxidative stress, which is a hallmark of the diabetic retina. This idea
is based on our recent report that the redox status of capillary cells is driven by
spermine-dependent oxidation (J Physiol 587:2233, 2009); spermine is a polyamine that
is abundant in retinal capillaries (J Physiol 573:483, 2006) and is catabolized to H2O2
and other potent oxidants. Here, we tested the hypothesis that spermine plays a key
role in making retinal capillaries particularly prone to oxidant overload.
Methods: In microvascular complexes freshly isolated from non-diabetic and diabetic
rat retinas, trypan blue dye exclusion was used to quantify H2O2-induced cell death
in the capillary network and in the feeder vessels that link capillaries to myocyteencircled arterioles. Diabetes was induced by streptozotocin. Standard statistical
methods were used.
Results: Dose-response relationships for H 2O2-induced cell death demonstrated
that capillaries are more vulnerable to oxidants than feeder vessels. Namely, in
capillaries the EC50 for H2O2 was 9 μM, which was significantly less than the EC50 of
15 μM for feeder vessels. Indicative of spermine having an important role, the EC50
for H2O2-induced capillary cell death was significantly increased to ~40 μM when
microvessels were pre-incubated in DFMO, a spermine synthesis inhibitor. Although
DFMO also lessened the vulnerability to H2O2 of the feeder vessels, we found that
at H2O2 concentrations between 8 and 15 μM, the DFMO-sensitive component of the
H2O2-induced cell death was ~2-fold greater in capillaries than in feeder vessels. Here,
we also assessed the effect of H2O2 on diabetic microvessels. Consistent with our
reports that diabetes increases spermine in retinal feeder vessels, the sensitivity of
diabetic feeder vessels to H2O2 was significantly increased via a spermine-dependent
mechanism. However, despite the diabetes-induced increase in feeder vessel sensitivity
to oxidative stress, cells in diabetic capillaries remained significantly more vulnerable
to H2O2 than feeder vessel cells.
Conclusions: Endogenous spermine renders retinal capillaries particularly vulnerable
to oxidant-induced cell death. Because oxidative stress is likely to play a role in diabetic
retinopathy, our finding that the inhibition of spermine synthesis protects against
oxidant toxicity suggests a new therapeutic strategy to minimize microvascular cell
death in the diabetic retina.
CR: M. Fukumoto, None; D.G. Puro, None.
Support: EY12505 and RPB.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1444-1445
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1876 - D757
Control of Ryanodine Receptor Mediated Calcium Signaling by Presenilins in
Mouse Retinal Ganglion Cells
1877 - D758
Control of Ryanodine Receptor Mediated Calcium Signaling by Calsenilin in
the Mouse Retina
A. Payne, S. Kaja, P. Koulen. Ophthalmology, UMKC School of Medicine, Kansas
City, MO.
M.G. Grillo, P. Koulen. Ophthalmology, Univ MO - Kansas City School of Medicine,
Kansas City, MO.
Purpose: Ca2+ homeostasis and intracellular Ca2+ release are tightly controlled and
contribute to a variety of cellular and synaptic functions in the neural retina. While in
the invertebrate retina, ryanodine receptors (RyRs) have been described to contribute
to light adaptation processes, little is known about their role in the mammalian retina,
especially in retinal ganglion cells (RGCs), which are pivotal for information processing
and signal integration and are affected by neurodegenerative diseases of the eye. The
goal of the present study was to determine the regulatory role of presenilins for RyR
mediated calcium signaling in the mouse RGCs.
Methods: Immunofluorescence studies were performed to determine the expression
and localization of RyR and presenilins in isolated mouse RGCs and in vivo. Control
of presenilins over RyR-mediated Ca2+ release from intracellular stores was measured
using optical imaging techniques and single channel electrophysiology.
Results: Immunocytochemical analyses revealed a distinct subcellular distribution
of RyRs in RGCs and a co-distribution with presenilins. Addition of presenilins to
the cytosolic face of the RyR resulted in a significant potentiation of RyR activity.
The channel properties and Ca 2+ dependence of the RyR were maintained while
mean current and open probability were significantly potentiated in the presence
of presenilins.
Conclusions: Our results indicate that RyRs and presenilins are co-expressed in
RGCs, that they interact through direct binding and that their interaction results in the
potentiation of intracellular Ca2+ release and Ca2+ induced Ca2+ release. As such, this
interaction controlling the gain of intracellular Ca2+ release mechanisms represent a
suitable pharmacological target in diseases of the retina involving Ca2+ dyshomeostasis.
CR: A. Payne, None; S. Kaja, None; P. Koulen, None.
Support: This study was supported in part by NIH grants EY014227, RR022570,
AG010485, AG022550 and AG027956 and the Felix and Carmen Sabates Missouri
Endowed Chair in Vision Research (P.K.).
Purpose: Calsenilin, also known as KCHIP 3 and DREAM, is a calcium binding protein
with a diversity of functions including transcriptional regulation, A-type potassium
channel modulation, and presenilin processing. Recently calsenilin expression has
been detected in rodent and primate retina. Ryanodine receptors (RyR) are intracellular
calcium induced calcium release channels located on intracellular calcium stores and
are involved in various signaling pathways. All three RyR subtypes have been observed
in the retina. Changes in intracellular calcium mediated by RyRs control neuronal
functions including ontogenesis, neurotransmission and cell death in physiologyical
and pathophysiological states. In the brain, calsenilin expression levels affect both
neuronal survival and intracellular calcium levels.
Methods: The expression and localization of RyR and calsenilin was determined with
immunochemistry in mouse retina neurons. Functional effects of the interaction of
RyRs and calsenilin were analyzed with single channel electrophysiology.
Results: RyR and calsenilin were co-localized intracellularly in retina neurons most
notably retinal ganglion cells. Binding of calsenilin to the cytosolic face of the RyR
had a biphasic effect on RyR activity. At resting cytosolic calcium levels mean current
and open probability of the RyR were attenuated while at higher cytosolic calcium
levels mean current and open probability of the RyR were significantly potentiated.
Conclusion: Data from the present study suggest that calsenilin has the potential
to control the intracellular Ca 2+ concentration in retinal neurons through direct
interaction with RyRs. Furthermore, the direct interaction of calsenilin and the
intracellular calcium induced calcium release channels appears capable of influencing
the gain of Ca2+ signaling pathways indicating potential roles in retina physiology,
pathophysiology and neuroprotection.
CR: M.G. Grillo, None; P. Koulen, None.
Support: This study was supported in part by NIH grants EY014227, RR022570,
AG010485, AG022550 and AG027956 and the Felix and Carmen Sabates Missouri
Endowed Chair in Vision Research (P.K.).
1878 - D759
Cell Adhesion Peptide RGDSP and Laminin Support Proliferation and
Migration of Postnatal Retinal Stem Cells in a 3D Hydrogel Culture System
1879 - D760
3D-Structure of Light- and Dark-Adapted Rod Photoreceptors
G.A. Zampighi1A, C. Schietroma1A, L.M. Zampighi1B, N. Brecha2. ANeurobiology,
B
Physiology, 1UCLA School Medicine, Los Angeles, CA; 2Neurobiology, Univ of
California-Los Angeles, Los Angeles, CA.
M.W. Djojosubroto1, M. Eberhardt1, T.P. Kraehenbuehl2, M. Tekaya1, M.P. Lutolf 2,
J.A. Hubbell2, Y. Arsenijevic1. 1Gene Therapy & Stem Cell Biology, Jules Gonin
Eye Hospital, University of Lausanne, Lausanne, Switzerland; 2Institute of
Bioengineering, Federal Institute of Technology, Lausanne, Switzerland.
Purpose: Retinal stem cells (RSCs) can be isolated from radial glia population of the
newborn mouse retina (Angénieux et al., 2006). These RSCs have great capacity to renew
and generate neurons including cells differentiated towards the photoreceptor lineage
(Mehri-Soussi et al., 2006). However, our published results showed poor integration
and survival rate after cell grafting into the retina. The uncontrollable environment of
retina seems to be the problem. To bypass this, we are trying to generate hemi-retinal
tissue in vitro that can be used for transplantation.
Methods: Expanded RSCs were seeded in a mixture of poly-ethylene-glycol (PEG)polymer-based hydrogels crosslinked by peptides that also serve as substrates for
matrix metalloproteinases. Different doses of crosslinker peptides were tested. Several
growth factors were studied to stimulate cell proliferation and differentiation.
Results: Cells were trapped in hydrogels and cultured in the presence of FGF2 and
EGF. Spherical cell clusters indicating proliferation appeared within several days, but
there was no cell migration within the gel. We then added cell adhesion molecules
integrin ligand RGDSP, or laminin, or a combination of both, into the gel. Cells grown
with laminin showed the best proliferation. Cells grown with RGDSP proliferated a
few times and then started to spread out. Cells grown with the combination of RGDSP
and laminin showed better proliferation than with RGDSP alone and larger spreadouts than with laminin alone. After stimulations with first FGF2 and EGF, and then
only FGF2, some cells showed neuronal morphology after 2 weeks. The neuronal
population was assessed by the presence of neuronal marker b-tubulin-III. Glial cells
were also present. Further characterizations are undergoing.
Conclusions: RSC can grow and migrate in 3D hydrogel with the addition of FGF2,
EGF, RGDSP and laminin. Further developments are necessary to form a homogenous
tissue containing retinal cells.
CR: M.W. Djojosubroto, None; M. Eberhardt, None; T.P. Kraehenbuehl, None; M.
Tekaya, None; M.P. Lutolf, None; J.A. Hubbell, None; Y. Arsenijevic, None.
Support: None
Purpose: To identify the changes in the 3D-structure of rod photoreceptor ribbon
synapses resulting from the fusion of thousands of synaptic vesicle during darkadaptation.
Methods: Retinas of C57Bl/6J mice that were dark-adapted for 5-15-30-60-180 minutes
were prepared for thin sectioning and electron tomography. For each experimental
condition, we collected three conical series that were aligned using fiduciary markers
and reconstructed using the weighted back projection algorithm. After refinement by
projection matching, the resulting 3D-maps were studied by volume rendering and
density segmentation methods.
Results: In the dark, ribbon synapses between rod axons and endings of horizontal
and bipolar cells underwent rapid but transient changes. These included: a) a mean
decrease of 5.1±1.0 μm3 in the axon’s volume, b) an increase of 11.5±1.0 μm2 in the surface
area of the area of synaptic contact, and c) an increase in the number of “omega”
figures representing fused vesicles (from ~190 to ~1,280 per terminal). In contrast,
the “docked” vesicular pool that was comprised of 450-480 hemi-fused vesicles per
terminal and occupied 3.1±1.0 μm 2 of “active zone” did not change significantly with
respect to light-adapted controls. More importantly, this “docked” pool was located
along the entire area of synaptic contact between rod photoreceptors and horizontal
cells, not just at the ribbon’s base.
Conclusion: Massive synaptic vesicle fusion and glutamate release involves two types
of synapses that coexist in the same rod axon terminal; one centered on the ribbon
(from base to zenith) for fast and transient release and another along the entire region
of contact for slow and sustained release.
CR: G.A. Zampighi, None; C. Schietroma, None; L.M. Zampighi, None; N. Brecha,
None.
Support: Grants from Jules Stein Eye Institute and NIH-EY04410 (GZ) and EY15573,
VA Senior Career Scientist Award (NB).
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1876-1879
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1880 - D761
Cell Proliferation in the Ciliary Body After Experimental Retinal Detachment
A.M. Suburo, M. Olivera, G. Luzzani, M.M. Castañeda, M.A. Cubilla. Cell and
Molecular Medicine, Universidad Austral - FCB, Pilar, Argentina.
Introduction: Retinal detachment (RD) is a severe condition with poor functional
recovery even after successful surgical reattachment. Undesired outcomes reflect both
retinal remodeling and growth of epi- and sub-retinal membranes. Astrocytes, Müller
cells, microglia and retinal pigment epithelial (RPE) can contribute to membranes.
However, membrane development is not completely understood. Here, we report the
appearance of minichromosome protein 2 (MCM2), a marker of cycling cells during
G1 phase, after experimental RD.
Methods: 5-week old Balb-c mice received general and topical anesthesia, following
the ARVO Statement for the Use of Animals. A nasal RD was produced by sub-retinal
injection of hyaluronic acid. 2, 3 or 5 days after surgery, animals were anesthetized for
examination of the eye fundus and perfusion fixation. Cryosections were incubated
with antibodies against MCM2, the macrophage/microglia marker F4/80, and
the intermediate filament nestin. They were studied with immunoenzymatic or
immunofluorescent procedures.
Resultados: In control animals, neither the neural retina nor the RPE showed
MCM+ nuclei. Some labeled nuclei appeared in the ciliary body. F4/80+ cells
were very few, and Müller cell endfeet showed low nestin immunostaining.
Numerous MCM2+ nuclei appeared in the detached retina, the detached
RPE and the nasal ciliary body. Within the detached retina, MCM2+ nuclei
occupied random positions. Their distribution resembled that of F4/80+
cells appearing after RD. Nestin immunostaining of Müller cells increased
after RD, but co-localization with MCM2 nuclear labeling was not observed.
The nasal ciliary body displayer a large increase of MCM2+ nuclei. Lower numbers
of MCM2+ nuclei appeared in the temporal region of the ciliary body, distant
from the detached retina. MCM2+ ciliary cells also showed cytoplasmic nestin.
Sub-retinal membranes were found in about 30% of RD eyes. These membranes usually
showed an anatomical conection with the ciliary body and contained numerous
MCM2+ and nestin+ cells.
Conclusions: Nuclear MCM2+ immunoreactivity detected cycling G1 cells. These cells
appeared in regions that are known to be involved in epi- and sub-retinal membrane
formation. Proliferation in the ciliary body, together with nestin expression in ciliary
MCM2+ cells, suggests that RD would also activate putative retinal progenitors. In
addition, anatomical connections between the ciliary body and sub-retinal membranes
indicate that cells derived from the ciliary body could perhaps contribute to membrane
formation.
CR: A.M. Suburo, None; M. Olivera, None; G. Luzzani, None; M.M. Castañeda,
None; M.A. Cubilla, None.
Support: MINCYT PICT21399/2004; Fundación Fiorini 2009
1882 - D763
The Role of FAK, Pyk2 and Fyn in Pretarget Sorting and Topographic Mapping
of the Visual System
S.L. Moseley1, J. Schlessinger2, H.E. Beggs1. 1Ophthalmology, University of California,
San Francisco, San Francisco, CA; 2Pharmacology, Yale University, New Haven, CT.
Purpose: Precise maping of the retina to the Superior Colliculus is essential for correct
visual perception. After presorting in the optic tract, counter gradients of ephrins
and Eph receptors are important regulators of precise topographic mapping at the
SC. However, little is known about how these cues translate into specific responses
within the RGC growth cone. Non-receptor tyrosine kinases are likely candidates for
regulating these growth cone responses. In this study we investigate the hypothesis
that FAK, it’s close family member Pyk2, and Fyn serve as signaling nodes in RCG
projections to enable precise retinotopic map formation in mice.
Methods: FAK was deleted in the retina by crossing Nestin:Cre and Six3:Cre driver
lines to the Floxed-FAK allele. To investigate genetic redundancy, double mutants of the
conditional FAK knockout with Pyk2 or Fyn mutants were generated. To trace retinal
projections to the Superior Colliculus in vivo, DiI was microinjected into specific retinal
quadrants using a picospritzer. Retinal explants were cultured adjacent to specific
guidance molecules to validate in vivo findings and examine growth cone morphology.
Results: FAK and Fyn single mutants both display major defects in pretarget sorting
of axonal projections. This results in defective mapping across the medial lateral axis
of the Superior Colliculus. Pyk2 mutants are also defective in medial/lateral targeting
but pretarget sorting is grossly normal. All three single mutants displayed defects
in targeting at sites where ephrinA/EphA gradients predominate. Additionally,
FAK mutant axons do not respond to ephrinA2 as a repulsive cue in vitro. Results of
synergistic interactions between FAK, Pyk2 and Fyn will be discussed.
Conclusions: Our results reveal a new role for FAK and Fyn as key regulators of
pretarget sorting in the visual system. Additonally, FAK plays an important role
in growth cone responses to ephrin-A signaling at the Superior Colliculus, likely
functioning redundantly with Pyk2. These data suggest the intriguing possibility
that pretarget sorting is regulated by non-receptor tyrosine kinases downstream of
Eph receptors and ephrins on adjacent axons.
CR: S.L. Moseley, None; J. Schlessinger, None; H.E. Beggs, None.
Support: This work was supported by NIH grants (EY017379 and EY002162) and
Knights Templar. HB is a recipient of the RPB Career Development Award.
1881 - D762
Control of Inositol 1, 4, 5-Trisphosphate Receptor Mediated Calcium Signaling
by Beta-Actin in Mouse Retina Ganglion Cells
P. Koulen, H. Xin, S.-Y. Hwang. Ophthalmology/Vision Research Center, University
of Missouri - Kansas City, Kansas City, MO.
Purpose: Inositol-1, 4, 5-triphosphate receptors (IP3Rs) are intracellular Ca2+ channels
known to be involved in several intracellular signaling pathways. IP3R mediated
changes in cytosolic Ca2+ concentrations control neuronal functions ranging from
synaptic transmission to differentiation and apoptosis and interface with components
of the cytoskeleton. Similarly, retinal ganglion cell physiology and pathophysiology
is determined by cytosolic Ca2+ transients. Determining the function of biophysically
distinct IP3Rs and their control by beta-actin in retina ganglion cells provides necessary
information on the molecular substrates of IP3R mediated Ca 2+ signaling in these
interneurons that are affected by retinopathies.
Methods: The expression and distribution of IP3Rs, beta-actin and related signaling
molecules was determined with immunochemistry. Functional effects of the
pharmacologic and molecular biological modulation of IP3Rs and beta-actin were
analyzed with optical imaging of intracellular Ca2+ concentrations and single channel
electrophysiology in mouse retinal ganglion cells.
Results: Beta-actin and IP3Rs were colocalized intracellularly in retina ganglion
cells. Binding of beta-actin to the cytosolic face of the IP3Rs significantly potentiated
IP3Rs single channel activity. In and in parallel a IP3R channel. In isolated retinal
ganglion cells, pharmacologic inhibition or molecular biological downregulation of
beta-actin expression led to an attenuation of IP3R-mediated intracellular Ca2+ signals
that showed a significant correlation with the beta-actin dependent changes in the
biophysical properties of IP3Rs.
Conclusion: Data from the present study suggest that components of the cytoskeleton
control the intracellular Ca2+ concentration in retinal neurons through direct interaction
with IP3Rs. Furthermore, these components of the cytoskeleton appear capable of
influencing the gain and sensitivity of Ca2+ signaling pathways indicating potential
roles in retina physiology, pathophysiology and neuroprotection.
CR: P. Koulen, None; H. Xin, None; S.-Y. Hwang, None.
Support: This study was supported in part by NIH grants EY014227, RR022570,
AG010485, AG022550 and AG027956 and the Felix and Carmen Sabates Missouri
Endowed Chair in Vision Research (P.K.).
1883 - D764
Proteomics Analysis of Cultured Human Retinal Vascular Endothelial Cells
A. Ando1A, Y. Mitsuma1A, T. Katano1B, S. Ito1B, T. Nishimura1A, K. Takahashi1A.
A
Ophthalmology, BMedical Chemistry, 1Kansai Medical University, Moriguchi,
Japan.
Purpose: Several different anti-glaucoma agents, especially prostaglandin analogues
(PGs), have been reported to have a retina blood flow improvement effect, though the
mechanism remains unclear. We used proteomics analysis with two-dimensional gel
electrophoresis (2D-PAGE) to determine how PGs influence RVE cells.
Methods: Cultured human RVE cells (ACBRI 181, Applied Cell Biology Research
Institute) were grown on attachment factor in CS-C culture medium. Following
quantification with a 2-D Quant Kit, whole protein was extracted from the cells at
sub-confluence with cell lysis buffer (including 8 M urea), then 100 μg of each sample
was loaded onto ImmobilineTM Dry Strips (pH 3-10). Next, isoelectric electrophoresis
(first dimension) was performed using gradient increased voltage to 8000 V with an
IPGphor (General Electric). Following isoelectric electrophoresis, second dimension
electrophoresis with ExcelGelTM 2-D (12.5%) was performed. Gels were stained with
Coomassie Brilliant Blue and the numbers of separated protein spots were examined
using PDQuest software (Bio-Rad). Furthermore, the gene expression of prostaglandin
F2alpha (FP) receptor in RVE cells was investigated by RT-PCR.
Results: RT-PCR revealed gene expression of the FP receptor. Protein extracted from
the RVE cells was separated by 2D-PAGE into 205 separate spots.
Conclusions: Proteomics analysis using 2D-PAGE was helpful to examine changes
in the protein profile of RVE cells for elucidation of the mechanism(s) of retinal blood
flow improvement by anti-glaucoma agents.
CR: A. Ando, None; Y. Mitsuma, None; T. Katano, None; S. Ito, None; T. Nishimura,
None; K. Takahashi, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1880-1883
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1884 - D765
Study on the Spatial Variation of Damage to Rabbit Retina Following IschemiaReperfusion Injury
1885 - D766
Detecting Acute Inner Retinal Excitotoxic Injury in Mice Using Diffusion MRI
Q. Yang1, W. Guo2. 1Ophthalmology department, Eye & ENT hospital of Fudan
University, Shanghai, China; 2Ophthalmology Department, Eye & ENT Hospital of
Fudan University, Shanghai, China.
1
Purpose: To investigate the spatial variation of damage to rabbit retina following
ischemia-reperfusion injury.
Methods: Acute ischemia in the retina was induced in rabbits by increasing intraocular
pressure to 140 mmHg for a period of 60 minutes. Thereafter, the eyes were reperfused
at normal intraocular pressure for 24 hours. These retinas were examined with light
and transmission electron microscopy, and terminal deoxynucleotidyl transferase
biotin-dUTP nick end labeling (TUNEL) staining was performed to detect apoptosis.
The damage was compared among four parts of the retina (upper unmyelinated region,
myelinated region, visual streak, and lower unmyelinated region).
Results: In TUNEL assays, the percentage of cells that underwent apoptosis was
significantly higher in the visual streak than in the other parts of the retina (P<0.05),
while few TUNEL-positive cells were observed in the upper unmyelinated region.
Histopathologic findings by electron microscopic examination further revealed cellular
edema, vacuolization and chromatin condensation in the visual streak, whereas the
ultrastructure of the upper unmyelinated region was least affected by the ischemiareperfusion injury.
Conclusions: Our results suggest that visual streak of the rabbit retina is the most
vulnerable part after ischemia-reperfusion injury.
CR: Q. Yang, None; W. Guo, None.
Support: Project supported by the National Natural Science Foundation of China
(Grant No.30772372 ).
J. Chen1A, C.-W. Chiang1B, S.-K. Song1C. AInternal Medicine, BChemistry, CRadiology,
Washington University, St. Louis, MO.
Purpose:Cerebral excitotoxic injury leads to acute decrease of apparent diffusion
coefficient (ADC) detected by in vivo diffusion MRI. We examined the feasibility of
detecting retinal excitotoxic injury using the same technique.
Methods:Forty-five 2-month old male C57BL/6 mice were imaged in vivo at 11.7T
followed by cross-sectional histology using previously reported method1. Each mouse
was examined at baseline, 3-hour, 1-, 3-, or 7-day post intravitreal injection of 0.5uL
5mM NMDA or 0.5uL saline (n>=5 for each group except n=3 for saline 3- and 7-day).
The ADC map was calculated from diffusion weighted images. The three MR-detected
retinal layers were respectively assigned to NFL/GCL/IPL, INL/OPL, and ONL/IS/OS.
Results:NMDA excitotoxicity resulted in substantial retinal swelling at 1-day (Fig.
1). However, an acute increase of inner retinal diffusion weighted MR signal (Fig 1)
reflecting a 30% decrease of ADC in INL/OPL (Fig. 2) was observed as early as 3-hour,
consistent with previous report in cerebral cytotoxic edema. ADC in NFL/GCL/IPL
exhibited a transient increase suggesting death of ganglion cells that are sensitive to
NMDA excitotoxicity. ADC in ONL/IS/OS was unchanged at all times because the
pre-synaptic photoreceptor cells are resistant to NMDA excitotoxicity.
Conclusions:Decreased ADC was observed before detectable retinal swelling in
NMDA induced retinal excitotoxic injury.
1.Chen J et. al, Magn Reson Med, 2008
CR: J. Chen, None; C.-W. Chiang, None; S.-K. Song, None.
Support: NIH R21 EY018914; Washington University DRTC DK20579
1886 - D767
In vivo Imaging of Retinal Structure With Spectral Domain Optical Coherence
Tomography in a Mouse Model of N-Methyl-N-Nitrosourea-Induced Retinal
Degeneration
H.-C.H. Wang1, A. Muniz1, D.R. Clarkson2, H. Alayon1, D.J. Golden1, P.R. Edsall2, A.
Akers1, B.E. Stuck1. 1U.S. Army Medical Research Detachment, Brooks City-Base, TX;
2
Northrop Grumman, San Antonio, TX.
Purpose: To study the progression of retinal degeneration induced by N-methyl-Nnitrosourea (MNU) in a mouse model with high-resolution, spectral domain optical
coherence tomography (SD-OCT) and light microscopy.
Methods: C57BL/6 mice received a single intraperitoneal injection of MNU at 60 mg/
kg. The progression of the retinal degeneration was investigated at 0, 3, 5, 8 and 10 days
after MNU treatment by SD-OCT for cross-sectional imaging of retinal structures.
Multiple scan averaging was applied to enhance the image of retinal structure. Retinal
histology was performed for each time point to correlate the structure findings
observed by the SD-OCT images. To identify the apoptotic cells, TUNEL assays were
performed on the frozen retinal sections at selected time points (0, 1, 3, 5 days) after
the MNU treatment.
Results: Following MNU treatment, the retinal thickness progressively decreased
with thinning of the outer nuclear layer (ONL) as shown by high quality of SD-OCT
images of the mouse retinas. At day 8 after MNU treatment, all the ONL was lost. The
sequential decrease in the retinal thickness as observed by the SD-OCT images was
confirmed and characterized with the histological data. Moreover, the detachment
of the retinal pigment epithelium (RPE) from Bruch’s membrane after the MNU
treatment observed in the SD-OCT images was confirmed by the histological analysis.
The thinning of the ONL was due to the selective destruction of photoreceptors by
the MNU. This result first suggested by the SD-OCT imaging was verified by the
observation that TUNEL- positive cells were located at the ONL.
Conclusions: The SD-OCT system enabled real-time, non-invasive imaging of the
mouse retina. This method allowed in vivo monitoring of the dynamic changes of
retinal structures in a mouse model of MNU-induced retinal degeneration. The in-vivo
SD-OCT imaging system bridges the gap between research and clinical observations.
SD-OCT holds great promise for being an important tool for assessing and monitoring
the progression of retinal degeneration and evaluating treatment approaches for
retinal trauma and disease.
CR: H.-C.H. Wang, None; A. Muniz, None; D.R. Clarkson, None; H. Alayon,
None; D.J. Golden, None; P.R. Edsall, None; A. Akers, None; B.E. Stuck, None.
Support: U.S. Army Military Operational Medicine Research Program (MOMRP)
1887 - D768
Characteristics of Growth and Death of Adult Rat Retinal Ganglion Cells in
Culture
J.P. Wood, G. Chidlow, T. Mammone, R.J. Casson. Ophthalmic Research Laboratories,
SA Institute of Ophthalmology, Adelaide, Australia.
Purpose: To perform a spatiotemporal characterisation of the growth of adult rat
retinal ganglion cells in culture and to closely observe the responses of these cells to
lethal influences as a prelude to assessing the protective or regenerative properties
of test agents.
Methods: Retinal dissociates from 8 week old Sprague-Dawley rats were incubated
in a medium containing the growth factors, BDNF, bFGF and CNTF (as previously
characterised by Pang et al, BMC Neuroscience 8:11). Retinal ganglion cells were
disseminated from other neurons by positive-immunolabelling for neurofilament
(NFs), Thy 1.1, tau and MAP1b. Growth characteristics were determined by using a
Biostation 1M live cell imaging system: measurements were recorded from these cells of
neurite growth for up to 30 days. In order to determine the death characteristics of these
cells in culture, they were treated with the excitotoxin, N-methyl-D-aspartate (NMDA),
the apoptotic inducer, staurosporine (STSN), and with oxygen-glucose deprivation
(OGD). As a test agent, the peptide factor, osteopontin (OPN) was also evaluated
for influences on the latter parameters as well as on changes in immunolabelling of
MAP2 and GAP43.
Results: Retinal ganglion cells had observable neurite growth from day 1-2 in vitro
(mean lengths of major neurites: days 0-3, 0-10μm; days 5-8, 25-50μm; days 12-30,
100-250μm). In most cases, an obvious axon was easily distinguishable from other
neurites. Cells showed positive immunolabelling for NFs, tau, Thy 1.1 and MAP1b.
Both NMDA and STSN caused a shrinking of cell perikarya which was followed
by an rapid and striking retraction of major neurites just before a sudden cell body
contraction characteristic of apoptotic death. OGD (60 minutes), conversely caused
degeneration to occur concurrently throughout the the length of the neurites, 12-24
hours after replacing cells into normal growth conditions. The peptide factor, OPN
caused cells to undertake an increased level of neurite formation and branching, an
elevation in labelling for MAP2 and GAP43, and was able to delay death resulting
from OGD, but not NMDA or STSN.
Conclusions: In the present system healthy ganglion cell growth and development
was observed in culture for up to 30 days. Live imaging of induced death showed that
cells died very differently depending on the lethal stimulus. These data, and those
demonstrating that OPN was able to delay cell death may have implications for the
treatment of diseases resulting from ganglion cell death in situ.
CR: J.P. Wood, None; G. Chidlow, None; T. Mammone, None; R.J. Casson, None.
Support: NHMRC grant 565202; ORIA
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1884-1887
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1888 - D769
Characterization of Four Distinct Transgenic Zebrafish Lines that Express GFP
and Crb2b-Targeting siRNA Genes Under the Control of the RH2-2 Promoter
1889 - D770
Vitreo-Retinal Interface (VRI) Injury After Acceleration Impulse in a Porcine
Model
X. Wei, J. Zou, S. Bonaffini. Ophthalmology, University of Pittsburgh, Pittsburgh, PA.
R. Mandiga1,2, T.L. Gosen2, S.S. Huang1,2. 1Department of Ophthalmology and Visual
Sciences, University Hospitals Eye Institute, Cleveland, OH; 2Department of
Ophthalmology, Case Western Reserve University School of Medicine, Cleveland,
OH.
Purpose: The inner segments of vertebrate photoreceptors are enriched with polarity
scaffold proteins, such as Crumbs. Many of these polarity proteins are required to
maintain the integrity of a variety of tissues. To investigate the functions of Crumbs
2b (Crb2b) in photoreceptors, the authors attempted to generate transgenic zebrafish
lines to suppress Crb2b expression in green cones.
Methods: Four distinct hairpin siRNA-crb2b transgenes that target different regions of
the crb2b mRNA were individually introduced into the zebrafish genome with an I-SceI
meganuclease-based transgenic approach. The RH2-2 promoter was used to drive the
expression of these transgenes. Each transgene construct also contains a RH2-2-driven
GFP reporter. Positive transgenic lines were identified by both GFP expression and
PCR confirmation of the siRNA-crb2b transgenes. Confocal immunomicroscopy was
then used to analyze the expression patterns of Crb2b and GFP in the retina.
Results: Eleven transgenic fish lines were identified in this study. However, none
display apparent downregulation of Crb2b expression in the photoreceptor layer.
The expression patterns of the GFP reporter vary among different transgenic lines.
Here, the authors report detailed characterization of the GFP expression patterns in
four different lines.
Conclusions: Our study suggests that different strategies are most likely needed for
efficient RNAi-mediated gene silencing in transgenic animal models. Nevertheless,
the four transgenic zebrafish lines are useful tools to study the development of the
photoreceptors and brain in zebrafish.
CR: X. Wei, None; J. Zou, None; S. Bonaffini, None.
Support: NIH Grant EY016099
1890 - D771
Diabetes-Associated Hyperglycemia and Hyperosmolarity Do Not Adversely
Affect Cell Growth or Viability of Immortalized Rat Retinal Müller (rMC-1)
Cells
Purpose: To identify a threshold for VRI injury after anterior-posterior (A-P) and lateral
vector acceleration impulse in a fresh porcine globe model.
Methods: Fresh porcine globes were shipped overnight from Sioux-Preme Packaging (Sioux
City, IA) on ice within 24 hours of harvest. As per our protocol to generate a model that
mimicked human adult vitreous, each eye was injected with 0.3cc of 0.25% trypsin-EDTA
(Invitrogen Carlsbad, CA) and incubated for 2 hours. A pendulum induced A-P or lateral
vector acceleration impulse on each globe suspended within a housing box attached to an
Endevco 751-10 accelerometer (San Juan Capistrano, CA) to generate impulses of 60g, 100g,
200g, 300g, 400g, and 500g. Each globe was dissected and inspected for gross VRI injury.
Results: In the A-P vector group, 10% of the eyes at 60g (n=10), 20% at 100g (n=10), 60% at
200g (n=10), 54.5% at 300g (n=11), 80% at 400g (n=15) and 90% at 500g eyes (n=20) showed VRI
injury. Under 400g, VRI injury was limited to 1 quadrant of up to 3 holes at the vitreous
base. Above 400g, VRI injury included up to 5 holes, small horseshoe tears, or dialyses in
one quadrant of the vitreous base. In the lateral vector group, 10% of the eyes at 60g (n=10),
40% at 100g (n=10), 60% at 200g (n=10), 70% at 300g (n=10), 90% at 400g (n=10) and 90% at 500g
(n=10) showed VRI injury. Under 400g, VRI injury included up to 5 holes or multiple tears in
multiple quadrants of the vitreous base. Above 400g, VRI injury was extensive from 4 to 16
holes, multiple dialyses, and horseshoe tears located around the vitreous base.
Conclusions: The threshold for VRI injury lies between 60g and 100g after both A-P and lateral
vector acceleration impulse injury and was localized to the vitreous base in our model. The
injury pattern varied by impulse vector. A-P impulse consistently produced single quadrant
relatively minor VRI injury, while lateral impulse produced more extensive multiple quadrant
injury regardless of amplitude. A proposed explanation for more extensive injury in the
lateral group is that the eye is better equipped to handle the shearing A-P vector impulse
by benefiting from a firm attachment of the vitreous base to the optic nerve head and retinal
vessels and a buffering effect of the lens-iris diaphragm.
CR: R. Mandiga, None; T.L. Gosen, None; S.S. Huang, SurModics, Inc., I; Digital Healthcare,
Inc., I; University Hospitals Eye Institute, E; Philip F. and Elizabeth G. Searle – Suber Huang
Chair Professorship, E; i2i Innovative Ideas, Inc., E; Bausch & Lomb, C; Merck & Co., Inc., C;
SurModics, Inc., C; Second Sight, C; Therapeutic Nanoparticle and Molecular Imaging, C;
Digital Healthcare, Inc., C; American Academy of Family Physicians, C; Retinal Dis Image
Analysis Reading Center/Case, C; Neurotech – REDIARC Fundus Reading, C; Lux Bio –
REDIARC Fundus Reading, C; Alcon – REDIARC Fundus Reading, C; Pfizer – REDIARC
Fundus Reading, C; Schering Plough – REDIARC Fundus Reading, C; VRT – Vitreo Retinal
Technologies – REDIARC, C; American Retina Foundation, C; Diabetic Retinopathy Clinical
Research Network (DRCR), C; MacuSight – REDIARC Fundus Reading, C; American Academy
of Ophthalmology, C; NEHEP/NEI/NIH, C.
Support: SEARLE Retinal Research Endowment
1891 - D772
Initial Increase of ERG Amplitudes in Type 2 Diabetes is Reversed by
Normalization of Glycemia
L.E. Johnson1, M.-T. Perez1,2, M. Larsen1,3. 1Dept. of Ophthalmology, Glostrup
Hospital, Glostrup, Denmark; 2Dept. of Ophthalmology, Lund University, Lund,
Sweden; 3Kennedy Center, Glostrup, Denmark.
E.C. Steele, Jr., P. Brooks, J. White, B. Bodo. Biology Department, Morgan State
University, Baltimore, MD.
Purpose: Retinal Müller glial cells provide many critical support functions for both
neuronal and vascular cells in the retina. It has been proposed that loss of these support
functions might precede and contribute to the progressive dysfunction and loss of
neurons and vascular cells observed in later stages of diabetic retinopathy. Previously
published data support the notion that such a loss of Müller glial cell functions might
occur via hyperglycemia-mediated direct and acute apoptosis of retinal Müller glial
cells. In this study, we re-tested this hypothesis directly using immortalized rat Müller
(rMC-1) cells (kind gift of Dr. V. Sarthy).
Methods: The cell growth rate of rMC-1 cells was quantitatively compared over a
72 hour time course in three different media: euglycemic and normosmolar control
(5mM glucose), hyperglycemic and hyperosmolar (25mM glucose), and euglycemic
and hyperosmolar control (5mM glucose + 20mM mannose). The gross health status
of rMC-1 cells was also compared in these same media over the same time course
using the trypan blue exclusion assay.
Results: rMC-1 cells grew with indistinguishable rates in all three media. No significant
number of rMC-1 cells exhibited trypan blue staining in any media condition.
Conclusions: Our results challenge the parsimonious hypothesis that diabetesassociated hyperglycemia compromises Müller cell support functions simply via
acute cell death. However, our data do not preclude nor discourage the possibility
that hyperglycemia and/or hyperosmolarity may result in acute and direct changes
in the expression or functional profiles of retinal Müller cells. The identification of
such changes, which may play an important role in the progressive pathophysiology
of diabetic retinopathy, is the focus of ongoing research in our laboratory.
CR: E.C. Steele, Jr., None; P. Brooks, None; J. White, None; B. Bodo, None.
Support: NIH Grants EY018346, RR171581, and GM58904; NSF Grant 0506066
Purpose: To examine functional and physiological changes in the retina during the
development of hyperglycemia in a rat model of type 2 diabetes and how glycemic
control affects these parameters both acutely and long term.
Methods: Zucker diabetic fatty (ZDF) rats were compared with lean control rats using
full field electroretinography (ERG) from 8 to 14 weeks of age as their fasted blood
glucose levels increased to around 20 mM. ZDF rats were then either left untreated
or given a long-acting insulin analog once daily from 17 weeks of age to reduce their
glucose levels to <10 mM. ERGs were performed in all 3 groups until 22 weeks. The
expression of glial fibrillary acidic protein (GFAP) and metallothioneins I and II (MTI+II) was examined in retinas obtained at 23 weeks of age.
Results: ZDF rats showed >10% higher scotopic a- and b-wave amplitudes (p<0.05 and
p<0.05, respectively) from 12 weeks of age and about 10% delays in the implicit times
of all oscillatory potential (OP) wavelets (p<0.01) from 14 weeks of age compared to
lean counterparts. Administration of insulin normalized amplitudes within 3 hours,
whereas the OP latencies were unchanged. Long term insulin treatment resulted in
some normalization of OP latencies but a- and b-wave amplitudes became subnormal
(p<0.05). GFAP overexpression was observed in hyperglycemic ZDF retinas in Müller
glial cells whereas MT-I+II were mainly accumulated in the innermost retina. The level
of expression of these proteins was normalized by insulin treatment.
Conclusions: The upregulation of GFAP and MT-I+II is likely to result from increased
stress on the retina caused by high glucose levels. The development of hyperglycemia
over time, however, also led to increasingly higher a- and b-wave amplitudes, indicative
of the retina’s ability to adapt to chronic metabolic challenges. While insulin treatment
caused some of the observed changes to reverse, lower than normal amplitudes were
eventually observed. These contradictory results may be related to the paradoxical
effect of normalization of blood glucose, which improves the long-term prognosis in
diabetic retinopathy, but produces in some patients an initial worsening of retinopathy.
CR: L.E. Johnson, None; M.-T. Perez, None; M. Larsen, None.
Support: Juvenile Diabetes Research Foundation, Glostrup Hospital, Novo Nordisk
Foundation, KMA, Stiftelse Synskadade f.d. Malmöhus Län, Crafoordska Stiftelsen,
Torsten o Ragnar Söderbergs Stiftelser.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1888-1891
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1892 - D773
Covalent Docking of All-Trans Retinal to Rhodopsin Predicts Binding
Interactions of the Metarhodopsin II and Metarhodopsin III States
1893 - D774
Differential Regulation of Coexpressed Opsins
B.A. Battelle1, R. Gonzalez1, E.M. García1, D.R. Dugger, Jr. 2, K.E. Kempler1. 1Whitney
Laboratory, University of Florida, St Augustine, FL; 2Ophthalmology, University of
Florida, Gainesville, FL.
R.M. Harris, A. Hanneken, A.J. Olson. Molecular Biology, The Scripps Research
Institute, La Jolla, CA.
Purpose:Crystal structures of rhodopsin not been generated for many of the photoactive
intermediates, including the all-trans retinal bound state of Metarhodopsin II (M2) and
the inactive, storage form of Metarhodopsin III (M3). We constructed computational
models of both the active and inactive conformations to predict the binding interactions
of all-trans retinal within the retinal binding pocket.
Methods:Using new features in AutoDock 4.0 modeling software (Morris et al. J
Comp Chem 2009), all-trans retinal was covalently attached to lysine 296 and docked
as a flexible residue within the retinal binding pocket of both the active and inactive
conformations of opsin to predict the structure of M2. All-trans-15-syn-retinal was
covalently docked in a similar manner to predict the structure of M3.
Results:A 5 kcal/mol difference in the docking energy of all-trans retinal favored the
docking to the active conformation over docking to the inactive conformation. The
model places the beta-ionone head of all-trans retinal next to methionine 207 on transhelix membrane 5 (TM5), which breaks contact with tryptophan 265 and is consistent
with NMR results of Ahuja et al. (JBC 2009). The all-trans-15-syn-retinal model of
the M3 state showed that the twist in the Schiff base causes the ligand to reform the
salt bridge with glutamate 113 and make contact with tryptophan 265 similar to the
11-cis-retinal binding which inactivates rhodopsin.
Conclusions:The energy difference between the docking of covalently attached alltrans retinal to the active and inactive conformations of opsin suggest a ratchet
mechanism which shifts the equilibrium towards the activated M2 state after 11-cis
retinal is converted to all-trans. Conversion of all-trans retinal to all-trans-15-synretinal (which occurs when a photon of blue light is absorbed) causes rhodopsin
to switch to the inactive M3 retinal storage state. The docking in the M3 state has
similarities to the 11-cis binding, which explains the shift in equilibrium towards
the inactive conformation.
CR: R.M. Harris, None; A. Hanneken, None; A.J. Olson, None.
Support: STSI grant U54RR025774
Opsin coexpression is more common than previously thought. But the functional
relevance of opsin coexpression is mostly unknown as is whether the levels of
coexpressed opsins can be differentially regulated. Three opsins are express in Limulus
lateral eye (LE): opsins1 and 2, which are 99% identical to one another and called here
Ops1-2, and Ops5, which is only 45% identical to Ops1-2. Ops1-2 and 5 are coexpressed
in Limulus LE photoreceptors.
Purpose: to determine relative levels of Ops1-2 and Ops5 in LE photoreceptors and
test whether their relative levels in the photosensitive membrane (rhabdom) change
with a diurnal rhythm or are influenced by the circadian clock.
Methods: Antibodies specific for Ops1-2 and 5 were applied to Western blots of SDS
solubilized LE membranes and known amounts of the antigens against which the
antibodies were raised. Immunoreactivity (ir) was visualized with chemiluminescence,
and the Ops-ir in the membranes was compared to that of the antigen standards.
Immunocytochemistry and confocal microscopy was used to assay relative levels of
Ops1-2- and 5-ir in rhabdoms of LEs (1) dissected at three different times of the day
from animals maintained in natural illumination. (2) dissected at night from darkadapted LEs that either received or were deprived of normal circadian clock input.
Results: At night, when most Ops-ir in the LE is in the rhabdom, the ratio of Ops5 to
Ops1-2 in LE membranes is about 1:5. Immunocytochemistry showed that rhabdomeral
Ops1-2-ir falls during the day to about 50% of its nighttime level while the rhabdomeral
level of Ops5 is unchanged. Thus, during the day, the ratio of Ops5 to Ops1-2 at the
rhabdom is estimated at about 1:2.5. We also found that rhabdomeral Ops1-2-ir is
significantly (35%) lower in nighttime LEs deprived of circadian clock input compared
to control eyes with clock input, while Ops5 levels are not significantly reduced.
Conclusions: In LE photoreceptors Ops1-2 is more abundant than Ops5, but the level
of Ops5 is sufficiently high to contribute significantly to the photoresponse, especially
during the day. The relative levels of Ops1-2 and 5 at the rhabdom change with a
diurnal rhythm and are influenced by the circadian clock. Ops1-2 levels fluctuate
more than Ops5. This is the first evidence that the levels of coexpressed opsins can
be differentially regulated.
CR: B.A. Battelle, None; R. Gonzalez, None; E.M. García, None; D.R. Dugger, Jr.,
None; K.E. Kempler, None.
Support: NSF-IOS 0517273, NSF-REU 0648969, NIH-EY08571
1894 - D775
An Inducible System for Controlling Gene Expression in Transgenic Xenopus
Rods
1895 - D776
Monitoring Rhodopsin Trafficking in Live Cells
J. Sammons1A, S.D. McAlear1B, A. Marsh1B, A.K. Gross1B,1A. ACell Biology, BVision
Sciences, 1University of Alabama at Birmingham, Birmingham, AL.
X. Zhuo, M. Haeri, B.E. Knox. Biochemistry & Molecular Biology and
Ophthalmology, SUNY Upstate Medical University, Syracuse, NY.
Purpose: To develop an inducible expression system in transgenic Xenopus rods.
Method: Three expression systems were investigated for use in Xenopus rods: 1)
TOPTK-i (Denayer et al., 2006, FEBS Lett. 580, 393), 2) LTRi (Tara et al.,2007 Cell, 130, 363)
and 3) a novel modification of Gal4/UAS (Hartley et al., 2002, PNAS, 99, 1377). In the
novel system, a driver, GAL4-GR-EGFP, was expressed under control of the Xenopus
opsin promoter. This driver encodes the Gal4 transcriptional activation-DNA binding
domains with the glucocorticoid receptor (GR) translocation domain at the carboxyl
terminus. The GR domain enables dexamethasone-dependent nuclear translocation
and thus inducible transcriptional activity through Gal4-UAS interactions. The reporter
construct contained a UAS-hsp promoter controlling the gene of interest. Expression
was measured quantitatively using live cell confocal microscopy.
Results: Preliminary experiments indicated that our novel system was suitable for
induction of gene expression in transgenic Xenopus rods. By contrast, TOPTK-iGFP
expressed GFP without induction and LTRi-EGFP was not inducible in Xenopus. Using
the novel system and a rhodopsin-mCherry fusion reporter, gene expression was
recorded in the rod outer segment for at least a month, which is the time needed for
disks to completely traverse the length of the outer segment. We observed fluorescent
protein only after dexamethasone treatments. Multiple inductions produced bands of
fluorescent protein that were spaced in the outer segment according to the treatment
length, with expression returning to background levels quickly (~2 days) after
drug removal. Kaplan banding of rhodopsin assisted the temporal analysis of gene
expression with this reporter.
Conclusion: We have created an inducible system with robust and reliable control of
gene expression in transgenic Xenopus. Although the mosaic expression, a common
phenomenon associated with transgenes, still exists in this inducible system,
quantitative analysis of fluorescence in individual cells permits a detailed analysis
of gene expression. Moreover, the efficient induction and low background expression
will permit future experiments using toxic or regulatory genes.
CR: X. Zhuo, None; M. Haeri, None; B.E. Knox, None.
Support: National Eye Institute/NIH (EY011256, EY012975); Research to Prevent
Blindness
Purpose: We have expressed rhodopsin fused to photoactivatable GFP with the
addition of rhodopsin’s C-terminal 8 amino acids (rho-paGFP-1D4), and studied its
localization, biochemical and spectral properties in order to use as a tool in trafficking
studies in cultured cells and in transgenic Xenopus laevis.
Methods: A synthetic gene encoding rho-paGFP-1D4 was transfected into COS cells or
into IMCD cells, a polarized ciliated mouse cell line derived from the inner medullary
collecting duct. The fusion protein was purified from COS cells, reconstituted with
11-cis retinal and solubilized. UV/Visible spectra were taken in the dark and after
exposure to light to monitor for proper folding of the fusion protein. The localization
of the expressed protein in both transiently transfected COS and IMCD cells was
monitored using immunocytochemistry and by photoactivation of the fusion protein.
Stable IMCD cell lines expressing low amounts of rho-paGFP-1D4 were prepared to
monitor ciliary localization of the fusion protein and trafficking in cultured polarized
cells. Transgenic Xenopus laevis expressing rho-paGFP-1D4 were prepared to examine
the localization and trafficking of the photoactivated protein in rod cells using 2-photon
fluorescence confocal microscopy.
Results: Rho-paGFP-1D4 forms a rhodopsin-like pigment with 11-cis retinal, and
undergoes appropriate spectral changes after exposure to visible light. Additionally,
the fusion protein traffics to the cilium in stably transfected polarized IMCD cells
and to the rod outer segments of transgenic X. laevis. The paGFP moiety of the fusion
protein is able to be photoactivated in individual rods from transgenic X. laevis.
Conclusions: These data suggest that rho-paGFP-1D4 has similar folding and
photoisomerization properties to those of wild type rhodopsin, and is properly
localized in polarized cells. Thus it is functionally similar to wild type rhodopsin and
can be used to measure trafficking in cultured cells or transgenic retinas.
CR: J. Sammons, None; S.D. McAlear, None; A. Marsh, None; A.K. Gross, None.
Support: NIH grant EY015048, EyeSight Foundation of Alabama and Karl Kirchgessner
Foundation
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1892-1895
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1896 - D777
Light-Dependent Interaction of Interphotoreceptor Retinoid-Binding Protein
(IRBP) With Xenopus Cone Outer Segments
1897 - D778
Generation of a Transgenic Mouse Expressing a Ca2+ Biosensor to Investigate
Cone Photoreceptor Ca2+ Homeostasis
M. Garlipp1,2, F. Gonzalez-Fernandez1,2. 1Ophthalmology & Ross Eye Institute,
University at Buffalo, Buffalo, NY; 2Medical Research, Veterans Affairs Medical
Center, Buffalo, NY.
T. Wei1, K. Koeppen2, T. Ott3, N. Rieger2, B. Baumann2, T. Euler1, O. Griesbeck4,
T. Ladewig1, B. Wissinger2. 1Center for Integrative Neuroscience, Institute for
Ophthalmic Research, University of Tuebingen, Germany; 2Molecular Genetics
Laboratory, Centre for Ophthalmology, University of Tuebingen, Germany;
3
Transgenic Animals Core Facility, University Medical School, Tuebingen,
Germany; 4Max-Planck-Institute for Neurobiology, Muenchen-Martinsried,
Germany.
Purpose: Our long-term interest is to understand how IRBP targets the delivery
and uptake of 11-cis retinal, all-trans and 11-cis retinol between the cells bordering
the interphotoreceptor matrix (IPM). For example, IRBP has significant activity in
promoting the outer segment delivery, and release of 11-cis retinal and all-trans retinol
respectively. Although much attention has been given to the ease with which IRBP
can be removed from the IPM by aqueous extraction, there are hints in the literature
that not all of the IRBP is available for such extraction. Here, we ask whether IRBP
demonstrates a physiologically relevant interaction with structures in the retina.
Methods: Xenopus laevis were selected for these studies due to the large size of their
photoreceptors, and ability to detach the retina in light and dark. Full-length Xenopus
IRBP (XIRBP) was expressed in a soluble form in E. coli and purified by a combination
of anion exchange, Ni2+ affinity, and size exclusion chromatography. XIRBP was labeled
with Alexa-647 at a 1:1 molar ratio. Anti-Xenopus IRBP serum raised in rabbits was
used to localize native IRBP. Our experiments detected “wash-resistant” IRBP, and sites
of exogenous IRBP binding. Neural retinas from light- or dark-adapted adult animals
were detached under HBSS (pH 7.4) and washed 3x in 5 ml. Retinas from dark adapted
animals were detached and handled under infrared light. Wash-resistant IRBP was
detected by indirect immunofluorescence using a secondary antibody conjugated to
Alexa-647. In controls, the retina was detached under 4% paraformaldehyde (with/wout
0.5% glutaraldehyde) and not washed. In other experiments, retinas were incubated
with XIRBP-647, ovalbumin-647, or unconjugated Alexa-647. Sections were examined
in cross-section or whole mounts.
Results: Wash resistant IRBP mainly labeled photoreceptor outer segments (OS),
predominantly cones. Whereas in unwashed retinas matrix staining was diffuse and
intense. Similar results were observed in exogenous IRBP experiments. XIRBP-647
staining was observed in cone OS. Ovalbumin-647 and unconjugated Alexa-647
dye showed minimal fluorescence. Dark adapted retinas in both paradigms showed
reduced staining.
Conclusions: IRBP interacts with the cone OS in a light dependent manner. On going
experiments are aimed at defining whether the interaction is to the out segment or
its matrix sheath.
CR: M. Garlipp, None; F. Gonzalez-Fernandez, None.
Support: EY09412; RPB Unrestricted Grant to the Department of Ophthalmology;
VA Merit Review Award
Purpose: Ca2+ plays a crucial role in modulating the photoresponse as well as in
glutamate release at the photoreceptor synapse and there is growing evidence that Ca2+
is involved in photoreceptor apoptosis in hereditary retinal dystrophies. To monitor
[Ca2+] fluctuations underlying those processes in mammalian cone photoreceptors we
generated a transgenic mouse line that express a Ca2+ biosensor specifically in cones.
Methods: We generated a plasmid construct containing the sequence of the Ca2+
biosensor TN-XL under the control of the human red opsin promoter. The linearized
construct was injected into the pronuclei of fertilized oocytes, which were then
implanted into pseudo-pregnant mice. The presence and copy number of the transgene
were determined by PCR. The retinal expression of the biosensor was studied using
confocal microscopy and immunohistochemistry, and the functionality of the biosensor
was tested using confocal Ca2+ imaging.
Results: In the founder mice the copy number of the transgene ranged from 1 to
45. In their progeny we identified one mouse line that expressed the biosensor in a
subset of retinal photoreceptors, immunostaining with antibodies against L/M opsins
confirmed that the biosensor is expressed in cones. We performed initial Ca2+ imaging
experiments, in which changing the external [Ca2+] in the presence of ionomycin led
to fluorescence changes that were small but showed the expected polarity.
Conclusions:We generated a transgenic mouse line that selectively expresses a Ca2+
biosensor in cones, allowing us to address questions such as the role of internal [Ca2+]
changes in cone degeneration using mice models for cone dystrophy in humans.
Preliminary Ca2+ imaging data suggest that the biosensor is functional in cone
photoreceptors.
CR: T. Wei, None; K. Koeppen, None; T. Ott, None; N. Rieger, None; B. Baumann,
None; T. Euler, None; O. Griesbeck, None; T. Ladewig, None; B. Wissinger, None.
Support: Tiston und Charlotte Kerstan Stiftung
1898 - D779
The C-Terminal Tail of Rds is Necessary for Establishment and Maintenance of
Photoreceptor Outer Segment Structure
1899 - D780
Functional and Anatomic Consequences of Subretinal Dosing in the
Cynomolgus Macaque
K. Boesze-Battaglia1A, A. Bragin1A, R. Dierova1A, M. Damek-Poprawa1B. ABiochemistry,
B
Microbiology, 1University of Pennsylvania, Philadelphia, PA.
T.M. Nork1, C.B.Y. Kim1, J.N. Ver Hoeve1, C.A. Rasmussen1, P.E. Miller1, H.D. Wabers1,
R.R. Dubielzig1, C.J. Murphy2, R.J. McCulloh 3, B.J. Christian3. 1Comparative Ophth Res
Labs (CORL), Univ of Wisconsin-Madison, Madison, WI; 2Comparative Ophth Res
Labs (CORL), Univ of California Davis, Davis, CA; 3Covance Laboratories, Madison,
WI.
Purpose: Any one of over 150 mutations within the PRPH2 gene result in a broad
variety of late onset progressive retinal dystrophies characterized by abnormal
photoreceptor structure. The pathogenic mechanism(s) underlying retinal degeneration
slow (rds) mediated retinal dystrophies are unknown. RDS belongs to a family proteins
characterized by regions known as intrinsically disordered domains (IDD). IDDs are
random structures which are stabilized by interactions with one or several binding
partners, in the case of RDS this domain is the multi-functional C-terminus. In these
studies we analyze a recently developed in vivo mouse model designed to determine
the role of the C-terminal tail of RDS in establishment and maintenance of outer
segment (OS) structure.
Methods: A transgenic mouse colony was generated on an rds-/- background that
express a FLAG-tagged version of the ROS polypeptide lacking the terminal ten
residues (RdsΔ10FLAG). The transgene, RDSΔ10FLAG, containing mouse PRPH2 cDNA
truncated by 30 nucleotides, was directed to rods by a 4.5-kb rhodopsin promoter.
Removal of this region abolishes MREG binding to RDS and enhances C-terminal
mediated membrane perturbations. Mice expressing either one or two copies of the
transgene on a null rds-/- background were analyzed in these studies. We determined
whether photoreceptor structure (assessed by light and electron microscopy) and
function (assessed by Electroretinograms) were correlated with modifications of the
RDS C-terminus.
Results: The RDSΔ10FLAG gene product was expressed as a covalently linked dimer that
resolved into a monomer in the presence of reducing agents based on western blot
analysis. It complexed with ROM-1 as indicted by co-immunoprecipitation studies.
The Rds-/- mice do not produce any OSs. Expression of a single copy of the RdsΔ10FLAG
transgene could partially rescue this phenotype, as small nubby OSs where formed.
Moreover, expression of RDSΔ10FLAG did not alter gross OS morphology as indicted in
EGFP positive sections stained with anti-opsin. The degree of structural instability
of the RDSΔ10FLAG mutant decreased relative to wild type as predicted by its PONDR
score. These results suggest less structural flexibility in the absence of the terminal
ten residues.
Conclusions: We propose that the IDD RDS C-terminal domain plays a direct role
in the biogenesis of OSs. Moreover, the C-terminus is specifically required for the
stabilization of OS structure.
CR: K. Boesze-Battaglia, None; A. Bragin, None; R. Dierova, None; M. DamekPoprawa, None.
Support: EY-10420, EY-18705
Purpose: There is growing interest in subretinal delivery of therapeutically
targeted viral vectors to treat ocular disease. In this study, we characterized the
functional and anatomic consequences of the subretinal injection per se using
multifocal electroretinography (mfERG), optical coherence tomography (OCT),
immunohistochemistry (IHC) and transmission electron microscopy (TEM).
Methods: The right eyes of 3 cynomolgus macaques were given subretinal injections
(100 µl) of Balanced Saline Solution (BSS™). The control (left) eyes received intravitreous
injections (100 µl) of BSS. Fundus photography, OCT and mfERG were obtained before
and immediately after injection and again 9 and approximately 35, 60 and 90 days
later. IHC for GFAP, rhodopsin, S-cone opsin and M/L-cone opsin, and TEM of the
retina were also done.
Results: Subretinal blebs included the superotemporal macula. Expected mild retinal
hemorrhage occurred at the injection sites. On the day of injection, mfERG was similar
to baseline in the eyes given intravitreous injections. However, there was transient
suppression of mfERG responses in the bleb and also in other non-bleb regions. The
retinas were re-attached by 2 days after injection. Partial recovery of mfERG amplitudes
occurred by 9 days post-injection and was largely, but not completely recovered after
90 days. OCT showed a marked decrease in the inner segment/outer segment (IS/
OS) line in the region of the former blebs. There was no change in GFAP and S-cone
staining but the rhodopsin and M/L-cone opsins were partially displaced into the inner
segments. TEM revealed disorganization of the outer segment rod (but not cone) disks.
Conclusions: Subretinal injections were well tolerated although some residual
effects remained after 90 days. Transient suppression of mfERG responses spatially
remote from the bleb was unexpected. While, the mfERG responses nearly recovered,
disorganization of the outer segment rod disks remained and may explain the
decrement in the IS/OS line on OCT seen in the region of the former blebs.
CR: T.M. Nork, None; C.B.Y. Kim, None; J.N. Ver Hoeve, None; C.A. Rasmussen,
None; P.E. Miller, None; H.D. Wabers, None; R.R. Dubielzig, None; C.J. Murphy,
None; R.J. McCulloh, None; B.J. Christian, None.
Support: NIH R01EY014041, NIH P30EY016665, AHAF, RRF, RPB
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1896-1899
Monday, May 3, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 1876 - 1902 / D757 - D783
265. Retinal Biology and Physiology
Organizing Section: RC Contributing Section: RE
1900 - D781
α2-Macroglobulin Promotes MMP-2 Activation, Actin Cytosqueleton
Remodeling and Cell Migration in the MIO-M1 Müller Cell Line Mediated by
LRP1
P.F. Barcelona, G.A. Chiabrando, M.C. Sanchez. CIBICI-Dpto de Bioq Clinica FCQ
UNC, Cordoba, Argentina.
Purpose: Müller cells (MC) are known to undergo functional and morphological
changes and to produce matrix metalloproteinases (MMPs) during retinal angiogenic
diseases. In previous studies we have demonstrated that MC expresses the α2Macroglobulin (α2M) receptor, LDL receptor-related protein 1 (LRP1), which mediates
the α2M-induced MMP-2 activity and cell migration. In addition, it is well known
that the trimolecular complex MT1-MMP/TIMP-2/proMMP-2 plays a key role in the
migratory regulation of cells. Herein we investigated the subcellular distribution
of MT1-MMP/TIMP-2/proMMP-2 complex and actin cytosqueleton remodeling in
the MIO-M1 Müller cell line stimulated with α2M. In addition, we evaluated the cell
migration induced by α2M on different matrix-protein-coated surfaces.
Methods: Transient transfection of the human MIO-M1 cells with the vector MT1MMP-GFP was performed using Lipofectamine 2000. Transfected cells cultured in
the presence of α2M were maintained in culture using Dulbecco’s modified Eagle’s
medium (DMEM). The effect of α2M on subcellular distribution of the trimolecular
complex proteins and cytosqueleton of actin was evaluated by immunofluorescence (IF)
and confocal microscopy. The cell migration of MIO-M1 was determined by woundhealing assay on collagen or laminin-coated surface using time-lapse video microscopy.
Results: Under α2-M stimulation MT1-MMP was localized at the cell surface of MC.
In agreement with a role for MMP-2 in cell motility, its pericellular expression was
principally associated with actin motile structures such as filopodia and lamellipodia.
Furthermore, MMP-2 and TIMP-2 partially colocalized at the periphery of the MC, a
pattern that was prevented by RAP, a ligand binding antagonist of LRP1. Finally, this
MMPs regulation was also accompanied by an increase of cell motility on collagen- or
laminin-coated surfaces.
Conclusions: All together, these data demonstrate that α2M/LRP1 system is involved in
the MIO-M1 cell migration, which could act as a linker between pericellular proteolysis
and the actin cytoskeleton.
CR: P.F. Barcelona, None; G.A. Chiabrando, None; M.C. Sanchez, None.
Support: CONICET; SeCyT; FONCyT
1901 - D782
Control of Müller Glial Cell Morphology by Focal Adhesion Kinase Impacts
Visual Function in Mice
H.E. Beggs, C. Bouquet, G. Lambright, H. Yang, G. Nielsen, D. Yasumura, M.T. Matthes,
J.L. Duncan, M.M. LaVail. Ophthalmology, University of California, San Francisco,
San Francisco, CA.
Purpose: Focal adhesion kinase (FAK) is an important signaling node that has been
shown to play a crucial role in nervous system development as well as in orchestrating
neuronal and glial cell morphology. However, its role in retinal function in vivo and
its subsequent impact on visual processing has not been studied.
Methods: Retinal-specific deletion of FAK was accomplished by crossing the FAK-flox
line to mice expressing Cre recombinase under control of the Six3 promoter. Retinal
function was analyzed by electroretinography (ERG) and optomotry. Individual
fills of Müller glial cells were accomplished by subretinal injection of adenoviruses
containing eGFP and Cre in a FAK-floxed background, and 3D reconstruction was
performed with Imaris software. Measurements of light transmission were performed
in retinal explants with a Zeiss Pascal microscope.
Results: FAK deletion in the retina (but not the RPE or lens) resulted in animals with
impaired retinal function as measured by ERG. Specifically, these animals displayed
a 40% reduction of the scotopic (dark adapted) a- and b-wave response amplitudes,
and a significantly decreased optokinetic behavioral reflex. Surprisingly however,
histological analysis did not reveal any gross morphological abnormalities, and
photoreceptor sensitivity was shown to be normal, thus offering no explanation for
the functional deficits observed. Upon closer examination, we found a unique defect
in the shape of Müller glial cell endfoot processes, with the FAK mutant displaying
a more simplified vitreal segment and a subsequent 2-fold reduction in surface area.
Since Müller glia have been shown to act as optical fibers, with their funnel-shaped
endfeet serving as light collectors (Franze et al, 2007, PNAS), we examined whether
this morphological change could account for the ERG defects in FAK mutants.
Remarkably, we found that the FAK mutant retinas transferred 2.2 times less light to
the photoreceptors than controls, and this decrease correlated with the changes in
ERG response amplitudes.
Conclusion: This study demonstrates an unexpected function for FAK in controlling
Müller glial endfoot morphology. Importantly, we have shown that this morphological
change corresponds to decreased light transmission in the retina, which highlights
the importance of Müller glia as light guiding elements in the optical path.
CR: H.E. Beggs, None; C. Bouquet, None; G. Lambright, None; H. Yang, None; G.
Nielsen, None; D. Yasumura, None; M.T. Matthes, None; J.L. Duncan, None; M.M.
LaVail, None.
Support: This work was supported by RPB, FFB, TMMS and NIH grants EY017379,
EY002162, EY01919.
1902 - D783
Mapping of Substrate Degradome of Calpain by a New Technology of
Functional Proteomics
W. Li, N.B. Caberoy, G. Alvarado. Ophthalmology, Univ of Miami Miller Sch of Med,
Miami, FL.
Purpose: Calpain plays a critical role in cell death in retinal diseases, including retinitis
pigmentosa and glaucoma. The pathways regulated by calpain are traditionally
elucidated on a case-by-case basis with daunting challenges. Consequently, calpainregulated pathways in retinal diseases are poorly defined. The purpose of this study
is to elucidate substrate degradome of calpain to facilitate the delineation of its role
in retinal cell death.
Methods: Newly-developed open reading frame (ORF) phage display was used as
a technology of functional proteomics to elucidate calpain substrates. ORF phage
display cDNA library with C-terminal biotin tag was generated from mouse eye,
bound to immobilized streptavidin, washed and eluted with calpain 2. Eluted phages
were amplified and used as input for the next round of selection. After three rounds
of functional selection, individual phage clones were analyzed for their activity as
the substrates of calpain 2, identified by sequencing and independently verified.
Results: New substrates of calpain 2, including Son cell proliferation proteins
(Son), CCR4-NOT transcription complex subunit 3 (Cnot3), Ring finger protein 146
(Rnf146), catenin delta 2 (Ctnnd2), caldesmon 1 (Cald1) and thymopoietin (Tmpo), were
systematically and efficiently elucidated by ORF phage display. Their roles as calpain
substrates were independently verified with purified recombinant substrate proteins.
Conclusions: These data demonstrated that calpain 2 has broad substrate specificities
in the eye and may regulate a number of pathways during retinal apoptosis. Moreover,
ORF phage display as a powerful technology for unbiased mapping of substrate
degradomes for calpain and other proteases will provide in-depth understanding of
protease biology in ocular physiology and disease pathogenesis.
CR: W. Li, None; N.B. Caberoy, None; G. Alvarado, None.
Support: NIH Grant R01EY016211 and P30EY014801
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
1900-1902
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2233 - A413
DNA Demethylation in Retinal Neurocytes Contributes to the Up-Regulation of
DNA Repair Protein, Ku80
J. Zhuang1, Y. Ye1, F. Li1, J. Ge2, K. Yu1. 1Zhongshan Ophthalmic Center, Sun Yatsen University, Guangzhou, China; 2Glaucoma, Zhongshan Ophthalmic Center,
Guangzhou, China.
Purpose: Ku80 plays a critical role in DNA repair. However, Ku80 is silenced in mature
neurocytes. This study aimed to investigate the mechanism of Ku80 silencing and if
Ku80 plays a role in DNA repair in retinal neurocytes.
Methods: Ku80 expression was measured in the retina of fetus, neonatal and adult
mice via immunofluorescence. The primary retinal neurocytes were treated with
5-azacytidine (5-Aza) and analysed by RT-PCR and Western Blot. Analysis of DNA
methylation in Ku80 promoter was performed. DSBs repair efficiency in primary retinal
neurocytes after treated by H2O2, was assayed by staining with γ-H2AX.
Results. Ku80 is only expressed along the retinal ventricular surface of rapidly
proliferating cells in the fetus. No expression of Ku80 and Ku70 is observed in the
retina after birth. Demethylation by 5-Aza activates Ku80 expression in vitro, while
methylation of -179bp in Ku80 promoter induces Ku80 silencing in retinal neurocytes.
Ku80 reactivation in retinal neurocytes by 5-Aza enhances DNA integrity after
treatment with H2O2.
Conclusions: Ku80 might be a target for reactivation to increase retinal neuronal
DNA repair.
CR: J. Zhuang, None; Y. Ye, None; F. Li, None; J. Ge, None; K. Yu, None.
Support: Supported by the grants from the National Natural Science Foundation
(Project: 30970923; 30872811), Guangdong Province Natural Science Foundation (Project:
7001573).
2234 - A414
Increased DNA Methylation in Several Animal Models of Retinitis Pigmentosa
P. Farinelli1, B. Arango-Gonzalez2A, J. Kaur2B, F. Paquet-Durand3, P.A. Ekstrom4.
1
Ophthalmology, Clinical Sciences, Lund, University of Lund, Lund, Sweden;
A
Division of Experimental Ophthalmology, BDivision of Ophthalmology, 2Centre
for Ophthalmology, Tuebingen, Germany; 3Experimental Ophthalmology, Institute
for Ophthalmic Research, Tuebingen, Germany; 4Ophthalmology, Clinical Sci
Lund, Lund University, Lund, Sweden.
Purpose: Gene repression by DNA methylation is a well documented epigenetic
phenomenon, that seems to be involved in a number of cellular responses. However, so
far there is only little information whether DNA methylation is also a component of the
mechanisms that lie behind retinal degenerations, such as Retinitis Pigmentosa. Here
we wanted to see whether there were any indications of altered DNA methylation in the
degenerating photoreceptors that characterize this disease and therefore investigated
several animal models for Retinitis Pigmentosa.
Methods: Retinas were collected from either rd1, rd2 or wild type background C3H
mice, as well as from either P23H-1 and S334ter-3 rhodopsin mutant rats or wild type
background CD rats at relevant age (postnatal day 11, 21, 20, 12 respectively for rd1, rd2,
P23H-1, S334ter-3). The specimens were fixed, cryo-sectioned and immunolabeled for
5-methyl cytosine, either alone or in combination with TUNEL staining for dying cells.
Results: Methylated DNA was only very rarely found in the photoreceptor layers
of wild type retina either from mouse or rat. By contrast, the outer nuclear layers in
all of the four models showed distinct staining in a subset of photoreceptors, that
corresponded to the expected number of degenerating cells of the respective age.
Furthermore, colabelling in rd1 retina showed that the staining for methylated DNA
overlapped with TUNEL positive cells to a major extent.
Conclusions: DNA hypermethylation was detected in degenerating photoreceptors
regardless of the different kinds of mutations in the various models, and with
characteristics that strongly suggested a relation with the cell death mechanism. These
findings are therefore compatible with increased DNA methylation as an important
and maybe also a general step in the cell death processes that occur in photoreceptors
burdened with inherited degeneration.
CR: P. Farinelli, None; B. Arango-Gonzalez, None; J. Kaur, None; F. Paquet-Durand,
None; P.A. Ekstrom, None.
Support: Swedish Research Council, Medicin; KMA; Torsten och Ragnar Söderbergs
Stiftelser; Kerstan Foundation, Stiftelsen f. synskadade i f.d. Malmöhus län
2235 - A415
Phototransduction and Photoreceptor Cell Death Are Closely Related in a
Zebrafish Model of Autosomal Dominant Retinitis Pigmentosa
2236 - A416
Intravenous Injection of Sodium Iodate Causes a Central-To-Peripheral
Gradient of Retinal Pigment Epithelial (RPE) and Photoreceptor Loss
T. Nakao, M. Tsujikawa. Ophthalmology, Osaka Univ Med School, Suita, Japan.
W. Wang, J.N. Brodfuehrer, L. Zhou, D.C. Dean, M.A. McCall, H.J. Kaplan.
Ophthalmology & Visual Sciences, University of Louisville, Louisville, KY.
Purpose: Many mutations which cause inherited retinal diseases such as retinitis
pigmentosa (RP) have been identified. However, the precise mechanism of photoreceptor
cell death affected by such diseases remains unknown. We previously demonstrated
that photoreceptor cells started to decrease from 5 days after fertilization and that
photoreceptor cell death is markedly accelerated by light exposure in Q344X transgenic
zebrafish. The purpose of our study was to determine whether phototransduction
cascade accelerates photoreceptor cell death in transgenic zebrafish with one of the
rhodopsin mutations at position 344 (Q344X), which is associated with autosomal
dominant retinitis pigmentosa (ADRP) in human.
Methods: Transgenic zebrafish with the rhodopsin Q344X mutation associated with
ADRP in human were used for analysis. We determined the number of surviving
rod photoreceptors in the Q344X transgenic fish while transducin alpha and the
rod cGMP-phosphodiesterase beta subunit (PDE6B) were suppressed with antisense morpholinos. We compared the number of surviving photoreceptor cells of the
morpholino groups to that of the control groups. Photoreceptors were visualized in
the zebrafish rhodopsin promoter-driven green fluorescent protein (GFP) fish line,
and the surviving photoreceptors on the cryosections were counted by means of
fluorescent microscopy 5 days after fertilization.
Results: Suppression of transducin significantly increased the number of surviving
photoreceptor cells (p = 0.0159, control group: n = 14, average = 25.9, SD = 9.56,
morpholino group: n = 15, average = 34.7, SD = 11.3), but suppression of PDE6B markedly
accelerated the photoreceptor cell death (p = 0.000787, control group: n = 14, average =
50.6, SD = 18.4, morpholino group: n = 15, average = 30.5, SD = 8.53).
Conclusions: Blocking of phototransduction upstream of phosphodiesterase has a
protective effect on photoreceptors affected by ADRP. However, suppression of PDE6B
reduces the number of surviving photoreceptor cells in Q344X transgenic zebrafish.
CR: T. Nakao, None; M. Tsujikawa, None.
Support: None
Purpose: Intravenous (IV) injection of NaIO3 selectively targets and eliminates the
RPE in a dose dependent manner. As a secondary consequence of RPE damage,
retinal photoreceptors undergo degeneration and retinal function measured by the
electroretinogram (ERG) declines. Although NaIO3 has been used previously to
produce RPE damage in many species, the uniformity of its effect across the retina
has not been examined. We compared the effect of NaIO3 on RPE between central and
peripheral retina and correlated this with photoreceptor apoptosis. Retinal and visual
function were measured using ERG and the optiokinetic response (OKR), respectively.
Methods: C57B6 (WT) mice (n = 10) were administered 25mg/kg NaIO3 IV. Visual
function was assessed before NaIO3 treatment with the OKR and at 3 days and 2 and
6 weeks post-injection (PI). Both dark- and light-adapted ERGs were used to assess
retinal function at 2 and 6 weeks PI. At six weeks, mice were euthanized and eyes were
enucleated and fixed in 4% paraformaldehyde. The eyes were embedded in paraffin
and 4μm thick sections were cut and stained either with H&E for nuclei counts or
immunohistochemistry for apoptosis. The data for central and peripheral regions of
the inner and outer nuclear layers were compared.
Results: At 25 mg/kg, NaIO3 causes patchy loss of RPE and this loss is more severe
in the central retina. Concomitant with RPE loss, there is increased apoptosis of
photoreceptors in the central retinal, which also is reflected in a decline in the number
of nuclei in the ONL. Apoptosis was not evident in the INL and there was no significant
change in the number of nuclei in the INL. There was a severe reduction in the ERG
of NaIO3 treated mice and in their OKR.
Conclusions: NaIO3 induces a central-to-peripheral gradient of RPE loss, which leads
to a similar gradient of apoptosis of underlying photoreceptors.
CR: W. Wang, None; J.N. Brodfuehrer, None; L. Zhou, None; D.C. Dean, None; M.A.
McCall, None; H.J. Kaplan, None.
Support: Research Prevent Blindness Inc., New York; Discovery Eye Foundation;
Kentucky Research Challenge Trust Fund (HJK)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2233-2236
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2237 - A417
Gestational Lead Exposure (GLE) Enhanced Age-Related Retinal Degeneration
and Outer Plexiform Layer (OPL) Remodeling
2238 - A418
Relationship Between High Resolution Optical Coherence Tomography,
Microperimentry and Autofluorescence in Retinal Distrophy Patients
S. Chaney1, R. Hao1, J.E. Johnson, Jr. 2, R. Hamilton1, S. Mukherjee1, W. Xiao1, D.A. Fox1.
1
University of Houston, Houston, TX; 2University of Houston-Downtown, Houston,
TX.
G. Lo Giudice1, L. Pinello2, M. Mazzarolo2, P. Radin1, V. de Belvis2, M. Tavolato1, A. Galan1.
1
Ophthalmology, San Antonio Hospital, Padova, Italy; 2Department of Pediatrics,
Pediatric Low Vision Centre, Padova, Italy.
Purpose: GLE increases retinal progenitor cell proliferation and the number of rods and
bipolar cells in adult (2 months old: 2m) mice. However, by 12-15m, GLE retinas compared
to age-matched controls have thinner outer (ONL) and inner nuclear layers (INL), a
decreased number of Chx10-immunoreactive (IR) bipolar cells (BC), and no change in
the number of cones (ARVO 2009). Our goal was to characterize further the specificity of
retinal cell loss and examine OPL remodeling in 15m control and GLE retinas.
Methods: C57BL/6 female mice were exposed to water or a 55 ppm lead solution
throughout gestation and until postnatal day 10: equivalent to human gestation period.
Fixed-frozen vertical sections from central retina of 2, 12 and 15m animals were labeled
with DRAQ5 (nuclear stain) and anti-rhodopsin, -Chx10, -PKCα (rod BCs) and -cyclin
D3 (CyD3: Müller glial nuclei) antibodies and processed for confocal studies and image
analysis. Western blots determined whole retinal protein levels of rhodopsin, Chx10, PKCα,
CyD3 and glutamine synthetase (GS) for 2, 12 and 15m control and GLE mice. Confocal
studies using 15m control and GLE retinas examined OPL remodeling and BC sprouting
using anti-PKCα, -plasma membrane calcium-ATPase (PMCA: labels rod spherules),
-vesicular glutamate transporter 1 (VGluT1) and -M-cone arrestin (M-CAr) antibodies.
Results: In controls, the ONL and INL thickness decreased from 2 to 12-15m. In 12-15m
GLE retinal sections, relative to age-matched controls, the thickness of the rhodopsin-IR
ONL decreased and number of PKCα-IR cells decreased, whereas the number of CyD3-IR
Müller glial cells was not significantly different. Western blot analyses in 12-15m GLE
retinas, relative to age-matched controls, revealed that rhodopsin, Chx10, PKCα and GS
protein levels decreased. In 15m GLE retinas, relative to age-matched controls, the number
of M-CAr-IR cone pedicles was not significantly different, whereas the number of ectopic
BCs sprouting into the ONL increased and the OPL was disorganized and thinned.
Conclusions: These results reveal that GLE enhanced age-related retinal degeneration
and OPL remodeling. We postulate that the increased aerobic metabolic demand in GLE
retinas resulting from the 25-30% increase in rods and BCs is not sustainable during
aging and that this results in selective and enhanced apoptotic cell death and synaptic
layer remodeling in the rod-mediated pathway. These findings suggest that children with
GLE have an increased risk for age-related retinal degeneration and visual disturbances.
CR: S. Chaney, None; R. Hao, None; J.E. Johnson, Jr., None; R. Hamilton, None; S.
Mukherjee, None; W. Xiao, None; D.A. Fox, None.
Support: Supported by NIH Grants ES012482, EY07551, EY07024 and UH SGP.
Purpose: To investigate relationship between macular morphology and visual function
in young patients with retinal dystrophy comparing high resolution, fourier domain
optical coherence tomography (FD-OCT), fundus autofluorescence (FAF), fundusrelated sensitivity (microperimetry), and correlate these with visual acuity (VA).
Methods: Eleven retinal dystrophy patients underwent FD-OCT imaging using
CirrusOCT (Carl Zeiss Meditec, Dublin, CA) and FAF of the macular area. All patients
were also studied by means of microperimetry, which automatically analyses macular
light differential threshold and fixation pattern. The thickness of retina (RT), inner
retinal layer (IRL), and outer retinal layer (ORL) and photoreceptor (PR) layers were
averaged over both 6-mm (macular) and 1.5-mm (foveal). Correlations were sought
between central transverse photoreceptor loss, central foveal thickness, VA, FAF
patterns and microperimetry.
Results: Among the 22 eyes with retinal dystrophy (6 patients with cone-rode
dystrophy, 1 with cone dystrophy, and 1 with bull’s eye maculopathy), OCT was
capable of visualizing and quantifying regions with loss of the central photoreceptor
layer in the foveal region. OCT scans revealed macular thinning and marked loss of
the choriocapillaris. The retinal pigment epithelium was relatively more thinned
centrally than peripherally. ORL OCT thickness profile shows marked thinning of the
macular area ORL thickness. Patients without clinically evident central atrophy had
small, focal parafoveal defects. A correlation was detected between VA and transverse
PR loss (P=0.03). A statistically significant association of central foveal thickness with
VA ( P=0.001) but not with transverse PR loss was observed. FAF showed a discrete
arched line of increased autofluorescence surrounding an area with slightly decreased
autofluorescence corresponding to the foveal area. Patients with abnormal FAF was
significantly associated with poor VA and ultrastructural abnormalities as detected
by FD-OCT (P=0001). Macular sensitivity was also significantly impaired over the
foveal region and within the ring, and preserved outside the ring. A relative instability
of fixation pattern characterized by predominantly eccentric fixation was observed.
Conclusions: The present study demonstrates the ultrastructural changes assessed
with simultaneous FAF and FD-OCT and their relationship with visual outcome
and microperimetry.
CR: G. Lo Giudice, None; L. Pinello, None; M. Mazzarolo, None; P. Radin, None; V.
de Belvis, None; M. Tavolato, None; A. Galan, None.
Support: None
2239 - A419
CNTF Induces Shortening of Cone Outer Segments
2240 - A420
Long-Term Effects of Radio Frequency Electromagnetic Fields Emitted by
Mobile Phones on The Macular Structure and Functions
Z. Wang, R. Wen, X. Xia, D. Huang, L. Luo, Y. Li. Bascom Palmer Eye Institute,
University of Miami, Miami, FL.
Purpose: We previously reported that ciliary neurotrophic factor (CNTF) induces
shortening of rod outer segments and down-regulates rod phototransduction
machinery. In the present work, we studied the influence of CNTF on cone outer
segments (COS).
Methods: Adult balb/c mice were housed in 50 lux in-cage illumination (12:12
light:dark). CNTF-treated eyes were injected intravitreally with recombinant human
CNTF protein (6 µg in 3 µl PBS). Fellow eyes were injected with PBS (3 µl). Eyes were
collected at 3, 6 days, or 3 weeks after injection. Vibratome sections (100 µm) were
cut. COS were identified by immunostaining with antibodies against either red/green
opsin, or blue opsin, and examined by confocal microscopy.
Results: Significant shortening of COS was found in retinas treated with CNTF.
The blue COS were 29.0% and 30.2% shorter in retinas treated with CNTF for 3 and
6 days, respectively, than PBS-treated controls (P<0.001). The red/green COS were
31.4% and 33.1% shorter in retinas treated with CNTF for 3 and 6 days, respectively,
than PBS-treated controls (P<0.001). Three weeks after CNTF injection, no significant
difference was found in blue COS between CNTF- and PBS-treated retinas, whereas
outer segments of red/green cones were slightly longer (8.4%, P<0.001) in the CNTFtreated retinas than in PBS-treated controls.
Conclusions: CNTF treatment induces significant shortening of COS in both blue and
red/green cones, very similar to its effects on the length of rod outer segments. In a
separate study, we found that bright habitat light also induces shortening of cone outer
segments. Together, these data suggest the same underlying mechanism that mediates
CNTF-induced changes in cone outer segments and light-induced cone plasticity.
CR: Z. Wang, None; R. Wen, None; X. Xia, None; D. Huang, None; L. Luo, None; Y.
Li, None.
Support: NIH grant EY-018586, JEK grant 08KN-09, Hope for Vision, Foundation
fighting blindness, NIH center grant P30-EY014801, and RPB.
M. Kucukevcilioglu1A, G. Gokce1A, G. Sobaci1A, Y. Karslioglu1B. AOphthalmology,
B
Pathology, 1Gulhane Military Medical Academy, Ankara, Turkey.
Purpose: Biological potential hazards of radio frequency electromagnetic fields (EMF)
emitted by GSM mobile have been the subject of controversy for decades. The proximity
of a mobile phone to the human eye raises the question as to whether electromagnetic
fields (EMF) affect the visual system which generates magnetic fields when stimulated
with a variety of visual cues. This study aims to explore the long-term effects of EMF
in structural and functional status of the macula.
Methods: Sixteen males aged 20 to 22 years old who were from the same location,
and in the habit of using mobile phone (Nokia, 900 MHz) half an hour per a day
during the past 5 years at least were enrolled. They had no systemic or ocular disease
or complaint, and had 20/20 vision in both eyes with brown-colored irides. They
underwent Fransworth Munsell 100 Hue test, recovery time for light discrimination
at the foveola after photostress testing using Humprey® perimeter, central foveolar
thichness (CFT) analysis by optical coherence tomography (OCT, Stratus™), and the
macular pigment density analysis using image processing and analysis software
written in Java programming language to make all of the images comparable to
measure pixel intensity values. Each eye was tested randomly and separately, and
evaluated by the examiner who was blind to the preferred site of the subject’s head
for calling. Test results in both eyes were compared to elucidate the effect of laterality
in EMF.
Results: Mean age was 20.6±0.65 years, and mean duration for mobile phone use
7.5±0.2.1 years. Results of all parameters measured were within the normal range
in both eyes, and there was no difference between the eyes regarding FM 100 Hue
values (p=0.85), CFT values (p=0.45), recovery time after the photostress (p=0.78), and
mean gray values and modal gray values for the macular pigment optical density
(p=0.51 and p=0.76).
Conclusions: Long-term exposure to 900 MHz emitting mobile phone seems to have
no harmful effect in the macular structure and functions.
CR: M. Kucukevcilioglu, None; G. Gokce, None; G. Sobaci, None; Y. Karslioglu,
None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2237-2240
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2241 - A421
Exploring Retinal Markers of Diabetic Neuropathy
A. Moavenshahidi1, G.P. Sampson1, N. Pritchard1, K. Edwards1, D. Vagenas1, A. Russell2,
R.A. Malik 3, N. Efron1. 1Institute of Health and Biomedical Innovation, Queensland
University of Technology, Kelvin Grove, Australia; 2Department of Diabetes and
Endocrinology, Princess Alexandra Hospital, Woolloongabba, Australia; 3Division
of Cardiovascular Medicine, The University of Manchester, Manchester, United
Kingdom.
Purpose: To investigate the application of retinal nerve fibre layer (RNFL) thickness
as a marker for severity of diabetic peripheral neuropathy (DPN) in people with
Type 2 diabetes.
Methods: This was a cross-sectional study whereby 61 participants (mean age 61 [41-75
years], mean duration of diabetes 14 [1-40 years], 70% male) with Type 2 diabetes and
DPN underwent optical coherence tomography (OCT) scans. Global and 4 quadrant
(TSNI) RNFL thicknesses were measured at 3.45mm around the optic nerve head of one
eye. Neuropathy disability score (NDS) was used to assess the severity of DPN on a 0
to 10 scale. Participants were divided into three age-matched groups representing mild
(NDS=3-5), moderate (NDS=6-8) and severe (NDS=9-10) neuropathy. Two regression
models were fitted for statistical analysis: 1) NDS scores as co-variate for global and
quadrant RNFL thicknesses, 2) NDS groups as a factor for global RNFL thickness only.
Results: Mean (SD) RNFL thickness (µm) was 103(9) for mild neuropathy (n=34),
101(10) for moderate neuropathy (n=16) and 95(13) in the group with severe neuropathy
(n=11). Global RNFL thickness and NDS scores were statistically significantly related
(b=-1.20, p=0.048). When neuropathy was assessed across groups, a trend of thinner
mean RNFL thickness was observed with increasing severity of neuropathy; however,
this result was not statistically significant (F=2.86, p=0.065). TSNI quadrant analysis
showed that mean RNFL thickness reduction in the inferior quadrant was 2.55 µm per
1 unit increase in NDS score (p=0.005). However, the regression coefficients were not
statistically significant for RNFL thickness in the superior (b=-1.0, p=0.271), temporal
(b=-0.90, p=0.238) and nasal (b=-0.99, p=0.205) quadrants.
Conclusions: RNFL thickness was reduced with increasing severity of DPN and the
effect was most evident in the inferior quadrant. Measuring RNFL thickness using
OCT may prove to be a useful, non-invasive technique for identifying severity of DPN
and may also provide additional insight into common mechanisms for peripheral
neuropathy and RNFL damage.
CR: A. Moavenshahidi, None; G.P. Sampson, None; N. Pritchard, None; K. Edwards,
None; D. Vagenas, None; A. Russell, None; R.A. Malik, None; N. Efron, None.
Support: National Health and Medical Research Council, George Weaber Foundation
2243 - A423
Neural Cell Attitudes in Ischemic Retinas Evoked by Ischemia/Reperfusion and
by Venous Cauterization
J.-M. Shin, F.-S. Quan, J.-H. Lee, M.-H. Chun, S.-J. Oh. Medicine of Anatomy, Catholic
University, Seoul, Republic of Korea.
Purpose: For pathogenic mechanisms of the glaucomatous neural degeneration, a
variety of experimentally evoked ischemic models were used. The present study was
aimed to investigate whether the attitudes of the neural cells differentially appeared
in ischemic retinas, which were evoked by ischemia/reperfusion and by venous
cauterization, respectively.
Methods: Elevated intraocular pressure (EIOP) model was made by air injection
through anterior chamber with 120 mmHg pressure for 1 hour and reperfusion using
Sprague-Dawley rats. Venous cauterization was done on three episcleral veins. Neuronal
cells were analyzed by calbindin, nNOS and ChAT immunohistochemistry, and glial
cells by Griffonia simplicifolia Isolectin (GSI) B4 and GFAP immunohistochemistry.
Results: Calbindin was expressed in the horizontal cells, amacrine cells and ganglion
cells at normal state. In EIOP retina, calbindin expression is slightly down-regulated
in the ganglion cells than those in normal and cauterization. nNOS was expressed
in the amacrine and displaced amacrine cells and a population of the bipolar cells
at normal. nNOS expressed bipolar cells are slightly increased in cell number of the
cauterized retina. ChAT was expressed in the amacrine and displaced amacrine cells.
In EIOP retina, ChAT expression is slightly down-regulated than those in normal and
cauterization. In normal, GSIB4 labeled microglia appeared in the inner plexiform layer
(IPL) near the blood vessels in addition to the endothelial cells. Microglia appeared
even in the IPL of the early EIOP (to 1week), those in cauterized retina were in the IPL.
GFAP expression was in the astrocytes in the nerve fiber layer and the ganglion cell
layer at normal. GFAP expression in EIOP retina was in the radial processes of Mueller
glial cells additionally, and that in cauterization similar but gradually down-regulated.
Conclusions: These results suggest that there is no large specific neuronal cell
activation but remarkable glial cell activation in the rat retina in response to ischemia/
reperfusion injury or venous cauterization.
CR: J.-M. Shin, None; F.-S. Quan, None; J.-H. Lee, None; M.-H. Chun, None; S.-J.
Oh, None.
Support: This study was supported by the Korea Science and Engineering Foundation
Grant (KOSEF 20090065405) and by the Ministry of Knowledge Economy, Republic
of Korea (10030064)
2242 - A422
Quantification of Retinal Detachment-Induced Apoptosis by Flow Cytometry in
an Experimental Rat Model
P. Tsoka1,2, M.K. Tsilimbaris1,2. 1Institute of Vision and Optics, University of Crete,
Heraklion, Greece; 2Eye Clinic, University Hospital, Heraklion, Crete, Greece.
Purpose: The primary purpose of this study was to evaluate the potential to quantify
the retinal detachment-induced apoptosis in the retina of Sprague-Dawley rats in an
accurate quantitative way by using flow cytometry.
Methods: Retinal detachment was performed on the right eye of deeply anesthetized
animals. The detachment was induced by a sub-retinal injection of sodium hyaluronate.
Rats were sacrifised at 72 hours and the eyes were enuclated to achieve retinal
dissection. Tissue dissociation was accomplished with trypsin. Trypsin action was
blocked and the cells were mechanically dissociated into a single-cell suspension by
gentle pippeting. The cells then were incubated with Neural Cell Adhesion Molecule
and finally with annexin-V-FITC/Propidium Iodide (PI). At least 100.000 cells were
analyzed with a FACScalibur and FlowJo software. Cell debris were exluded from
analysis by gating with forward scatter and side scatter as indicators.
Results: Quantification of the retinal detachment-induced apoptosis in the neuronal
cells of the retina was possible using flow cytometry. In this study the mean value of
the early apoptotic cells (annexin positive/PI negative) was 22.4% (second picture) while
in the control eye was 6.28% (first picture). The experiments were repeated ten times.
Conclusions: Flow cytometry can be used to quantify the apoptotic neuronal cells
after retinal detachment. It is quick and precise and it is also possible to study and
quantify apoptosis/retinal cell type. Flow cytometry will be very useful in future in
studies in neuroprotection as also in quantification of apoptosis during time.
CR: P. Tsoka, None; M.K. Tsilimbaris, None.
Support: None
2244 - A424
The Long Term Effect of Hypoxic-Ischemia in Immature Eyes
H.-M. Huang1, Y.-C. Chang2. 1Ophthalmology, Kaohsiung Chang Gung Memorial
Hospital, KSOHSIUNG, Taiwan; 2Neuro-Pediatrics, Kaohsiung Chang Gung
Memorial Hospital, Ksohsiung, Taiwan.
Purpose: Visual loss associated with brain damage is now the most common cause
of visual impairment in children. At present, the single most common cause of
visually impaired brain injury in children is perinatal hypoxic- ischemia. The ocular
abnormalities and visual dysfunction associated with hypoxic-ischemia injury were
found in these patients. The hypoxia-ischemia insult affecting the optic radiation has
now become the principal cause of visual impairment and dysfunction in children
born prematurely. However, the eye is susceptible to hypoxia ischemic damage in
utero and the optic radiation damage was not the only cause of visual dysfunction. We
hypothesize that hypoxic ischemic insult in the immature eyes may play a significant
role in the visual impairment of these brain damaged children.
Methods: Hypoxia- ischemia (HI) eye injury was produced in postnatal day 7 (P7)
rats by right common carotid artery ligation followed by hypoxia with 8% O2 for 2hrs.
After HI 24 hrs (P8), 48 hrs (P9), 72 hrs (P11), 7 days (P14), 14 days (P21) and 21 days (P33),
we evaluate the histological changes in eyes and brains by H&E stain. At the same
time, immunohistochemistry staining including hypoxyprobe, TUNEL and cleaved
caspase-3 for these eyes slides was performed to explore the possible mechanisms in
this model. Functional assessment was done from P14 due to lid fissure opening in this
period of time by electroretinography (ERG). Pancaspase inhibitor-VAD intraperitoneal
injection was performed after HI 1 hour in P7 and the eyes were analyzed in P14.
Results: After HI in P7, hypoxyprobe staining showed increased density in the
ipsilateral retinas. A marked reduction in inner retinal thickenss was observed from P8
to P33, and ERG recorded the amplitude of b wave were significant lower in ipsilateral
eyes, comparing with contralateral or naïve eyes. TUNEL positive cells increasing from
P8 to P9 then dramatic decreasing from P14 to P21 and activated caspase-3 staining
showed the same temporal profile. However, i.p VAD only provided moderate but not
complete protection from hypoxic-ischemia in immature retinas
Conclusions: Theses results indicated HI caused significant retinal damage in
immature eyes. The HI model may provide another method to understand the
mechanism about the HI induced immature eyes injury and further treatment will
be studied for rescuing eyes in these immature infants.
CR: H.-M. Huang, None; Y.-C. Chang, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2241-2244
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2245 - A425
Tissue Reconstruction in Chicken Retinal Reaggregates Depends on Müller
Glial Cells and is Supported by Fgf-2
2246 - A426
The Up-Regulation of the Reticulon Protein Nogo-A/RTN4-A Does Not Change
the ER Stress Response in Axotomized Retinal Ganglion Cells
F. Frohns, P.G. Layer. Zoology, TU Darmstadt, Darnstadt, Germany.
V. Pernet1, S. Joly2, F. Christ1, J.L. Martin3, M.E. Schwab1. 1Neuromorphology, ETH/
University of Zurich, Zurich, Switzerland; 2Ophthalmology, University Hospital
Zurich, Zurich, Switzerland; 3Loyola University Medical Center, Maywood, IL.
Purpose: 3D-reaggregates from embryonic chicken retina are histotypically structured,
presenting rosettes with photoreceptors and areas comparable to the inner retina,
which surround cell-free neuropil spaces. Here we have analysed the cell-by-cell
development of these areas resembling the formation of an inner plexiform layer
(furtheron called IPL areas). In particular, roles of Müller cells and of FGF-2 were
investigated.
Methods: Retinal spheroids were reaggregated in rotation culture from dissociated
retinal cells of six days old chicken embryos. Cells were treated without/with 25
ng/ml FGF-2 for the whole culture period of up to 10 days. Sizes and shapes of
spheres were analysed and quantified by light microscopy of sphere whole-mounts.
Formation of IPLareas was investigated by immunohistochemistry of sphere cryosections,
including antibodies to vimentin, transitin and glutamine synthetase (GS) to follow
the development of immature and mature Müller cells, and several antibodies for
amacrine cells, but also BrdU uptake and RT-PCR studies.
Results: IPL areas are first announced by DAPI-free open spaces on div2, which are
strongly stained by vimentin and transitin, but not yet GS. As these IPL areas enlarge,
vimentin+ processes become radially directed towards the centre of these open spaces.
Around div6, these IPL areas become concentrated under the surface of spheres, where
they are detectable even from the outside by their pushing out of bumpy structures
(the spheres are now potato-shaped). MC specific staining, incl. GS has increased and
is highly organised within IPL areas, being in close contact with diffusely displaced
amacrine cells within IPLareas. Occasionally, large IPLareas fuse with each other, eventually
forming a continuous IPL underneath the sphere surface. Thereby, MCs appear to
have a leading role. FGF-2 strongly accelerates this process, whereby its addition
at any time has an immediate effect and can be limited at any time by its removal.
Added at div10, FGF-2 strongly stimulated the proliferation of a ring of MCs, which
parallels the formation of the outer IPL ring, directly showing that the fusion process
depends on the action of MCs.
Conclusions: Retinal spheroids are suitable to analyse basic constraints of IPL
formation of the chicken retina. MCs have an active role in spatially organising the
space of IPL areas. FGF-2 appears to stimulate these processes by acting directly upon
MCs. It should be noted, that these processes occur in almost complete absence of
ganglion cells, showing that IPL formation does not depend on GCs.
CR: F. Frohns, None; P.G. Layer, None.
Support: Deutsche Forschungsgemeinschaft (La 379/12-1)
2247 - A427
Effects of the Pesticide Rotenone on the Rat Retina: Degeneration of
Photoreceptors, Impairments in Dopaminergic Neurons and Loss of Synaptic
Connectivity
Purpose: In the present study, we investigated the role of neuronal Nogo-A in
axotomized retinal ganglion cells (RGCs) and its possible influence on the ER stress
response in vivo.
Methods: Nogo-A was observed by immunohistochemistry on retinal cross sections
and on retinal flat mounts from C57Bl6 male mice. RGCs and Mueller cells were
identified using β3-tubulin and glutamine synthetase antibodies respectively. Adult
mice were axotomized and retinal flat-mounts were double labeled for Nogo-A and
β3-tubulin between 3 and 14 days after injury. To visualize apoptotic RGCs annexin
V was injected in the vitreous body at 7 or 10 days following axotomy. The ER stress
markers CHOP and Bip, as well as ATF3 and c-jun, were followed by quantitative RTPCR in injured retinal lysates. RGC survival was evaluated after intraocular injections
of different adeno-associated viruses 2 (AAV2). AAV2 treatments were performed
4 weeks before axotomy to block the intracellular up-regulation of Nogo-A (AAV2.
ShRNA-Nogo-A) or to enhance Nogo-A gene expression (AAV2.Nogo-A) relative to
a control virus containing the green fluorescent protein (GFP) gene.
Results: Nogo-A was increased in 55% of RGCs at 7 and 14 days after optic nerve
transection. The protein and mRNA levels of CHOP were strongly and selectively
enhanced in RGCs, 3 and 5 days post axotomy, suggesting a fast ER stress induction
prior to the RGC loss. Surprisingly, big cells up-regulating Nogo-A protein presented
a low level of the CHOP protein, suggesting a negative correlation between the ER
stress-associated apoptosis and the elevation of Nogo-A post-lesion. Moreover, Nogo-A
immunopositive cells were not labeled by annexin V at 7 days, implying that the
increase of Nogo-A may be protective against apoptosis. However, the blockade of
Nogo-A up-regulation with an AAV2.shRNA-Nogo-A did not alter the increase of
CHOP mRNA compared with the AAV.GFP treatment 5 days after injury. The density
of surviving RGCs was not significantly changed by inhibiting Nogo-A. In contrast,
the exogenous increase of Nogo-A after AAV2.Nogo-A treatment exacerbated the RGC
death but did not affect CHOP mRNA levels in intact or injured retinas.
Conclusions: Our data show that Nogo-A and the ER stress are quickly up-regulated
in RGCs after optic nerve lesion. We show that Nogo-A does not seem to influence the
ER stress induction and apoptosis triggered by the optic nerve injury.
CR: V. Pernet, None; S. Joly, None; F. Christ, None; J.L. Martin, None; M.E. Schwab,
None.
Support: Swiss National Science Foundation Grant 31-63633.00
2248 - A428
Soluble Adenylyl Cyclase (sAC) Promotes Survival and Axon Growth in Retinal
Ganglion Cells (RGCs)
R.G. Corredor1A, J.L. Goldberg1B. ABascom Palmer Eye Inst/Neurosci, BBascom Palmer
Eye Institute, 1University of Miami Miller Sch of Med, Miami, FL.
J. Esteve-Rudd, L. Fernández-Sánchez, P. Lax, J. Martín-Nieto, N. Cuenca. Fisiología,
Genética y Microbiología, Universidad de Alicante, Alicante, Spain.
Purpose: Rotenone is a pesticide that inhibits the mitochondrial respiratory chain
complex I (NADH oxidase). The activity of this complex is usually reduced in the
brain of Parkinson disease patients, and mutations in genes encoding some of its
components are associated with Leber hereditary optic neuropathy. Patients suffering
from these diseases exhibit retinal deficiencies, including altered electroretinograms
(ERGs) and nerve fiber thinning. The aim of this work was to address the cellular
and physiological impairments taking place in the retina of rotenone-treated rats.
Methods: Adult Sprague-Dawley rats were subcutaneously injected with rotenone
(3 mg/kg) or vehicle every 48 h during 2 months. Thereafter, ERGs were recorded
and rats sacrificed. Eyes were enucleated and retinas processed for protein extraction
or immunohistochemistry. Proteins were subjected to immunoblotting analysis
with tyrosine hydrolase (TH) antibodies. Retinal sections and whole-mounts were
incubated with antibodies against specific neuronal markers, and visualized by
immunofluorescence confocal microscopy.
Results: A thinning of the outer nuclear and plexiform layers was observed,
corresponding to a decrease of photoreceptor cell number and synaptic contacts
between photoreceptors and bipolar or horizontal cells, respectively. As well, the
ERGs of rotenone-treated rats exhibited significantly lowered amplitudes of a and
b waves. We also found that levels of TH were decreased by ~70% in the retina, and
that the dopaminergic cell number and plexus were greatly reduced, together with
their synaptic contacts with AII amacrine cells.
Conclusions: Our results indicate that rotenone strongly damages photoreceptors and
synaptic contacts with their postsynaptic neurons, as well as the retinal dopaminergic
system, eliciting retinal neurodegeneration. Thus, rotenone treatment provides a
suitable model of retinal degeneration induced by mitochondrial impairment-derived
oxidative stress.
CR: J. Esteve-Rudd, None; L. Fernández-Sánchez, None; P. Lax, None; J. MartínNieto, None; N. Cuenca, None.
Support: MEC (BFU2006-00957/BFI), MSyC (RETICS RD07/0062/0012), FUNDALUCE
and ONCE.
Purpose: Mammalian RGCs do not regenerate their axons, and die after optic nerve
injury or ischemia, or in glaucoma and other diseases, leading to permanent visual
loss. Previous work has shown that an increase in electrical activity or in levels
of intracellular cAMP can enhance trophic responsiveness and promote survival
and axonal growth of RGCs. However it is not known which adenylyl cyclases are
responsible for elevating cAMP in response to electrical stimulation (ES). Here we
investigate whether sAC is present in RGCs and promotes survival and axon growth
in vitro.
Methods: Immunostaining, western blot and RT-PCR were used to investigate
expression and localization of sAC in RGCs. Cultured rat postnatal RGCs were
tested with the sAC agonist bicarbonate, and the sAC antagonist 2-hydroxyestradiol
for effects on survival and axon growth, quantified using time lapse microscopy
and axon-tracing at 48 hour in vitro, in the presence or absence of ES and/or the
transmembrane adenylyl cyclase(TmAC) activator forskolin. Intracellular levels of
cAMP were quantified using ELISA.
Results:ES improved survival of RGCs and promoted axon growth in vitro,
even in the presence of forskolin. We detected sAC transcripts in purified RGCs
by RT-PCR. By western blotting, one band was detected at approximately 50Kd.
Immunostaining showed sAC reactivity in the cytoplasm, nucleus and axons of RGCs.
The sAC activator bicarbonate increased intracellular levels of cAMP, stimulated RGC
survival, and enhanced axon growth when compared with cells in bicarbonate-free
media. Interestingly, bicarbonate also enhanced survival and axon growth of RGCs
in the presence of forskolin or the cell-permeable cAMP analog 8-bromo-cAMP.
Pharmacological inhibition of sAC dramatically decreased RGC survival and axon
growth.
Conclusions:sAC activation increases and sAC inhibition decreases RGC survival and
axon growth in vitro. Since electrical stimulation also enhanced RGC survival and axon
growth in the presence of TmAC activation, we hypothesize that additional cAMP may
be generated in response to activity by the calcium- and bicarbonate-sensitive sAC.
CR: R.G. Corredor, None; J.L. Goldberg, None.
Support: American Heart Association, James and Esther King Biomedical Research
Foundation, National eye institute P30, Unrestricted grant from Research to Prevent
Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2245-2248
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2249 - A429
The Glutamate Transporter Inhibitor, DL-Threo-Beta-Benzyloxyaspartate (DLTBOA), Prevents Neurochemical Effects but Not Neurotoxicity Yielded in the
Retina by Elevated Intraocular Pressure (IOP)-Induced Ischemia/Reperfusion
in Rat
R. Russo1, F. Cavaliere1, G.P. Varano1, G. Bagetta1,2, M. Corasaniti3, L.A. Morrone1,2,2.
1
Pharmaco-Biology, University of Calabria, Cosenza, Italy; 2Sect. Neuropharmacol.
Norm. Pathol. Neur. Plasticity, Cosenza, Italy; 3Pharmacobiol. Science, University
Magna Graecia, Catanzaro, Italy.
Ischemic phenomena are common features of retinal pathological conditions, including
glaucoma, anterior ischemic optic neuropathy and retinal vessels occlusion (see
Osborne et al., 2004, Eye 18:1075-1084). Several studies suggest that excitotoxicity
occurs during retinal ischemia leading to retinal ganglion cells (RGCs) death.
Purpose: Using a model of retinal ischemia induced by transient elevation of IOP
we investigated on the role of excitatory amino acid transporters (EAATs) in the
extracellular changes of glutamate (GLU) in the vitreous.
Methods: Retinal ischemia was induced in the eye of adult Wistar rats by acutely
increasing the IOP. Extracellular GLU was monitored in the vitreous before, during
and after pressure-induced ischemia using a microdialysis technique (Nucci et al., 2005,
Neurotoxicology, 26: 935-941). DL-TBOA or coenzyme Q10 (CoQ10) were administered
intravitreally. RGCs labeling by intracollicular injection of FluoroGold was used for
evaluation of cellular survival.
Results: Extracellular level of GLU increased by 70% in the first 10 min of ischemia
with a larger and significant (P<0.05 vs pre-ischemia) increase during reperfusion.
Administration of DL-TBOA (500 μM) minimized the accumulation of GLU, though it
worsened the delayed RGCs loss typically observed under these conditions (DL-TBOA
50 ±10.5 vs control 25.9± 4.2 %; n= 3). Importantly, pre-ischemic treatment with the free
radical scavenger CoQ10 reduced extracellular GLU raise (86% reduction vs control)
seen during reperfusion and prevented RGCs loss (CoQ10 12±3.8 vs control 25.9± 4.2 %).
Conclusion: Our data suggest that derangement of the mechanisms underlying
glutamate transport is a key event triggering excitotoxicity in the ischemic retina and
provide useful pharmacologic information for the correct use of DL-TBOA as a tool
to study ischemia/reperfusion mechanisms in in vivo settings.
CR: R. Russo, None; F. Cavaliere, None; G.P. Varano, None; G. Bagetta, None; M.
Corasaniti, None; L.A. Morrone, None.
Support: None
2251 - A431
Differential Localization of Pax6-Positive Mueller Cell Nuclei After LightInduced Photoreceptor Degeneration
2250 - A430
Mechanisms of Neuroinflammatory Activation by the Neural Retina Following
Light-Induced Retinal Degeneration
M.V. Rutar1,2, R. Natoli1,2, K. Valter1,2, J.M. Provis1,2. 1The Research School of Biology,
The Australian National University, Canberra, Australia; 2ARC Centre of Excellence
in Vision Science, Canberra, Australia.
Purpose: To investigate the expression and localization of potent inflammatory
markers from the chemokine and complement system pathways in the neural retina
following photoreceptor death induced by excessive light.
Methods: SD rats were exposed to 1000lx of light for up to 24hrs, after which
some animals were kept in dim light (5 lux) to recover. At specific time points
during exposure (1, 3, 6, 12, 17, and 24hrs) and following exposure (3 and 7 days),
animals were euthanized and retinas processed. The expression of the monocyte
chemoattractant chemokine CCL-2, and an array of complement system components
(C1s, C3, and C5) and receptors (C1qR, C3aR, C5aR), were assessed by qPCR (n=4),
immunohistochemistry (n=3), and in situ hybridization (n=3). In conjunction, counts
were made of monocytes on retinal cryo-sections immunolabeled with ED1 (n=3), and
photoreceptor cell apoptosis was assessed using TUNEL labeling (n=5). Statistical
significance was determined using the Students t-test and One-way ANOVA.
Results: Up-regulation of CCL-2 gene expression was evident in retinal tissue,
and reached a maximum at 24hrs, which correlated with the increase (p<0.05) in
photoreceptor cell death. Immunohistochemistry and in situ hybridization on retinal
cryo-sections revealed that CCL-2 is expressed by Müller cells, predominately in
regions of heavy photoreceptor degeneration. In conjunction, a significant (p<0.05)
localized recruitment of monocytes to the choroidal and retinal vascular supplies from
24hrs exposure was observed. A significant up-regulation (p<0.0001) of complement
genes C3, C1s, C3aR, C1qR, and C5aR was observed during and following the course of
light exposure, correlating with significant increases in photoreceptor death (p<0.001).
Conclusions: Our data indicate that the retina actively contributes to the guidance
of the neuroinflammatory response following retinal injury, through the local
expression of inflammatory factors from both chemokine and complement pathways.
Characterization of the immune response of the neural retina is crucial in clarifying
the underling pathogenesis of inflammation in retinal degeneration.
CR: M.V. Rutar, None; R. Natoli, None; K. Valter, None; J.M. Provis, None.
Support: Australian Research Council Centres of Excellence Program, Ophthalmic
Research Institute of Australia.
2252 - A432
Electrophysiologial Changes After Acute Retinal Damage Induced by Blue Light
S. Joly1A, V. Pernet1B, C. Grimm1A. AOphthalmology- Lab for Retinal Cell Biol, BBrain
Res. Inst., Univ. of Zurich/ETH Zurich, 1University of Zurich, Zurich, Switzerland.
Purpose: Pax6 is a transcription factor expressed by multipotent progenitors in the
retina during development. It is also a stem cell marker and its expression can be
re-initiated in adult Mueller cells, for example after NMDA excitotoxicity. The aim
of the present study is to follow the pattern of expression of Pax6 in a mouse model
of light-induced photoreceptor degeneration and to determine if Mueller cells can
proliferate and dedifferentiate after light-induced photoreceptor cell death.
Methods: Adult 129S6/SvEvTac mice were exposed to 13,000 lux of white light
for 2 hours to induce photoreceptor degeneration. Intraperitoneal injections of
bromodeoxyuridine (BrdU; 50 µg/g) were performed daily for 4 consecutive days,
starting immediately after the offset of the light exposure. For immunohistochemical
analysis, mice were perfused at different time points after light exposure (N=3 per
group). Pax6 protein levels were evaluated by Western-blotting.
Results: By immunohistochemistry, Pax6 was detected in amacrine, horizontal and
Mueller cells of adult, unexposed mice. Starting at one day after light exposure,
Pax6-positive Mueller cell nuclei appeared in the upper half of the inner nuclear
layer (INL), close to the outer nuclear layer (ONL). Total retinal Pax6 protein levels
were slightly increased after light exposure. Although light exposure led to Mueller
cell gliosis and to the expression of the mitosis marker phospho-histone 3 (PH3) in
some Mueller cells, BrdU was only incorporated into nuclear DNA of photoreceptors.
BrdU colocalized with ligase-4 positive nuclei suggesting that BrdU incorporation
reflected DNA repair rather than DNA duplication. Although intravitreal injections
of fibroblast growth factor 2(FGF2)/insulin slightly increased the appearance of Pax6
positive Muller cell nuclei in the upper INL after light exposure, the effect did not
differ from PBS/BSA control injections.
Conclusions: Light-induced retinal degeneration caused the appearance of Pax6positive Mueller cell nuclei in the upper INL/lower ONL of the mouse retina.
Interestingly, a similar localization of Pax6-positive nuclei was also detected in the
rd10 mouse, a model for inherited photoreceptor degeneration. The expression of
PH3 and the lack of BrdU incorporation suggest that Mueller cells attempt but fail
to enter the cell cycle in the degenerating retina. Further experiments are ongoing to
clarify the role of the differential localization of Pax6-expressing Mueller cell nuclei
in retinas undergoing photoreceptor degeneration.
CR: S. Joly, None; V. Pernet, None; C. Grimm, None.
Support: Swiss National Science Foundation 3100A0-105793
M. Rostkjaer, L.E. Johnson, L. Kessel. Department of Ophthalmology, Glostrup
Hospital, Glostrup, Denmark.
Purpose: Acute phototoxic damage by short wavelength light may occur as an
unwanted side-effect when visualizing the retina for therapeutic purposes, such as
during prolonged vitreoretinal surgery. Furthermore, phototoxic damage is believed
to be a risk factor for the development of age-related macular degeneration. The
purpose of the present study was to examine the electrophysiological changes over
time after blue light induced acute retino-phototoxic damage in Brown Norway rats.
Methods: Male Brown Norway rats were exposed to a blue laser at 445 nm on one
eye for 10 minutes. The time course of changes in retinal function and morphology
was assessed weekly for 4 weeks using full-field electroretinography (ERG) with 30
min dark adaptation and funduscopy before the rats were euthanized and the eyes
used for histology.
Results: Acute effects were seen on the exposed eyes using scotopic ERG where
amplitudes were severely reduced after phototoxic injury despite dark adaptation
whereas photopic responses were only somewhat affected. One week after phototoxic
injury scotopic ERG showed slightly decreased a- and b-wave amplitudes in exposed
eyes when compared with unexposed, whereas no major differences were observed on
the photopic ERG. This difference remained consistent for all following measurements
up to 4 weeks after exposure. In parallell, funduscopic examination revealed white,
edematous lesions immediately after damage. Four weeks post-injury, the lesions were
barely visible macroscopically but a slight change in the pigmentation of the fundus
was observed in the injured region.
Conclusions: Our results indicate that phototoxic retinal damage can be detected
electrophysiologically immediately after exposure and that the ERG of the exposed
eyes recovers to a plateau within a week. The action spectrum of phototoxic damage
implies that the damage mechanism for short wavelength irradiation is related to
the absorption by rhodopsin but we found no evidence of expansive damage to the
photoreceptors as seen by ERG.
CR: M. Rostkjaer, None; L.E. Johnson, None; L. Kessel, None.
Support: Danish Advanced Science Technology Foundation, the BIOP Center, and
a proof-of-concept grant from the University of Copenhagen and the Technological
University of Denmark
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2249-2252
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2253 - A433
Müller Cell Upregulate the Expression of SAP97 in Light-Injured Rat Retina
2254 - A434
Retinal Light Damage Induced by Ultraviolet Light in Albino Rats
H. Ren, G. Xu. Ophthalmology department of EENT Hospital, Fudan University,
Shanghai, China.
S. Kaidzu1, T. Okuno2, M. Tanito1, A. Ohira1. 1Ophthalmology, Shimane Univ Sch of
Medicine, Izumo, Japan; 2National Institute of Occupational Safety and Health,
Kawasaki, Japan.
Purpose:To determined whether scaffolding protein family member synapseassociated protein 97 (SAP97) is involved in the Müller cell response to blue light injury.
Methods: Sprague-Dawley rats were exposed to intense blue light for 24 h. Transmission
electron microscopy(TEM) was performed to evaluate the outer retina edema in lightinjured retina.Cryosections and single isolated Müller cells were immunostained
with SAP97, aquaporin-4(AQP4)and inwardly rectifying potassium channel Kir4.1
antibodies to detect the immunolocalization changes by confocal microscopy.Western
blot and quantitative real-time PCR(qRT-PCR) were applied to evaluate the retinal
SAP97,AQP4 and Kir4.1 protein and mRNA levels respectively.
Results: In light-injured rats, obvious intracellular edema in the outer retina was
observed by TEM. SAP97 was upregulated and concentrated in the outer nuclear
layer after photic injury (Fig.1). The immunostaining of the AQP4 and Kir4.1 proteins
were increased in the outer retina after light treatment which was similar with those
changes of SAP97. Compared with control rat retina, retinal SAP97 mRNA in the lightexposed group was upregulated and maintained at a relatively high level. Whereas
the mRNA levels of both Kir4.1 and AQP4 were increased at d1(the first after light
exposure) and then declined at d2 and d3.Western blot showed that SAP97 and AQP4
protein levels were increased in d3 compared to control group(P<0.05),whereas the
alteration of Kir4.1 protein level had no statistical significance.
Conclusion:Upregulation of SAP97 coincide with the redistribution of AQP4 and
Kir4.1,suggesting that SAP97 play a major role in recruitment of such channels in
light-induced outer retina edema.
Purpose: Ultraviolet light (UV) is hazardous compared with visible light to the eye.
However, the damaging effect(s) of UV on the retina in in vivo has not been fully
examined. In this study, we tested the stimulation-response relationship between the
UV exposure dose and the resultant damage in rat retinas at different UV wavelengths.
Methods: Under deep anesthesia, the left eyes of 5-week-old Sprague-Dawley albino
rats (n=6 for each condition) were exposed to five narrow-band lights with 10 nm
in bandwidth at wavelengths of 330, 340, 360, 380 and 400 nm, using a xenon lamp
source with bandpass filters (Asahi Spectra Co., Ltd., Tokyo, Japan). The right eyes,
left unexposed to light, served as controls. For each wavelength, rats were exposed to
5 or 6 different doses (retinal radiant exposure) ranging from 0.6 to 170 J/cm 2 to obtain
stimulation-response relationship. The retinal radiant exposure was determined by
multiplying the measured corneal radiant exposure by the combined transmittance
of the rat lens and cornea. Seven days after the exposure, flash electroretinograms
(ERGs) were recorded and the radiant exposure that causes 50 % reduction in ERG
b-wave amplitudes (ED50) was calculated for each wavelength of light. Both eyes
were enucleated and retinal sections containing the whole retina including the optic
disc were stained with hematoxylin-eosin (H & E) and outer nuclear layer (ONL)
thickness was measured.
Results: Compared to unexposed eyes, significant reductions in a- and b-wave ERG
amplitudes and in ONL thickness were observed for all wavelengths of light tested.
ED50 of 330, 340, 360, 380, and 400 nm were calculated to be 4.14, 5.04, 7.06, 17.10 and
57.5 J/cm 2 in a-wave and 2.89, 4.10, 5.87, 13.3 and 43.2 J/cm 2 in b-wave respectively.
Conclusions: Stimulation-response relationship curve in UV-induced retinal
damage were obtained in rats. As expected, retinal damage induced by UV depends
on wavelength and radiant exposure. Compared to longer wavelengths, shorter
wavelengths cause more severe retinal damage.
CR: S. Kaidzu, None; T. Okuno, None; M. Tanito, None; A. Ohira, None.
Support: None
CR: H. Ren, None; G. Xu, None.
Support: National Basic Research Program of China (973 program), (2007 CB512205),
National Basic Research Grants of China (30872825, 2008) and Plan of the Best
Disciplines Leaders in Shanghai (09XD1400900)
2255 - A435
The Effect of an Inducer of Bip, an ER-Resident Molecular Chaperone, on LightInduced Retinal Damages
2256 - A436
Redox Modulation is Neuroprotective for Photoreceptor Death After Photic
Injury
T. Nakanishi1A, S. Imai1A, Y. Inokuchi1A, K. Tsuruma1A, M. Shimazawa1A, Y. Monguchi1B,
H. Sajiki1B, T. Kudo2, H. Hara1A. ABiofunctional Evaluation, Molecular Pharmacology,
B
Medical Chemistry, 1Gifu Pharmaceutical University, Gifu, Japan; 2Psychiatry,
Osaka University Graduate School of Medicine, Osaka, Japan.
C.J. Lieven1, L.A. Levin2. 1Ophthalmology and Visual Sciences, University of
Wisconsin, Madison, WI; 2Ophthalmology, Univ Montreal/Univ Wisconsin,
Montreal, QC, Canada.
Purpose: Recent evidence suggests that endoplasmic reticulum (ER) stress is activated
during light-induced retinal damage. A preferential inducer of 78 kDa glucoseregulated protein (GRP78)/immunoglobulin binding protein (BiP; Bip inducer X, BIX)
has the protective effect against ER stress-induced cell death. Here, we investgated
changes of ER stress-related factors in retina after the light exposure and effect of BIX
on light-induced retinal cell death in mice.
Methods: To examine the ER stress activation, we measured time-dependent changes of
ER stress-related factors in retina after light exposure using quantitative RT-PCR: The
expressions of GRP78/BiP, C/EBP-homologous (CHOP), calreticulin, ER degradation
enhancing α-mannosidase like protein (EDEM), asparagine synthetase (ASNS), 94
kDa glucose-regulated protein (GRP94), and 52 kDa repressor of the inhibitor of the
protein kinase (p58IPK) were measured. Retinal light damage was induced by exposure
to constant white fluorescent light for 3 h at an illumination of 8000 lux. To evaluate
the effect of BIX on retinal damages, a- and b-wave amplitudes of the dark-adapted
electroretinogram (ERG) were recorded at 5 days after light exposure, then outer
nuclear layer (ONL) thickness was measured on hematoxylin-eosin-stained sections.
BIX (5 nmol/eye) was intravitreously injected at 6 h before light exposure.
Results: RT-PCR revealed that all ER stress-related factors except ASNS were
increased after light exposure. The a- and b-wave amplitudes of the dark-adapted
ERG were reduced at 5 days after light exposure, and BIX treatment significantly
prevented the retinal dysfunctions as compared with those in vehicle-treated group.
In histological analysis, the light exposure reduced ONL thickness, and treatment
with BIX significantly inhibited the ONL atrophy as compared with that in vehicletreated group.
Conclusions: These data indicate that ER stress may play a pivotal role in light
exposure-induced retinal damage, and treatment with BIX may prevent the retinal
damage.
CR: T. Nakanishi, None; S. Imai, None; Y. Inokuchi, None; K. Tsuruma, None; M.
Shimazawa, None; Y. Monguchi, None; H. Sajiki, None; T. Kudo, None; H. Hara, None.
Support: None
Purpose: Light exposure is a risk factor for age-related macular degeneration and
retinitis pigmentosa. Exposure to intense light in animal models of these diseases
speeds the degenerative process, possibly mediated by increased oxidative stress.
We previously showed that redox modulation towards a reducing environment with
tris(2-carboxyethyl)phosphine (TCEP) rescues retinal ganglion cells from axonal
damage in vitro and in vivo. We hypothesized that a similar therapeutic mechanism
would be neuroprotective in a photic injury model of retinal degeneration.
Methods: Male Wistar rats 6 to 10 weeks of age were reared on a 12-hour light/12-hour
dark cycle. Rats were dark adapted for 16 hours, then administered TCEP (28.6 mg/
kg) or saline i.p. immediately prior to exposure to uniform, omnidirectional white
fluorescent light at 10 klux for 10 hrs. After exposure, rats were returned to housing for
5 days prior to sacrifice. Eyes were fixed, embedded in glycomethacrylate, sectioned
at 2 microns, and stained with toluidine blue. NIH ImageJ was used to measure the
thickness and area of the outer nuclear layer (ONL). Thickness measurements at
regular intervals were compared by ANOVA.
Results: Photic injury led to significant thinning of the inferior ONL (mean thickness
17.8 ± 2.9 vs. 34.3 ± 0.7 µm, p = 0.004), and nearly complete obliteration of the superior
ONL (3.1 ± 0.6 vs. 37.1 ± 0.8 µm, p < 0.001 ). Pre-treatment with TCEP decreased thinning
both inferiorly and superiorly (28.2 ± 4.3 and 8.0 ± 2.2 µm, respectively; p < 0.001).
Conclusions: The systemic administration of the disulfide reducing agent TCEP
decreases photoreceptor loss after photic injury. This is consistent with light-induced
oxidation of certain proteins containing reduced sulfhydryls serving as a critical
signal for photoreceptor death in the photic injury model. Disulfide formation in
these proteins may be novel therapeutic targets in eye diseases exacerbated by light.
CR: C.J. Lieven, None; L.A. Levin, Wisconsin Alumni Research Foundation, P.
Support: NIH EY R21EY017970, P30EY016665, Retina Research Foundation, Research
to Prevent Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2253-2256
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2257 - A437
Yellow Intraocular Lens Implantation Prevents Light-Induced Retinal Damage
in Mice
M. Ebinuma1, T. Kurihara1A, M. Omoto2,3, K. Noda1A,4, S. Kubota1A, Y. Ozawa1A, S.
Shimmura2,3, S. Ishida4,1A, K. Tsubota2. AOphthalmology, 1Laboratory of Retinal Cell
Biology, Tokyo, Japan; 2Ophthalmology, Keio University School of Medicine,
Tokyo, Japan; 3Ophthalmology, Laboratory of Corneal Cell Biology, Tokyo, Japan;
4
Ophthalmology, Hokkaido University Graduate School of Medicine, Sapporo,
Japan.
Purpose: Although the inhibitory effect of yellow filter on light-induced retinal damage
was already shown, there is no study reported on the effect of yellow intraocular lens
(IOL) implanted after lens extraction, which mimics human post operative status. After
establishing a novel model of IOL implantation in mice, we determined the effect of
yellow IOL in comparison with colorless IOL.
Methods: Extra-capsular crystalline lens extraction was performed to 6-week-old
BALB/c male mice. Ultraviolet (UV)-blocking colorless IOL or blue light- and UVabsorbing yellow IOL was implanted into lens capsule. IOL-implanted mice were
exposed to 5,000 lux white light for 24 hours. Retinal damage was evaluated by TUNEL
staining, outer nuclear layer (ONL) thickness measurement and electroretinography
(ERG).
Results: TUNEL-positive cells in yellow IOL-implanted mice were significantly (P <
0.05) reduced compared with colorless IOL-implanted mice. Compared to colorless
IOL, yellow IOL implantation led to significant (P < 0.05) reduction of ONL thickness
and ERG amplitude.
Conclusions: Yellow IOL implanted after lens extraction has protective effects against
light-induced retinal damage.
CR: M. Ebinuma, None; T. Kurihara, research materials, F; M. Omoto, research
materials, F; K. Noda, research materials, F; S. Kubota, None; Y. Ozawa, None; S.
Shimmura, None; S. Ishida, None; K. Tsubota, None.
Support: None
2258 - A438
Glucocorticoid Receptors in Light-Induced Retinal Degeneration
M.A. Cubilla, M.M. Castañeda, G.A. Luzzani, A.M. Suburo. Cell and Molecular
Medicine, Ciencias Biomedicas. Universidad Austral, Pilar, Argentina.
Purpose: Glucocorticoids (GCs) block photoreceptor cell death in inherited and lightinduced retinal degenerations. Steroids can exert potent antiapoptotic effects through
different pathways. However, GC-dependent survival pathways in the retina are still
controversial. Therefore we evaluated the effects of GC receptor (GR) inhibition on
light-induced photoreceptor degeneration and sought a correlation between these
effects and levels of the antiapoptotic protein Bcl-XL.
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.
They were divided in two groups: those returning to standard illumination, and
those exposed to 1,500 lux during 2-4 days. Animals from both groups received
mifepristone (MFP, a GR inhibitor) or dexamethasone (DEX) or saline. Photoreceptor
degeneration was evaluated by histology, cleaved caspase-3 (CC3) immunostaining and
opsin protein levels. GR and Bcl-XL was immunochemically detected in cryosections
and Western blots.
Results: In normal mice, GR-ir appeared in cell nuclei from all retinal layers.
Light-exposed retinas showed stronger immunostaining. After 2 days under
1,500 lux, retinas displayed shortening of photoreceptor outer segments and
decreased opsin. Damage after 4 days included loss of photoreceptor cell nuclei and
decrease of opsin below detection levels. Opsin decrease at 2 days was prevented
by DEX and increased by MFP administration. GR inhibition also increased the
number of CC3+ cell nuclei. Opsin levels were unchanged in animals remaining
under standard illumination, with or without DEX or MPF administration.
Animals exposed to 1,500 lux during 4 days and returned to standard
illumination for another 6 days showed partial recovery of opsin levels.
Opsin recovery did not occur in mice receiving MFP during light exposure.
Bcl-XL levels decreased after 2 days under 1,500 lux. This effect was prevented by
DEX. Administration of MFP determined a large reduction of Bcl-XL levels, even in
animals remaining under standard illumination.
Conclusions: Our observations indicate that MFP increased photoreceptor damage and
death after exposure to toxic light levels. GR-mediated activation of the antiapoptotic
molecule Bcl-XL would be an important component of photoreceptor response to light
injury. Retinas deficient in Bcl-XL might be abnormally sensitive to apoptotic stimuli.
CR: M.A. Cubilla, None; M.M. Castañeda, None; G.A. Luzzani, None; A.M. Suburo,
None.
Support: MINCYT PICT 21399/2004
2259 - A439
Measuring Antioxidant Protection in a Rat Model of Light Induced Retinal
Damage
2260 - A440
Müller Glia and Phagocytosis of Constant Intense Light-Damaged
Photoreceptors
D.T. Organisciak1, R.M. Darrow1, L.S. Barsalou1, J.C. Lang2. 1Petticrew Research
Laboratory, Department of Biochemistry & Molecular Biology, Wright State
University, Dayton, OH; 2Alcon Research, Ltd., Fort Worth, TX.
T.J. Bailey, S.L. Fossum, J.E. Montgomery, D.R. Hyde. Dept of Biological Sciences,
University of Notre Dame, Notre Dame, IN.
Purpose: Antioxidants are known to reduce the extent of light-induced retinal
degeneration in animal models, but comparing effectiveness is difficult. We treated
rats with a variety of natural or synthetic antioxidants, at various concentrations, to
determine their relative efficacy in preventing retinal light damage.
Methods: Sprague-Dawley rats were reared in darkness for 40 days and then treated
with intense green light (490-580nm; 1200 lux) for 4 hrs, beginning at 9:30 am. One hr
before light, rats were treated (1X IP) with: ascorbic acid, α-tocopherol, various forms
of rosemary from the plant Rosmarinus officinalis or dimethylthiourea (DMTU). Some
rats were given rosemary 1 hr after the start of light. Following light treatment, rats
were returned to darkness for 14 days. Photoreceptor cell loss was then determined
by measuring rhodopsin and retinal DNA in light exposed and unexposed rats. For
each dose of antioxidant, protective efficacy was calculated from the average recovery
of rhodopsin and DNA in experimental vs. control animals. The effects of light and
antioxidants on protein markers of oxidative stress, retinal heme oxygenase-1 (HO-1)
and carboxyethylpyrrole (CEP) lipid-protein adducts, were determined by Western
analysis.
Results: The concentration of antioxidant required to achieve 50% efficacy in light
damage was 250 and 200 mg/kg body weight for ascorbic acid and DMTU. Rosemary, in
an oil extract or powder form, provided the same level of protection with 2-10 fold lower
doses, α-tocopherol was ineffective. The active fractions of rosemary were 5-10% by
weight, respectively, and it was most effective when given before light onset. In retinal
extracts, rosemary and DMTU reduced the levels of immunoreactive HO-1 and CEP.
Conclusions: Comparisons of antioxidant efficacy, based on rhodopsin and DNA
recovery, provide a direct index of protection against light-induced retinal damage.
Rosemary is more effective than other natural or synthetic antioxidants in preventing
retinal light damage and appears to do so by reducing oxidative stress during light
exposure.
CR: D.T. Organisciak, Alcon Research, Ltd., F; R.M. Darrow, None; L.S. Barsalou,
None; J.C. Lang, Alcon Research, Ltd, E.
Support: Research Funding Alcon Ltd. Fort Worth TX and Ohio Lions Eye Research
Foundation.
Purpose: Intense constant light treatment of dark-adapted albino zebrafish results
in photoreceptor apoptosis. Subsequently, many, but not all Müller glia in the lightdamaged retina dedifferentiate and proliferate to produce retinal progenitor cells,
which continue to proliferate, migrate from the inner nuclear layer to the outer nuclear
layer and replace the lost photoreceptors. Although a robust regeneration response
is seen in zebrafish, only meager cell proliferation is found in damaged mammalian
retinas. The mechanism by which Müller glia are triggered to respond to damage is
not known. We tested whether engulfment of apoptotic cell bodies are required to
initiate the Müller glial proliferative response after injury.
Methods: Dark-adapted albino zebrafish were intravitreally injected with control saline,
that either contained or lacked a small molecule inhibitor of phagocytosis, L-serineO-phosphate (L-SOP) and were placed in constant intense light. Treated fish retinas
were analyzed for cell death, by TUNEL, and proliferation, by immunohistochemistry.
The ability of L-SOP to inhibit Müller glial cell proliferation was also compared for
potential side effects with metabotropic glutamate receptors using the agonist L-2amino-4-phosphonobutyrate (L-AP4), or antagonist L-2-amino-3-phophonopropanoic
acid (L-AP3).
Results: Müller glia endocytosed both TUNEL label and a rod-specific marker in light
damaged retinas. Engulfment of apoptotic photoreceptor cell bodies correlated with
Proliferating cell nuclear antigen (Pcna) expression in Müller glial cells. Injection of
L-SOP, L-AP4, or L-AP3 failed to neuroprotect the photoreceptors in light-damaged
retinas. L-SOP, but not L-AP4 or L-AP3, however, showed reduced numbers of Pcnapositive Müller glia in the light-damaged retina.
Conclusion: No evidence was found for the involvement of metabotropic glutamate
receptor signaling in retinal regeneration. The Müller glia proliferative response to
retinal damage is due, at least in part, to the engulfment of apoptotic cellular debris.
CR: T.J. Bailey, None; S.L. Fossum, None; J.E. Montgomery, None; D.R. Hyde, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2257-2260
Monday, May 3, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 2233 - 2263 / A413 - A443
284. Retinal Degenerative Diseases
Organizing Section: RC Contributing Section: GL
2261 - A441
Cumulative Light Distribution on the Human Retina Under Natural Viewing
Conditions
2262 - A442
Light-Induced Cone Photoreceptor Plasticity
Y. Li, Z. Wang, D. Huang, L. Luo, X. Xia, R. Wen. Bascom Palmer Eye Institute,
University of Miami, Miami, FL.
R.A. Bone1A, J.C. Gibert1A, J.T. Landrum1B. APhysics, BChemistry and Biochemistry,
1
Florida International University, Miami, FL.
Purpose: Since light has been implicated in the etiology of age-related macular
degeneration (AMD), the purpose of our study was to measure the cumulative light
distribution on the human retina over extended periods. Our hypothesis was that light
distributions would peak in the macula where AMD damage is most pronounced.
Methods: A head-mounted eye-tracker captured the subject’s field of view with a video
camera (60 frames/s), superimposed the gaze position, and continuously recorded pupil
size. Fifteen subjects, ages 18-65, participated. Five, who were familiar with the study,
were assigned to a control group; 10, who were naïve, formed a test group. In phase
1, subjects viewed a sequence of photographic images projected on a screen. In phase
2, they were seated before a computer monitor and allowed to browse the Internet,
and in phase 3, they moved freely around the lab and exterior of the building. As a
check, the control group was specifically instructed to gaze at bright features in the
field of view and, in a second test, at dark features. The rest of the participants were
allowed to gaze freely. Based on the subject’s gaze position within each movie frame,
and the corresponding pupil diameter, we calculated the cumulative light distribution
over 5 to 15 min periods on a ~20o(H)×14o(V) area of the retina centered on the fovea.
Results: Relative retinal light distributions maps were obtained for all 15 subjects. The
data were quantified by plotting the relative intensity of light versus retinal position.
For the control group, cumulative retinal light distributions peaked and dipped in
the fovea when the subjects gazed at bright or dark features respectively in the field
of view. The light distributions obtained from the test group in phase 2, but not in
phases 1 and 3, showed a tendency to peak in the macula.
Conclusions: At this stage of our investigation, we have not been able to confirm
our hypothesis that the macula receives more light than the periphery under general
viewing conditions. However, specific tasks, such as working in front of a computer
monitor, appear to expose the retina to a higher illuminance in the macular region,
and this could have implications as far as development of AMD is concerned.
CR: R.A. Bone, None; J.C. Gibert, None; J.T. Landrum, None.
Support: NIH Grant SC3GM083671
Purpose: It has been well documented that the length of rod outer segments (ROS) is
influenced by light history. Animals reared in total darkness have longer ROS, and
those in bright habitat light have shorter ROS. In the present work, we studied the
influence of habitat light on the length of cone outer segments (COS).
Methods: Adult balb/c mice were housed in 50 lux in-cage illumination on a 12:12
light:dark cycle. One group of animals were kept in total darkness for 10 days and
another group were kept in 400 lux (12:12 light:dark) for 10 days. Eyes were collected
and cut to obtain thick sections of 100 µm on a vibratome. COS were identified by
immunostaining with antibodies against either red/green opsin, or blue opsin, and
examined by confocal microscopy.
Results: COS lengths in control animals are 14.96± 1.29 µm (blue cones, mean±SD,
n=90) and 15.15±1.85 µm (red/green cones, n=87). Significant increases in COS length
in both blue cones (29.1%, P<0.001) and red/green cones (25.4%, P<0.001) are found in
retinas from animals after 10 days in total darkness, compared with controls in 50 lux
habitat light. On the other hand, exposure to 400 lux habitat light for 10 days induced
significant decreases in COS length in blue cones (34.7%, P<0.001) and in red/green
cones (37.6%, P<0.001), compared with controls.
Conclusions: Our data demonstrate that the length of cone outer segments is regulated
by light history in the same way as rod outer segments. In a separate study, we found
that CNTF also regulates the length of cone outer segments. Together, these data
suggest a same underlying mechanism that mediates light-induced cone plasticity
and CNTF-induced changes in cone outer segments
CR: Y. Li, None; Z. Wang, None; D. Huang, None; L. Luo, None; X. Xia, None; R.
Wen, None.
Support: NIH grant EY-018586, JEK grant 08KN-09, Hope for Vision, Foundation
fighting blindness, NIH center grant P30-EY014801, and RPB.
2263 - A443
670 nm Photobiomodulation Attenuates Light-Induced Retinal Degeneration
J.T. Eells1, S. Gopalakrishnan1, B. Abroe1, R. Albarracin2, K. Valter-Kocsi2. 1Health
Sciences, Univ of Wisconsin - Milwaukee, Milwaukee, WI; 2ARC Center for
Excellence in Vision Science, Australian National University, Canberra, Australia.
Purpose: Irradiation by light in the far-red to near-infrared (NIR) region of the
spectrum (photobiomodulation; PBM) has been demonstrated to attenuate the severity
of degenerative retinal diseases in experimental and clinical studies. The cytoprotective
actions of NIR-PBM have been attributed to improved mitochondrial energy production
and the upregulation of cytoprotective factors. The purpose of this study was to
test the hypothesis that a brief course of 670 nm photobiomodulation would protect
against the loss of retinal function, prevent mitochondrial dysfunction and attenuate
photoreceptor loss in the light damaged retina.
Methods: The eyes of adult female Sprague Dawley rats (250 g) were treated with 670
nm light for 90 sec at 50 mW/cm 2 (fluence = 4.5 joules/cm2 ) using a light emitting diode
array (Quantum Devices, Inc). Rats were treated once per day for 5 days prior to light
damage [LD] (1500 lux for 24 h). Sham-treated LD rats were restrained for 90 sec, but
not exposed to 670 nm PBM. At day 7 following LD, retinal function was examined
by ERG. At day 14, eyes were enucleated. One eye was prepared for histological
evaluation and the retina from the other eye was removed, snap frozen and stored at
-80 C for future analysis of mitochondrial function and antioxidant concentrations.
Results: 670 nm photobiomodulation protected against LD-induced loss of rod and
cone function. In sham-treated LD animals rod-mediated b-wave amplitude was 320
+ 78 μV compared to 450 + 65 μV in NIR-treated LD rats. The rod-mediated b/a ratio
was 14 + 4 in sham-treated LD animals and 22 + 6 in NIR-treated LD animals (p <
0.05). The cone-mediated b-wave in LD rats was 103 + 9 μV compared to 165 + 25 μV
(p < 0.05) in NIR-treated LD rats.
Conclusions: Previous studies in our laboratory have demonstrated neuroprotective
and retinoprotective actions of 670 nm PBM light treatment in vitro and in vivo. The
present study extends these findings to include protection against light-damage
induced photoreceptor dysfunction. Given the reliance of photoreceptors on
mitochondrial oxidative metabolism and the evidence for LD-induced mitochondrial
injury, we speculate that the observed retinoprotective actions of NIR PBM are due, in
part, to the prevention of photoreceptor mitochondrial dysfunction and the induction
of mitochondrial repair in retinal light damage.
CR: J.T. Eells, None; S. Gopalakrishnan, None; B. Abroe, None; R. Albarracin,
None; K. Valter-Kocsi, None.
Support: Foundation Fighting Blindness TA-NP-0709-0465-UWI and the Australian
Research Council CE0561903
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2261-2263
Tuesday, May 4, 8:30 AM - 10:15 AM Floridian A Paper Session Program Number Range: 2489 - 2495
305. Cellular and Molecular Basis of Retinal Diseases
Organizing Section: RC
2489 - 8:30AM
Loss of Cell-Cell Contact Initiates Epithelial-Mesenchymal Transition and
Proliferation of Retinal Pigment Epithelial (RPE) Cells
2490 - 8:45AM
Modeling an Inherited RPE Disorder With Human Induced Pluripotent Stem
Cells
S. Tamiya1A,1B, L. Liu1A, D.C. Dean1A, H.J. Kaplan1A. AOphthalmology & Visual Sciences,
B
Biochemistry and Molecular Biology, 1University of Louisville, Louisville, KY.
D.M. Gamm1A, S. Howden1B, L.S. Wright1C, B.R. Pattnaik1D, A. Verhoeven1C, E.E.
Capowski1C, R.L. Shearer1C, J. Thomson1B, J.S. Meyer1C. AOphthalmology and Visual
Sciences, Waisman Center, Eye Research Institute, BMorgridge Institute, CWaisman
Center, DPediatrics, Ophthalmology and Visual Sciences, 1Univ of Wisconsin,
Madison, WI.
Purpose: Epithelial-to-mesenchymal transition (EMT) and proliferation of RPE cells
has been implicated to play a role in the development of proliferative vitreoretinopathy
(PVR). However, molecular mechanisms that initiate EMT remain elusive. Studies were
conducted to examine the role of cell-cell contact in regulating EMT and proliferation
of RPE cells.
Methods: Porcine RPE cells were isolated as sheets and cultured in vitro on lens capsule.
Cell morphology was examined by light and electron microscopy. Western blotting
and immunostaining were used to follow protein expression. Cell proliferation was
assessed by BrdU incorporation.
Results: RPE cells in the center of the sheet maintained cell-cell contact and retained
a differentiated phenotype. Disruption of cadherin function in these cells resulted in
loss of cell-cell contact and concomitant induction of mesenchymal marker protein
expression and cell proliferation. RPE cells at the edge of the sheet migrated away
from the sheet, underwent EMT and initiated proliferation. While TGF-beta is thought
to be a classic inducer of EMT, it was unable to initiate EMT in RPE cells maintaining
cell-cell contact. However, it induced a change to alpha-smooth muscle actin positive
myofibroblasts in cells that had already undergone EMT.
Conclusions: EMT and the onset of proliferation in RPE cells is initiated by loss of
cell-cell contact. TGF-beta cannot initiate EMT or proliferation of RPE cells maintaining
cell-cell contact but appears to play an important secondary role downstream of
EMT in inducing transition to a myofibroblast phenotype - a phenotype linked to
the development of fibrotic complications.
CR: S. Tamiya, None; L. Liu, None; D.C. Dean, None; H.J. Kaplan, None.
Support: University of Louisville Intramural Research Incentive Grant (ST); The
Kentucky Research Challenge Trust Fund (HJK); Research to Prevent Blindness Inc.;
The Kentucky Lions Eye Foundation
Purpose: The ability of human induced pluripotent stem cells (hiPSCs) to generate specific
retinal cell types has recently been established. One highly anticipated use of hiPSCs is as
a means to model cell autonomous disease processes. In this study, we sought to generate
and characterize a hiPSC line derived from a patient with an RPE-based inherited retinal
degenerative disease.
Methods: hiPSCs were differentiated towards a retinal fate using a previously described
method. Within 2 months of differentiation, uniform pigmentation was observed in a
significant fraction of hiPSC-derived neurospheres. Pigmented neurospheres were manually
isolated, plated on laminin and expanded in the presence of FGF2 and EGF. hiPSC-RPE
produced in this manner was compared to human embryonic stem (ES)- and fetal-derived
RPE using PCR, ICC and functional assays. To demonstrate the potential of hiPSCs to model
human disease, iPS cells were derived from a patient with gyrate atrophy, differentiated
into RPE and tested for ornithine aminotransferase (OAT) activity.
Results: hiPSCs were differentiated towards a retinal fate using a previously described
method. Within 2 months of differentiation, uniform pigmentation was observed in a
significant fraction of hiPSC-derived neurospheres. Pigmented neurospheres were manually
isolated, plated on laminin and expanded in the presence of FGF2 and EGF. hiPSC-RPE
produced in this manner was compared to human embryonic stem (ES)- and fetal-derived
RPE using PCR, ICC and functional assays. To demonstrate the potential of hiPSCs to model
human disease, iPS cells were derived from a patient with gyrate atrophy, differentiated
into RPE and tested for ornithine aminotransferase (OAT) activity.
Conclusions: This study confirms that RPE derived from hiPSCs is similar to that derived
from human ES and prenatal sources. Furthermore, patient-specific hiPSCs can be used to
produce retinal cell types that retain disease-causing functional defects. As such, hiPSCs
should prove useful for studying the pathophysiology of some human retinal diseases, as
well as for screening small molecules for therapeutic effects.
CR: D.M. Gamm, None; S. Howden, None; L.S. Wright, None; B.R. Pattnaik, None; A.
Verhoeven, None; E.E. Capowski, None; R.L. Shearer, None; J. Thomson, None; J.S. Meyer,
None.
Support: FFB Wynn-Gund Research Acceleration Award, Lincy Foundation, RPB McCormick
Scholar Award, Walsh Foundation, Retina Research Foundation, Heckrodt Foundation, and
NICHD P30 HD03352
2491 - 9:00AM
Consequences of Defective Outer Segment Morphogenesis for Rod
Photoreceptor Gene Expression
2492 - 9:15AM
TLR4-Dependent, TNF-α Mediated Oxidative Stress/Mitochondrial DNA
Damage of Photoreceptor as an Innate Immune Response
Y.V. Sharma1, R. Cojocaru2, M. Brooks2, A. Scott2, A. Swaroop2, A.F. Goldberg1. 1Eye
Research Institute, Oakland University, Rochester Hills, MI; 2N-NRL, National Eye
Institute, Bethesda, MD.
M.K. Ko, N.A. Rao. Opthalmic Pathology, Doheny Eye Institute, Los Angeles, CA.
Purpose: Inherited defects in retinal photoreceptor outer segment (OS) structure
impair function, disrupt relationship with the retinal pigment epithelium (RPE),
compromise cell viability, and result in a wide variety of progressive retinal
degenerative diseases. We are investigating gene expression in the retinal degeneration
slow (rds; aka prph2) mouse model; rds lacks peripherin/rds, and is unable to elaborate
photoreceptor OSs. We utilized a dissociated retinal preparation, in conjunction with
microarray screening, to assay how gene expression in rod photoreceptors responds
to development in the absence of peripherin/rds and OSs.
Methods: Rods were labeled with GFP by crossing a transgenic line of Nrl-GFP mice
onto a congenic line of rds mice. Fluorescence activated cell sorting (FACS) was used
to enrich rod photoreceptors from dissociated retinas of rds and WT control mice,
prior to and after the stage at which OSs are normally elaborated (~P10). Purified
mRNA was subjected to 4-5 Affymetrix GeneChip hybridizations (mouse Genome
430 V2.0 chips). Statistical validation of the raw data was performed using Agilent’s
Genespring GX software, to select transcripts with a minimum average fold change
of >2 (p-value <0.05). Gene regulation networks were constructed using Ingenuity
pathway analysis, and further validations were done using Taqman analysis.
Results: The microarray data indicates that the rds defect causes large scale gene
expression changes at time points just before the onset of OS elaboration. Close to
700 transcripts show differential regulation at P9, in contrast to only 118 in case of P6.
Functional annotation clustering identified the gene networks most affected as the
ones related to: lipid transport and metabolism, disc morphogenesis, intraflagellar
transport, and tight junction signaling.
Conclusion: This study tracks the broad based response of rod photoreceptor gene
expression to the loss of peripherin/rds protein, and identifies major genetic networks
which are significantly perturbed. These data provide a framework for further
understanding the role of peripherin/rds in photoreceptor structure and viability.
CR: Y.V. Sharma, None; R. Cojocaru, None; M. Brooks, None; A. Scott, None; A.
Swaroop, None; A.F. Goldberg, None.
Support: E. Matilda Ziegler Foundation for the Blind
Purpose: Our previous studies showed oxidative stress in the photoreceptor
mitochondria during the early phase of experimental autoimmune uveitis even
before the infiltration of inflammatory cells. Moreover, complete Freund’s adjuvant
(CFA) alone, injected subcutaneously, induced the innate immune response of TLR4
expression in the retina. Herein, we investigated whether TLR4 activation causes
retinal photoreceptor oxidative stress as an innate immune response.
Methods: To screen the differential gene regulation profiles in oxidative stress and
apoptosis, we used PCR super-array in the retina of B10.RIII mice injected with CFA
compared to control groups on day 5 post-injection (p.i). CFA-mediated TLR4 activation,
oxidative stress, mitochondrial DNA (mtDNA) damage, and apoptosis were determined
in MyD88, TLR4, TNF-α, and caspase 7 knockout (KO) mice using quantitative real-time
PCR (qPCR), ELISA, Western blot analysis, and immunohistochemistry.
Results: Oxidative stress-related and apoptosis genes were up-regulated in retinas
of CFA-injected mice compared to controls on day 5 p.i without any inflammatory
cell infiltration in retina and uvea. mtDNA damage and oxidative stress, confirmed
by qPCR and 8-OHdG immunostaining, were shown especially in inner segments
of photoreceptors of CFA-injected B10.RIII; however, there was no damage in MyD88
KO mice. Along with up-regulation of oxidative stress related genes, TLR4-mediated
TNF-α expression, shown in the retina and serum, was co-localized with TNFR1
and 8-OHdG; however, CFA-mediated iNOS was significantly reduced in TNF-α KO
mice. There were CFA-mediated induction of caspase 7 and caspase 8 and further
activation of caspase 3 was attenuated by caspase 8 inhibitor in vivo and significantly
blocked in caspase 7 KO mice.
Conclusions: TLR4-mediated innate immune response, mainly via TNF-α, resulted in
oxidative stress and apoptotic phenomenon in the photoreceptor, in which depended
on MyD88-mediated pathway but not depended in adaptive immunity. Among upregulated apoptotic genes, caspase 7 may be a major executioner in the induction of
mitochondrial pathway-mediated apoptosis.
CR: M.K. Ko, None; N.A. Rao, None.
Support: NIH Grant EY019506 and EY 017347
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2489-2492
Tuesday, May 4, 8:30 AM - 10:15 AM Floridian A Paper Session Program Number Range: 2489 - 2495
305. Cellular and Molecular Basis of Retinal Diseases
Organizing Section: RC
2493 - 9:30AM
Light Prevents Exogenous 11-cis Retinal From Maintaining Cone
Photoreceptors in Chromophore-Deficient Mice
2494 - 9:45AM
Subcellular Localization of Usher Syndrome Proteins in the Human Retina
J. Fan1A, R.K. Crouch1B, M. Kono1B. AOphthalmology-Storm Eye Inst, BOphthalmology,
1
Medical Univ of South Carolina, Charleston, SC.
Studies have shown that cones degenerate in chromophore-deficient mouse models
for Leber Congenital Amaurosis (LCA), but exogenous supplementation of the native
11-cis retinal chromophore can inhibit this degeneration, suggesting that 11-cis retinal
could be used as a therapeutic agent for preserving functional cones in patients with
LCA. However, the treated mice were maintained in the dark.
Purpose: To determine the effect of light/dark cycles on the cones of 11-cis retinaltreated Rho -/-Rpe65 -/- mice.
Methods: 11-cis Retinal was introduced into Rho -/-Rpe65 -/- mice at P10 as a single
subcutaneous injection mixed with Matrigel. The mice were maintained in either
normal cyclic light/dark or constant dark conditions. Cone function was determined
by electroretinography (ERG), and morphology assessed by fluorescence microscopy.
Cross sections at P21 were used to visualize cone opsin localization, and flat-mounted
retinas used to determine the number of cone opsin-containing cells at P25 and
P30.
Results: As expected, the 11-cis retinal-treated mice raised in constant dark shows
improved cone ERG signals, improved cone opsin localization and increased cone
photoreceptor cell number when compared with untreated mice. However, 11-cis
retinal-treated mice raised in cyclic light did not show any of the improvements seen
with the dark-reared mice.
Conclusion: Thus, 11-cis retinal by itself will not be a good therapeutic candidate for
preserving cones in LCA due to the need for total darkness following treatment.
CR: J. Fan, None; R.K. Crouch, None; M. Kono, None.
Support: NIH Grants R01-EY019515 (MK) and R01-EY004939 (RKC); Research to
Prevent Blindness (RKC and MUSC)
U. Wolfrum1, T. Goldmann1, N. Overlack1, C. Mueller2, J.M. Vetter2, K. Nagel-Wolfrum1.
1
Inst of Zoology, Cell & Matrix Biology, Johannes Gutenberg Univ of Mainz,
Mainz, Germany; 2Dept. of Ophtalmology, University Med Center Mainz, Mainz,
Germany.
Purpose: The human Usher syndrome (USH) is the most frequent cause of inherited
combined deaf-blindness. It is assigned to three clinical types and 12 genetically
heterogeneous subtypes. USH is characterized by profound inner ear defects and
Retinitis pigmentosa. In contrast to USH patients, USH rodent models undergo, if at all, a
very mild retinal degeneration. So far, there is no explanation for this difference in the
retinal phenotype. One possible explanation is that primates and rodent photoreceptor
cells differ in structure and in the subcellular distribution of individual USH proteins.
Here, we tested the latter hypothesis by analyzing the subcellular localization of
USH1/2 molecules in human retinas.
Methods: We assessed the expression of USH1/2 proteins by Western blots and
by immunohistochemistry on cryosections through human retinas. Furthermore,
subcellular localization was analyzed using a combination of high resolution
immunofluorescence and immunoelectron microscopy.
Results: All USH1/2 proteins are expressed in photoreceptor cells of human retinas.
As shown in rodents, USH1/2 proteins are associated with the ciliary region or
localize to the outer segment of human photoreceptors. However, staining intensities
of USH proteins significantly differed between rods and cones. In addition, a subset
of USH proteins were identified in calycal processes which are prominent features
of human photoreceptors.
Conclusions: There are significant differences between rodents and humans in the
expression of USH1/2 proteins in retinal photoreceptors which can explain the lack
of retinal USH phenotype in mice. In human defects in USH molecules may lead to
disorganization of the USH protein network in calycal processes and thereby to the
destabilization of photoreceptor outer segments.
CR: U. Wolfrum, None; T. Goldmann, None; N. Overlack, None; C. Mueller,
None; J.M. Vetter, None; K. Nagel-Wolfrum, None.
Support: FAUN, Fofö University of Mainz, Foundation Fighting Blindness
2495 - 10:00AM
Elongation of Very Long Chain Fatty Acids by Elovl4
S. Logan1, M.-P.G. Agbaga2,3, R.S. Brush2,3, A.O. Edwards4, R.E. Anderson2A,3. 1Cell
Biology, University of Oklahoma HSC, Oklahoma City, OK; AOphthalmology/
Cell Biology, 2Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK; 3Opthalmology,
Dean A. McGee Eye Institute, Oklahoma City, OK; 4Institute of Molecular Biology,
University of Oregon, Eugene, OR.
Purpose: Mutations in the ELOVL4 gene lead to autosomal dominant Stargardtlike macular dystrophy (STGD3). These frame-shift mutations result in premature
termination of ELOVL4 protein, loss of its carboxy-terminal endoplasmic reticulum
(ER) retention motif, and subsequent mislocalization. Whether the nonsense mutation
(5 bp deletion) contributes to disease progression by altering the enzymatic activity of
ELOVL4 is unknown and thereby limits the development of therapeutic interventions.
The purpose of this study is to establish the condensation activity of wild type ELOVL4,
which is the step specifically catalyzed by ELOVL4 during fatty acid elongation, and
determine if mutant ELOVL4 retains this activity.
Methods: Wild type (WT), STGD3 mutant, and a fusion construct containing the
5 bp deletion and the WT ER localization signal were N-terminally tagged with
hemagglutinin (HA) and expressed in HEK293 cells. Cellular expression and
localization were confirmed by immunohistochemistry and Western blotting. In vitro
elongation assay was performed by supplementing transfected cells with 20:5n3 or
34:5n3. Following treatment, lipids were converted to fatty acid methyl esters (FAMES)
and analyzed by gas chromatography-mass spectrometry (GC-MS). Microsomal
fractions from cells over-expressing ELOVL4 were isolated and elongase activity
was assayed in the presence of NADPH/NADH, 26:0-CoA, 34:5n3-CoA, and 2[14C]malonyl-CoA. Omitting NADPH/NADH from the reaction revealed condensation
activity alone. FAMES were generated and separated by C-18 reverse phase HP-TLC,
and incorporated radioactivity was visualized by autoradiography.
Results: Wild-type ELOVL4 localized to the ER while mutant protein was aggregated
and mislocalized to other cellular compartments. Addition of the WT ER localization
signal in the mutant protein resulted in localization of the mutant to the ER. Wild type
ELOVL4 showed elongation of 20:5n3 and 34:5n3 in culture, whereas no elongation
was seen in cells expressing either STGD3 or ER fusion of the mutant. In microsomal
reactions, wild type ELOVL4 mediated condensation and elongation of 26:0 and
34:5n3.
Conclusions: ELOVL4 is involved in the condensation reaction in very long chain
fatty acid biosynthesis. Further experiments determining mutant activity is crucial
to gain a better understanding of its role in Stargardt3 pathogenesis.
CR: S. Logan, None; M.-P.G. Agbaga, None; R.S. Brush, None; A.O. Edwards,
None; R.E. Anderson, None.
Support: NIH Grants EY014467, EY00871, EY04149, EY12190, and RR17703; Foundation
Fighting Blindness and Research to Prevent Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2493-2495
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2637 - A444
In vitro Characterization of Retina-Committed Bone Marrow-Derived
Progenitor Cells
2638 - A445
Priming Bone Marrow-Derived CD34+ Cells With the Matricellular Protein
Cysteine-Rich Protein 61 (Cyr61/CCN1) Regulates Their Commitment to the
Endothelial Cell Lineage Through the Integrin and Wnt Signaling Pathways
S.G. Lecaude, R.S. Zulliger, S. Wolf, V. Enzmann. Department of Ophthalmology,
Inselspital, University of Bern, Bern, Switzerland.
Purpose: The bone marrow contains hematopoietic stem cells but also other stem
and progenitor cell types that are not committed to the hematopoietic lineage. The
purpose of this study is to isolate and characterize bone marrow-derived progenitor
cells and to determine their differentiation potential towards retinal pigment epithelial
(RPE) cells.
Methods: Bone marrow was harvested from tibias and femurs of three weeks
old GFP C57BL/6 mice. First, mature hematopoietic cells were removed using a
cocktail of lineage antibodies coupled with paramagnetic beads (MACSTM) or by flow
cytometry (FACS). Second, the cell populations of interest were separated by FACS.
These cells were then grown in direct coculture with low passage murine RPE cells
for 10 days. Expression profile of the progenitor cells in co-culture was assessed by
immunocytochemistry and after additional FACS separation by real time qPCR.
Results: Lineage depletion of the bone marrow performed by FACS and MACS
yielded similar results with a lineage negative population representing 1.45±0.07% and
2.81±0.02% of the bone marrow population, respectively. Therefore the MACSTM system
was adopted as the method of choice. After 10 days of co-culture the GFP+ progenitor
cells adopted an RPE-like elongated morphology and immunocytochemistry showed
expression of RPE markers such as RPE65 and MITF. The number of GFP+ cells
harvested by FACS after co-culture ranged from 2.56±3.39% to 71.28±0.28% of the
input cell number. The yield was depending on the progenitor cell type, its attachment
properties to the culture dish and proliferation or lack thereof. RTqPCR analysis
showed the expression of RPE markers (RPE65 and MITF) and neuronal markers
(GFAP, TUBB3, MAP2).
Conclusions: The progenitor cells isolated from the bone marrow showed the
expression of RPE markers and RPE-like morphology after co-culture with RPE
cells. Further studies are pivotal to assess the functional properties of these progenitor
cells pre-differentiated in vitro. These cells might be of interest for therapeutic use in
degenerative pathologies such as AMD.
CR: S.G. Lecaude, None; R.S. Zulliger, None; S. Wolf, None; V. Enzmann, None.
Support: SNF grant 310000-119894 and Velux Foundation Grant
B. Chaqour1A, M.B. Grant2, A. Hasan1A, H. Liu1A, D. Lazarro1B. ACell Biology,
B
Ophtalmology, 1SUNY Downstate Medical Center, Brooklyn, NY; 2Pharmacology
and Therapeutics, University of Florida, Gainesville, FL.
Purpose: Development and remodeling of retinal blood vessels involves, at least in
part, the recruitment and commitment of progenitor stem cells to vascular cell lineage.
This complex process is imperfectly recapitulated in ischemic retinal diseases. The
Cysteine-rich protein 61 (Cyr61/CCN1), is an inducible extracellular matrix protein with
the potential of coordinating the execution of multiple angiogenic and vasculogenic
programs involving vascular progenitor cells. This study examined the effects of Cyr61
on the differentiation of hematopoietic stem cells (HSCs) to endothelial cells.
Methods: The ability of recombinant (r) Cyr61 protein to induce cell adhesion,
migration and differentiation of HSCs was examined using multiple in vitro assays.
The functional significance of various integrin chain subtypes and their signaling
pathways was examined using specific pharmacological inhibitors, neutralizing
antibodies and pathway-specific microarrays.
Results: Exposure of HSCs to rCyr61 induced their adhesion, migration, capillarylike structure formation and expression of specific endothelial cell markers. Cyr61
activated Pyk2, a focal adhesion kinase linked to integrin activation. Cyr61-induced
cell adhesion was dependent on α6β1 while cell migration involved both α4 β1 and α6β1
integrin chains. Furthermore, Cyr61 activated the Wnt signaling cascade pathway as
demonstrated by dephosphorylation of glycogen synthase kinase-3β and differential
expression of multiple components of the Wnt pathway including Wnt proteins, their
receptors and downstream targets such as Myc, fibronectin and cyclin D2. Inhibition
of Wnt signaling reduced both cell adhesion and capillary-like structure formation
upon cell exposure to rCyr61.
Conclusions: Priming with rCyr61 regulates HSC lineage commitment through
stimulation of their vasculogenic potential , suggesting possible therapeutic utility
of rCyr61 in retinal vascular diseases.
CR: B. Chaqour, None; M.B. Grant, None; A. Hasan, None; H. Liu, None; D. Lazarro,
None.
Support: 1R21EY019387-01A1
2639 - A446
Hematopoietic Stem Cell Integration and Differentiation Within the Murine
Retina
2640 - A447
Investigating Phenotypic Characteristics and Heterogenity of Pericytes Around
Retinal Vessels in Rodents
T. Asami, Y.-H. Chen, W. Dailey, L.Y. Ho, M. Cheng, K.A. Drenser. Associated Retinal
Consultants, Beaumont Eye Institute, Royal Oak, MI.
R. Funk1A, J. Jaszai1B, D. Corbeil1B, U. Schumann1A, D. Wittig1A. AInst of Anatomy,
B
Biotec, 1TU Dresden, Dresden, Germany.
Purpose: Several studies have shown that hematopoietic stem cells (HSC) have the
ability to differentiate into non-hematopoietic cell types. We describe our initial
experience in isolating a specific collection of HSCs called side population (SP) cells,
exposing them to various factors found in the Wnt-signaling pathway, to see if they
integrate and differentiate into cell types normally found within the murine retina.
Methods: Flow cytometry was used to isolate the SP cells from the bone marrow
of transgenic mice that express enhanced green fluorescent protein (EGFP) derived
from the C57BL/6 strain. These “green” SP cells were injected into the vitreous of
non-transgenic C57BL/6 mice alone as well as with various adjuncts including norrin
or wingless-type MMTV integration site family member 3a (Wnt-3a) protein in the
presence or absence of plasmin enzyme. The mice were then euthanized at various time
points, the eyes were enucleated, fixed, embedded in OCT, frozen sections are made
for immunofluorescent stains and evaluated by using fluorescent microscope.
Results: Fluorescent microscopy shows that SP cell integration into the ganglion
cell layer as well as inner nuclear layer of the murine retina is both feasible and
reproducible. Immunohistochemistry techniques show that the SP cells which
incorporate into the murine retina have neural progenitor cell markers. Mice receiving
injections with SP cells and (Norrin or Wnt3a) have a higher SP cell number in the
retina compared to those injected with SP cells alone. It also appears that Norrin
may play an important role in delaying SP cell degradation. Plasmin, when used as
an adjuvant to intravitreal injections, appears to allow for greater penetration of the
SP cells into the retina.
Conclusions: Our preliminary results show that it is possible to have SP cells integrate
and differentiate within the murine retina after intravitreal injection. In an attempt to
enhance this process, we plan future investigations in which we will make additional
injections of growth factors at periodic time points after SP cell injection. Further
research is necessary before stem cells can be used in the treatment for diseases that
cause severe retinal degeneration or vision loss.
CR: T. Asami, None; Y.-H. Chen, None; W. Dailey, None; L.Y. Ho, None; M. Cheng,
None; K.A. Drenser, None.
Support: None
Purpose: Mural (perivascular) cells, including pericytes help to maintain vessel
stability and have an inhibitory effect on endothelial cell proliferation. In different
organs the coverage of microvessels shows a significant difference. Retinal capillaries
have a greater pericytic coverage than capillaries in many other organs. In diabetic
retinopathy a significant decrease in the pericytic coverage has been reported.
Interestingly, isolated mural cells of microvessels are endowed with Mesenchymal
Stem Cell (MSC)-like traits capable of multilineage differentiation. Thus, MSC-like
pericytes around retinal vessels could potentially represent a source for tissue
regeneration. To establish a framework for future comparative and functional/
regeneration studies we investigated the phenotypic features, development/agerelated changes of pericytes and addressed their potential heterogeneity in situ on
retinal vessels in two rodent species.
Methods: Rat and murine retinae were investigated at the end of the second postnatal
week shortly after the release of the fusion of the eyelids and in young adult animals.
In whole mount preparations and horizontal cryosections of these samples pericytes
were identified based on the expression of NG2, a chondroitin sulfate proteoglycan.
In double staining experiments, the samples were co-stained with a panel known
stem/progenitor cell markers (e.g. CD146, Sox2) and were subsequently analyzed
by epifluorescence.
Results: Our results show a distinct distribution pattern of perivascular NG2-positive
cells around the retinal microvessels in postnatal and young adult rodents. Double
staining in rats also reveals a phenotypic heterogeneity of the cells in adult perivascular
cells based on expression of CD146. These data will be discussed.
Conclusions: Future studies are needed in order to assess the potential transdifferentiation capacity and enrolment in regenerative processes both in situ and on
isolated and transplanted pericytes.
CR: R. Funk, None; J. Jaszai, None; D. Corbeil, None; U. Schumann, None; D.
Wittig, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2637-2640
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2641 - A448
In vitro Parallel Plate Flow Chamber Assessment of Endothelial Progenitor Cell
Homing
2642 - A449
Generation of Retinal Pigment Epithelial Cells from Human Induced
Pluripotent Stem Cells
J.M. Barnett1,2, H.S. Toma1, G.W. McCollum1, J.S. Penn1,2. 1Ophthalmology and Visual
Sciences, Vanderbilt Eye Institute, Nashville, TN; 2Department of Pharmacology,
Vanderbilt University, Nashville, TN.
M. Lehmann1A, T.U. Krohne1A, D.F. Friedlander1A, A.L. Dorsey1A, W. Li1B, S. Ding1B, M.
Friedlander1A. ACell Biology, BChemistry, 1The Scripps Research Institute, La Jolla,
CA.
Purpose:Bone marrow-derived endothelial progenitor cells (EPCs) have been shown
to play a significant role in the neovascularization of vascular diseases. In many of
these diseases, including diabetic retinopathy and age-related macular degeneration,
elevated stromal derived factor-1 (SDF-1) is observed in the neovascular tissue, and this
is believed to play an important role in EPC homing function. Little is known about
other factors that might contribute. This study sought to develop an in vitro system of
evaluating EPC homing capacity in conditions relevant to ocular disease states.
Methods:Bone marrow was isolated from 4-6 week old Brown Norway rats and a
population of EPCs was identified using the markers CD34, CD133 and CXCR4 in
fluorescence-activated cell sorting experiments. EPCs were labeled with quantum
dot conjugated acetylated LDL. Labeled EPCs and mature endothelial cells (ECs)
were placed into a parallel plate flow chamber (PPFC) [Glycotech Corporation] in
separate experiments. In the first, cells were allowed to flow at 15 dynes/cm2 shear
stress over exposed extracellular matrix (ECM) and in the second, over conditioned
endothelial monolayer (EML). The ECM conditions tested were hyaluronic acid (HA) or
chondroitin sulfate (CS) with and without SDF-1. EMLs were conditioned by a 24-hour
pretreatment in either hypoxia (0% oxygen) or normoxia (20.9% oxygen).
Results:In the ECM studies, EPCs were found to be 3-fold (p<0.001) more adherent to
HA than CS, and this adherence was increased 67% (p<0.01) with the addition of SDF-1
to HA. EPC adherence to HA + SDF-1 was reduced by 56% (p<0.01) by incubating the
EPCs with anti-CXCR4 for 10 minutes prior to their addition to the PPFC. Compared
to the ECs, EPCs were 59% (p<0.01) more adherent to HA alone, and 2.6-fold (p<0.001)
more adherent to HA + SDF-1. In the EML studies, EPC adherence was increased by
33% (p<0.05) when EML were treated with hypoxia relative to normoxia.
Conclusions:The in vitro system has demonstrated adherence of EPCs and ECs to
surfaces chosen to model damaged blood vessel walls found in ocular neovascular
diseases. This high throughput system can now be used to further characterize homing
in more specific EPC subpopulations and to define the roles of EPCs in pathological
disorders of the eye.
CR: J.M. Barnett, None; H.S. Toma, None; G.W. McCollum, None; J.S. Penn,
None.
Support: EY07533, AG031036, EY08126 and a Challenge Award from Research to
Prevent Blindness
Purpose: The recent development of technologies capable of producing induced
pluripotent stem cells (IPS) from adult somatic tissues may permit their use to generate
autologous retinal pigment epithelium (RPE) grafts for the treatment of diseases like
age-related macular degeneration (AMD). Current reprogramming techniques require
retroviral transduction of four factors that include oncogenes such as c-Myc. Given
the associated risk of tumor formation, alternative technologies that eliminate the use
of these factors would be advantageous. We evaluated two and four factor-derived
human IPS for their capacity to differentiate into RPE cells.
Methods: IPS were generated from human fibroblasts (IMR-90) using standard four
factor lentiviral transduction (Oct4, Klf4, Sox2, c-Myc) or alternatively, from primary
human epidermal keratinocytes by transduction with only two factors (Oct4, Klf4)
and additional treatment with small molecules (CHIR99021, tranylcypromine).
Human embryonic stem cells (H1) were used for comparison. Cells were subjected
to a variety of different culture conditions, and RPE cell differentiation was assessed
by morphological, molecular, and functional parameters.
Results: RPE cells could be differentiated from four factor-derived IPS and embryonic
stem cells. Cells exhibited polygonal morphology and strong pigmentation, formed
epithelial monolayers with intracellular tight junctions (ZO-1 positive), and expressed
RPE-specific markers (RPE65, CRALBP, bestrophin). Moreover, cells phagocytosed
photoreceptor outer segments and demonstrated apical-to- basolateral fluid transport
in vitro. Cells were expanded over several passages and quickly regained RPE cell
morphology after reaching confluency. Two factor-derived human IPS were also
generated from somatic cells, differentiated into pigmented cells, and analyzed for
RPE characteristics.
Conclusions: We demonstrate the differentiation of somatic human cell-derived
IPS into cells with RPE-specific morphology and function. Further optimization of
differentiation efficiency is crucial for future therapeutic application of IPS-derived
RPE cells as autologous grafts in diseases like AMD.
CR: M. Lehmann, None; T.U. Krohne, None; D.F. Friedlander, None; A.L. Dorsey,
None; W. Li, None; S. Ding, None; M. Friedlander, None.
Support: Grants from the National Eye Institute ( EY11254) and the MacTel Foundation
to MF; TUK is supported by a fellowship from the German Research Foundation
(KR 2863/6-1).
2643 - A450
Evaluation of Human Y 79 Cell Lines for Putative Stem Cell Properties by
Single Cell Assay and Gene Expression
2644 - A451
Sirt1 Involvement in Mouse and Human Retinal Development and Function
M.S. Balla1A, G.K. Vemuganti1A, C. Kannabiran1B, S.G. Honavar1A. AKallam Anji Reddy
Campus, BKallam Anji Reddy Molecular Genetics Lab, 1LV Prasad Eye Institute,
Hyderabad, India.
Purpose: Cells from tumors like retinoblastoma are suspected to have two kinds of cell
populations, one being quiescent stem like cells, and another dividing cells. In order
to investigate and quantify the populations, we studied the human Rb cell line Y79 for
their clone forming ability, differential gene expression and cell cycle status.
Methods: Y79 cell line was maintained in RPMI with 10% FCS. Single cell assay was
performed to evaluate clone-forming ability. One million cells were stained and
analyzed for CD133 by Flow cytometry and sorted for CD133+/CD133 - populations.
These two subpopulations were then evaluated for cell cycle status by propidium
iodide labeling, and differential expression of putative stem/progenitor cell markers
ABCG2, PROX1 by RT-PCR.
Results: Clone forming ability was noted in 26.6±3.8% cells and CD133 expression in
79.7±1.3% of the cells. RT-PCR analysis of the CD133 - population showed the expression
of PROX1, which was not detected in the CD133+ cells. Majority of CD133 - population
(83.3±4.1%) were in G0/G1 phase, while CD133+ cells were predominantly (81.1±10.6%)
in S, G2/M phase as assessed by PI staining.
Conclusions: The Y79 cell line showed presence of cells with clone- forming ability
and differential expression of CD133, thus supporting the existence of putative stemlike cells. Expression of PROX1 and quiescence of CD133 - cells, further substantiate
this hypothesis.
CR: M.S. Balla, None; G.K. Vemuganti, None; C. Kannabiran, None; S.G. Honavar,
None.
Support: ICMR, C-TRACER, Hyderabad Eye Research Foundation, CSIR
S.C. Maloney1, E. Antecka1, T. Granner1, B.F. Fernandes1, B.A. Tucker2, M.E. Orellana1,
M. Eghtedari1, M. Young2, M.N. Burnier, Jr.1. 1Ophthalmology, McGill University,
Montreal, QC, Canada; 2Ophthalmology, Schepens Eye Research Institute, Boston,
MA.
Purpose: Recent evidence suggests that SIRT1 may play a significant role in development
of the central nervous system as well as during pathological neurodegeneration. The
purpose of this study was to investigate the expression of SIRT1 in developing,
adult and degenerating mouse and human retinas and to study the effects of SIRT1
knockdown on retinal development genes.
Methods: Immunostaining for SIRT1 was performed on the following tissues: two
post-natal day 1 (P1) mouse eyes, 14 normal adult B57BL6 mouse eyes (P41-P347), 20
Rhodopsin-knockout (RHO -/-) mouse eyes (P42-P336), two human fetal eyes (10 weeks
gestation), six normal adult human eyes from four donors, and peripheral retinal
sections from three human retinitis pigmentosa (RP) patients. Knockdown of SIRT1
via siRNA in mouse retinal progenitor cells (mRPCs) was performed followed by
PCR to evaluate changes in the following retinal development genes: Nestin, PAX6,
CRX, and Nrl.
Results: Fetal human, adult human, and adult mouse retinas all had cytoplasmic
staining for SIRT1 in all layers, with the exception of P176 mouse eyes, which were
negative in the ONL and photoreceptors. Additionally, P1 mouse eyes had two distinct
nuclear layers, with the inner layer staining positive and the outer layer negative. Two
of the three human RP cases showed staining in remaining retinal layers, while the
third patient showed staining only in the inner plexiform and ganglion cell layers.
RHO -/- mouse eyes demonstrated expression of SIRT1 in all remaining retinal layers
at all time points, with the exception of the P302 eyes which revealed a predominantly
negative inner nuclear layer with sparse positive cells. SIRT1 siRNA studies showed
significant decreases in CRX, PAX6 and Nestin in cells treated with SIRT1 siRNA
compared to controls. No significant changes were observed in Nrl expression.
Conclusions: Expression of SIRT1 in the developing and adult retinas, coupled with
siRNA results from mRPCs, suggests a probable role for this protein in normal retinal
development and function. The possible role of SIRT1 in retinal degeneration is less
clear and warrants further investigation.
CR: S.C. Maloney, None; E. Antecka, None; T. Granner, None; B.F. Fernandes,
None; B.A. Tucker, None; M.E. Orellana, None; M. Eghtedari, None; M. Young,
None; M.N. Burnier, Jr., None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2641-2644
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2645 - A452
Inhibition of Notch Pathway Enhances Photoreceptor Commitment From
Cultured Retinal Stem Cells
2646 - A453
Induced Pluripotent Stem Cells (iPSCs) Generate Both Early and Late Born
Retinal Neurons
P. Montavon, Y. Arsenijevic. UGTSCB, University of Lausanne, Lausanne,
Switzerland.
S. Parameswaran, S. Balasubramanian, N. Babai, W.B. Thoreson, I. Ahmad.
Ophthalmology, University of Nebraska Med Ctr, Omaha, NE.
Purpose: Consequently to the principle that photoreceptors have to be at a very
precise development stage to be successfully transplanted (MacLaren 2006), we are
trying to mimic this development stage in vitro using retinal stem cells. The latter
one isolated from the newborn mouse retina, derived from the radial glia population,
which were previously isolated and characterized in our laboratory. We developed
a protocol to commit these cells to the photoreceptor fate, but even if the percentage
of cells expressing photoreceptor markers is high (30%), the differentiation process
is incomplete so far (Merhi-Soussi 2006).
Methods: In order to ameliorate photoreceptor differentiation, we hypothesized
that the Notch pathway may interfere with this process by either promoting glia
commitment, or maintaining an undifferentiated state. We are thus using a gammasecretase inhibitor (DAPT), which inhibits Notch receptor cleavage and thus Notch
activation. DAPT was used either during the whole differentiation stimulation, or
only during a restricted period in two various retinal stem cell lines (RSC AA and
RSC MP1).
Results: RT-PCR performed during cell proliferation, showed the same positive
expression in both cell lines for the following genes: Math3, Six3, Hes1, NeuroD, Pax6 and
Notch1. Additionally, Mash1, Hes5, Prox1, Crx and Otx2 were detected in both cell lines
but with a stronger expression in RSC MP1. Opposite results were obtained for Chx10.
Nrl, Peripherin/RDS, GFAP and Math5 were detected neither in RSC AA, nor in RSC MP1.
The constant presence of DAPT i) leads to a 233% (RSC AA) or 900% (RSC MP1) increase
in peripherin/RDS-positive (photoreceptor marker) cells, compared to controls (no
DAPT, n=3, P<0.02) along with a 68% (RSC AA) or 80% (RSC MP1) decrease in GFAPpositive cells (n=3, P<0.04), ii) modifies the ratio between uni-/bi- (23%) and multi(77%) polar peripherin/RDS-positive cells to 45% and 55%, respectively, for both cell
lines and iii) reduces by 50% the total cell number during the whole differentiation
process for both cell lines.
Conclusions: We are now exploring whether this reduction in total cell number is
due to inhibition of cell proliferation or to cell death and whether photoreceptor
differentiation is promoted instead of glial induction. We also want to confirm the
results obtained with DAPT with RSCs isolated from Notch1-loxP mice. Such protocol
may help to better mimic photoreceptor development, but this needs to be confirmed
by genomic and proteomic profile analyses.
CR: P. Montavon, None; Y. Arsenijevic, None.
Support: Velux Foundation and Rotary Club
Purpose: The direct reprogramming of somatic cells to a pluripotent state holds
significant implications in treating intractable degenerative diseases by ex-vivo cell
therapy. These reprogrammed cells can also serve as a model for diseases and discovery
of drugs and genes. Here, we examined the depth of retinal potential of mouse iPSCs
to determine their usefulness in formulating stem cell approaches to understand and
treat retinal degenerative diseases.
Methods: Mouse fibroblast iPS cell line, iPS-MEF-Ng-20D-17 (Okita et al., 2007, Nature
448: 313-317) was subjected to previously described, neural differentiation protocol
for ES cells (Zhao et al., 2002, BBRC 297(2):177-84). Following neural induction, cells
were cultured in the presence of Noggin and FGF2 to enrich retinal progenitors. These
cells were cultured in simulated environments of early and late retinal histogenesis
to examine their potential to generate a range of retinal cell types on biochemical,
molecular and physiological criteria.
Results: The neural induction and expansion significantly altered the global gene
expression in iPSCs and caused an up-regulation of transcripts corresponding to eyefield genes. Neurally induced iPSCs when cultured in the E14 retinal cell condition
medium responded by down regulating retinal progenitor markers and activating
the expression of the regulators and markers of retinal ganglion cells (RGCs). The
iPSC-derived RGCs elaborated processes and interacted with cells in the explants of
superior colliculus, a target of RGCs in the retina. The cells when cultured with PN1
retinal cell CM responded by activating the expression of regulators and markers
of rod photoreceptors. When transplanted intravitreally in neonatal pups, a small
subset of these cells incorporated in the outer nuclear layer and expressed rhodopsin.
We also observed that induced iPSC possess the potential to differentiate along
cone photoreceptor lineage when exposed to the environment simulating early
histogenesis.
Conclusions: Mouse fibroblast iPSCs can generate a wide range of retinal cell types.
This depth of retinal potential suggests that they may support stem cell approaches
to understand and treat a wide range of degenerative retinal diseases, from glaucoma
to age-related macular degeneration.
CR: S. Parameswaran, None; S. Balasubramanian, None; N. Babai, None; W.B.
Thoreson, None; I. Ahmad, None.
Support: The Lincy Foundation, Pearsons Foundation, Nebraska Department of
Health and Human Services, and Research to Prevent Blindness.
2647 - A454
Jagged- and Wnt-Activated Muller Stem Cells Participate in Photoreceptor
Regeneration
2648 - A455
Adherent Cultivation of Neural Progenitors From the Adult Human Ciliary
Body and Peripheral Retina
C.B. Del Debbio, S. Bhattacharya, S. Balasubramanian, A. Chaudhuri, S. Parameswaran, I.
Ahmad. Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, NE.
E.O. Johnsen1, R.S. Kolberg1, G. Petrovski2, M.C. Moe1, B. Nicolaissen1. 1Centre of Eye
Research, Deptartment of Ophthalmology, Oslo University Hospital, University
of Oslo,, Oslo, Norway; 2Department of Ophthalmology and Biochemistry and
Molecular Biology, University of Debrecen, Debrecen, Hungary.
Purpose: Muller cells in the mammalian retina are latent stem cells, capable of
differentiating into early (ganglion cells) and late (rod photoreceptors) born retinal
neurons (Das et al., 2006, Dev. Biol. 299:283). This raises the prospect of Muller cell-based
regenerative repair of diseased retina. Here, we demonstrate a targeted activation of
Muller stem cells through Notch and Wnt pathways and that a small subset of activated
cells possess potential to differentiate along photoreceptor lineage
Methods: Activation of Muller stem cells and their regenerative capacity was examined
in two different experimental paradigms: (1) In controlled conditions that consisted of
retinal explants from wild type and rd1 mice (2) In S334ter rats where photoreceptor
degenerate due to termination mutation in opsin gene. Notch and Wnt pathways were
activated in Muller stem cells by Jagged1 peptide and Wnt3A, respectively. Activation
was monitored by proliferation, SP cell phenotype and progenitor properties of Muller
cells. Following 4 days of activation, BrdU or genetically-tagged Muller cells were
chased for another 4 days for their migration and differentiation along photoreceptor
lineage
Results: Treatment of the retina with Jagged1/Wnt3A/Jagged1+Wnt3A led to an
increase in the number of BrdU-positive cells expressing Muller cell markers. This
was accompanied by an increase in the expression of transcripts corresponding to
transducers of Notch and Wnt pathways and progenitors markers. The number of
Muller SP cells also increased in response to treatments. These changes were abrogated
in the presence of inhibitors of Notch and/or Wnt pathways. In the chase phase of the
experiments a subset of BrdU-tagged Muller cells were observed to have migrated
to the outer nuclear layer. A rare population of the migrated Muller cells expressed
immunoreactivities corresponding to rod photoreceptors in both experimental
paradigms. Hoechst dye efflux technique was used to confirm and corroborate the
differentiation of activated Muller cells along photoreceptor lineage
Conclusions: Our results demonstrate Notch and Wnt pathway-mediated activation of
Muller stem cells towards regenerative purposes in different models of photoreceptor
degeneration
CR: C.B. Del Debbio, None; S. Bhattacharya, None; S. Balasubramanian, None; A.
Chaudhuri, None; S. Parameswaran, None; I. Ahmad, None.
Support: The Lincy Foundation, Pearsons Foundation, Nebraska Department of
Health and Human Services, and Research to Prevent Blindness.
Purpose: Isolation and cultivation of progenitor cells from the adult human ciliary
epithelium (CE) and retina may have a future potential in cell-based therapy of retinal
diseases. Recently, the concept of CE as a niche for retinal stem cells in humans
has been challenged (Moe et al., 2009; Cicero et al., 2009). In the present study, we
investigated whether adherent cultivation could effectively expand neural progenitors
from the adult human CE and peripheral retina (PR), and their potential for neural
differentiation.
Methods: The PR and CE were dissected from adult post-mortem donor eyes after
approval by the Regional Committees for Medical and Health Research Ethics. The
tissue was dissociated into single cells and cultivated as 1) adherent cells with 1%
FCS + EGF/FGF or 2) as spheres with EGF/FGF and no serum. Differentiation was
performed with removal of growth factors and 3% FCS. Fixed cells were studied
by immunocytochemistry, quantitative RT-PCR, and scanning/transmission
electronmicroscopy.
Results: When cultivated under adherent conditions cells with an elongated
morphology from both the adult human PR and CE could be expanded for several
passages. Our preliminary data show that at P3, 54% of the PR and 74% of the CE
cells stained positive for nestin, a marker of neural progenitors. None of the PR cells
contained pigment, while 21% of the nestin-positive CE cells contained pigment. Both
adherently cultivated PR and CE cells could form secondary spheres after removal of
FCS. After induced differentiation, both PR and CE progenitors differentiated into
cells with neuronal properties.
Conclusions: Cells with properties of neural progenitors, isolated from both the adult
human peripheral retina and ciliary body epithelium, could be effectively isolated
and expanded using adherent cultivation.
CR: E.O. Johnsen, None; R.S. Kolberg, None; G. Petrovski, None; M.C. Moe, None; B.
Nicolaissen, None.
Support: The Norwegian Association of the Blind and Partially Sighted, the
Blindmission IL, the Research Council of Norway, Ullevål University Hospital and
Rikshospitalet University Hospital.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2645-2648
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2649 - A456
Molecular Characterization of Human Retinal Progenitor Cells During Long
Term Culture
2650 - A457
GDNF Augments Proliferation and Reduces Apoptosis of Murine Retinal
Progenitor Cells
J. Yang, P. Gu, H. Klassen. Gavin Herbert Eye Institute, Ophthalmology Department,
University of California, Irvine, Orange, CA.
J. Wang, J. Yang, H. Klassen. Gavin Herbert Eye Institute, Department of
Ophthalmology, University of California - Irvine, Orange, CA.
Purpose: Human retinal progenitor cells (hRPC) are a potential therapeutic agent
in the setting of retinal degeneration, however, supplies of these cells are restricted
by a limited capacity for growth in culture. We sought to extend the limits of hRPC
growth and provide a characterization of heavily passaged hRPCs, including karyotpe
analysis and molecular evidence of phenotypic potential.
Methods: Neural retinal tissue of 18 weeks GA was minced and enzymatically
digested. Cells were seeded into proliferation medium containing either DMEM:F12or Ultraculture-based medium, supplemented with N2, Glutamax, EGF (20 ng/ml)
and bFGF (20 ng/ml). Cells were passaged at 80-90% confluence every 4-6 days,
continuously, for 4 months. Total RNA was isolated on days 17, 22, 27, 38, 48, 72 and
99, and subjected to quantitative PCR analysis. At passage 6, cells were fixed with
4% PFA and immunolabeling performed. Karyotype and FISH were performed at
passage 19.
Results: Human RPCs were still proliferating after 4 months of continuous culture in
DMEM:F12-based medium. Expression of markers associated with progenitors (nestin,
vimentin, Ki-67, CD9, CD81, FUT4) as well as lineage (β3-tubulin, PKC-α), immune
function (β2-microglobulin, HLA-DPa1), development (c-myc, KLF4), apoptosis
(caspase1, caspase3, annexinV) and GDNF were up-regulated at progressive time
points. In contrast, Sox2, Six6, Notch1, Map2, recoverin, and GFAP were downregulated. Compared to human fibroblasts, Sox2 and Map2 were much more highly
expressed by hRPCs. Immunolabeling showed more than 95% of cells to be positive
for progenitor cell markers (nestin, vimentin), 50% for the proliferation marker Ki-67,
and 5% for GFAP. In addition, karyotype and FISH showed 86% of cells to be normal
and 14% tetraploid (8% aneuploid considered upper limit of normal) at passage 19.
Conclusions: hRPCs derived from the fetal retina can be expanded extensively,
although not indefinitely, in vitro. After long term growth in culture, the majority of
cells continue to express progenitor markers and retain a normal karyotype, as well
as the ability to express markers of neuronal and glial lineage in vitro.
CR: J. Yang, None; P. Gu, None; H. Klassen, None.
Support: Lincy Foundation, Discovery Eye Foundation
Purpose: Glial cell line-derived neurotrophic factor (GDNF) is neuroprotective in
the retina and retinal progenitor cells (RPCs) can deliver this cytokine, particularly
as a transgene product. While GDNF alone does not sustain RPC proliferation, it
may confer advantages when combined with epidermal growth factor (EGF), as we
investigate here.
Methods: RPCs, originally from neonatal GFP-mice, were assessed under different
conditions corresponding to the presence or absence of EGF (20 ng/ml) and/or GDNF
(10 ng/ml). Growth curves were calculated and sphere formation analyzed using an
IncuCyte live-cell imaging system. Apoptosis was assessed via caspase-3 activity
assay; marker expression via quantitative RT-PCR. Western blotting was quantified
by densitometry.
Results: RPCs exhibited exponential growth in either EGF or EGF+GDNF, with
the latter condition showing a measurable advantage. Live-cell monitoring of both
conditions revealed that initial aggregates formed as early as 5.5 hr from collision
and adherence of dissociated RPCs, as opposed to clonal proliferation. Spheres
subsequently enlarged in size and number in both conditions, with more reaching
threshold criteria for cross-sectional area in the EGF+GDNF condition. Caspase-3
activity levels were markedly higher in the absence of EGF (p < .05) and the no-growthfactor condition was higher than GDNF alone (p < .05). EGF and EGF+GDNF conditions
were equivalent. Expression of most progenitor-associated markers remained stable
following 5 days treatment, as did the majority of precursor and lineage-related
markers, although with EGF+GDNF there were marginal increases in Ki-67 (1.07
fold), hes5 (1.03 fold), mash1 (1.12 fold) and vimentin (1.20 fold). At the protein level,
Ki-67 was increased in EGF+GDNF by 1.19±0.0095 fold.
Conclusions: The addition of GDNF to EGF-based proliferation medium augments
the proliferation of RPCs in association with increased Ki-67 expression. GDNF
reduces RPCs apoptosis compared to complete growth factor withdrawal. These data
support the feasibility of generating GDNF-producing RPCs for use in therapeutic
models of retinal disease.
CR: J. Wang, None; J. Yang, None; H. Klassen, None.
Support: Lincy Foundation, Discovery Eye Foundation
2651 - A458
Vsx2 (Chx10) Expression Identifies a Retinal Progenitor With Neurogenic
Potential in Human Prenatal Eye Cultures
2652 - A459
RE-1 Silencing Transcription Factor (REST) as a Potential Target for RNAi in
Muller Glia Derived Retinal Stem Cells
L.S. Wright1A, I. Pinilla2, J.M. Clermont1A, J.S. Meyer1A, K.A. Wallace1A, E.E. Capowski1A,
D.M. Gamm1A,1B. AWaisman Center, BOphthalmology and Visual Sciences,
1
University of Wisconsin, Madison, WI; 2Ophthalmology, Hospital Univ Miguel
Servet, Zaragoza, Spain.
J. Phillips1A, D.C. Otteson1B. ACollege of Optometry, BOptometry, 1University of
Houston, Houston, TX.
Purpose: To examine a proliferating Vsx2+ progenitor population in primary human
prenatal retinal cultures and to determine the neurogenic potential of these cells.
Methods: Retinal progenitor cells were prepared from postmortem human prenatal
eyes at approximately 60 to125 days of gestation and cultured as neurospheres
in serum-free medium supplemented with mitogens. Cells were prepared for
immunocytochemical analysis, immunostained for markers of neural and retinal
proliferation and differentiation, and compared to cryosections from aged-matched
whole eyes.
Results: A population of Vsx2+ proliferating cells was present in primary neurosphere
cultures derived from human prenatal retina. These cells retained multiple
characteristics of retinal progenitor cells found in situ, including Ki67, Sox2, nestin
and Notch expression. Upon withdrawal of mitogens, there was an increase in Ascl1+
expression which co-localized with retinal progenitor markers. Treatment with the
Notch inhibitor, DAPT, increased Ascl1 expression and PKCα immuno-positive
cells, but did not alter the percentage of cells expressing recoverin. After 1 month in
culture, very few cells expressed either Vsx2 or Ascl1, and DAPT treatment no longer
demonstrated an effect on neuronal differentiation.
Conclusions: Under serum-free conditions, a population of Vsx2+ late retinal
progenitor cells can be identified and propagated for a limited time in vitro. The
presence of Vsx2 in retinal progenitor cells correlated with maintenance of neurogenic
potential regardless of the culture method employed. Furthermore, DAPT-induced
enhancement of neural differentiation did not occur following loss of Vsx2 expression
in long-term cultures. Thus, Vsx2 is useful for identifying proliferative progenitor
cells with neurogenic potential in human prenatal retina cultures.
CR: L.S. Wright, None; I. Pinilla, None; J.M. Clermont, None; J.S. Meyer, None; K.A.
Wallace, None; E.E. Capowski, None; D.M. Gamm, None.
Support: NIH Grant PD30HD03352, Foundation Fighting Blindness, Lincy Foundation,
Research to Prevent Blindness Foundation
Purpose: Despite evidence showing the neurogenic potential of Muller cells, only
a fraction of cells turn on neuronal genes. The repressor element (RE)-1 silencing
transcription factor (REST) represses neuronal genes and neuronal differentiation in
non-neuronal tissues. The purpose of this study is to examine Rest mRNA and protein
expression in cultured mouse Muller cells and the mouse retina, and to target Rest
mRNA for RNAi knockdown using an inducible shRNA lentiviral construct.
Methods: Quantitative RT-PCR was used to analyze Rest expression in embryonic (E17),
neonatal (P0) and adult mouse retinas and conditionally immortalized Muller cells
(ImM10) under conditions that promote growth, neurosphere formation and neuronal
differentiation. REST and Co-REST protein expression in ImM10 cells and adult retina
was analyzed by immunocytochemistry. For RNAi, 3 shRNAs targeted against Rest
and control inserts containing Gapdh shRNA or lacking a shRNA insert were subcloned
into a Tet-inducible, lentiviral construct with a red fluorescent protein (RFP) reporter.
Transduced Muller cells were puromycin selected (6μg/ml) and promoter activity was
induced by 1μg/ml doxycycline.
Results: Developing and mature retina and cultured Muller cells expressed Rest
mRNA. Expression was decreased in neurospheres, but returned to original levels in
differentiation conditions. REST antibody prominently labeled nuclei and filamentous
processes of Muller cells in culture and in the mature retina, where they also labeled
nuclei in the INL, fibers in the nerve fiber layer, and both plexiform layers. Monoclonal
Co-REST antibody labeled Muller cell nuclei in vitro and nuclei of Muller cells, rod
bipolar cells and horizontal cells in the mature retina, where perinuclear Co-REST
staining was also present throughout the ONL, particularly in cones. In transduced,
puromycin selected Muller glia, 100% of cells expressed the RFP reporter following
doxycycline induction, but the 3 shRNA constructs tested to date did not significantly
knockdown REST mRNA.
Conclusions: REST is expressed in Muller glia and expression persists under conditions
previously shown to upregulate neuronal gene expression in Muller-derived stem
cells. This suggests that cells maintaining a non-neuronal phenotype are also present
under current culture conditions. Ongoing studies are focused on developing effective
shRNAs to knock-down Rest mRNA using RNAi to promote increased neuronal
differentation of Muller glia derived stem cells.
CR: J. Phillips, None; D.C. Otteson, None.
Support: T32 EY007024, AOF Ezell Fellowship, P30 EY007551
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2649-2652
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2653 - A460
Negative Feedback Restricts Neural Cell Production in the Retinal-Ciliary
Margin of Adult Mouse Retina
2654 - A461
Ciliary Neurotrophic Factor Regulation of Gfap Expression in Mouse Muller
Glia-Derived Neurospheres
T. Kiyama, S.W. Wang. Ophthalmology and Visual Science, The University of Texas,
Houston, TX.
K.M. Beach1A, J. Phillips1B, D.C. Otteson1A. AOptometry, BCollege of Optometry,
1
University of Houston, Houston, TX.
Purpose: It is not clear whether a mammalian retina contains a proliferative region
similar to the ciliary margin zone (CMZ) of an aquatic vertebrate. Our study showed
that cells continue to proliferate at the retinal-ciliary margin in challenged mouse
retinas indicating a potential that cell cycle can be reactivated in the region. Therefore,
we hypothesize that the mammalian retinal-ciliary margin retains proliferative
potency and proliferation can be triggered by retinal shortages. Based on the paradigm
that a specific retinal cell type inhibits the formation of the same type, we followed
the response of the retinal-ciliary margin of mouse retnas with different degrees of
retinal ganglion cell (RGC) loss at different developmental and adult stages.
Methods: To test the hypothesis, we traced the response of retinal-ciliary margin in
a series of retinas with a spectrum of RGC loss. Cell proliferation, marked by BrdU
and phosphohistone-3, as well as the activity of math5, a gene essential for RGC
production, were examined at given different points.
Results: The math5 activity was prolonged to different degrees in the retinal-ciliary
margin corresponding to the severity of RGC loss. Briefly, the less there are the RGCs,
the longer the math5 is active in the retinal-ciliary margin. Also, we found that most
cells in the ciliary margin are proliferative up to one month old, the latest time point
we have examined.
Conclusion: Cells in the mouse retinal-ciliary margin retains the proliferative
potential. Such a potential can be activated by lifting inhibition suggesting RGCs
generate a negative feedback circuit for the production of new neurons.
CR: T. Kiyama, None; S.W. Wang, None.
Support: NEI Grant 1R01EY018352-01A1
Purpose: Muller glia are a source of stem cells in the mammalian retina. In vivo, ciliary
neurotrophic factor (CNTF) upregulates glial acidic fibrillary protein (Gfap) expression
in Muller glia via phosphorylation of STAT3. We examined STAT3 phosphorylation
and Gfap expression in mouse Muller-derived retinal stem cells following CNTF
treatment or fibroblast growth factor 2 (FGF2)-induced differentiation.
Methods: Neurospheres were generated from conditionally immortalized mouse
Muller cells (ImM10) by culturing in serum-free Neurobasal medium with epidermal
growth factor (EGF) + FGF2. STAT3 phosphorylation was assayed by Western blots
of total proteins from CNTF treated neurospheres or dissociated mouse retinal cells.
Gfap expression was assayed by quantitative RT-PCR in total RNA isolated from
neurospheres treated with CNTF (0, 2, 20, and 200 ng/ml, 24 hrs) or differentiated
neurospheres (primed with FGF2, 5 days, followed by growth factor withdrawal, 5
days). RT-PCR data was analyzed using Relative Expression Software Tool (REST).
Results: FGF2-induced differentiation of Muller glial-derived neurospheres resulted
in a 9.3-fold increase in Gfap mRNA expression (p=0.03) vs. neurospheres. CNTF did
not alter Gfap mRNA expression in neurospheres at any concentration tested (p=0.28,
0 vs. 200 ng/ml). Although CNTF increased phosphylation of STAT3 in dissociated
retinal cells by 3.6-fold, only a 1.31 increase in p-STAT3 was detected in CNTF treated
neurospheres.
Conclusions: The minimal changes in STAT3 phosphorylation and lack of significant
increase in Gfap expression in Muller-derived neurospheres treated with CNTF
was unexpected. These results suggest that Gfap upregulation during FGF2
induced differentation may result from activation of alternate signaling pathways.
Understanding the mechanisms regulating glial activation in Muller-derived stem
cells will be important in identifying strategies to block gliogenesis and promote
neurogenesis.
CR: K.M. Beach, None; J. Phillips, None; D.C. Otteson, None.
Support: NIH Grant T35 EY007088 (KB), NIH Grant T32 EY007024 (JP), UH New
Faculty Grant (DCO)
2655 - A462
Activation of Id Genes by BMP Signaling Influences Proliferation and Cell Fate
Choices of Retinal Progenitors
2656 - A463
Unfolded Protein Response in Mouse Stage-Specific Retinal Progenitor Cells
H.K. Yip, Y. Du. Anatomy, Univ of Hong Kong Fac of Med, Hong Kong SAR, China.
Purposes: Bone morphogenetic proteins (BMPs) are multi-functional growth factors,
and are well-known for regulating eye development in diverse aspects. It is also known
that BMP signaling is essential for embryonic development and cellular functions
such as cell growth, fate determination, differentiation and apoptosis. Inhibitors of
differentiation (Id) proteins have emerged recently as the critical targets of BMPs.
Id proteins are known to be important mediators of BMPs and the regulation of Id
protein by BMPs is mainly through a Smad-dependent pathway. Our previous work
showed that BMPs and BMPRs were co-expressed with Ids mainly in the ganglion
cells and amacrine cells in the inner nuclear layer (INL) of the adult retina, suggesting
a possible role for Id in inner retina development and in the terminal differentiation
and/or maintenance of INL interneurons and ganglion cells in the adult. In this study,
we used retinal progenitor cells (RPCs) from mouse embryos to study (1) the regulation
of Id expression by BMPs; and (2) the effects of BMPs on retinal cell differentiation.
Methods: Expression of Id1-3 gene and protein was examined by Q-PCR and western
blot respectively in retinal progenitor cell (RPC) culture prepared from E14.5 mouse
embryos in the presence or absence of mouse recombinant BMP-4. Activation of Smad
proteins was detected by western blot analysis in RPCs incubated with or without
noggin. Id promoter activity was measured by luciferase assay. In addition, the effect
of BMP-4 on the differentiation of RPCs was examined by immunohistochemistry.
Results: Significant increases of Id1-3 mRNA and protein expression levels were
observed in the RPCs after treatment with BMP-4, and BMP-4 also activated the Id1
promotor to drive luciferase expression in the RPCs. A decrease in PRC proliferation
accompanied these effects. Phosphorylation of Smad proteins was detected 30 min
after the addition of recombinant BMP-4. These responses were abolished when
cells were co-treated with noggin, a BMP antagonist. Immunoflurorescent staining
demonstrated the translocation of phospho-Smad1/5/8 into the nucleus of RPC upon
BMP-4 stimulation. Furthermore, BMP-4 promoted RPCs to differentiate into neuronal
lineage.
Conclusions: These results demonstrate for the first time a potentially new pathway
for regulating retinogenesis through the interactions between BMP-4, Ids and their
downstream signaling molecules.
CR: H.K. Yip, None; Y. Du, None.
Support: General Research Fund from Hong Kong Research Grant Council
(GRF10208603) and The University of Hong Kong Seed Funding Program for Basic
Research (200611159203)
L. Xu, T.-K. Ng, C.-P. Pang, H.-F. Yam. Ophthalmology and Visual Sciences, The
Chinese University of Hong Kong, Hong Kong, Hong Kong.
Purpose: To investigate the patterning of unfolded protein responses (UPR) in retinal
progenitor cells in mice at early or late embryonic (E) stages and compared with retinal
cells from postnatal (PN) stages.
Methods: Encleated eyes from Balb/C mice of age at E12, E18 and PN7 were dissected
to obtain retina. Dissociated single retinal cells were expanded in neural basal medium
containing N2 supplement, epidermal growth factor and basic fibroblast growth factor
for neurosphere formation. At day 7, neurospheres were collected, dissociated to
single cells and treated with 5 μg/ml tunicamycin for 2 hours to induce ER stress by
preventing global N-linked glycosylation. The expression of UPR markers, BiP/GRP78,
calreticulin, GADD153/CHOP and retinal differentiation marker, neuron-specific
enolase, was examined by western blotting and immunofluorescence, respectively.
Apoptosis rate was represented by percentage of TUNEL positive cells.
Results: Protein analyses showed that, in retina tissues, the level of BiP expression
induced in E12 cells was about 12-fold more than that in E18 and PN7 cells. Assay of
molecular chaperones, including calreticulin and protein disulfide isomerase, showed
similar changes. GADD153/CHOP level was also found higher in E12 versus E18 or
PN7 cells. E12 cells under tunicamycin treatment had the least induced apoptosis,
whereas PN7 cells had the highest apoptotic rate. In addition, most treated E12 cells
were positive for neuron-specific enolase. The level was reduced in E18 cells.
Conclusions: Early progenitor cells possessed a specific mechanism to protect against
ER stress. The upregulation of BiP and molecular chaperones helped the cells resolve
the ER stress problem and maintained the stem cell survival. Our result demonstrated
a differential UPR patterning in early embryonic progenitor cells when compared to
late progenitor cells. A different adaptive mechanism may be recruited to confront
with the ER overloading situation in cells of early embryonic stages.
CR: L. Xu, None; T.-K. Ng, None; C.-P. Pang, None; H.-F. Yam, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2653-2656
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2657 - A464
Induction of RPE Cells From Monkey iPS Cells
2658 - A465
Adherent Differentiation of Human Embryonic Stem Cells Promotes
Generation of Retinal Pigment Epithelial Cells
S. Okamoto, H. Kamao, M. Mandai, M. Takahashi. Laboratory for Retinal
Regeneration, Riken Center for Developmental Biology, Kobe, Japan.
Purpose: The induced pluripotent stem (iPS) cell is expected as a powerful tool for
research of regenerative medicine. Previously we reported that monkey embryonic
stem (ES) cell-derived retinal pigment epithelial (RPE) cells rescued photoreceptor
cells after transplantation into RCS rats (Haruta et al. IOVS, 2004). In this report we
produced iPS cell lines from monkeys to investigate their ability to differentiate
into retinal cells.
Methods: The fibroblasts derived from cynomolgus monkey abdominal skin were
infected with retroviruses carrying oct3/4, sox2, klf4 and c-myc genes. They were
cultured in ES cell maintaining medium on STO feeder cells. Next, the established
monkey iPS cells were induced to differentiate into RPE cells by culturing with the
supernatant of PA6, a mouse stromal cell line. The property of the differentiated RPE
cells were analyzed.
Results: After one month from retroviral infection, some colonies appeared among
the fibroblasts. The colonies were picked up and expanded as cell lines. These cell
lines showed monkey ES cell-like morphology and expressed ES cell markers such as
alkaline phosphatase, Nanog and SSEA-4. By making teratoma in SCID mice, these
cells were confirmed to have the ability to differentiate into three germ layers. In
addition, these iPS cells had capability of differentiating into retinal cells including
RPE cells with pigment and polygonal shapes. These cells expressed PRE cell-specific
markers and exhibited phagocytotic function in vitro.
Conclusions: We established iPS cells lines from monkey skin fibroblast. The RPE
cells derived from these monkey iPS cells can be used for autologous or allogenic
transplantation to evaluate the immune rejection and their function in vivo.
CR: S. Okamoto, None; H. Kamao, None; M. Mandai, None; M. Takahashi, None.
Support: The Project for Realization of Regenerative Medicine (MEXT)
2659 - A466
Derivation of Photoreceptor Precursors From Adult Human Müller Stem Cells
H. Jayaram1,2, S. Singhal1, B. Bhatia1, P.T. Khaw1,2, G.A. Limb1. 1Ocular Biology &
Therapeutics, UCL Institute of Ophthalmology, London, United Kingdom;
2
Moorfields Eye Hospital, London, United Kingdom.
H. Vaajasaari1, T. Ilmarinen1, H. Hongisto1, R. Suuronen1,2, H. Uusitalo3, H. Skottman1.
1
Regea - Inst. for Regenerative Medicine, University of Tampere, Tampere, Finland;
2
Tampere University Hospital, Department of Eye, Ear and Oral Diseases, Finland;
3
Medical School, SILK, University of Tampere, Tampere, Finland.
Purpose: Human embryonic stem cell (hESC) derived retinal pigment epithelial
(RPE) cells can provide a cell model for various retinal diseases or drug discovery and
essentially, a regenerative cure for vision-threatening diseases. The utilization of hESC
derived RPE cells for these purposes requires an efficient differentiation method. In
this study, the efficiency of the adherent differentiation was compared to suspension
(EB) differentiation in the generation of the RPE cells from hESCs.
Methods: Human ESCs (Regea 08/023) were derived and maintained without serum
and animal feeder cells. The differentiation was performed by plating the hESCs
on protein coating or by culturing them as cell aggregates in suspension using a
serum-free medium without any growth factors. The differentiation efficiency of the
methods was compared by using qRT-PCR. The maturation of the cells was confirmed
by RT-PCR and immunocytochemistry.
Results: First pigmented cells were observed by day 14 with both differentiation
methods. qRT-PCR data demonstrated that the adherent differentiation of the hESCs
enhanced the expression of the essential genes for RPE development (PAX6, RAX,
SIX3, MITF) compared to EB differentiation. Furthermore, the expression of neural
retinal marker, CHX10, was down-regulated in adherent differentiation compared
to EB differentiation. The RPE cell maturation was confirmed at gene level (MITF,
RPE65, BEST, PEDF, PMEL, tyrosinase) and at protein level (MITF, CRALB, Bestrophin,
ZO1).
Conclusions: The adherent differentiation method was more efficient for the generation
of RPE cells than the EB method. The constituents responsible for the differences
between the two methods are currently under investigation.
CR: H. Vaajasaari, None; T. Ilmarinen, None; H. Hongisto, None; R. Suuronen,
None; H. Uusitalo, None; H. Skottman, None.
Support: Academy of Finland, the Competitive Research Funding of the Tampere
University Hospital, TGSBB,Evald and Hilda Nissi Foundation, Sokeain ystävät
Foundation and two private donations
2660 - A467
Sox11b Expression in the Zebrafish Eye Suggests a Role in Retinal Development
and Rod Regeneration
A.C. Morris1, D. Mari2, J.M. Fadool2. 1Biology, University of Kentucky, Lexington, KY;
2
Biological Science, Florida State University, Tallahassee, FL.
Purpose: Adult human Müller stem cells represent a potential source of cells for use
in cell-based therapies to treat retinal degenerative diseases. This project aimed to
develop in vitro methods to differentiate these cells towards a photoreceptor fate.
Methods: Cells were cultured for various periods of time in the presence of extracellular
matrix (basement membrane proteins (bMP) or laminin) and combinations of
various growth and differentiation factors including retinoic acid, taurine, fibroblast
growth factor, insulin like growth factor and DAPT. Gene expression of markers
of photoreceptor precursors (CRX, NRL, Nr2e3) was studied by RT-PCR, whilst
analysis of phenotypic differentiation was performed by immunohistochemistry
and confocal microscopy.
Results: A significant increase in the expression of Crx, Nrl and Nr2e3 was observed
in Müller stem cells cultured on laminin or bMP in the presence of retinoic acid,
taurine, insulin like growth factor, fibroblast growth factor or DAPT. All individual
factors induced an increased expression of these genes but combinations of some of
these factors had a more significant effect not only on gene expression (CRX, p=0.001,
NRL, p=0.0285) but also on the phenotypic changes observed on these cells after 5-7
days in culture.
Conclusions: It has been shown that post mitotic retinal progenitors are ontogenetically
ideal to replace photoreceptors in experimental models of photoreceptor cell death.
This study shows that Müller stem cells can be induced to differentiate towards
photoreceptor precursors in vitro by using a short time protocol based on extracellular
matrix substrates and exogenous factors. It suggests that this strategy may potentially
be used to generate a population of ontogenetically suitable cells for transplantation
into models of photoreceptor degeneration that could in principle be scaled up under
GMP conditions and readily translated into human therapies.
CR: H. Jayaram, None; S. Singhal, None; B. Bhatia, None; P.T. Khaw, None; G.A.
Limb, None.
Support: Medical Research Council (UK) (G0701341)
Purpose: Unlike mammals, the retinas of teleost fish display remarkable regenerative
ability in response to experimental damage or genetic manipulation. We are interested
in identifying factors that regulate photoreceptor regeneration in the zebrafish
retina. This will increase our understanding not only of the molecular mechanisms
of adult retinal regeneration, but may also uncover genes that are important for
photoreceptor development during embryogenesis. In a microarray analaysis of
retinal RNA purified from wild type zebrafish and from zebrafish that experience
rod degeneration, members of the sox family of transcriptional regulators displayed
increased expression in response to rod degeneration. Of these, sox11b demonstrated
the largest increase over wild-type levels. Therefore, we evaluated the expression
pattern of sox11b in adult retinas from wild type, rod-deficient, and cone-deficient
zebrafish, as well as during embryonic retinal development.
Methods: sox11b expression in the zebrafish retina was examined by RT-PCR and in
situ hybridization of embryonic, larval and adult retinal cryosections. Cell proliferation
in the retina was visualized by immunodetection of BrdU incorporation. Localization
of sox11b expression in different retinal cell types was determined by immunolabeling
with antibodies to cell-type specific markers. Expression patterns of sox11b were
compared in wild type, transgenic XOPS-mCFP (rod-deficient), and mutant pde6cw59
(cone-deficient) retinas.
Results: In the developing zebrafish eye, sox11b expression was observed throughout
the retinal neuroepithelium at 24 hours post fertilization (hpf). Thereafter, sox11b
expression became progressively restricted to the persistently neurogenic marginal
zone (CMZ), in a pattern very similar to that of the proliferation marker PCNA. At
5 dpf, sox11b expression was observed in the CMZ and in a few cells located in the
outer nuclear layer (ONL). In the wild type adult retina, sox11b expression was very
low. However, in rod-deficient XOPS-mCFP retinas, sox11b expression was observed
in numerous rod precursor cells at the base of the ONL. sox11b expression did not
appear to co-localize with BrdU-positive rod progenitor cells.
Conclusions: Our results suggest that sox11b plays a role in both embryonic retinal
development and in the generation of rod photoreceptors in adult zebrafish in response
to rod degeneration. In the mature retina, sox11b may function after rod progenitor
exit from the cell cycle.
CR: A.C. Morris, None; D. Mari, None; J.M. Fadool, None.
Support: NIH Grant EY017753
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2657-2660
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2661 - A468
Ex Vivo Gene Expression of Retinal Differentiation Transcription Factors
Induces a Photoreceptor Phenotype in Mouse Neural and Embryonic Stem Cells
M.A. Fields, L. Vickers, H. Cai, J. Gong, S. Tsang, L. Del Priore. Ophthalmology,
Columbia Univ Eye Inst, New York, NY.
Purpose. Embryonic stem cell (ESC) transplantation is a promising therapeutic
approach for the replacement of degenerated retinal cells in patients with age-related
macular degeneration (AMD) and other retinal degenerations. Previously we have
used microarray analysis to identify key transcription factors in murine retinal
development. Herein we perform ex vivo gene therapy to express these transcription
factors in ESC to induce their differentiation into photoreceptors.
Methods. Retinal samples were collected from E11 to adult murine eyes and
developmental transcription factors expressed at different development time
points were identified by microarray analysis. cDNA of identified transcription
factors [namely cone-rod homeobox (Crx), orthodenticle homeobox 2 (Otx2), neural
retina leucine zipper (Nrl), neurogenic differentiation 1 (NeuroD1) and neurogenic
differentiation 4 (NeuroD4)], were cloned into the pCDH-EF1-MCST2A expression
vector containing a red fluorescent protein (RFP) or green fluorescent protein (GFP)
reporter gene. Mouse neural stem cells or mouse ESD3 cells were plated on laminin
cultured at 37°C, 5% CO2 in DMEM supplemented with 10% FBS, β-mercaptoethanol
and retinoic acid. Purified DNA was transfected into mouse neural stem cells or mouse
ESD3 cells. Various neural marker antibodies and fluorescence microscopy were used
to identify expression of neural/photoreceptor markers.
Results. Transfection with Crx and NeuroD1 induced phenotypical changes in embryonic
stem cells and neural stem cells demonstrated by positive immunofluorescence
staining for neural markers β-tubulin III, NF-200 and photoreceptor markers Nrl,
Crx, and recoverin. Conversely, we did not observe similar effects from transfection
of Otx2 or Nrl into neural stem or ESD3 cells.
Conclusions. Microarray analysis reveals high expression levels of transcriptions
factors involved in mouse retinal development, consistent with the previous reports
which suggest that the time between embryonic day 18 and postnatal day 5 appears
to be critical for photoreceptor differentiation in the neural retina. Ex vivo gene
expression of selected genes into mouse neural stem cells and mouse embryonic stem
cells direct stem cells to differentiate into a neural/photoreceptor phenotype.
CR: M.A. Fields, None; L. Vickers, None; H. Cai, None; J. Gong, None; S. Tsang,
None; L. Del Priore, None.
Support: NIH Grant EY13933, Research to Prevent Blindness, Robert L. Burch III
Fund, Retina Society, the Foundation Fighting Blindness
2662 - A469
Identification of Müller Glia Proliferation Signaling Pathways
C.M. Nelson, R.A. Gorsuch, D.R. Hyde. Department of Biological Sciences and Center
for Zebrafish Research, University of Notre Dame, Notre Dame, IN.
Purpose: Intense light exposure causes photoreceptor apoptosis in dark-adapted adult
zebrafish. After 16 hours of light-treatment, maximal cell death is observed, followed
by Müller glia reentering the cell cycle to yield transiently amplifying pluripotent
neuronal progenitor cells that migrate to the ONL and differentiate into photoreceptors.
The signals that mediate the initiation of Müller glia proliferation remain unknown.
We hypothesize that the dying photoreceptors generate a trans-acting signaling
molecule that induces the regeneration response. This analysis attempts to identify
the photoreceptor-generated signal that is required for regeneration.
Methods: To determine if dying photoreceptors produce a proliferation signal, adult
albino zebrafish were either undamaged or light-damaged for 16 hours, their retinas
were homogenized and the lysate was injected into healthy eyes. Lysate-injected eyes
were subsequently harvested for immunohistochemical analysis of PCNA, which
labels dividing cells. Undamaged and light-damaged protein lysates were analyzed by
2D gel electrophoresis and MALDI-TOF mass spectrometry to identify candidate signal
proteins. These candidate proteins were immunolocalized in light-damaged retinas
to determine their spatial and temporal expression patterns during photoreceptor
apoptosis and regeneration. Inhibiting candidate protein expression revealed the role
of these signaling pathways in initiating the Müller glia regeneration response.
Results: Injection of light-damaged retinal lysates significantly increased the
number of dividing Müller glia relative to control lysate-injected eyes. Proteomic
techniques identified increased expression of specific signaling pathway proteins
in the light-damaged lysate, which suggests they are the signals that induce Müller
glia proliferation.
Conclusions: A trans-acting signal is generated by dying photoreceptors that stimulates
Müller glia proliferation. Proteins from distinct candidate signaling pathways were
identified that increase in expression in dying photoreceptors, which may induce
Müller glial-derived photoreceptor regeneration.
CR: C.M. Nelson, None; R.A. Gorsuch, None; D.R. Hyde, None.
Support: NIH R01-EY018417, R21-EY018919, Center for Zebrafish Research
2663 - A470
Effect of Anti-Vascular Endothelial Growth Factor Antibody on the
Differentiation of Retinoblastoma Cells
2664 - A471
Stem Cells Play a Key Role in the Formation of the Human Choroidal
Vasculature and Mural Cells
C.S. Cho1, J. Kim1, K.-W. Kim2, Y. Yu1, J. Kim1. 1Ophthalmology, Seoul National
University Hospital, Seoul, Republic of Korea; 2College of Pharmacy and Research
Institute of Pharmaceutical Sciences, Seoul National University, Seoul, Republic of
Korea.
P. Hu1, L. Baxter1, M. Koina2, J.R. McColm1, J. Dahlstrom2, E. Bean2, T. Chan-Ling1.
1
Department of Anatomy, University of Sydney, Camperdown, Australia;
2
Department of Pathology, Australian National University, Canberra, Australia.
Purpose: To evaluate whether anti-vascular endothelial growth factor (VEGF) antibody
affects the differentiation of retinoblastoma cells
Methods: Human retinoblastoma cells, SNUOT-Rb1, were differentiated by treatment
of 0.1% bovine serum albumin (BSA), and then treated by anti-VEGF antibody
(Bevaxizumab) for 48 hours. To determine the differentiation of SNUOT-Rb1, the
neurotrophin receptors (Trk A, Trk B) and the neuronal differentiation markers (Shank1,
2) were detected by Western blot analysis and reverse transcriptase-polymerase chain
reaction (RT-PCR), respectively. Furthermore, extracellular signal-regulated kinases
1 and 2 (ERK 1/2) phosphorylation was also detected.
Results: Treatment of anti-VEGF antibody did not affect to cell viability, but attenuated
neurite outgrowth of 0.1% BSA treated-retinoblastoma cells. mRNA of Shank 1 and
2 were decreased by anti-VEGF antibody treatment, which might be mediated by
inhibition of ERK 1/2 phosphorylation. Furthermore, Trk A expression was decreased
by anti-VEGF antibody treatment.
Conclusions: Anti-VEGF antibody treatment might inhibit the differentiation
of retinoblastoma cells. Therefore, anti-VEGF antibody should be careful to treat
to the developing eyes, because of the effect of anti-VEGF antibody on neuronal
differentiation.
CR: C.S. Cho, None; J. Kim, None; K.-W. Kim, None; Y. Yu, None; J. Kim, None.
Support: None
Purpose: To undertake the first complete study of human choroidal vascular formation
& determine the role played by stem cells.
Methods: Human foetal eyes aged 8-40 weeks gestation (WG) were examined.
Choroidal & retinal wholemounts & histological sections were examined using
antibodies for stem & precursor cell populations: Vimentin, CD14, CD34, CD39 &
αSMA; the vasculature: CD39, CD34, CD31 & Factor VIII; & mural cells: αSMA, Desmin,
NG2, Calponin & Caldesmon. Endothelial proliferation was examined by doublelabelling with BrdU & CD34. TEM and H & E histology was also undertaken.
Results: Vimentin+ mesenchymal precursor cells were evident in the region of
the incipient choroid at 9 WG, & down regulation of Vimentin was evident with
maturation. CD14+ vascular precursor cells (VPCs) were evident in the choroidal
stroma throughout foetal life. CD39+/CD34+ VPCs were evident within the choroidal
stroma from 10 WG, interspersed amongst CD39+ solid vascular chords in the central
one-third of the choroid surrounding the optic nerve head. SMA+ mural precursor
cells (MPCs) were scattered & isolated over the primordial vascular tree at 12WG.
Non vascular-associated αSMA+/CD34+/--/ NG2+/desmin- presumed MPCs were
associated with immature choroidal blood vessels at 12, 18 & 20 WG. Calponin &
caldesmon were expressed only on the large vessels.
Conclusions: We conclude that formation of the human choroid takes place via
transformation from Vimentin+ mesenchymal precursor cells to CD14+/CD39+/
αSMA+ precursor cells representing the monocytic vascular and mural cell lineages
respectively. Vasculogenesis plays a greater role in formation of human choroid
than previously reported but angiogenesis also contribute to vascular density in
the formation of the human choroid. We have shown that CD44+ stem cells give rise
to αSMA+ smooth muscle cells & CD39+ vascular endothelial cells in the human
choroid.
CR: P. Hu, None; L. Baxter, None; M. Koina, None; J.R. McColm, None; J. Dahlstrom,
None; E. Bean, None; T. Chan-Ling, None.
Support: NHMRC Project Grants & Fellowship (#464859, #57100), Baxter Charitable
Foundation, Macular Vision Loss Support Society, Rebecca L. Cooper Medical Research
Foundation
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2661-2664
Tuesday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 2637 - 2668 / A444 - A475
316. Stem Cells: Proliferation, Differentiation and Gene Regulation
Organizing Section: RC Contributing Section: VN
2665 - A472
The CYP4A-20-HETE System in Regulation of Cord Blood Derived Endothelial
Progenitor Cells
2666 - A473
Cathepsin L in Bone Marrow-Derived Endothelial Progenitor Cells is Required
for Choroidal Neovascularization
J. Sheng1A, A.M. Guo1B, A.-A. Syed1C, B. Janic1C, P.A. Edwards1D, A.G. Scicli1A. AEye Care
Services, BWomen’s health Services, CRadiology, DDept of Ophthal and Eye Care
Services, 1Henry Ford Health System, Detroit, MI.
N. Shimada1A, K. Ohno-Matsui1A, T. Yoshida1A, M. Mochizuki1A, I. Morita1B.
A
Ophthalmology, BCellular Physiological Chemistry, 1Tokyo Medical & Dental
Univ, Tokyo, Japan.
20-hydroxyeicosatetraenoic acid (20-HETE) is an arachidonic acid metabolite
produced by Cytochrome P450 (CYP) ω-hydroxylases. We have reported that 20HETE is angiogenic and 20-HETE synthase inhibitors decrease and even suppress
angiogenesis induced by growth factors. In human, the more critical 20-HETE
synthases are CYP4A11/4A22 and CYP4F2. Endothelial progenitor cells (EPC) are
an important component of neovascularization processes.
Purpose: We studied whether the CYP4A-20-HETE system can contribute to the
regulation of EPC.
Methods: Progenitor cells were isolated from human umbilical cord blood and were
identified by AC133 and CD34 expression.
Results: We found that these EPC contained both immuno-reactive CYP4A11 and
CYP4F2. RT-PCR showed that the mRNA’s coding for these enzymes were also present.
When EPC were incubated with arachidonic acid, they formed and secreted 20-HETE.
These results indicate that EPC contain an active CYP-20-HETE system. Furthermore,
stimulation with 20-HETE increased EPC proliferation and migration indicating that
20-HETE can influence EPC functions. When matrigel plugs containing 20-HETE
were injected sc in nude mice, a marked angiogenic response was observed. To
study whether the injection of labeled EPC iv results in their accumulation into the
20-HETE-containing plug, we used iron-labeled EPC and determined the presence of
the labeled EPC in the plug by MRI and immunohistochemistry. These labeled EPC
accumulate into the site of 20-HETE induced neovascularization, which suggests that
20-HETE may induce the homing of EPC. Tube formation by EC seed in matrigel is
an in vitro model of angiogenesis. We found that co-culture of EC and EPC results
in the tube formation and this effect was inhibited by inhibitors of cytochrome P450
4A/F ω-hydroxylases and by a 20-HETE competitive antagonist. This suggests that
20-HETE is involved in EPC-induced tube formation.
Conclusions: The results suggest that the CYP4A-20-HETE system is involved in
regulation of EPC functions associated to angiogenesis.
CR: J. Sheng, None; A.M. Guo, None; A.-A. Syed, None; B. Janic, None; P.A. Edwards,
None; A.G. Scicli, None.
Support: None
Purpose: Age-related macular regeneration is characterized by intraocular
neovascularization. The ideal goal of the treatment is to prevent the invasion of new
vessels into the avascular tissue through a matrix barrier. The purpose of this study
was to determine the role of cathepsin L, a matrix degrading enzyme, in choroidal
neovascularization (CNV).
Methods: Laser irradiation was performed to induce CNV in wild type C57BL
mice which received bone marrow transplantation from cathepsin L gene-deficient
mice or wild type mice. Two weeks after laser irradiation, serial sections were
cut from the enucleated eyeball and the area of CNV was measured. To identify
the cathepsin L-expressed cells during the CNV development, the sections were
immunocytochemically analyzed using different cell markers.
Results: Transplantation of bone marrow from cathepsin L-deficient mice into wild
type mice significantly reduced the degree of CNV. Immunohistochemical analyses
demonstrated that VE cadherin-positive endothelial progenitor cells (EPCs), but
not CD43-positive or Iba-1 positive cells, were the major cells contributing to the
production of cathepsin L.
Conclusions: These data indicate that cathepsin L-expressed EPCs from bone marrow
plays a critical role in CNV and suggest a potential therapeutic approach of targeting
cathepsin L for CNV.
CR: N. Shimada, None; K. Ohno-Matsui, None; T. Yoshida, None; M. Mochizuki,
None; I. Morita, None.
Support: None
2667 - A474
Decreased Clonogenic Capacity of Endothelial Progenitor Cells (EPCs) in Age
Related Macular Degeneration (AMD)
2668 - A475
Nerve Growth Factor Promotes Endothelial Progenitor Cell-Mediated
Angiogenic Responses
F. Scotti1A, A. Maestroni1B, S. Maestroni1B, U. Introini1A, M. Setaccioli1A, P. Rama1C, F.
Bandello2, M. Lorenzi 3, G. Zerbini1B. ADepartment of Ophthalmology, BComplications
of Diabetes Unit, Division of Metabolic and Cardiovascular Sciences, CDepartment
of Ophthalmology, Cornea and Ocular Surface Unit, 1San Raffaele Scientific
Institute, Milan, Italy; 2Department of Ophthalmology, University Vita-Salute, San
Raffaele Scientific Institute, Milan, Italy; 3Schepens Eye Research Institute and
Department of Ophthalmology, Harvard Medical School, Boston, MA.
C.S. Jadhao1A, A.D. Bhatwadekar1A, M.E. Boulton1B, J.J. Steinle2, M.B. Grant1A.
A
Pharmacology and Therapeutics, BAnatomy and Cell Biology, 1University
of Florida, Gainesville, FL; 2Ophthalmology, Univ of Tennessee Hlth Sci Ctr,
Memphis, TN.
Purpose: The major cause of severe vision loss in AMD is choroidal neovascularization
(CNV). The aim of this ongoing study is to learn whether active CNV is accompanied by
changes in circulating EPC.
Methods: The study of AMD patients with new-onset CNV scheduled for intravitreal
ranibizumab (IVR), is enabling us to observe in the same patient the relationship of EPC to
evolving stages of CNV. To date, we completed data collection in 11 AMD subjects (6F/5M,
age 74±8 yrs) undergoing the recommended cycle of IVR (3 monthly injections). Blood
samples were obtained at baseline, and at 4, 30, and 120 days after the first injection. The 120
days sample was collected 30 days after the final IVR. Nine subjects matched for age (69±6
yrs) and gender (5F/4M) but without AMD provided control baseline data. We measured
(i) the number of circulating EPCs (CD45dim, CD34+, VEGFR-2+) by flow cytometry; (ii) their
clonogenic capacity by the Hill’s assay; and (iii) the plasma levels of VEGF and SDF-1, the
two cytokines known to mobilize EPC, by ELISA.
Results: Clinical improvement occurred in 6 of the 11 patients. At baseline, the number of
circulating EPC and the VEGF and SDF-1 plasma levels were similar in AMD patients and
controls. In a subset of patients, the number of EPC responded to IVR but was not correlated
with the clinical response. The striking observation is the much lower clonogenic capacity
of EPCs obtained at baseline from AMD patients when compared to controls (1.97±1.14 EPC
colonies /106 mononuclear cells, vs 5.06±1.42 in controls, mean±SD, P=0.0001). At 120 days,
the clonogenic capacity of EPCs from patients who did not improve clinically remained
low (1.90±1.19 vs1.85±0.89 at baseline); whereas that of the EPCs from patients who did not
improve appeared to increase (2.83±1.53 vs 2.07±1.40 at baseline).
Conclusions: . The decreased clonogenic capacity of EPCs from AMD patients may reflect
the increased cardiovascular risk that is being reported in this population. However, its
apparent association with active CNV may be revealing that CNV signals to the function
of circulating EPCs.
CR: F. Scotti, None; A. Maestroni, None; S. Maestroni, None; U. Introini, None; M.
Setaccioli, None; P. Rama, None; F. Bandello, None; M. Lorenzi, None; G. Zerbini, None.
Support: International Agency for the prevention of blindness (IAPB)
Purpose: Retinal neuronal cells are dysfunctional in diabetes and in response to injury
express nerve growth factor (NGF) which, in some settings, can be pro-angiogenic. We
postulated that the NGF that accompanies retinal injury/ischemia will a) contribute
to an “angiogenic switch” in CD34+ cells taking them from beneficial reparative cells
to cells that participate in pathological angiogenesis and b) have little effect on the
resident endothelial cells of the retinal vasculature.
Methods: CD34+ cells were isolated from healthy volunteers (n=10) using
immunomagnetic separation. Human retinal endothelial cells (HREC) were obtained
from retinas of healthy human donors (n=3). Cell proliferation in response to NGF (1,
2 and 4pM) was determined by cell counts for CD34+ cells and MTT assay for HREC.
Migration of CD34+ cells and HREC was studied using a modified Boyden chamber
assay. NGF stimulated CD34+ cell incorporation into HREC tubule formation was
evaluated using 3-dimensional (3D) extracellular matrix. NGF receptor activation
was assessed by Western blot analysis.
Results: NGF induced a dose-dependent increase (1.5-3-fold; p<0.001) in proliferation
of CD34+ cells at 24 hrs compared to controls but had no effect on HREC proliferation.
However, NGF stimulated HREC migration in a dose-dependent manner. Pretreatment
of CD34+ cells with NGF increased migration to SDF-1 (p<0.05). NGF-treated CD34+ cells
enhanced in vitro tube formation by HRECs 2-fold (p<0.05) as assessed by increases
in numbers of sprouts in a 3D model of angiogenesis Western blotting of cell lysates
with phospho-specific antibodies revealed phosphorylation of TrKA (receptor for
NGF) in CD34+ and HRECs. NGF stimulation resulted in activation of Akt in HRECs
and ERK1 in CD34+ cells while pretreatment with LY294002 (PI3K inhibitor) and
PD98059 (MAPK inhibitor) blocked this phosphorylation response by 1.2-fold and
5-fold respectively.
Conclusion: NGF differentially regulates the angiogenic potential of CD34+ cells
and resident endothelial cells and may contribute to the angiogenic switch in CD34+
cells which is associated with the pathological neovascularization in diabetic
retinopathy.
CR: C.S. Jadhao, None; A.D. Bhatwadekar, None; M.E. Boulton, None; J.J. Steinle,
None; M.B. Grant, None.
Support: NIH grants 2RO1 EY012601-08 , 2RO1 EY007739-17, R01 EY018358
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2665-2668
Tuesday, May 4, 1:45 PM - 3:30 PM Floridian A Symposium Program Number Range: 2945 - 2951
341. Neuroprotection: Mechanisms and Promise of Future Therapies - Minisymposium Organizing Section: RC Contributing Section: BI
2945 - 1:45 PM
Introduction
2946 - 1:50 PM
Functional Rescue Of Cone Photoreceptors In RP Models : A Translational
Approach
J.D. Ash. Ophthalmology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK.
J.-A. Sahel1,2. 1Inserm- UPMC-CNRS UMR-S 968, Institute de la Vision, Paris, France;
2
Ophthalmology, Pierre and Marie Curie Medical School, Paris, France.
CR: J.D. Ash , None.
Support: None
Identification of Rod-derived Cone Viability Factor (RdCVF 1 and 2) by our group
(Leveillard et al, Nat Gen 2004), provides clues for understanding one of the key
mechanisms leading to cone function loss in RP and more importantly for rescue by
protein and gene therapies. Studies from mice inactivated for these factors demonstrate
the dependance of cones upon these factors, particularly in pathologic paradigms.
Demonstration of cone function preservation in a Rhodopsin mutant RP model, and
the correlation with cone outer segments morphology indicates that this marker could
indicate how efficiency could first be demonstrated alongside with functional testing
in affected patients, using high resolution images analysis (OCT, adaptive optics).
CR: J.-A. Sahel, patent co-inventor, P; Fovea Pharmaceuticals, R; Novartis, Fovea
Pharmaceuticals, Sanofi-Aventis, F; Imagine Eyes, Allergan, F; Fovea Pharmaceuticals,
I; Fovea Pharmaceuticals, C.
Support: FFB, EU-FP6-EVI-GENORET, INSERM, ANR,, Fondation Voir et Entendre,
Federation des Aveugles de France
2947 - 2:05 PM
Molecular Safety Mechanisms For Photoreceptor Protection
2948 - 2:20 PM
Mechanisms of Neuroprotection by the Wnt signaling pathway
C. Grimm. Lab for Retinal Cell Biology, Dept. Opht, University of Zurich, Zurich,
Switzerland.
A.S. Hackam. Ophthalmology, Bascom Palmer Eye Institute, Miami, FL.
To preserve vision, the retina has established several molecular safety mechanisms
for the protection of photoreceptor cells. Prominent members of these mechanisms
are cytokines of the IL-6 family of proteins like ciliary neurotrophic factor (CNTF) or
leukemia inhibitory factor (LIF) and factors induced by hypoxia like erythropoietin.
To make these endogenous rescue pathways available for efficient neuroprotective
therapies for patients, we focus on the understanding of the regulatory mechanisms
and on the identification of the elements controlling the endogenous retinal response
to stress or photoreceptor injury.
CR: C. Grimm, None.
Support: SNF Grant 31003A-117760
The canonical Wnt pathway regulates a wide range of essential processes in embryonic
and adult tissues. The importance of Wnt signaling in the retina is increasingly
being recognized. Wnt ligands control retinal neuron viability and differentiation,
regeneration and stem cell proliferation. We recently demonstrated that Wnt signaling
is upregulated during rod and cone death in the rd1 mouse and Wnt ligands rescued
photoreceptors from oxidative stress in culture. Wnt-mediated neuroprotection
was found to be indirect and involved activation of the canonical Wnt pathway
in Muller glia. Wnt signaling was also neuroprotective to RGC cells exposed to
high pressure, in this case acting directly on the rescued cells. A combination of
genomic analyses and experimental confirmation was used to identify Wnt signaling
target genes. Intriguingly, a large number of growth factors and their receptors
were found to be direct and indirect targets of Wnt, suggesting that Wnt controls
multiple neuroprotective pathways. In an effort to determine cellular pathways
that regulate Wnt, we recently demonstrated that a receptor for the innate immune
system suppressed Wnt activity in Muller glia. The novel interaction between innate
immunity and Wnt pathways adds an additional layer of complexity to Wnt-mediated
neuroprotection, and may be significant to the pathology of diseases, such as AMD,
in which chronic activation of the innate immune response is a contributing factor.
CR: A.S. Hackam, None.
Support: NEI R01EY017837, Karl Kirchgessner Foundation, NEI P30EY014801, RPB
Career Dev Award, RPB Unrestricted grant (BPEI)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2945-2948
Tuesday, May 4, 1:45 PM - 3:30 PM Floridian A Symposium Program Numbe Range: 2945 - 2951
341. Neuroprotection: Mechanisms and Promise of Future Therapies - Minisymposium Organizing Section: RC Contributing Section: BI
2949 - 2:35 PM
Photoreceptor Neuroprotection is negatively regulated by Protein Tyrosine
Phosphatase-1B
2950 - 2:50 PM
Hydrogen Sulphide: A Neuroprotective Agent In Retinal Ischemia And
Oxidative Stress Or Light-induced Damage To Retinal Cells.
R.V. Rajala. Ophthal/Dean McGee Eye Inst, Univ of Oklahoma Hlth Sci Ctr,
Oklahoma City, OK.
N.N. Osborne1,2. 1Nuffield Lab of Ophthalmology, University of Oxford, Oxford,
United Kingdom; 2Fernandez-Vega, Instituto Oftalmologico, Oviedo, Spain.
Protein-tyrosine phosphatase 1B (PTP1B) has been implicated in the negative regulation
of insulin signaling. Studies from our laboratory over the past decade have shown the
existence of a novel insulin receptor (IR) signaling pathway in photoreceptor cells.
The IR activation is functionally important for rod survival, since its deletion from
rods resulted in the loss of neuroprotective survival signaling. We have demonstrated
the importance of downstream effectors of IR in the photoreceptor neuroprotection.
Further PTP1B activity negatively regulated the neuroprotective survival signaling in
the retina. Our studies suggest that rhodopsin signaling is coupled to the activation
of IR signaling in rods. One of the challenging questions in the retina research is
how mutations in human rhodopsin gene slowly disable and eventually disrupt
photoreceptor functions. Our studies suggest that a defect in the photobleaching of
rhodopsin and mutation in rhodopsin gene enhances the activity of PTP1B and this
activated activity could down regulate the IR survival signaling.
CR: R.V. Rajala, None.
Support: NIH grants EY016507, EY00871, RR17703, and Research to Prevent Blindness, Inc.
The recent discovery that hydrogen sulphide (H2S) is an endogenously produced
gaseous secondary messenger capable of modulating many physiological processes,
much like nitric oxide, prompted us to investigate the potential role of H2S as a
retinal neuroprotective agent. In the current study we use dithiolethiones (kindly
provided by Dr. Piero Del Soldato, Milan, Italy) as H2S donors and show that such
substances attenuate the effect of retinal ischemia as well as oxidative and light-induced
injury to a transformed line of cells (RGC-5 cells) in culture. Ischemia was delivered
to rats by elevation of the intraocular pressure above the systolic blood pressure.
Partial damage to the retina after seven days was determined by a combination of
procedures which included analysis of electroretinograms, immunohistochemistry
and changes in the retinal content of proteins and mRNAs known to be associated with
ganglion cell function and apoptosis. Most of the changes caused by ischemia were
significantly attenuated by intravitreal injection of a H2S donor directly after ischemia.
Both light (400-700nm, intensity 1000 lux) and hydrogen peroxide caused death to
RGC-5 cells in culture over a period of 24-48 hours in a time and dose-dependent
manner, respectively. Light and hydrogen peroxide-induced RGC-5 cell death is by
different forms of apoptosis but they are both attenuated by the H2S donor, ACS1.
These initial findings demonstrate that donors of H2S may be value in the treatment
of various retinal dysfunctions where oxidative stress, light or ischemia is implicated
as causative factors.
CR: N.N. Osborne, None.
Support: None
2951 - 3:05 PM
Neuroprotective Redox Driven Signal Transduction Pathways In The Retina
T.G. Cotter. Biochemistry Dept, University College Cork, Cork, Ireland.
Purpose : To investigate the role of hydrogen peroxide as a cell survival promoting
molecule in retinal photoreceptors and how recombinant FGF mediates its
neuroprotective effects through redox signalling.
Methods: Tunel staining was used to identify cells undergoing apoptosis at each of
the time points examined. Western blotting was used to look at PI3k/Akt and Nox
protein expression patterns. Hydrogen peroxide and related reactive oxygen species
were measured using fluorescent probes such as 2’,7’ dichlorodihydrofluorescin
diacetate and dihydroethidium. Retinal explant cultures, light induced and the rd10
models of retinal degeneration were used in the study. Confocal microscopy and
immunohistochemistry were used to look at retinal staining patterns.
Results : This study demonstrated that 661W cells, a retina-derived photoreceptor
cell line, and mouse retinal explants rapidly produced a burst of hydrogen peroxide
following the exposure of cells and explants to external stress. The burst of hydrogen
peroxide induced a pro-survival response by up-regulating signalling through
the PI3k/Akt pathway for a period of up to 120 mins. The source of the hydrogen
peroxide is one of the members of the Nox family of proteins and could be inhibited
by diphenyleneiodonium chloride. This stress response, pro-survival generation of
hydrogen peroxide was also seen in the rd10 mouse model of retinal degeneration
particularly at the early stages of photoreceptor loss. Recombinant FGF also stimulated
the production of hydrogen peroxide as part of its cell survival promoting properties in
both retinal explants and in in vivo models. Surprisingly FGF stimulated the production
of hydrogen peroxide via activation of the COX pathway. Specific COX inhibitors
blocked hydrogen peroxide production and the protective effects of FGF.
Conclusions : The generation of hydrogen peroxide either as a result of a stress response
or via FGF stimulation induces a cell survival effect due in part to its down stream
activation of the PI3k/Akt pathway in retinal photoreceptor cells. This highlights the
importance of redox signalling on retinal cell survival.
CR: T.G. Cotter, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
2949-2951
Tuesday, May 4, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 3140 - 3158 / A476 - A494
353. Stem Cells: Translational Research
Organizing Section: RC
3140 - A476
Photoreceptor Differentiation From Porcine Induced Pluripotent Stem Cells
3141 - A477
Effect of Ndp Treatment After Neurotoxic Injury in the Rat Retina
L. Zhou1, D.C. Dean1, T. Ezashi2, R.M. Roberts2, H.J. Kaplan1. 1Ophthalmology &
Visual Sciences, University of Louisville, Louisville, KY; 2Animal Sciences and
Biochemistry, University of Missouri, Columbia, MO.
Y.-H. Chen1A, W. Dailey1, M. Cheng2, K.A. Drenser1A,3. AAssociated Retinal
Consultants, 1Beaumont Eye Institute, Royal Oak, MI; 2Oakland University, Eye
Research Institute, Auburn Hills, MI; 3Oakland University, Eye Research Institute,
Rochester, MI.
Purpose: Because retinal regeneration is limited in higher vertebrates, stem cells are
being utilized to create progenitors for use in cell transplants in models of retinal
damage. We have generated a genetic porcine model of retinitis pigmentosa (RP)
utilizing the P23H rhodopsin mutation, which is common in human patients with
RP. As an initial step toward developing cells for transplant into this large animal
model, induced pluripotent stem cells (iPSC) were generated from porcine skin
fibroblasts utilizing retroviral expression of embryonic stem cell genes. These iPSC
were then used in cell culture experiments for generation of cells expressing markers
of photoreceptor differentiation.
Methods: A protocol for photoreceptor differentiation (Lamba et al. Proc Natl Acad
Sci USA. 2006;103:12769-74) was adapted for porcine iPSC differentiation in culture.
Porcine iPSC were cultured as floating aggregates in nonadherant plates in media
containing the Wnt inhibitor Dkk1, the BMP inhibitor Noggin, and insulin-like
growth factor (IGF-1) for three days, and then the aggregates were transferred to
poly-D-lysine plus matrigel coated plates to allow for attachment of the aggregates.
Twenty days after transfer to the poly-D-lysine plus matrigel coated plates, cells were
fixed and immunostained.
Results: Following the differentiation protocol, many of the adherent cells showed
neuronal morphology and these cells immunostained for the general neuronal marker
β III-tubulin. Additionally, approximately 50% of the adherent cells immunostained
for the photoreceptor marker recoverin.
Conclusions: These studies demonstrate that porcine iPSC can efficiently differentiate
into cells expressing a photoreceptor marker in culture. Thus, these cells should provide
a source of cells for transplant into the porcine model of retinitis pigmentosa.
CR: L. Zhou, None; D.C. Dean, None; T. Ezashi, None; R.M. Roberts, None; H.J.
Kaplan, None.
Support: None
Purpose: Stimulation of the Wnt pathway has been shown to increase retinal neural
progenitor cells in injured retina. We sought to evaluate the effect of a retina specific
Wnt ligand, norrin, for its effects on injured retina.
Methods: Four-week-old male Sprague-Dawley rats were treated with phosphate
buffered saline (PBS), NMDA, or NMDA+ norrin. Eighteen hours after receiving
intravitreal injections of 400 nmol of NMDA, the treatment eyes received intravitreal
injections of 125ng NDP and the untreated eyes received intravitreal PBS injections.
Control eyes were injected with PBS only at each experimental time point. Tissues
were fixed at 1, 2, 4 and 7 days after the first injection (PBS in control and NMDA in
study group). The eyes were sectioned, and immunolabeled with Chx10 and Pax 6 to
evaluate the presence of retinal neural progenitor cells.
Results: NMDA injured retina resulted in increased numbers of progenitor cells.
Many of these cells co-expressed Chx10 and Pax 6 compared to the PBS treated eyes
at day 7. A significant difference was not noted at days 1, 2, or 4. Eyes treated with
NMDA+ norrin also showed a significant increase in retinal neural progenitor cells
compared to PBS only eyes. However, a statistically significant difference was not
seen between eyes treated with NMDA alone and eyes treated with both NMDA+
norrin at day 7.
Conclusions: Our preliminary results showed that single NDP treatment did not
significantly increase retinal progenitors in NMDA damaged retina at day 7. This may
have been due to the use of a relatively high dose of NMDA used to induce neurotoxic
injury or a short half life of the NDP after injection into the retina. Therefore, we have
initiated further studies to evaluate the effect of multiple injections with NDP and
a longer time course.
CR: Y.-H. Chen, None; W. Dailey, None; M. Cheng, None; K.A. Drenser, None.
Support: None
3142 - A478
Induction of Ocular Tissues From Human Pluripotent Stem Cells on the
Amniotic Membrane Matrix
3143 - A479
Transcorneal Electrical Stimulation Induce the Proliferation of Progenitor-Like
Cells in the Ciliary Body of Adult SD Rats
M. Ueno1,2, Y. Nakai1,2, M. Matsumura2, M. Takahashi 3, Y. Sasai2, S. Kisnoshita1.
1
Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan;
2
Organogenesis and Neurogenesis Group, Center for Developmental Biology
RIKEN, Kobe, Japan; 3Laboratory for Retinal Regeneration, Ctr for Developmental
Biology RIKEN, Kobe, Japan.
Y. Wang1, M. Zhang2, X. Rong2, X. Wang2, X. Mo2. 1Ophthalmology, Shanghai EENT
hospital,Fudan University, Shanghai, China; 2Ophthalmology, Shanghai EENT
Hospital,Fudan University, Shanghai, China.
Purpose: Driving human pluripotent stem cells (PS cells), including embryonic stem
(ES) cells and induced pluripotent stem (iPS) cells, to differentiate into ocular tissues
is a key technology for the ophthalmological application of regenerative medicine.
We previously reported a differentiation technique using the matrix components
of the human amniotic membrane (hereafter referred to as ‘‘denuded hAM’’) which
induces the neural conversion of mouse and human ES cells. The purpose of this
present study was to investigate whether human PS cells differentiate to ocular
tissues on denuded hAM.
Methods: hAMs encasing the fetus within the human female uterus were obtained
at the time of Caesarean section after obtaining proper informed consent from both
parents and in accordance with the tenets of the Declarations of Helsinki. To prepare
the denuded hAM for culture, the matrix was carefully removed from its overlying
epithelium, and then transferred to cell culture plates. Human PS cells were then
seeded onto the denuded hAM and cultured in KSR (Invitrogen Corp., Carlsbad,
CA)-containing Glasgow-MEM (Invitrogen) medium at 37°C under 5% CO2 .
Results: Both human ES cells and human iPS cells formed large colonies and
differentiated into neural precursors at an efficiency of approximately 80%, when
cultured on the denuded hAM in the serum-free medium (amniotic membrane matrixbased ES cell differentiation or AMED). AMED-induced neural tissues developed to
pigmented cells which were positive for Pax6 and showed actin bundles; consistent
with the characteristic of retinal pigment epithelium. Light-reflecting lentoid tissues
were also found in the culture. These tissues were alpha-crystallin positive, consistent
with the nature of lens cells.
Conclusions: The AMED culture uses a non-cellular inductive material derived from
an easily available human tissue. Thus, AMED should provide a highly practical and
versatile system for generating ocular tissues from human ES cells and human iPS
cells for clinical applications.
CR: M. Ueno, None; Y. Nakai, None; M. Matsumura, None; M. Takahashi, None; Y.
Sasai, None; S. Kisnoshita, None.
Support: Grant-in-aid from MEXT, the Kobe Cluster Project and the Leading
Project
Purpose: In this study we investigated the effect of transcorneal electrical stimulation
(TES) on the activation of progenitor-like cells residing in adult mammal’s ciliary
body (CB).
Methods: Adult male Sprague Dawley (SD) rats received electrical stimulation
for three days via a contact electrode applied to the cornea and another reference
electrode puncture into subepithelium of the head. Biphasic rectangular current
pulses (1 ms, 100uA/200uA) at 20 Hz were delivered by an electrical stimulation
system for 1h per day. Proliferative activity of cells in the CB was assessed by
Bromodeoxyuridine (BrdU) expression respectively on seven, ten and fourteen days
after TES. Immunocytochemistry in combination with confocal microscopy were
performed to identify the phenotype and migration of these proliferating cells.
Results: There were few BrdU labeled cells in the CB of control group as well as the
group received TES of 100 µA current pulses at any time point. However, in group with
TES of 200 µA current, some BrdU-positive cells were found in CB on the seventh day
after TES. And the number of BrdU labeled cells were significantly increased fourteen
days after TES. In addition, Nestin/BrdU double staining cells were detected at this
time, indicating that the proliferating cells were progenitor-like cells. These cells,
however, were restricted to the ciliary marginal region, suggesting that the divided
progenies did not migrate to the adjacent neural retina.
Conclusions: These results suggest that TES can activate the proliferation of
progenitor-like cells in the CB of adult SD rats. So, it raised the possibility that we
could also promote the proliferation and differentiation of these cells in diseased
status for cell based treatment.
CR: Y. Wang, None; M. Zhang, None; X. Rong, None; X. Wang, None; X. Mo,
None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3140-3143
Tuesday, May 4, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 3140 - 3158 / A476 - A494
353. Stem Cells: Translational Research
Organizing Section: RC
3144 - A480
Transplantation of Differentiated Embryonic Stem Cells for Re-Population of
Retinal Ganglion Cells in the Adult
3145 - A481
Infiltrating Monocyte-Derived Macrophages Enhance Progenitor Cell Renewal
in the Adult Retina Following an Insult
Y.J. Liao1A, Y.-W. Chen1A, J. Weimann1B, L. Recht1B. AOphthalmology, BNeurology,
1
Stanford, Stanford, CA.
A. London, E. Itskovich, M. Schwartz. Neurobiology, Weizmann Institute of Science,
Rehovot, Israel.
Purpose: The mammalian central nervous system has limited regenerative abilities. As
a result, injury and disease typically lead to irreversible neuronal loss and permanent
functional impact. Stem cell transplantation, despite being in its infancy, promises the
greatest potential for re-population and re-engineering of neural circuit. Embryonic
stem (ES) cells are totipotent cells that can undergo unlimited self-renewal and
differentiate into any adult cell type. We have previously injected differentiated ES cells
into the visual cortex, which led to prominent axon projections to the dorsal superior
colliculus, which is important for pupillary response and visually guided behavior.
Next, we tackled regenerative therapy to replenish retinal ganglion cells.
Methods: We grew enhanced green fluorescent protein-positive ES cells on a confluent
layer of MS5 cells. After 6 days of co-culture, we generated colonies of neural precursor
cells, with few cells expressing Oct3/4 (an ES cell marker), 80% expressing nestin
(proliferative marker), and 30% expressing class-III beta-tubulin, an early marker for
retinal ganglion cells. We transplanted differentiated ES cells into adult wild-type
mice and performed morphometric analyses.
Results: We found that compared to subretinal injection, intravitreal injection was
technically easier and led to superior transplantation efficacy. Transplantation was
also enhanced by induction of retinal fate by treating ES cells at days 1 and 3 with
noggin, a BMP inhibitor, and dkk1, a Wnt inhibitor. Lastly, retina lasering, a technique
already well established in humans for treatment of diabetic retinopathy and other
diseases, dramatically enhanced stem cell survival and caused elaboration of neuritelike processes which extended over 800-microns from the peripheral retina to the
optic nerve head.
Conclusions: Regenerative therapy using embryonic stem cells is feasible in adult
mammals. Different techniques, including intravitreal injection of retinal progenitors
and retina lasering provide a permissive environment for robust stem cell survival
and extension of processes.
CR: Y.J. Liao, None; Y.-W. Chen, None; J. Weimann, None; L. Recht, None.
Support: Career Award in Biomedical Sciences from Burroughs Wellcome
Foundation
Purpose: To test whether retinal insult at adulthood, in a model of glutamate
intoxication, awakens the quiescent retinal progenitor cells (RPC), and if so, whether
infiltrating monocyte-derived macrophages affect progenitor cell renewal following
retinal insult.
Methods: Adult mice, subjected to retinal insult in a model of Glutamate intoxication,
were administrated intra-vitreously with the cell proliferation marker 5-bromo2-deoxyuridine (BrdU). Proliferation of RPC and recruitment of distinct myeloid
populations were analyzed by immunohistochemistry and flow-cytometry.
Bone-marrow chimeras were used to characterize these myeloid populations and
specifically to identify the infiltrating monocyte-derived macrophages. Depletion
and augmentation of these infiltrating cells were performed in order to evaluate their
contribution to RPC renewal following an insult.
Results: Glutamate intoxication stimulated proliferation of Pax6+ and SOX2+ RPC
in the adult ciliary body and changed the relative contribution of distinct myeloid
populations in the retina. Specifically, retinal insult resulted in the infiltration of
CD11b+ /CX3CR1+/Gr1+ blood borne monocyte-derived macrophages to the damaged
retinal ganglion cell layer. Enhanced accumulation of this monocytic population at
the damaged area augmented RPC proliferation, while ablation of these cells resulted
in a reduced renewal of RPC.
Conclusions: Retinal insult evokes proliferation of the dormant adult RPC and
recruitment of distinct myeloid populations to the retina. Specifically, monocytederived macrophages infiltrate to the damaged retina following the insult and enhance
progenitor cell renewal.
CR: A. London, None; E. Itskovich, None; M. Schwartz, None.
Support: The Glaucoma Foundation, New York
3146 - A482
Optimisation of Cellular Scaffolds for Neural Retinal Cell Replacement Using
Human M[[Unsupported Character - &#971;]]ller Stem Cells
3147 - A483
Generation of New Cone and Rod Photoreceptors in Models of Retinal
Degeneration by Transplantation of Crx-Positive Precursor Cells
L.M. James1A, S. Singhal2A, H. Jayaram2B, P.T. Khaw3, G.A. Limb1B. AORBIT, BOcular
Biology and Therapeutics, 1Institue of Ophthalmology UCL, London, United
Kingdom; AOcular Biology and Therapeutics, BOcular Biology & Therapeutics,
2
UCL Institute of Ophthalmology, London, United Kingdom; 3Director, Research
& Development, Moorfields Eye Hospital & UCL Inst Ophth, London, United
Kingdom.
M. Baron1, J. Lakowski1, J.W. Bainbridge2,3, A. Barber2, R.A. Pearson2, R.R. Ali2,3, J.C.
Sowden1. 1Developmental Biology Unit, UCL Institute of Child Health, London,
United Kingdom; 2Genetics Department, UCL Institute of Ophthalmology, London,
United Kingdom; 3NIHR Biomedical Research Centre for Ophthalmology, London,
United Kingdom.
Purpose: Adult human Müller stem cells can be made to differentiate into retinal
neurons in vitro. These cells may be potentially used for retinal transplantation to
replace neurons damaged as a result of retinal degeneration. This study aimed to
investigate the use of Collagen type I as a source of biomaterial to build cellular
scaffolds using Müller stem cells.
Methods: Nanofibre mats of Collagen type I were used to culture Müller stem cells in
the presence of the Notch1 inhibitor DAPT (N-[N-(3, 5-Difluorophenacetyl)-L-alanyl]S-phenylglycine t-butyl ester) and FGF2 for 1 week. Cellular attachment and neural
morphology were examined by phase microscopy and scanning electron microscopy.
Expression of the retinal ganglion cell markers HuD and Islet-1 was examined by
confocal microscopy.
Results: Cells cultured for one week on nanofibre mats of Collagen type-I in the
presence of DAPT and FGF2 displayed long processes characteristic of neural
morphology. These cellular processes were more pronounced when cells were cultured
on thin Collagen nanofibre mats than when cultured on thick mats. Cells cultured
on these scaffolds also showed immunostaining for the retinal ganglion cell markers
HuD and Islet-1.
Conclusions: Nanofibre mats of Collagen type I provide the mechanical and physical
properties necessary for cell delivery into the degenerated retina. These mats support
adhesion and spreading of human Müller stem cells as well as neurite formation
upon differentiation into the ganglion cell phenotype. Further studies will explore
the practical application of these scaffolds in cell based therapies to repair damaged
retina.
CR: L.M. James, None; S. Singhal, None; H. Jayaram, None; P.T. Khaw, None; G.A.
Limb, None.
Support: Biomedical Research centre for Ophthalmology Moorfield’s eye hospital,
MRC
Purpose: The irreversible loss of photoreceptors during retinal degeneration is
a leading cause of blindness in the developed world. Repair of such damage by
retinal cell therapy may be a feasible treatment option, as transplanted rod precursor
cells have been shown to integrate into diseased retinae and restore some visual
function. As human vision is cone dependent, we investigated the competency of
early photoreceptor precursors to give rise to both rod and cone photoreceptors after
transplantation into mouse models of retinal degeneration.
Methods: Crx (cone-rod homeobox), a key regulator of vertebrate photoreceptor
development, is expressed in post-mitotic rod and cone cells. Immature photoreceptors
expressing a Crx.gfp transgene were isolated from embryonic and postnatal retinae
by flow cytometry. GFP+ cells were transplanted sub-retinally in adult wildtype and
degenerating retinae (RetGC1-/-, Crb1rd8/rd8; models of Leber’s congenital amaurosis).
Three weeks after transplantation, GFP+ photoreceptors in the recipient outer nuclear
layer (ONL) were counted and cones identified by immunohistochemistry for RxRgamma
and cone arrestin.
Results: Crx+ precursors efficiently integrated into wildtype and degenerating retinae.
The transplanted Crx.gfp cells acquired mature photoreceptor morphologies in the
ONL of the recipient retina. Whilst the majority of integrated cells adopted a rod fate,
a proportion of embryonic donor cells developed into cone photoreceptors. These
cone cells displayed characteristic features (cone pedicles, nuclear structure, position
within ONL) and expressed RxRgamma and cone arrestin. A significant increase in
cone integration efficiency was observed after transplantation in the cone-deficient
RetGC1-/- retina.
Conclusions: Embryonic Crx.gfp donor cells develop into cone and rod photoreceptors
after transplantation into the adult wildtype and degenerating retina.
CR: M. Baron, None; J. Lakowski, None; J.W. Bainbridge, None; A. Barber, None; R.A.
Pearson, None; R.R. Ali, None; J.C. Sowden, None.
Support: Macula Vision Research Foundation, Fight for Sight, the Medical Research
Council UK (G03000341), Wellcome Trust and the Royal Society
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3144-3147
Tuesday, May 4, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 3140 - 3158 / A476 - A494
353. Stem Cells: Translational Research
Organizing Section: RC
3148 - A484
Development of Müller-Like Cells After Subretinal Transplantation of Feline
Red Fluorescent Retinal Progenitors in Abyssinian Cat Hereditary Retinal
Degeneration
K. Narfstrom1,2, J. Yang3, G.P. Lewis 4, R. Bragadottir5, G. Luna4, S.K. Fisher4, R. Whiting6,7,
I.-K. Kong8, H.J. Klassen 3. 1Vet. Medicine & Surgery, College of Vet. Med., Univ. of
Missouri, Columbia, MO; 2Ophthalmology, Mason Eye Inst., Univ. of MissouriColumbia, MO; 3Ophthalmology, Gavin Herbert Eye Inst., Univ. of California,
Irvine, CA; 4Neuroscience Research Inst., Univ. of California, Santa Barbara, CA;
5
Ophthalmology, Oslo Univ. Hospital, Oslo, Norway; 6Vet. Medicine & Surgery,
Univ. of Missouri, Columbia, MO; 7Biological Engineering, Univ. of MissouriColumbia, Columbia, MO; 8Dept. of Animal Sciences, Gyeongsang Natl. Univ.,
Jinju, Republic of Korea.
Purpose: To determine if feline red fluorescent retinal progenitor cells (RPCs)
survive transplantation, integrate, and develop into retinal cell types in feline retinal
dystrophy.
Methods: RPCs from neural retinas of embryonic day 45 RFP-transgenic cats were
passaged 3 times in DMEM/F12, N2 supplement, GlutaMax, 20ng/ml EGF and 20ng/
ml bFGF. Aseptic vitreo-retinal surgery was performed on six 6-17 months-old cats
with early stage rod-cone degeneration and 100 µl of donor cells (500,000-750,000)
injected subretinally in one eye. Post-op examination included ophthalmoscopy,
bilateral full-field ERG, and immunohistochemistry using anti-red fluorescent protein
(ARFP), anti-rod opsin, anti-vimentin, anti-ezrin and peanut agglutinin. Four of the
cats were euthanized at 6 (n=1), 15 (n=2) and 63 (n=1) days post-op. Posterior eyecups
were fixed in 4% paraformaldehyde in Sorensen’s buffer.
Results: No ERG changes were observed 15-63 days post-op. RFP+ donor profiles
were observed within the subretinal space, along the retinotomy, within all cellular
layers of the neural retina, and the RPE. Integrated donor cells included examples
with striking Müller glial morphology and others suggestive of neuronal cell types.
IHC showed co-localization of ARFP together with vimentin and, to lesser extent,
anti-rod opsin.
Conclusions: Feline retinal progenitor cells survive following subretinal
transplantation, migrate within the dystrophic feline retina and develop into profiles
with features characteristic of the local cellular populations, including Müller glia.
CR: K. Narfstrom, None; J. Yang, None; G.P. Lewis, None; R. Bragadottir, None; G.
Luna, None; S.K. Fisher, None; R. Whiting, None; I.-K. Kong, None; H.J. Klassen,
None.
Support: Discovery Eye Foundation and Lincy Foundation
3149 - A485
Neuronal Progenitor Cells Derived From Human Persistent Fetal Vasculature
Are Good Candidate Cells for Optic Nerve Transplantation
G. Chen, M.A. Shatos, K. Lashkari. Schepens Eye Research Institute, Schepens Eye
Research Institute, Boston, MA.
Purpose: Persistent fetal vasculature (PFV) is caused by failure of the primary vitreous
and the hyaloidal vascular systems to regress during development. Our laboratory
has previously shown that PFV membranes are potential sources of progenitor cells.
We have characterized these cells in vitro and evaluated their potential use in optic
nerve transplantation.
Methods: PFV cells were subjected to IHC and qRT-PCR analysis for expression of
progenitors and neuronal markers, and gene expression. Calcium imaging was also
performed to evaluate the response profiles to different neurotransmitters. Results were
compared with human retinal progenitor cells (hRPC). For transplantation, PFVs were
labeled with AAV2-GFP and intravitreally injected into DBA/2J pigmentary glaucoma
mice. Mice were sacrificed at 30 days and confocal microscopy was performed.
Results: Cultured PFV cells highly express neural progenitor (Nestin, Ki67, Brn3a,
and MAP1b) and adult neuronal (beta-tubulinIII, NF200 and Thy1) markers. 25% of
cultured PFV cells uptake Brdu and have a stem-cell like morphlogy. Compared to
hRPCs, PFVc express higher copies of synapse-related genes transcripts (SNAP25,
NLGN4Y, STXBP1 and RAPSN). Calcium imaging revealed the following responses:
glutamate 75%, glycine 38%, GABA 33%, acetylcholine 26%, and dopamine 19%).
Following tranplantation, PFVs migrated into the inner retina and intraorbital portion
of optic nerve.
Conclusions: PFV cells express progenitor and adult neuronal markers and
morphology simlar to retinal ganglion cells. Intravitreal transplantation of PFV
cells into glaucomatous mice results in their migration and incorporation into sites
of retinal ganglion cell damage. These cells may be good candidates for cell-based
therapy for glaucomatous and other optic nerve damage.
CR: G. Chen, None; M.A. Shatos, None; K. Lashkari, None.
Support: funded by the Harvard Stem Cell Institute and the Canary Charitable
Foundation
3150 - A486
Improvement of Sodium Iodate-Induced Retinal Degeneration in Mouse by
Subretinal Transplantation of iPSCs-Derived NPCs
3151 - A487
Photoreceptor Reprogramming: A New Road Path of Translational Retinal Cell
Therapy
F. Gao1, Z. Qu2, Y. Guan2, L. Cui2, Y. Wu2, W. Li1,3, G.-T. Xu1. 1Tongji Eye Institute
and Department of Regenerative Medicine, Tongji University School of Medicine,
Shanghai, China; 2Laboratory of Clinical Visual Sciences, Institute of Health
Sciences, Shanghai Jiao Tong University School of Medicine and Shanghai
Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai,
China; 3Department of Ophthalmology, Drexel University College of Medicine,
Philadelphia, PA.
T.G. Qiu, D.A. Carter, D. Copland, A.A. Dick. Ophthalmology, University of Bristol,
Bristol, United Kingdom.
Purpose: To investigate the transplantation potential of neural precursor cells (NPCs)
that are derived from C57 mouse induced pluripotent stem cells (iPSCs) under the
subretinal space in retinal degeneration mouse model induced by Sodium Iodate
(SI).
Methods: A C57 mouse iPSC line was generated by ectopic expression of the four
define transcription factors Oct4, Sox2, Klf4 and cMyc in the mouse embryonic
fibroblasts. The mouse iPSCs were characterized by RT-PCR, immunofluorescence
and differentiation assays in vitro. Mouse iPSCs were induced to differentiate into
NPCs by the embryoid body (EB) formation method. The differentiated NPCs were
transplanted into subretinal space of SI-induced mouse which had been evaluated
by electroretinogram (ERG), histology (HE staining) and immunohistology (TUNEL
assay). The therapeutic effects of NPCs’ transplantation were monitored by ERG
and fundus examination. The abilities of NPCs in terms of survival, integration and
differentiation into retinal cells after subretinal transplantation were also examined
by histology and immunohistology.
Results: Mouse iPSCs exhibited similarity to mouse embryonic stem cells (mESCs)
in morphology and expression profile of characteristic pluripotency markers. The
mouse iPSCs were successfully differentiated into derivatives of all three embryonic
germ layers in vitro. Highly enriched cultures of NPCs expressing transcripts of key
regulatory genes of retinal development were developed from the iPSCs. The SIinduced retinal degeneration showed a time dependent severity. After transplantation
into mouse eyes, the NPCs survived, migrated and integrated into the host retina.
Furthermore, these transplanted NPCs can improve the retinal function of SI-induced
retinal degeneration.
Conclusions: The iPSCs, which are similar to ESCs, could be efficiently induced to
differentiate into NPCs. The subretinal transplantation of NPCs appears to improve
the function of degenerative retina induced by SI in mouse. These findings indicate
that iPSCs can be used for therapeutic applications in retinal degenerations.
CR: F. Gao, None; Z. Qu, None; Y. Guan, None; L. Cui, None; Y. Wu, None; W. Li,
None; G.-T. Xu, None.
Support: Ministry of Science and Technology 2007CB948004
2010 ARVO Abstract G. Qiu Dec. 2009
Tina Guanting Qiu1**, Debra A Carter1, Dave Copland1, Andrew A Dick1
Bristol Eye Hospital, University of Bristol, Bristol BS1 2LX United Kingdom
Lower Maudlin Street, Bristol, BS1 2LX, UK
Purpose: To introduce a unique concept of photoreceptor reprogramming to generate
high profile human photoreceptor lineage progenitors for retinal cell therapy.
Materials & Methods: Sixty-four pairs of post-mortem human eyes were obtained
from Bristol Eye Bank with research consent and ethical approval. A series of cell
isolation and stage specific purification tactics were developed to trigger the cell
reprogramming in vitro, and a unique stepwise in vitro cell manipulations in
combination with serum free define culture media were developed and validated.
The evolving cell behaviours were observed and documented by light microscopy.
Phenotypic and genotypic analysis was performed.
Results: The study first demonstrated that post-mitotic human photoreceptors be
reprogrammed toward a precursor stage expressing nestin and a proliferation marker
(Ki-67), and forming a robust neuronal network in vitro. qPCR confirmed the nestin
up-regulation in the reprogrammed cell population. The majority of the cultured cells
expressed photoreceptor cell markers (recoverin or rhodopsin positive) with negligible
glial lineage phenotypes. High resolution 3D confocal imaging demonstrated a unique
“tree-like” or “grape-like” structure of the cultured photoreceptor cells centring
around the neurosphere.
Conclusions: The unique concept of cell reprogramming (turn the clock back at a
certain limit) opens an alternative therapeutic pathway for regenerative medicine.
The ability to generate high profile human photoreceptor lineage progenitor cell from
adult tissue provides a valuable human cell source for photoreceptor replacement.
Acknowledgements: 1. National Eye Research Centre, UK (Financial Support); 2. Bristol
Tissue Eye Bank, UK; 3.Research Enterprise Development, University of Bristol, UK.
Patent (Pending): UK Application No. 0913109.5 **Inventor:Guanting Qiu. ** Corresponding
author: Guanting Qiu, MD PhD, University of Bristol, Email: guantingqiu@yahoo.com
Patent Filing No.: UK Application no. 0913109.5 (Pending)
CR: T.G. Qiu, UK Application No: 0913109.5, P; D.A. Carter, None; D. Copland,
None; A.A. Dick, None.
Support: National Eye Research Centre, UK
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3148-3151
Tuesday, May 4, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 3140 - 3158 / A476 - A494
353. Stem Cells: Translational Research
Organizing Section: RC
3152 - A488
Endothelial Progenitor Populations Participate in Revascularization of Ischemic
Retina
3153 - A489
Feasibility Study on Intravitreal and Subretinal Delivery of Adult Stem/
Progenitor Cells (MSCs) for Retinal Repair
A.V. Ljubimov1,2, S. Li Calzi 3, S. Caballero, Jr. 3, A. Millard4, D. Ammar4, M. Levi4, M.B.
Grant3. 1Ophthalmology Research Laboratories, Cedars-Sinai Medical Center,
Los Angeles, CA; 2Medicine, David Geffen School of Medicine at UCLA, Los
Angeles, CA; 3Pharmacology & Therapeutics, University of Florida, Gainesville, FL;
4
Medicine, University of Colorado Denver, Aurora, CO.
R.H. Rosa, Jr.1, G.W. Roddy2, U.C. Krause2, D.J. Prockop2. 1Dept of Ophthalmology,
Scott & White Eye Institute, Temple, TX; 2Institute for Regenerative Medicine, Texas
A&M Health Science Center, Temple, TX.
Purpose: To assess whether bone marrow stem cells transplanted to the embryonic
liver, as well as human CD34+ cells and late outgrowth endothelial cells (LOEC)
participate in vascular repair in the oxygen-induced retinopathy (OIR) and ischemiareperfusion injury mouse models.
Methods: Three approaches were utilized: 1) Intrauterine transplantation of mouse
gfp+, c-kit+, sca-1+ bone marrow stem cells (106) was performed into the livers of
mouse embryos (day 13 post conception) with a fine-needle syringe. 2) Pups that
underwent OIR model or were kept in room air were injected with fluorescently
labeled human CD34+ cells on postnatal day 12 and were sacrificed on day 17. Retinal
flatmounts were prepared to assess incorporation of progenitors into developing
neovascularization areas. 3) Mice undergoing ischemia-reperfusion injury model
were injected with human LOEC labeled with Qdot®-655 nm nanocrystals to allow
tracking. All mice were perfused prior to sacrifice with rhodamine-conjugated R.
communis agglutinin I to label vascular endothelial cells for subsequent visualization
by confocal microscopy.
Results: Liver-injected cells survived and grafting took place in the bone marrow
judged by the presence of gfp+ cells in femur bones. The expression of green-labeled
mouse gfp+ or human CD34+ cells was low in retinas of mice not exposed to the OIR
model, whereas these cells were present in areas of neovascularization in OIR and
co-localized with growing vessels. LOEC also associated with retinal vasculature in
areas of ischemic injury.
Conclusions: Intrauterine transplantation may be a useful tool to study the
contribution of bone marrow-derived cells to retinal development, injury repair,
and neovascularization. CD34+ cells and LOEC actively incorporate into the injured
retina. These cells may allow durable repair of injured retina and may serve as
potential therapeutic cell populations to relieve retinal ischemia in patients with
ischemic retinopathies.
CR: A.V. Ljubimov, None; S. Li Calzi, None; S. Caballero, Jr., None; A. Millard,
None; D. Ammar, None; M. Levi, None; M.B. Grant, None.
Support: NIH R01 EY13431, EY007739, EY012601, M01 RR00425, Winnick Family
Foundation, Eye Defects Research Foundation, OneSight Research Foundation
3154 - A490
The Common IL6 Signal-Transducing Receptor, gp130, is Implicated in
Endothelial Progenitor Cell Dysfunction in Diabetes
Purpose: The current understanding of mesenchymal stem cell (MSC) biology indicates
that MSCs are able to secrete anti-apoptotic, pro-growth, and/or anti-inflammatory
proteins depending on the specific need of the injured tissue in order to preserve function.
A limited number of in vivo studies with MSCs suggest a positive effect on the rescue of
retinal degeneration. The mechanism of rescue effect, however, is not clearly established
based on the published studies. We wish to determine the mechanism(s) by which
photoreceptors and/or RPE are rescued by MSCs. Herein, we evaluated the feasibility
of intravitreal and subretinal injections of MSCs in rodent and porcine experimental
models.
Methods: After IACUC approval, adult Long Evans rats were sedated with a ketamine
cocktail and redosed as needed and pigs (10-12 weeks old) were anesthetized with
isoflurane for the entire length of the procedure. Pupil-dilating drops (tropicamide
0.5-1%, phenylephrine 2.5%) were paced in both eyes. Retinal fundus photography was
performed with the RetCam II before and after the injections. Intravitreal injection of
GFP-labeled mouse MSCs (10 μl/200,000 cells/PBS in rats; 50μl/1,000,000 cells/PBS in
pigs; 30 gauge needle) was performed in the right eye followed by subretinal injection
of mouse mesenchymal stem cells (2μl/40,000 cells/PBS in rats; 10μl/200,000 cells/PBS
in pigs; 30 gauge needle) in the left eye using an operating microscope. The eyes were
enucleated, and the animal subjects were euthanized. The eyes were examined by light
and fluorescence microscopy.
Results: Retinal fundus photography and histologic studies demonstrated the delivery of
MSCs into the vitreous cavity and subretinal space in both rat and pig eyes. Intravitreal
injections were performed without complications in both species. Acute complications
with subretinal injections in both species included vitreous and subretinal hemorrhage
and retinal perforation.
Conclusions: Intravitreal and subretinal delivery of MSCs is technically feasible in both
small and large animal models. Potential complications with subretinal injections include
intraocular hemorrhage and retinal perforation. Future studies will evaluate the potential
benefit of high resolution ultrasound-guided microinjections of MSCs into the subretinal
space and the mechanism(s) of rescue of retinal degeneration by MSCs.
CR: R.H. Rosa, Jr., None; G.W. Roddy, None; U.C. Krause, None; D.J. Prockop, None.
Support: NIH Grant K08EY016143 (RHR), NCRR/NIH Grant P40 RR 17447 (DJP), Scott &
White Research Grants Program (RHR, DJP), Texas A&M Health Science Center (GWR)
3155 - A491
Outgrowth Endothelial Cells Potential for Reversing Ischaemic Retinopathy
S. Hazra1A, Y.P.R. Jarajapu1A, C.A. Lee2, M.E. Boulton1B, T.S. Kern2, J.D. Ash 3, M.B.
Grant1A. APharmacology and Therapeutics, BAnatomy and Cell Biology, 1University
of Florida, Gainesville, FL; 2Medicine, Case Western Reserve University, Cleveland,
OH; 3Ophthalmology, University of Oklahoma Health Sciences Center, Oklahoma
City, OK.
Purpose: gp130 controls the activity of a group of cytokines involved in inflammation
and cell survival. Since diabetes is typically associated with increased inflammation
and endothelial and endothelial progenitor cell (EPC) dysfunction, we asked whether
elimination of gp130 would prevent EPC dysfunction.
Methods: Mice carrying the gp130 allele with exon 16 flanked by loxP sites were
crossed with transgenic mice that express Cre recombinase under the control of the
Tie2 promoter. This genetic combination inactivates gp130 in greater than 95% of
vascular endothelial cells and bone marrow derived cells. gp130 knockout (KO) were
made diabetic for 6 months and compared to age-matched WT diabetic mice and
non-diabetic controls. Peripheral blood and bone marrow were obtained from WT
mice with 6 and 12 months of diabetes and age matched controls. Cell number (FACS),
cell proliferation (colony formation), iNOS/ eNOS/ nNOS, heme oxygenase-1 (HO-1),
PAI-1, MMP-2, and MMP-9 levels (RT-PCR), migration (modified boyden chamber),
and NO generation (DAF-FM fluorescence) was determined in EPCs.
Results: As diabetes duration increased, increasing numbers of EPCs were trapped
inside the bone marrow (p=0.0003). EPCs from 12 month diabetic mice demonstrated
50% reduction in colony formation compared to age matched control (p=0.0002). eNOS
mRNA expression decreased with increasing age in WT mice. iNOS mRNA was
increased in diabetic mice but not in WT. HO-1 mRNA increased 3 fold in aged WT
mice, but not in diabetics. Diabetic EPCs demonstrated defective migration and reduced
bioavailable NO at 6 and 12 months. Diabetic gp130 KO showed greater migratory
response and higher NO generation compared to diabetic WT mice (p=0.01).
Conclusions: The common cytokine receptor chain, gp130, plays a central role in loss
of EPC functions during diabetes and may offer a potential therapeutic target.
CR: S. Hazra, None; Y.P.R. Jarajapu, None; C.A. Lee, None; M.E. Boulton, None; T.S.
Kern, None; J.D. Ash, None; M.B. Grant, None.
Support: NIH grants 2RO1 EY012601-08 , 2RO1 EY007739-17, R01 EY018358
A.W. Stitt1, R.J. Medina1, C.L. O’Neill1, M.W. Humphreys2, T.A. Gardiner1. 1Centre for
Vision & Vascular Science, Queens University Belfast, Belfast, United Kingdom;
2
Northern Ireland Regional Genetics Centre, Belfast Health and Social Care Trust,
Belfast, United Kingdom.
Purpose: The purpose of this study was to isolate endothelial progenitor cells from
human peripheral blood, characterise a distinct endothelial progenitor cell population
named Outgrowth Endothelial Cells (OECs) and establish their potential as a novel
cell therapy for ischaemic retinopathy.
Methods: OECs were isolated and characterised using immunophenotyping,
genome-wide transcriptional profiling, and multiple in vitro functional assays to
assess interaction with retinal capillary endothelial cells and angiogenic activity.
OECs were delivered intravitreally in a mouse model of ischaemic retinopathy, and
flat mounted retinas were examined using confocal microscopy.
Results: Our data indicate that OECs are committed to an endothelial lineage, have
significant proliferative, and de novo tubulogenic potential. Furthermore, OECs are
able to closely interact with endothelial cells through adherens and tight junctions,
and integrate into retinal vascular networks in vitro. Using a murine model of retinal
ischaemia, it is demonstrated that OECs directly incorporate into the resident
vasculature, significantly decreasing avascular areas, concomitantly increasing
normovascular areas and preventing pathologic pre-retinal neovascularisation.
Conclusions: OECs have potential as therapeutic cells to vascularise the ischaemic
retina and prevent sight-threatening pathology.
CR: A.W. Stitt, None; R.J. Medina, None; C.L. O’Neill, None; M.W. Humphreys,
None; T.A. Gardiner, None.
Support: JDRF, MRC, GDBA
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3152-3155
Tuesday, May 4, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 3140 - 3158 / A476 - A494
353. Stem Cells: Translational Research
Organizing Section: RC
3156 - A492
Human Cord Blood Derived CD14 + Cells Promote Vascular, Glial, and
Metabolic Stabilization in a Model of Ischemic Retinopathy
3157 - A493
Mechanism of Stem Cells in Rescuing Vision in a Rodent Model of Retinal
Degeneration
V. Marchetti1, E. Aguilar1, O. Yanes1, D. Friedlander1, M. Wang1, G. Nemerow1, G.
Siuzdak1, K. Storm2, M. Friedlander1. 1Cell Biology, The Scripps Research Institute, La
Jolla, CA; 2Source MDx, Boulder, CO.
S. Wang1, B. Cottam1, B. Lu1, C.W. Morgans1, T.J. McFarland1, B. Appukuttan1, E.E.
Capowski2, D.M. Gamm 3, R.D. Lund1. 1Ophthalmology, Casey Eye Institute, OHSU,
Portland, OR; 2Waisman Center, University of Wisconsin-Madison, Madison, WI;
3
Ophthalmology and Visual Sciences, Univ of Wisconsin-Madison, Madison, WI.
Purpose: To use metabolomic and transcriptomic analysis, immunohistochemistry
and confocal microscopy to assess the trophic rescue effect of human umbilical cord
blood (HCB)-derived myeloid progenitor cells in a model of OIR.
Methods: CD14+ cells were freshly isolated from HCB, characterized by flow cytometry,
infected with Adenovirus 5-GFP, injected intravitreally and visualized by confocal
microscopy. Rescue was evaluated on p17 by quantifying areas of vaso-obliteration
and neovascularization (NV). A panel of 60 genes was used to analyze and quantify
the expression of human and mouse genes in mouse retinas after the injection of
human cells. Metabolites were extracted and identified in OIR and normal retinas
at p12, p15 and p18 and evaluated for the ability to induce toxicity and apoptosis of
astrocytes and endothelial cells in vitro and in vivo.
Results: On day 0 CD14+ cells are a heterogeneous population expressing both myeloid
progenitor (CD33 and CD44) and endothelial (VEGFR-2) cell specific antigens. Using
the OIR model, we show that Ad5-GFP CD14+ cells: (1) target sites of retinal NV and
significantly enhance vascular repair; (2) induce expression of mouse angiogenic
cytokines such as FGF a and b, IL-8 and TGFα in OIR retinas; (3) up regulate the
expression of human anti oxidative stress genes as Catalase 1, Beta-glucuronidase,
Superoxide dismutase-1; (4) reduce apoptotic events by expressing anti apoptotic
genes as BCL2, AKT1, IFI6, IFI1; and (5) decrease the production of toxic metabolites
such as β-epoxycholesterol and 7-ketocholesterol to levels observed in control, nonOIR retinas.
Conclusions: We have characterized human CB-derived CD14+ cells that target retinal
vasculature and provide trophic rescue in a model of OIR. Metabolomic analysis
confirmed the transcriptomic profile. CD14+ cells regulate events leading to the
production of several metabolites toxic for EC and glia in OIR retinas and continue
to express monocyte and dendritic cell markers (CD163, CD68, CD209, HLDRA) after
injection into the mouse retinas subjected to OIR.
CR: V. Marchetti, None; E. Aguilar, None; O. Yanes, None; D. Friedlander, None; M.
Wang, None; G. Nemerow, None; G. Siuzdak, None; K. Storm, None; M. Friedlander,
None.
Support: NIH Grant EY11254, NIH Grant EY017540, the MacTel Foundation
Purpose: Cell-based therapy has been shown to be effective in slowing down the
progression of retinal degeneration in animal models. However, the molecular
mechanisms underlying vision rescue are unclear. It is likely that both the injected
cells and the host retina undergo molecular changes post-transplant. Here we study
changes in expression of growth factor genes by human progenitor cells before and
after injection into the eye in a rodent model of retinal degeneration, as well as changes
in gene expression in host retinal neurons following the injection.
Methods: Human forebrain derived progenitor cells (hNPCctx) were injected into the
subretinal space of RCS rats at postnatal day (P) 21-23. The efficacy of injected cells
was examined by measurements of visual acuity. Retinal tissues and injected hNPCctx
were collected at P35, P60 and P90 using laser capture microdissection (LCM). RNA
from hNPCctx and retinal tissues were isolated and examined for expression of trophic
factors by polymerase chain reaction and immunohistochemistry.
Results: Subretinal injection of hNPCctx produced substantial rescue morphologically
and functionally. Trophic factors released by human donor cells, including IGF and
BDNF, were detected both in vitro and in vivo. However, at P90, BDNF was no longer
detected. The host retina expressed Apolipoprotein E in areas close to the donor cell
distribution.
Conclusions: This study demonstrates that vision rescue by injected hNPCctx is
correlated with the expression of trophic factors released by both donor cells and
the host retina. The cellular localization of these factors in the host retina is under
investigation.
CR: S. Wang, None; B. Cottam, None; B. Lu, None; C.W. Morgans, None; T.J.
McFarland, None; B. Appukuttan, None; E.E. Capowski, None; D.M. Gamm,
None; R.D. Lund, None.
Support: FFB, RPB
3158 - A494
Human Placenta Stem Cells as Potential Neuroprotective Mediators for the
Treatment of Degenerative Retinopathy
S.Z. Scalinci1,2, L. Scorolli1,3, F. Alviano 4, G. Lanzoni4, R. Costa4, C. Marchionni4, L.
Bonsi4, G. Bagnara4, P. Tazzari 5, L. Calzà6. 1Ophthalmology, University of Bologna
SOrsola Malpighi, Bologna, Italy; 2Department of Surgery and Transplants,, Centro
di Studio per l’Ipovisione, University of Bologna - Bologna, Italy; 3Department of
Surgery and Transplants,, Centro di Studio sul Glaucoma, University of Bologna
- Bologna, Italy; 4Department of Histology, Embriology and Applied Biology,
University of Bologna - Bologna, Italy; 5Immunohaematology and Transfusion
Medicine Service, S. Orsola Malpighi Hospital, University of Bologna - Bologna,
Italy; 6BioPharmaNet-DIMORFIPA, University of Bologna - Bologna, Italy.
Purpose: Placenta derived stem cells display intriguing features like phenotypic
plasticity and immunomodulatory properties. More recently they have been proposed
as “factories” of cytokines, growth factors and anti-apoptotic factors. In this study
we investigated in placenta-derived stem cells the expression of neurotrophic
and neuroprotective factors with a potential for the treatment of degenerative
retinopathy.
Methods: Stem cells were isolated from amniotic membrane, chorion leave and
Wharton’s Jelly. The populations were extensively characterized through flow
cytometry analysis and assessed for multidifferentiation potential. Gene expression
profile was studied with regard to nerve growth factor (NGF), brain derived
neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and glial derived
neurotrophic factor (GDNF).
Results: Placenta derived stem cells displayed considerable in vitro expansion
potential; they showed an immunophenotypical profile consistent with mesenchymal
stem cells; in addition they underwent in vitro osteogenic, adipogenic and angiogenic
commitment. Real-time PCR analysis evidenced notable expression levels of
neurotrophic factors with differences related to the source. Wharton’s jelly and chorion
cultures showed respectively the highest expression of BDNF and CNTF.
Conclusions: In the degenerative context of retinopathy, the retinal photoreceptive
epithelium could benefit from the neuroprotective effect of neurotrophic factors
released by placenta stem cells. In vivo experiments in a mouse model of retinopathy
are underway.
CR: S.Z. Scalinci, None; L. Scorolli, None; F. Alviano, None; G. Lanzoni, None; R.
Costa, None; C. Marchionni, None; L. Bonsi, None; G. Bagnara, None; P. Tazzari,
None; L. Calzà, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3156-3158
Tuesday, May 4, 3:45 PM - 5:30 PM Grand H Paper Session Program Number Range: 3499 - 3505
372. Hot Topics and Novel Concepts in AMD Research
Organizing Section: RC
3499 - 3:45PM
Histone Acetylation is a Major Mediator of VEGF-Induced Angiogenesis
N. Chan1, S. He2A, S.J. Ryan, Jr. 2B, D.R. Hinton 3. 1Pathology-DVRC 313, University of
Southern California, Los Angeles, CA; AOphthalmology-USC, BOphthalmology,
2
Doheny Eye Institute, Los Angeles, CA; 3Pathology, Keck School of Medicine USC,
Los Angeles, CA.
Purpose: Choroidal neovascularization (CNV) is a serious complication of age-related
macular degeneration (AMD). An imbalance of pro- and anti-angiogenic factors leads
to the formation of CNV. However, the mechanisms underlying the regulation of
vascular endothelial growth factor (VEGF) expression in intraocular angiogenesis
remain unclear. The current study was to investigate whether epigenetics, specifically
the inhibition of histone deacetylation, is involved in the regulation of expression of
VEGF and its signaling in human fetal retinal pigment epithelial (RPE) cells.
Methods: Cultured early passage human fetal RPE cells were used in the study. The
viability of RPE cells treated with the histone deacetylase inhibitor, trichostatin A
(TSA), was tested by the MTT assay. The expression of acetyl-histone H3 in RPE cells
treated with TSA (0μM, 0.1μM, 0.5μM or 1μM) for 24 hours and in mouse CNV sections
induced by laser was investigated by immunostaining and/or Western blot. The
expression of VEGF, PEDF, Flk-1, HIF-1α, PPARγ, caspase 3, p-p38 and p-Akt in RPE
cells was examined by Western blot after treatment with TSA (0μM, 0.05μM, 0.1μM,
0.3μM or 0.5μM) for 24 hours with or without 150μM of cobalt chloride for 6 hours.
Results: TSA induced a strong acetylation of the histone H3 protein in RPE cells
and mouse CNV lesion as revealed by Western blot and immunostaining. TSA also
inhibited RPE cell proliferation and arrested the cell cycle at G1 and S phase. Most
importantly, TSA significantly down-regulated the expression of VEGF and VEGF
receptor 2, and up-regulated the anti-angiogenic and neuro-protective factor, PEDF,
as demonstrated by Western blot. The decreased VEGF expression was consistent with
the reduced production of HIF-1α, as the concentration of TSA reached 0.3μM. TSA also
induced an increased phosphorylation of p38 and activation of caspase 3; however, the
phosphorylation of Akt was dramatically reduced by the exposure of TSA. In addition,
the anti-proliferative transcription factor, PPARγ, was up-regulated by TSA. The results
implicated that epigenetic events are involved in the regulation of angiogenesis and
histone acetylation is a major mediator of VEGF-induced angiogenesis in vitro in
RPE cells. Conclusion: TSA inhibits angiogenesis by down-regulating VEGF and
its signaling and up-regulating the anti-angiogenic factor, PEDF. Epigenetics play a
critical role in the regulation of angiogenesis and TSA can be a potential therapeutic
candidate for the treatment of CNV.
CR: N. Chan, None; S. He, None; S.J. Ryan, Jr., None; D.R. Hinton, None.
Support: NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel
Beckman foundation
3500 - 4:00PM
Sub-Classification of Exudative Age-Related Macular Degeneration Based on
Anti-VEGF Therapy Response
M.H. Nelson. North Carolina Macular Consultants, Winston-Salem, NC.
Purpose: This study proposes a new classification for Exudative Age-Related Macular
Degeneration. It is based on the clinical response to anti-VEGF medications.
Methods: 225 eyes of 175 patients with Exudative Age-Related Macular Degeneration
were evaluated by IVFA/ICG videoangiography and spectral domain OCT. Those with
evidence of subretinal neovascular membrane formation, either classic or progressive
occult, were treated with monthly Lucentis monotherapy. Clinical patterns of persistent
or recurrent intraretinal, subretinal or subRPE leakage were appreciated upon monthly
OCT and every three month IVFA/ICG over a 12-36 month period.All patients were
treatment naive.
Results: 40.1% of all patients had persistent leakage after anti-VEGF monotherapy. One
quarter (9.9%) of those patients were classified as PRIMARY anti-VEGF failures when
100% of all subretinal, intraretinal or subRPE leakage did not completely resolve after
three intravitreal injections of Lucentis. Three quarters (30.2%) of those patients were
classified as SECONDARY anti-VEGF failures when leakage completely resolved after
Lucentis injections, but later recurred. Most of these patients were treated with a ‘treat
and extend’ maintenance therapy with Macugen or Lucentis however several displayed
new neovascular processes or developed mature vessels that became resistant to antiVEGF therapy.
Conclusions: Persistent intraretinal, subretinal and subRPE leakage is common with
non-selective anti-VEGF monotherapy. Three classifications of response to this therapy
are offered:
1. Anti-VEGF SENSITIVE - 15% that respond quickly in a sustained pattern.
2. Anti-VEGF DEPENDENT - 75% who need chronic VEGF suppression to maintain a
clinical response.
3. Anti-VEGF RESISTANT - 10% that do not respond or incompletely respond.
ICG videoangiography revealed that all patients who are anti-VEGF failures have the
presence of arteriolarized subretinal neovascularization and/or polypoidal vasculopathy.
PRIMARY anti-VEGF failures were found in the anti-VEGF resistant group and required
combination therapy with intravitreal anti-VEGF medications , intravitreal triamcinolone
acetonide and PDT to achieve complete leakage resolution. SECONDARY failures were
found in the anti-VEGF DEPENDENT group, however, less than half required combination
therapy. Therefore, sub-classifying Exudative ARMD patients by their response to
anti-VEGF medication and by their ICG presentation creates an opportunity to perform
primary combination therapy which obviates the need for suboptimal monthly antiVEGF monotherapy.
CR: M.H. Nelson, Novartis Phamaceuticals, Inc., F; Novartis Pharmaceuticals, Inc., C;
Novartis Pharmaceuticals, Inc., R.
Support: None
3501 - 4:15PM
Multifactorial Contributions of Complement Initiation Pathways to Mouse
Laser-Induced Choroidal Neovascularization
3502 - 4:30PM
Regulation of the Master Anti-Oxidant Transcription Factor, Nrf2, Through a
Redox-Sensing Cysteine in the Ubiquitin Conjugating Enzyme, UbcM2
B. Rohrer1A, B. Coughlin1A, Q. Long1A, G.S. Gilkeson1B, S. Tomlinson1C, K. Takahashi2,
V.M. Holers3. AOphthalmology, BMedicine, CImmunology, 1Medical University of
South Carolina, Charleston, SC; 2Massachusetts General Hospital; Program of
Developmental Immunology, Harvard Medical School, Boston, MA; 3Medicine,
University of Colorado Health Sciences Center, Denver, CO.
S. Plafker, K.S. Plafker, L. Nguyen, M. Barneche, S. Mirza. Cell Biology, Univ of
Oklahoma Health Sciences Center, Oklahoma City, OK.
Purpose: Human genetic studies have demonstrated that polymorphisms in different
complement proteins each increase the risk for developing AMD. There are three
pathways of complement activation, classical (CP), alternative (AP), and lectin (LP),
which all activate a final common pathway. The proteins encoded by the risk genes
participate in the AP, CP or in the final common pathway. Here we tested which
pathway is essential in mouse laser-induced CNV.
Methods: CNV was analyzed using single knockouts (i.e., eliminating one complement
pathway at a time), followed by double knockouts, in which only the AP pathway
is present and the CP and LP are disabled, using molecular, histological and
electrophysiological readouts.
Results: First, single-gene knockouts were analyzed and compared to wildtype
mice; C1q-/- for CP, MBL-A/C-/- for LP, and CFB -/- for AP activation. Six days after the
laser-induced lesion, mice without a functional AP had reduced CNV progression
(P<0.001) and preserved ERG amplitudes, whereas those without a functional CP
or LP were indistinguishable from the wild type controls (P>0.3). Second, doublegene knockout mice were used to investigate whether AP signaling is sufficient for
triggering C3 activation and CNV development. While C1q-/- MBL-A/C-/- mice, having
only a functional AP, were found to be sensitive to developing CNV, the size of the
lesion was reduced when compared to C57BL/6 mice (P<0.05).
Conclusions: The analysis of complement initiation pathways in mouse laser-induced
CNV allows for the following conclusions. Comparing the single pathway knockouts
with those having only a functional AP showed (1) that the AP activation is necessary
but not sufficient for injury; and (2) that the LP and CP can compensate for each other,
requiring elimination of both to reveal an effect. While these data demonstrate a
unique role of the AP in the generation of complement-dependent injury in the RPE
and choroid, CP- and LP-triggered complement activation appears to contribute to the
disease. Improving our understanding of the local regulation of this pathway in the
retina is essential to developing improved treatment approaches for AMD.
CR: B. Rohrer, None; B. Coughlin, None; Q. Long, None; G.S. Gilkeson, None; S.
Tomlinson, None; K. Takahashi, None; V.M. Holers, Taligen Therapeutics, F; Taligen
Therapeutics, E; Taligen Therapeutics, P.
Support: NIH grants HL082485, UO1AI074503 and 1R21AI077081. Foundation Fighting
Blindness and Research to Prevent Blindness.
Purpose: Age-related macular degeneration (AMD) is a leading cause of vision
impairment and loss in the elderly. Clinical studies and animal models support
that chronic oxidative stress, which arises from an imbalance in the production and
elimination of reactive oxygen species (ROS), is a major etiological factor of AMD. Our
long-term goal is to develop strategies that protect the retina from the damaging effects
of ROS and AMD development. Our work focuses on the retinal pigment epithelium
(RPE). This cell layer is highly susceptible to ROS damage and atrophy of the RPE has
been implicated as a precipitating event for AMD. The RPE employs a host of defense
mechanisms to counter chronic oxidative stress and the transcription factor, Nrf2, is
a central coordinator of these defenses. Nrf2 induces the expression of enzymes that
neutralize intracellular ROS. Yet, a complete molecular understanding of how Nrf2
stability and function are regulated in RPE cells remains to be elucidated.
Methods: A combination of recombinant pulldowns, co-immunoprecipitations,
transcription assays, siRNA studies and mutant over-expression studies were
used.
Results: We have discovered a novel mechanism of Nrf2 regulation. We find that
UbcM2, a ubiquitin conjugating enzyme present in RPE cells, stabilizes and induces
the transcriptional activity of Nrf2 in a redox-dependent fashion. The mechanism
underlying this effect resides in a unique cysteine residue of UbcM2. To mimic oxidation
of this cysteine, we changed the cysteine to a phenylalanine. This mutant enzyme
constitutively activates Nrf2 and is currently being explored for its capacity to amplify
the endogenous anti-oxidant system in a mouse model of AMD. Complementary siRNA
studies revealed that UbcM2 knockdown in RPE cells prevents Nrf2 stabilization in
response to oxidative stress.
Conclusions: These findings support a model in which oxidation of a redox-sensing
cysteine in UbcM2 results in the enhanced activity of Nrf2. This work represents a new
paradigm by which components of the ubiquitin proteolytic system protect vulnerable
cells of the retina from the deleterious effects of oxidative stress. We anticipate that
this avenue of investigation will ultimately provide a molecular rationale for the
development of UbcM2-based ocular gene therapy for the treatment of AMD.
CR: S. Plafker, None; K.S. Plafker, None; L. Nguyen, None; M. Barneche, None; S.
Mirza, None.
Support: NIH Grant P20 RR024215-03, AHAF grant, Karl Kirchgessner Foundation
grant, OCAST grant
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3499-3502
Tuesday, May 4, 3:45 PM - 5:30 PM Grand H Paper Session Program Number Range: 3499 - 3505
372. Hot Topics and Novel Concepts in AMD Research
Organizing Section: RC
3503 - 4:45PM
Implication of the Small-G Protein Rac-1 in Amyloid Beta-Induced Oxidative
Stress and Retinal Cells Toxicity
3504 - 5:00PM
Peptide Hormone Hepcidin Regulates Retinal Iron Homeostasis
F. Lamoke1, M. Labazi1, F. Scarinci2, G. Ripandelli2, D.M. Marcus3, G. Buccafusco1, G.
Liou1, M. Bartoli1. 1Department of Ophthalmology, Medical College of Georgia,
Augusta, GA; 2IRCCS Fondazione GB Bietti, Rome, Italy; 3Suotheastretina, Augusta,
GA.
Purpose: Deposition of amyloid beta peptides (A-beta) in retinal drusen has been
implicated in the pathogenesis of age-related macular degeneration (ARMD). We have
previously shown that retinal overexpression of A-beta provokes time-dependent and
progressive retinal tissue damage and this effect correlated with induction of oxidative
stress. The small G protein Rac-1 is upstream a number of cellular responses to stress
conditions including generation of reactive oxygen species (ROS) and induction of
pro-inflammatory factors. In this study we determined Abeta effects in promoting
activation of Rac-1 in retinal tissue as well as in retinal pigmented epithelial cells
(RPE) exposed to Abeta.
Methods: Transgenic mice overexpressing amyloid precursor protein (APP) as well
as pre-senilin 1 (PS1) were used as model of Abeta-mediated retinal tissue injury.
Cultures of ARPE19 exposed to oligomers of Abeta 1-42, or the reverse peptide Abeta
42-1, were used as in vitro model of retinal cell toxicity. Rac-1 activity was assessed
by measuring, by immunoprecipitation analysis, ratio of Rac-1/ GTP binding and
by determining Rac-1 translocation to the plasma membrane by cell fractionation
and Western analysis. Rac-1 inhibition was achieved by adenovirus-mediated cell
transduction of the inactive mutant Rac-1N17. ROS production was determined by
fluorimetric assay using the fluorescent probe dichlorofluorescein (DCF).
Results: Exposure of ARPE19 cells to Abeta 1-42 oligomers stimulated Rac-1 binding to
GTP as well as its translocation to the plasma membrane. Overexpression of Rac-1N17
prevented Abeta-induced production of ROS as measured by DCF-based fluorimetric
assay. Finally, immunoprecipitation studies revealed that ratio of Rac-1/GTP (active
Rac-1) was increased in the retinal tissue of mice overexpressing Abeta and this
correlated with enhanced production of ROS, thus confirming the in vitro data.
Conclusions: Activation of the small G protein Rac-1 in the retinas of mice
overexpressing Abeta as well as in cultured RPE exposed to Abeta 1-42 oligomers may
represent a critical initial step in Abeta induction of pro-oxidant and pro-inflammatory
effects at both retinal cells and tissue levels.
CR: F. Lamoke, None; M. Labazi, None; F. Scarinci, None; G. Ripandelli, None; D.M.
Marcus, None; G. Buccafusco, None; G. Liou, None; M. Bartoli, None.
Support: Progetto Finalizzata Italian Ministry of Health - IRCCS Fondazione G.B.
Bietti - Dept. of Ophthalmology, Medical College of Georgia
Y. Song1, M. Hadziahmetovic1, A. Hunter1, J. Iacovelli1, N. Haddad1, S. Vaulont2, J.L.
Dunaief 1. 1Dept of Ophthalmology, University of Pennsylvania, Philadelphia, PA;
2
Institut Cochin, Université Paris Descartes, Paris, France.
Purpose: Age related macular degeneration (AMD) is the leading cause of blindness in
the elderly. Elevated iron levels in AMD retinas and early onset retinal degeneration in
mice and humans with hereditary iron overload suggest that iron induced oxidative
stress may exacerbate the disease. Studies on retinal iron regulation may explain how
iron accumulates in the retina and suggest novel therapeutic modalities. The iron
regulatory hormone hepcidin is secreted by the liver to regulate iron import in the
intestine. Here, we test whether hepcidin, which is produced by the retina (GnanaPrakasam, Biochemi J, 2008) may regulate retinal iron import.
Methods: Hepcidin knockout mouse retinas were studied by histology,
immunohistochemistry, Perls’ stain for iron, atomic absorption spectrophotometry
to quantify iron, and qPCR for iron regulatory genes. Hepcidin mRNA levels were
also assessed by qPCR in mice with retinal iron overload resulting from mutation in
ceruloplasmin and hephaestin or from iron injection into the eye. Hepcidin regulatory
proteins ERK and SMAD were also assessed by Western analysis following intraocular
iron injection.
Results: Hepcidin knockout mice have age-dependent retinal iron accumulation and
retinal degeneration with some features of AMD. Retinal hepcidin production was
increased in mice with elevated retinal iron levels, following ERK phosphorylation.
Retinal levels of hepcidin’s target, the iron exporting protein ferroportin, were
increased in hepcidin knockouts and decreased when hepcidin was upregulated by
iron injection into the eye.
Conclusions: Hepcidin regulates not just systemic iron levels but also functions
locally to control retinal iron homeostasis. Upregulated by IL6, hepcidin may cause
iron dysregulation in diseases involving retinal inflammation.
CR: Y. Song, None; M. Hadziahmetovic, None; A. Hunter, None; J. Iacovelli, None; N.
Haddad, None; S. Vaulont, None; J.L. Dunaief, None.
Support: NIH EY015240, International Retina Research Foundation, American Health
Assistance Foundation, Mackall Foundation Trust, FM Kirby Foundation
3505 - 5:15PM
Complement Fragment C5a Activates Retinal Pigment Epithelial Cells and
Contributes to the Pathogenesis of Age-Related Macular Degeneration
M. Hu, B. Liu, S. Chakrabarty, M. Casady, R.B. Nussenblatt. Laboratory of
Immunology, National Eye Institute, Bethesda, MD.
Purpose: To investigate complement fragment C5a’s effects on retinal pigment
epithelial (RPE) cells and how these effects may play a role in the pathogenesis of
age-related macular degeneration (AMD).
Methods: Human adult RPE and ARPE-19 cells were used in this study. An MTT assay
was used to detect cell growth and viability. A real-time polymerase chain reaction was
used to detect cytokine mRNA expression. C5a was added to RPE-PBMC co-cultures
and a tritiated thymidine incorporation assay was used to detect cell proliferation.
Results: C5a slightly inhibited RPE cell growth. C5a also down-regulated chemokine
receptors CX3CR1 and CXCR5 expression on RPE cells by 24 hours. In addition, C5a
abrogated RPE cells’ suppressive effects on T cells and this abrogation was reversible
with a C5aR antagonist. These results are seen in both primary human RPE cells
and ARPE-19 cells.
Conclusion: Our study demonstrates that C5a decreased RPE cell viability, a
finding that may explain the geographic atrophy seen in “dry” AMD. C5a downregulated certain chemokine receptors, which may lead to dysfunction of leukocyte
trafficking and decrease the elimination of macular debris. We provide evidence
that C5a abrogated RPE cells’ suppressive effects on T cells, which could increase
inflammation in the eye. Together, these findings suggest C5a might play a role in
the pathogenesis of AMD.
CR: M. Hu, None; B. Liu, None; S. Chakrabarty, None; M. Casady, None; R.B.
Nussenblatt, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3503-3505
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3689 - A495
3D Visualization and Assessment of Cone Photoreceptor Protection in Rodent
Model of Retinitis Pigmentosa by Antioxidant
3690 - A496
Neuroprotective Effect of an Anti-Oxidant, Lutein, on Light-Induced Retinal
Degeneration
R. Lai, G. Wu, D. Sao, J. Edelman. Biological Sciences, Allergan, Inc, Irvine, CA.
M. Sasaki1,2, Y. Ozawa1,2, T. Kurihara1,2, S. Kubota1,2, K. Yuki1,2, K. Noda1,3, S. Kobayashi4,
S. Ishida1,3, K. Tubota2. 1Laboratory of Retinal Cell Biology, Keio University School of
Medicine, Tokyo, Japan; 2Department of Ophthalmology, Keio University School
of Medicine, Tokyo, Japan; 3Department of Ophthalmology, Hokkaido University
Graduate School of Medicine, Hokkaido, Japan; 4Wakasa Seikatsu Co., Kyoto, Japan.
Purpose: Retinitis pigmentosa (RP) is a group of diseases characterized by genetic
mutations leading to rod photoreceptor cell death and subsequent loss of cone
photoreceptors. Previously, we had found that antioxidants such as α-tocopherol,
ascorbic acid, and α-lipoic acid were effective in preserving cone photoreceptors in
the heterozygous transgenic S334ter RP rat model. In this study, we established a
method to automate the cone photoreceptor cell quantification by combining Confocal
microscopy with Imaris 3D computer software. We then employed another antioxidant,
N-acetylcysteine (NAC), and tested its effectiveness in the homozygous S334ter RP
rat model, which exhibits faster onset and a more severe form of photoreceptor
degeneration.
Method: 1m and 2m old homozygote S344ter rats were fed with drinking water
containing 2mg/ml or 5mg/ml NAC. At 3m and 4m time points, retinas were isolated
from NAC treated animals and age-matched S334ter control rats and stained with
flourescent tagged PNA. Confocal microscopy was set up to detect and automatically
image cone photoreceptors in the retina, which was flatmounted with photoreceptor
side up. The cone photoreceptors were visualized and quantified using Imaris software
at various regions of the retina. Additionally, retinas isolated from the contralateral
eyes were processed, sectioned, H&E stained and evaluated for total photoreceptor
cell loss.
Result: Between days 30 and 120, there was significant loss of photoreceptors in
these homozygous S334ter rats. The number of photoreceptor nuclei was reduced to
about 1 to 2 rows, with significant loss of photoreceptors observed in the superior
retina. The degeneration of the cone photoreceptors was characterized by islands of
areas lacking PNA staining with loss of cone photoreceptors observed throughout
the superior retina and diffusely in the nasal and inferior regions. In animals treated
with NAC, the number of cone photoreceptors was significantly higher than in the
control animals at each time point. By day 120, NAC treated animals had 35% more
cone photoreceptors in the superior retina than the control S334ter rats.
Conclusions: Antioxidant NAC administrated orally was found to significantly
reduce the cone cell photoreceptor death in the homozygous transgenic S334ter rats.
Combined with previous data, this strongly suggests that oxidative stress plays a
major role in cone photoreceptor death in retinitis pigmentosa and that antioxidants
may be a potential therapy for retinitis pigmentosa.
CR: R. Lai, Allergan, E; G. Wu, Allergan, E; D. Sao, Allergan, E; J. Edelman, Allergan,
E.
Support: Allergan, Inc.
3691 - A497
Valproic Acid Prevents Photoreceptor Cell Apoptosis in Retinitis Pigmentosa
Mice by Increasing Bcl-2 Expression
Purpose: To investigate the neuroprotective effect of lutein on retinal neural damage
caused by photo-oxidative stress.
Methods: Mice were fed with laboratory chow with or without 0.1% lutein and exposed
to 5,000 lux fluorescent light for 3 hours. Retinal damage was estimated by analyzing
(1) number of TUNEL positive cells, (2) outer nuclear layer (ONL) thickness, and (3)
retinal function was measured by electroretinography (ERG).
Results: Light exposure induced increase in TUNEL positive cells in ONL, leading
to the reduction of ONL thickness and a-wave amplitude in ERG, as previously
reported. (1) However, lutein diet decreased the light-induced apoptosis in ONL
by 43.7% of the control (p<0.01). (2) Consistently, Lutein diet significantly avoided
the reduction of ONL thickness (p<0.05). (3) Furthermore, mice fed with lutein diet
showed higher level of a-wave amplitude in ERG than those fed with control chow
after light exposure (p<0.01).
Conclusions: Lutein contributed to preservation of better visual function by protecting
photoreceptor cells from apoptosis after light exposure. Lutein has a neuroprotective
effect in light-induced retinal degeneration.
CR: M. Sasaki, Wakasa Seikatsu Co., F; Y. Ozawa, Wakasa Seikatsu Co., F; T.
Kurihara, None; S. Kubota, None; K. Yuki, None; K. Noda, None; S. Kobayashi,
Wakasa Seikatsu Co., E; S. Ishida, None; K. Tubota, None.
Support: None
3692 - A498
Ciliary Neurotrophic Factor-Induced Changes in Retinal Müller Cells
V.P. Sarthy, C. Shum. Ophthal-Feinberg Med Sch, Northwestern University,
Chicago, IL.
L. Yang. Ophthalmology, Peking Univ First Hosp, Beijing, China.
Purpose: To observe the neuroprotective effect of valproic acid (VPA) on photoreceptor
cells in retina pigmentosa mice and to study the mechanism of the protection.
Methods: 48 neonatal C3H/HeJ mice were divided into two groups. One group
received daily intraperitoneal (i.p.) injection of valproic acid (100mg/Kg·d), and the
other received injection of same volume of saline. Mice were sacrificed on 7, 14, 21,
and 28 days separately. Eyes were taken and retinas were sectioned. Histochemistry,
TUNEL and immunofluorescence were conducted to analyze the retinal histology
and apoptosis. Real-time PCR and western blot were used to measure the expression
of Bcl-2 in retina.
Results: The differentiation of inner and outer nuclear layers was finished on day 7
and photoreceptor cells began to loss gradually in both groups. There is no significant
difference on the outer nuclear layer thickness and the count of photoreceptor cells
between two groups on day 7. The outer nuclear layer thickness and the count of
photoreceptor cells were higher in VPA group than in control group at other time
point after day 7. In the control group photoreceptor cell apoptosis began on day 7
and peaked on day 14, but in VPA group the photoreceptor cell apoptosis began on
day 14 and gradually decreased. The apoptotic cells count were much lower in VPA
group than in control on any time point. There was significantly difference. The mRNA
transcription level and the expression of Bcl-2 deceased slowly in both group, while
it is higher in VPA group than in control.
Conclusions: Valproic acid may prevent photoreceptor cell apoptosis in the early
stage of retina pigmentosa in mice, although it cannot fully stop the onset of retinal
degeneration. The mechanism of apoptosis prevention maybe associated with the
increasing of Bcl-2 expression.
CR: L. Yang, None.
Support: NationalNatural Science Foundation of China (30571987)
Purpose: Ciliary neurotrophic factor (CNTF) is a neuroprotective agent known to
retard retinal degeneration in several animal models of retinitis pigmentosa. The
molecular mechanisms underlying CNTF-mediated neuroprotection are currently
not understood. In a recent microarray analysis of GFP+-Müller cells flow-sorted
from CNTF-injected eyes, we were surprised to find that CNTF treatment leads to
transcriptional activation of several neurotrophin and proinflammatory cytokine
genes in Müller cells. One or more of these might be responsible for the neuroprotective
effect of CNTF. The study, however, did not distinguish genes that are directly induced
by CNTF from those induced by secondary factors acting back on Müller cells. The
goal of the present study was to examine the direct effect of CNTF on Müller cells.
Methods: We investigated the effect of CNTF on an established Müller cell line, rMC-1.
Actively growing rMC-1 cultures were treated with 50 nM CNTF for periods ranging
from 15 to 60 min. RNA was isolated and used to determine transcriptional profiles
of known growth factors, cytokines and neurotrophic factors using commerciallyavailable, quantitative real-time PCR array kits (SuperArray Bioscience Corporation,
Frederick, MD).
Results: We found that rMC-1 cells treated with CNTF expressed only a small number
of growth factors and cytokines or their receptors. At 15 min, the genes induced were
(-fold): CxCl1, 3.1; IL10, 4.9; CCl 2, 2.0; CxCR5, 2.4; IL6Rα, 2.0; IL2Rγ, 2.8 and STAT3, 2.0.
At 30 min, the profile changed to: IL6, 3.5; CxCl1, 2.5; IL10, 3.9 and Myc, 2.5. Finally, at
60 min, gene expression changes included: IL6, 3.6; CCl 2, 7.1; CCl7, 2.5; Myc, 2.5; BCl6,
2.2 and STAT3, 2.0. In addition, Fos was strongly induced in rMC-1 cells at all times:
5.9-fold at 15 min, 12.4-fold at 30 min and 4.3-fold at 60 min.
Conclusions: Our studies lead to the surprising finding that CNTF treatment leads
to transcriptional activation of only a small number of cytokines in Müller cells.
This result suggests that in situ, Müller cell response to CNTF is complex, and that it
involves both primary and secondary effects, which strongly increase the repertoire
of gene expression changes in Müller cells.
CR: V.P. Sarthy, None; C. Shum, None.
Support: NIH Grant EY019325, RPB Inc.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3689-3692
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3693 - A499
Neuro and Vascular Protective Effect of FeTPPs in NMDA-Model: Similarity to
Diabetes
M.M.H. Al-Gayyar, M.A. Abdelsaid, S. Matragoon, B.A. Pillai, A.B. El-Remessy.
Program in Clinical and Experimental Therapeutics, College of Pharmacy,
University of Georgia, Augusta, GA.
Purpose: We have previously shown neuro and vascular protective effects of
decomposing peroxynitrite using FeTPPs in models of diabetic and oxygen-induced
retinopathy. Intravitreal-injection of N-methyl-D-aspartic acid (NMDA) is an
established model to study acute retinal ganglion cell degeneration. This study will
elucidate the role of peroxynitrite in activating the apoptotic ASK-1 pathway and
alterating thioredoxin interacting protein (TXNIP) and thioredoxin (Trx), a major
cellular antioxidant and anti-apoptotic protein. Furthermore, we will investigate
the neuro/vascular protective effects of FeTPPs on glial activation and capillary
degeneration in NMDA model.
Methods: SD Rats received intravitreal-injection of either NMDA, NMDA and FeTPPs
or the control N-methyl-L-aspartic acid (NMLA). For short term (1 day), retinal
neurotoxicity was examined by TUNEL assay and ganglion cell (GC) count. Glial
activation and peroxynitrite were assessed by immunohistochemistry. Expression of
TXNIP, pp-38 and PARP and interaction of Trx/TXNIP and Trx/ASK-1 were analysed
by Western-Blot. For long term effects (7days), acellular capillaries and pericytes were
counted in retinal trypsine digest.
Results: Intravitreal-injection of NMDA caused significant increases in neuronal cell
death and reduced GC count compared with NMLA. This effects was associated with
enhanced nitrotyrosine and TXNIP expression which disrupt Trx-ASK-1 inhibitory
complex leading to release of ASK-1. Activation of ASK-1 proapoptotic pathway was
evident by increases in p-p38 and PARP expression in NMDA-injected retinas. NMDA
causes glial activation and capillary degeneration as indicated by prominent GFAP,
1.7-fold increase in acellular capillaries and 25 % reduction in number of preicytes.
Treatment with FeTPPs blocked these effects in NMDA-injected rats.
Conclusion: NMDA-induced retinal neuro/vascular injury is mediated in part by
increasing peroxynitrite and reducing the antioxidant defense which in turn activates
ASK-1 apoptotic pathway. In addition to neurotoxicity, NMDA-model can be useful tool
to study glial activation and capillary degeneration, hall marks of diabetic retinopathy
in much shorter time.
CR: M.M.H. Al-Gayyar, None; M.A. Abdelsaid, None; S. Matragoon, None; B.A.
Pillai, None; A.B. El-Remessy, None.
Support: SDG from AHA and CDA from JDRF
3694 - A500
Neuroprotection of Mouse Retina Ganglion Cells by N-Acylethanolamines
R.S. Duncan, H. Xin, P. Koulen. Ophthalmology, Univ MO - Kansas City Sch of Med,
Kansas City, MO.
Purpose: N-Acylethanolamines (NAEs) are lipids upregulated in response to cell
and tissue injury and some have been shown to be involved in neuroprotection.
Arachidonylethanolamide is a well characterized NAE that is an endogenous ligand
at cannabinoid and vanilloid receptors. The function and molecular target of other
NAEs that do not bind to these receptors, such as NAE 18:2, are unclear and have thus
prompted us to measure their neuroprotective properties in the retina. We hypothesized
that NAE 18:2 protects retinal ganglion cells from glutamate excitotoxicity.
Methods: Whole retinas were isolated from C57BL/6 mice and maintained as
organotypic cultures. Retinas were treated with NAE 18:2 over a range of physiologically
relevant concentrations for 6 hours followed by addition of an excitotoxic insult
with 100µM glutamate for 16 - 20 hours. Loss of neuronal viability and cell death
were assessed using terminal deoxynucleotidyl transferase (TdT)-mediated dUTP
nick-end labeling (TUNEL) histochemistry, cell specific immunohistochemistry and
fluorescence microscopy.
Results: Exposure of retinas to 100µM glutamate resulted in a dramatic increase in
retinal ganglion cell (RGCs) death. Preincubation of retinas with NAE 18:2 prior to
glutamate exposure resulted in a dose-dependent decrease in the number dead or
dying RGCs with high physiological concentrations reducing the number of dead or
dying RGCs to to that of the Glutamate vehicle control group.
Conclusions: The highly significant, dose-dependent reduction of glutamate-induced
RGC death by treatment with NAE 18:2 reveals a neuroprotective function for NAEs
that do not bind to cannabinoid and vanilloid receptors. These results provide a
rationale for the use of NAEs as potential therapeutic compounds in acute and chronic
neurodegenerative diseases of the retina.
CR: R.S. Duncan, None; H. Xin, None; P. Koulen, None.
Support: This study was supported in part by NIH grants EY014227, RR022570,
AG010485, AG022550 and AG027956 and the Felix and Carmen Sabates Missouri
Endowed Chair in Vision Research (P.K.).
3695 - A501
Protective Effect of Granulocyte Colony Stimulating Factor on Human Retinal
Endothelial Cell Injured by Oxidative Stress
3696 - A502
Β1-Integrin Activating Antibody HUTS-21 Exhibits Retinal Ganglion Cell
Neuroprotective Properties
H. Kojima, A. Otani, H. Nakamura, A. Oishi, Y. Usami, S. Nakagawa, N. Yoshimura.
Ophthalmology and Visual Sciences, Kyoto University Graduate School of
Medicine, Kyoto, Japan.
M.L. Bajenaru1, R.A. Santos1, B.A. Obesso1, R.G. Corredor2, E. Hernandez3, M.E. Fini4,
J.L. Goldberg5. 1Bascom Palmer Eye Institute, Miami, FL; 2Bascom Palmer Eye
Inst/Neurosci, University of Miami Miller Sch of Med, Miami, FL; 3Ophthalmic
Biophysics Center, Retina Group of Florida, Miami, FL; 4University of Southern
California, Los Angeles, CA; 5Bascom Palmer Eye Inst, University of Miami,
Miami, FL.
Purpose: Granulocyte colony stimulating factor (G-CSF) has been shown to have
direct protective effect in cerebral stroke model and light-induced retinal damage
model. In the present study, we investigated whether G-CSF has protective effect on
human retinal endothelial cell injured by oxidative stress and inhibits pathological
neovascularization in the mouse model of retinopathy of prematurity.
Methods: Human endothelial cells were exposed H 2O2 with or without G-CSF.
Apoptotic cells were detected with Annexin V staining. Oxygen induced retinopathy
(OIR) model of new born mice was used as a retinal angiogenesis model. G-CSF or
vehicle was injected systemically for consecutive 5 days prior to and during oxygen
exposure (between P6 and P10).To quantify the area of retinal neovasculareization
and vasoobliteration, we stained retinal flat mount with collagen IV at P17. The
neuroprotective effect was evaluated through electroretinography (ERG) and retinal
histology at P30.
Results: G-CSF decreased apoptotic cells by oxidative stress. Further, in mice model,
injection of G-CSF inhibits retinal pathological neovascularization and decrease the
area of vasoobliteration. The outer plexiform layer thickness was limited reduction,
and an electroretinogram confirmed the preservation of wave amplitudes in early
G-CSF-treated mice.
Conclusions: G-CSF protect retinal endothelial cells directly from oxidative stress and
inhibits pathological neovascularization and protects retinal function and structural
damage in the mouse of ROP. These findings may lead to a novel treatment strategy
for ischemic diseases of the retina.
CR: H. Kojima, None; A. Otani, None; H. Nakamura, None; A. Oishi, None; Y.
Usami, None; S. Nakagawa, None; N. Yoshimura, None.
Support: None
Purpose: RGC survival and neurite outgrowth are promoted by neurotrophins, and
extracellular matrix (ECM) molecules such as laminin. Degradation of laminin in the
ECM of RGC in experimental animal models relevant to retinal disease contributes
to RGC death. Integrins are the major ECM receptors. The purpose of this study is
to evaluate HUTS-21, a β1 integrin activating antibody as neuroprotective agent to
improve RGC regeneration after retinal ischemia reperfusion injury (RIRI).
Methods: Studies were performed in rat RGC cultures, and in a rat RIRI model. Purified
rat RGC cultures were prepared by immunopanning. Expression of β1 integrin, FAK,
and P-FAK was analyzed by immunofluorescence in RGC cultured on laminin, or polyD-lysine (PDL). RGC in culture were treated with β1 integrin activating, and blocking
antibodies, and FAK pharmacological inhibitors, genistein, and PP2. RGC survival,
and neurites were examined and counted by live cell imaging. RIRI was induced in
Sprague-Dawley rats by unilateral elevation of the intraocular pressure to 110 mm
Hg for 60 min. HUTS-21 activating antibodies were administered intravitreally in the
rat eye 30 min prior to RIRI. RGC loss was quantified in the retina after retrograde
labeling with FG. Expression and activation of β1 integrin, and FAK was determined
by western blotting in retinal extracts.
Results: We have previously identified β1 integrin and FAK as key regulators of the
integrin survival pathway, and neurite outgrowth in RGC. We showed this pathway is
disrupted after RIRI. When cultured on laminin RGC exhibited increased survival, and
neurite outgrowth. These properties correlated with an up-regulation of the integrin
survival pathway, and increased focal contacts on laminin, versus PDL. Treatment with
β1 activating antibodies HUTS-21 of RGC cultured on PDL mimics the neuroprotective
effect of laminin, and enhances the RGC survival, and neurite outgrowth. HUTS21 protective effect can be abolished by co-treatment with the FAK inhibitor PP2.
Intravitreal admistration of HUTS-21 in the rat RIRI model partially rescues RGC
death and prevents disruption of the β1 integrin survival pathway in RGC.
Conclusions: We have identified HUTS-21 as a potential neuroprotective agent that
promotes RGC survival, and regeneration after RIRI.
CR: M.L. Bajenaru, None; R.A. Santos, None; B.A. Obesso, None; R.G. Corredor,
None; E. Hernandez, None; M.E. Fini, None; J.L. Goldberg, None.
Support: AHA Grant 09SDG2280555
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3693-3696
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3697 - A503
Efficacy of Rho-Kinase Inhibition in Reducing Glial Reactivity and Promoting
Retinal Ganglion Cell (RGC) Survival in vitro
3698 - A504
Effects of Different Intravitreal Treatments on the Phagocytosis of Retinal
Dying Cells and Inflammation
A. Tura1, K.U. Bartz-Schmidt2, S. Henke-Fahle2. 1University Eye Hospital, Luebeck,
Germany; 2Centre for Ophthalmology, Tubingen, Germany.
G. Petrovski1A, E. Berenyi1B, M.C. Moe2, A. Vajas1A, L. Fesus1B, A. Facsko1A, A.
Berta1A. ADepartment of Ophthalmology, BDepartment of Biochemistry and
Molecular Biology, 1University of Debrecen, Debrecen, Hungary; 2Department of
Ophthalmology, Centre for Eye Research, University of Oslo, Oslo, Norway.
Purpose: To analyze the extent of Rho-kinase activity and the outcomes of Rho-kinase
inhibition in isolated RGCs and glial cells under neurotrophin (NT) and serum
deprivation, respectively.
Methods: Primary RGCs and glial cells were isolated from the retinas of newborn
rats by immunomagnetic separation and cultured with or without the Rho-kinase
inhibitor H-1152. The Rho-kinase activity was determined by an in vitro kinase assay
and the extent of apoptosis was analyzed by immunostaining, TUNEL assay, and by
measuring the neurite length. Cell proliferation in cocultures of astrocytes and Müller
cells (AMC) was detected by the MTT- and BrdU-tests whereas the reactivity of these
cells and the microglia seeded on fixed AMC was analyzed by immunostainings
and -blotting.
Results: NT deprivation resulted in an increase in Rho-kinase activity which was
associated with a considerable degree of axon retraction and caspase-3 cleavage in the
RGCs by 6h. However, the Rho-kinase inhibitor exerted a significant anti-apoptotic
effect which persisted after 24h. H-1152 also suppressed the proliferation of AMC and
reduced the upregulation of GFAP under serum deprivation possibly by interfering
with the Rho-kinase dependent phosphorylation of this protein. Likewise, the inhibitor
suppressed the upregulation of vimentin, endothelin, and fibronectin together with a
decrease in Rho-kinase activity. The serum-deprived AMC stimulated the activation
of microglia seeded on them as determined by the ameboid morphology the latter cell
type acquired despite being grown in an optimized medium. However, the microglia
seeded on serum deprived AMC treated with H-1152 exhibited a quiescent morphology,
suggesting that the Rho-kinase also regulates certain extracellular changes in the
activated AMC which in turn stimulate microglia activation.
Conclusions: The deprivation of NTs and serum results in an increase in Rho-kinase
activity in the RGC and AMC cultures, respectively. Accordingly, the inhibition of
Rho-kinase emerges as a promising approach for attenuating the glial reactivity and
promoting RGC survival under these unfavorable conditions.
CR: A. Tura, None; K.U. Bartz-Schmidt, None; S. Henke-Fahle, None.
Support: Fortüne 1765-0-0
Purpose: To study the effect of different intravitreal treatments on the phagocytosis
of dying retinal pigment epithelial cells and the inflammatory response associated
with it.
Methods: Two different death patterns were induced in vitro in ARPE-19 cells: death
through detachment from the extracellular matrix on polyHEMA coated surfaces
known as anoikis and UV-induced apoptosis. Two-colored phagocytic assays
were carried out where the phagocytes (human monocyte-derived macrophages)
engulfed the dying cells under different treatment modalities (triamcinolone (1uM),
bevacizumab (312.5ug/mL), pegaptanib (75ug/mL) and ranibizumab (125ug/mL)). Flow
cytometric analysis (FACS Calibur) was used to quantify the phagocytic process as
well as measure the released amounts of IL-1β, IL-6, IL-8, IL-10 and TNFalpha using
a Cytokine Bead Array.
Results: Macrophages engulfed the dying anoikic and apoptotic ARPE-19 cells at
a similar and increasing rate over 8 hours of co-incubation (11.2+/-1.7% and 10.5+/1.2% at 8 hours, respectively). Their phagocytic capacity increased by 2.1+/-0.3 times
during engulfment of both types of dying cells under triamcinolone treatment,
but remained unchanged for all other treatments. In the case of UV-induced dying
ARPE-19 cells, probably due to presence of secondary necrosis, increased IL-6 and
IL-1beta levels were detected which could be suppressed by triamcinolone, but not
the other three treatments.
Conclusions: The macrophage mediated clearance of different dying ARPE-19 cells
can serve as a good in vitro model for studying wet AMD and for testing different
pharmacological and inflammatory aspects of this process.
CR: G. Petrovski, None; E. Berenyi, None; M.C. Moe, None; A. Vajas, None; L. Fesus,
None; A. Facsko, None; A. Berta, None.
Support: Mecenatura Grant, University of Debrecen, Hungary
3699 - A505
FK506 Binding Protein 51 (FKBP51) Mediated Neuroprotection Against
Glutamate Excitotoxicity in 661w and HT22 Cell Culture
3700 - A506
Neuroprotection of Retinal Ganglion Cells in P23H Rats: A Way to Improve
Resolution in Retinal Prosthesis or Optogenic Approach
D.R. Daudt, III, T. Yorio. Pharmacol & Neurosci, UNT Health Science Center, Fort
Worth, TX.
L. Cadetti1,2, H. Lorach1, J. Degardin1, J.A. Sahel1,2, S. Picaud1,2. 1Institut de la Vision,
INSERM, Paris, France; 2Universite’ Pierre et Marie Curie (UPMC), Paris, France.
Purpose: Glaucoma is a progressive optic neuropathy characterized by loss of
retinal ganglion cells (RGC) and optic nerve degradation. Existing treatments focus
on lowering intraocular pressure (IOP); however, vision loss may still progress.
Neuroprotectant drugs are useful as an adjunct approach in preventing further loss of
RGCs; though, candidate genes are lacking. FK506, a widely used immunosuppressant
drug, has profound neuroprotective and neuroregenerative properties throughout the
central nervous system, including the eye. FK506 achieves these properties through
interaction with FK506 Binding Proteins (FKBP), including FKBP51. Currently, we
investigated FKBP51’s neuroprotective properties in two neuronal cell lines.
Methods: 661w cells were treated with various concentrations of FK506 and analyzed
through western blot analysis to determine changes in protein levels of FKBP51, NFκB,
and activated NFκB. Additionally, 661w and HT22 cell cultures were either stably
transfected with FKBP51 overexpression vectors or treated with FKBP51 targeted siRNA
and then challenged with glutamate-induced neurotoxicity. Cell death and apoptosis
were measured through calcein-AM/propidium iodide cell-surivival assay, caspase
3 dectection kit, and activated BAX protein levels. Furthermore, NFκB and activated
NFκB were analyzed as a factor of FKBP51 protein levels for each condition.
Results: FK506 increased FKBP51 protein levels in a stepwise manner. FK506 also
increased NFκB protein levels; however, it was difficult to determine activated NFκB
protein levels. The 661w and HT22 cell cultures, stably transfected cells overexpressing
FKBP51 and FKBP51 targeted siRNA caused changes to protein levels and cell
viability.
Conclusions: This data provides strong evidence that FKBP51 promotes antiapoptotic action in neuronal cell cultures when challenged with glutamate-induced
neurotoxicity. Additionally, it suggests a possible neuroprotective mechanism of
FKBP51 through NFκB.
CR: D.R. Daudt, III, None; T. Yorio, None.
Support: NIH Grant T32 AG020494
Purpose: Following photoreceptor loss, restoring vision with prosthetical microchip
devices or the optogenetic approach in diseases like retinitis pigmentosa or macula
degeneration requires a functional inner network or at least Retinal Ganglion Cells
(RGCs) connected to higher visual centres. Nonetheless, a significant loss of GCs was
observed in advanced retinitis pigmentosa (Humayun, 1999) as well as in different
animal models of the disease. We show that a small molecule (IDV007) pulled from
a screening on pure RGCs can prevent or slow down RGCs death after the complete
loss of photoreceptors.
Methods: P23H rats were administered IDV007 for 3 months because this molecule
was found to prevent RGC degeneration in pure retinal RGC culture. RGCs from
P23H rats and control rats were immunostained with an antibody directed against
Brn3A in both slices and retinal flat mount preparations.
Results: Rod photoreceptors degeneration is virtually complete in 6 months old
homozygous P23H rats whereas almost no photoreceptors are present in 9 months
old heterozygous and homozygous P23H rats. At 6 months, homozygous rats have
already lost 17 % of RGC (P<0.0315, two tailed T-test), and this loss is further increased
to 34% in 1 year old P23H rats (P<0.0001, two tailed T test). A similar loss was observed
in heterozygous P23H rats from 9 to 12 months. When IDV007 was administered to
heterozygous P23H rats, the RGC loss was slowed down. After 3 months, treated
animals had 17% more GCs than untreated ones (P<0.0111, two tailed T test).
Conclusions: In P23H rats, the RGC number decreases during and after photoreceptor
degeneration. Treatment with IDV007, which was identified for its RGC neuroprotection
in vitro, partially prevented the RGC loss in P23H rats. This study suggests that
improving the resolution of rehabilitating strategies (retinal prosthesis and optogenic
approach) could be achieved by preventing the RGC loss in patients affected by
retinitis pigmentosa.
CR: L. Cadetti, Patent, P; H. Lorach, None; J. Degardin, Patent, P; J.A. Sahel, FOVEA,
E; Patent, P; S. Picaud, FOVEA, E; Patent, P.
Support: FRM (Fondation pour la recherche médicale) , ANR Glaucome
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3697-3700
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3701 - A507
Stat3 is Essential for Chronic Stress-Induced Endogenous Protection of
Photoreceptors
3702 - A508
Modulation of Crystallin Network by Lipin1 is Associated With
Neuroprotection in the Mouse Retina
J.D. Ash1A, J. Wang1A, A.J. Chucair-Elliott2, Y. Ueki1B. AOphthalmology, BOklahoma
Center for Neuroscience, 1Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK;
2
Department de Biologia, Bioquimica y Farmacia, Universidad Nacional del Sur,
Bahia Blanca, Argentina.
E.E. Geisert, J.P. Templeton, N.E. Freeman-Anderson, C.W. Abner. Ophthalmology,
Univ of Tennessee Health Sci Ctr, Memphis, TN.
Purpose: We have previously found that gp130 activation, a common signaltransducing receptor for the IL-6 family of cytokines, is essential for stress-induced
endogenous protection of photoreceptors. Gp130 is believed to signal through three
signal transduction pathways including PI3K/Akt, Erk1/2, and the Jak/STAT3
pathways. The purpose of this study was to determine whether STAT3 and/or Akt
are required for endogenous protection of photoreceptors.
Methods: Retina-specific gp130 (gp130f/f; Chx10-cre+) and STAT3 (STAT3f/f ; Chx10-cre+)
knockout (KO) mice were generated using the Cre/lox system. Mice with total knockout
of Akt2 were also used. Endogenous protection was experimentally induced by
exposure to sublethal cyclic light (800 lux) for 6 days (preconditioning). Light damage
was induced with 3000 lux light for 4 hrs. Photoreceptor function and survival were
evaluated by electroretinography (ERG) and morphometric analyses. The effect of
gp130 or STAT3 loss on the rate of photoreceptor degeneration in a model of autosomal
dominant retinitis pigmentosa was assessed by crossing retina-specific gp130 KO
and STAT3 KO with VPP transgenic mice. STAT3 activation under chronic stress was
measured by Western blot.
Results: Wildtype mice and Akt2 knockout mice had robust preconditioning induced
protection from light damage as demonstrated by ERG and histology. In contrast,
mice lacking gp130 or STAT3 in the retina had significantly reduced protection of
photoreceptors. Mice lacking gp130 or STAT3 in the retina had normal photoreceptors
on their own. However, VPP mice lacking gp130 or STAT3 demonstrated significantly
faster degeneration than the parental VPP mice.
Conclusions: Our data show that the absence of gp130 or STAT3 in the retina impairs
stress-induced endogenous protection and increases sensitivity to both light- and
genetically-induced photoreceptor cell death. This study provides direct evidence
that gp130-STAT3 activation in the retina is essential for endogenous photoreceptor
protection from chronic stress. Data also suggests that activation of STAT3 can
compensate for the loss of Akt2, and that Akt2 is not playing a role in preconditioning
induced protection.
CR: J.D. Ash, None; J. Wang, None; A.J. Chucair-Elliott, None; Y. Ueki, None.
Support: R01 EY016459, P20 RR017703, P30 EY012190, Foundation Fighting Blindness,
and Research to Prevent Blindness
Purpose: In our ongoing efforts to define the molecular networks modulating neuronal
survival, we examine the effects of a candidate gene (Lpin1) previously identified
to affect neuronal survival and to alter genetic networks activated by optic nerve
crush (ONC).
Methods: Using ONC as a model of retinal injury we examined the effects of a naturally
occurring mutation in Lpin1 on ganglion cell survival. We counted the number of
neurons surviving 30 days after ONC in Lpin1 mutant mice (MT) and in control wildtype mice (WT). For this portion of the study we used 6 normal MT retinas along
with 6 ONC MT retinas. These were compared to counts from 5 normal WT retinas
and 6 ONC WT retinas. We also studied changes in gene expression using Illumina
microarray systems, comparing the Lpin1 MT with WT controls. The 6 groups (3
independent biological samples in each) include normal retinas (MT and WT), retinas
2 days after ONC (MT and WT), and 5 days after ONC (MT and WT).
Results: The mutation in Lpin1 provided significant neuroprotection with 49% of the
RGCs surviving in the MT mice and only 38% surviving in the WT control animals.
Our microarray analysis revealed that the crystallin network (Templeton et al., 2009
BMC Neuroscience 10:90-) was up-regulated in the MT mice and down-regulated in
the WT controls. Genes within this crystallin network include but are not limited
to: Cryaa, Cryab, Cryba1, Cryba2, Cryba4, Crybb1, Crybb2, Crybb3, Crygb, Crygc,
Crygn, Crygs, Grifin, Lim2, and Mip.
Conclusions: These results support the hypothesis that the upregulation of the
crystallin network is neuroprotective. Furthermore, it appears that we have identified
an upstream modulator of the crystallin network, Lipin1.
CR: E.E. Geisert, None; J.P. Templeton, None; N.E. Freeman-Anderson, None; C.W.
Abner, None.
Support: This project was supported by an unrestricted grant from Research to Prevent
Blindness, an NEI grant R01EY017841 and NEI Core Grant (EY13080).
3703 - A509
A Novel Mechanism for Photoreceptor Protection: Selective Apical Secretion of
αB Crystallin From Polarized Human RRE Cells
3704 - A510
Study of the Mechanism Involved in Lipopolysaccharide-Induced Protection
Against Light-Induced Retinal Damage in Rats
S. Parameswaran1, C. Spee1, S.J. Ryan1,2A, R. Kannan1, D.R. Hinton1,2B. 1Arnold and
Mabel Beckman Macular Research Center, Doheny Eye Institute, Los Angeles,
CA; AOphthalmology, BPathology, 2Keck School of Medicine of the University of
Southern California, Los Angeles, CA.
M.F. Lanzani, M. Bordone, J.J. López Costa, R.E. Rosenstein. Human Biochemistry,
University of Buenos Aires, Buenos Aires, Argentina.
Purpose: Our laboratory showed that the small heat shock protein αB crystallin, has
antiapoptotic properties in addition to its well known chaperone function. Our aim
herein was to identify αB crystallin as a secretory protein in human RPE cells and
characterize its asymmetry of secretion in human polarized RPE cells.
Methods: Early passage, confluent human fetal RPE cells grown in T75 flasks in DMEM
were switched to serum free medium and the extracellular medium was collected and
concentrated using filters. Exosomes were isolated using well established procedures.
Release of αB crystallin to the extracellular medium through exosomes was confirmed
by Western blot and immunogold transmission electron microscopy (TEM), using
CD63 as an exosome marker. Secretion was also examined in the presence of lipid raft
inhibitor (25µg/ml methyl-β-cyclodextrin) and classical protein transport inhibitor
brefeldin (BF 7µg/ml), tunicamycin (TM 5µg/ml) and exosome inhibitor dimethyl
amiloride (25µg/ml). Secretion of αB crystallin from the apical and basolateral domains
was determined in polarized RPE monolayers with TER >340 Ω.cm 2.
Results: Western blot analysis and immunogold TEM revealed that RPE cells secrete αB
crystallin into the extracellular medium. The secretion was by a nonclassical exosomal
pathway. Inhibiting lipid rafts reduced the αB crystallin secretion significantly (p<0.01
vs controls). However, transport inhibitors BF and TM did not affect secretion. αB
crystallin was predominantly secreted to the apical side toward the photoreceptors
and the neural retina with undetectable secretion to the basolateral, choroidal side.
In support, αB crystallin was found in the interphotoreceptor matrix (IPM) in retinal
sections by immunofluorescence and TEM.
Conclusions: Our data show that αB crystallin is secreted by RPE through a lipid
raft dependent exosomal pathway and the secretion is selective to the apical surface.
Secretion of αB crystallin into the IPM could serve to protect the photoreceptors
from injury.
CR: S. Parameswaran, None; C. Spee, None; S.J. Ryan, None; R. Kannan, None; D.R.
Hinton, None.
Support: NIH grants EY 02061, EY 03040 & grants from RPB & the Arnold & Mabel
Beckman Foundation
Objective: In a previous report, we showed that intravitreal injections of bacterial
lipopolysaccharide (LPS) induce a significant protection against light-induced retinal
damage. The aim of the present work was to analyze the mechanism involved in
this effect of LPS.
Methods: Male Wistar rats were intravitreously injected with 3 μg LPS in one eye
and vehicle in the contralateral eye. Fifteen min before and 6 h after LPS injection,
aminoguanidine (an inhibitor of inducible nitric oxide synthase, 100 mg/kg) was
intraperitoneally injected. 5-hydroxidecanoic acid (5HD, a mitochondrial K+ ATP
channel blocker, 40 mg/kg) was intraperitoneally injected 15 min before LPS,
whereas wortmannin (a phosphatidylinositol 3-kinase (PI3K) inhibitor, which supress
autophagy, 2µl, 0.2 mM) was injected in the vitreous. One day after injections, rats
were placed for 24 h in an open white acrylic box of 60 cm x 60 cm x 60 cm with 10
halogen lamps (10 V 50 W each) located on top. White light spectrum was used, and
lighting level was 10,000 lux. The lighting box was kept in an air-conditioned room,
and temperature was monitored and maintained at 24°C. All the animals received
food and water ad libitum. Subsequently, 7 or 14 days after light exposure, rats were
subjected to electroretinography and histological analysis.
Results: Constant light for 24 h induced a significant decrease in scotopic ERG
a- and b-wave amplitude. The light-induced decrease in both a- and b-wave
amplitude was significantly lower in eyes treated with 3μg LPS both at 7 and 14
days after light exposure. Moreover, significant alterations in the outer nuclear
and the outer plexiform layers and the pigment epithelium were observed in eyes
exposed to constant light. The injection of LPS reduced light-induced photoreceptor
degeneration and protected the outer plexiform layer and the pigment epithelium.
The injection of wortmannin (but not of aminoguanidine or 5HD) prevented
the functional and histological protection induced by LPS against light-induced
damage.
Conclusions: These results suggest that functional and histological protection against
the deleterious effects of constant light provided by LPS involves a PI3K/autophagy
dependent mechanism.
CR: M.F. Lanzani, None; M. Bordone, None; J.J. López Costa, None; R.E. Rosenstein,
None.
Support: anpcyt,conicet,uba
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3701-3704
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3705 - A511
Protection of Cone Photoreceptors by Oncostatin M (OSM) in the Transgenic
Rats Carrying the S334ter Rhodopsin Mutation
3706 - A512
NQO2: A Novel Target to Mediate Neuroprotection Against Retinal
Degeneration
X. Xia, Y. Li, Z. Wang, D. Huang, L. Luo, R. Wen. Bascom Palmer Eye Institute,
University of Miami, Miami, FL.
C.K. Hwang, H. Zhou, R. Haque, P.M. Iuvone. Ophthalmology, Emory University,
Atlanta, GA.
Purpose: Our previous study showed that loss of cone outer segments (COS) is an
early sign of cone degeneration. In this study, we examined the effects of OSM, a
member of the IL-6 family of cytokines, on the secondary cone degeneration in the
transgenic rats carrying the murine rhodopsin mutation S334ter.
Methods: The left eyes of S334ter rats were intravitreally injected with OSM (10 µg in 3
µl PBS) at postnatal day (PD) 20 and the right eyes with 3 µl PBS. Eyes were collected 10
days later. Retinas were stained with FITC-conjugated PNA (Peanut agglutinin) which
specifically labels COS, flat-mounted on slides, and examined by confocal microscopy.
Quantitative analyses were performed by counting PNA-positive cells with software
MBF-ImageJ and then calculating the densities of PNA-positive cells.
Results: Many round and irregularly shaped small PNA-negative areas were found
in PBS-treated controls, similar to what was found in untreated retinas, indicating
loss of cone outer segments. In eyes treated with OSM, however, the PNA-negative
areas were much smaller and in many cases completely disappeared. Quantitative
analyses showed that PNA-positive cells are 20% more in the retinas treated with
OSM than in the PBS-treated retinas (P=0.01 Student t-test).
Conclusions: Our results demonstrate that OSM significantly protects cone cells
from degeneration, similar to what was found in CNTF-treated retinas we reported
previously. Since OSM and CNTF are members of the IL-6 family of cytokines and
they share the same receptor complex of LIFRβ and gp130, our data suggest that the
protective effect of CNTF and OSM is mediated through the same mechanism.
CR: X. Xia, None; Y. Li, None; Z. Wang, None; D. Huang, None; L. Luo, None; R.
Wen, None.
Support: NIH grant EY-018586, JEK grant 08KN-09, Hope for Vision, Foundation
fighting blindness, NIH center grant P30-EY014801, and RPB.
Purpose: Oxidative stress-induced damage to the retinal pigment epithelium (RPE) is
thought to be involved in the development of AMD. In this study, we test the hypothesis
that NRH:quinone oxidoreductase 2 (NQO2) inhibitors protect human RPE cells
(ARPE-19) from oxidative stress. In addition, we determine if NQO2 inhibitors alter
the level of the tumor-suppressor protein, p53, which may limit the clinical utility of
NQO2 inhibitors in the treatment of AMD.
Methods: Total protein lysate of ARPE-19 cells was probed with antibody for NQO2.
ARPE-19 cells were treated daily with melatonin, the NQO2-specific inhibitor,
5-methoxycarbonylamino-N-acetyltryptamine (MCA-NAT), or vehicle for five days
and then exposed to 600 µM H2O2 for 16 hours. Cell viability was then determined by
measuring intracellular conversion of resazurin to resorufin (Cell Titer Blue Viability
Assay, Promega). Total protein lysate of ARPE-19 cells treated for 3 days with 10µM
MCA-NAT, 10µM resveratrol (a potent inhibitor of NQO2), or vehicle was probed
with antibody for p53.
Results: Immunoblot analysis confirms the expression of NQO2 protein in human RPE
cells. Melatonin or MCA-NAT protects human RPE cells from H2O2-induced oxidative
stress in a concentration-dependent manner with an EC50 of approximately 0.3 µM.
Pharmacologic inhibition of NQO2 does not detectably alter p53 levels in ARPE-19
cells compared to control cells in immunoblot analysis.
Conclusions: MCA-NAT protects ARPE-19 cells from oxidative stress at the same
potency as melatonin’s, suggesting that NQO2 is the target of melatonin to mediate
protection against oxidative stress. Contrary to what was observed in NQO2
knockdown cells or Nqo2 knockout mice, NQO2 inhibitors merely block the catalytic
action of NQO2 and do not reduce the level of p53. Therefore, NQO2 inhibition may
be a feasible treatment strategy for AMD.
CR: C.K. Hwang, None; H. Zhou, None; R. Haque, None; P.M. Iuvone, None.
Support: NIH Grant R01 EY04864-26, NIH Grant P30 EY006360, NIH Grant T32 GM
008602, Research to Prevent Blindness Grant
3707 - A513
The Possible Neuroprotective Effects of RoY Peptide Ligation to Membranal
Grp78 in the Ischemic Retina
3708 - A514
Photoreceptor Transplantation Rescues Cones by Reversing Cell Damage
Morphology
T. Goldstein1, O. Dratviman-Storobinsky2, N. Goldenberg-Cohen2,3, N. Goldenberg-Cohen1,
B. Hardy4, A. Raiter4. 1Sackler School of Medicine, Tel Aviv University, Ramat
Aviv, Israel; 2The Krieger Eye Research Laboratory, Felsenstein Medical Research
Center, Tel Aviv University, Petach Tiqwa, Israel; 3Pediatric Unit, Department of
Ophthalmology, Schneider Children Medical Center, Sacker School of Medicine,
Tel Aviv University, Petach Tiqwa, Israel; 4Laboratory of Cellular and Vascular
Immunology, Felsenstein Medical Research Center, Tel Aviv University, Petach
Tiqwa, Israel.
Y. Yang1, S. Mohand-said1,2, M. Simonutti1, S. Fouquet1, T. Léveillard1, J.-A. Sahel1,2,3.
1
Institut de la Vision, INSERM,UMR-S 968, UPMC University of Paris 06,
Paris, France; 2Ophthalmology Department, Centre Hospitalier National
d’Ophtalmologie des Quinze-Vingts, Paris, France; 3Institute of Ophthalmology,
University College of London, London, United Kingdom.
Purpose: Optic nerve injury causes severe axonal damage leading to apoptosis
of the retinal ganglion cells (RGCs) and consequent loss of vision. GRP78, a wellcharacterized endoplasmic reticulum (ER) chaperone, is present in all cells and
plays an important role as one of the initial components of the signaling cascade
that produces the unfolded protein response. In this study, we would like to evaluate
the neuroprotective effect of intravitreal injection of RoY, a 12 amino-acid peptide
that identified GRP78 following induction of retinal ischemia in mice, by crush.
This connection is increased under hypoxic conditions and is related to increased
cell survival.
Methods: Fifteen C57BL mice underwent right optic nerve crush immediately followed
by intraocular RoY peptid injection (n=10) or saline (n=5). The left eyes were untreated
and served as controls. Another control group underwent injection of RoY peptide
to a normal non injured eye (n=5). All the mice were analyzed histologically 21 days
following the injury.
Results: Mean RGC cell loss on day 21 was 53% in the group after crush injury without
injection of the peptide. In the intraocular injected RoY post crush, the cell loss was
reduced to 27%. No RGC loss was measured in the control group injected with RoY.
Conclusions: Histologically, we have demonstrated a neuroprotective effect of
intraocular RoY injection. The peptide prevented RGC loss after optic nerve crush
injury. These results encourage further studies of the mechanism and clinical uses
of the agent.
CR: T. Goldstein, None; O. Dratviman-Storobinsky, None; N. Goldenberg-Cohen,
None; N. Goldenberg-Cohen, None; B. Hardy, None; A. Raiter, None.
Support: The Isabel and Zanvyl Krieger Fund, Baltimore, MD
Purpose: We have recently shown that the Rod-derived Cone Viability Factor (RdCVF)
delays the loss of function of the cone by exerting a stabilizing effect on the morphology
of the cone outer segment in P23H transgenic rats. The purpose of the present study
is to extend this observation to the transplantation of normal photoreceptors into the
eye of rd1 mouse, the animal model of recessive retinitis pigmentosa (RP).
Methods: Transplantation was performed using 8-day-old photoreceptor layers
isolated by vibratome sectioning from wild type mouse (C57BL/6) retina and grafted
into the subretinal space of the rd1 mice (C3H/HeN) at 3 weeks of age. The cone
density of the host retinas was measured after labeling with the lectin PNA and
using a stereological method 3 weeks after transplantation. We also analyzed the
morphology of the cone outer segments.
Results: The transplantation of normal photoreceptors rescues the cones from the
rd1 mouse as shown by an increase of 21% compared to the contralateral untreated
retinas (P<0.001), which is consistent with our previous work. We also observed
that the transplantation protects the morphology of the cone outer segments from
damage linked to the Pde6b mutation. The cones of the treated rd1 eye present small
tip areas and longer cone outer segments. In the contralateral untreated rd1 eye, the
cone morphology is characterized by an increase in the area of the tip sheath and
shortening of cone outer segment, implying that the rd1 cone morphology is altered
prior to their death.
Conclusions: Healthy wild-type photoreceptor transplants can protect cones in rd1
mice by increasing cone number and reversing the damage to the cone morphology. Our
observation is consistent with results obtained by transplanting normal photoreceptors
into the P23H rat model of autosomal dominant RP or by performing injections of the
RdCVF protein in that latter model. Our results suggest that the mechanism underlying
the protective effect of the transplantation directly involves the maintenance of a
cellular structure essential for cone function.
CR: Y. Yang, None; S. Mohand-said, None; M. Simonutti, None; S. Fouquet, None; T.
Léveillard, None; J.-A. Sahel, None.
Support: INSERM - Université Piere & Marie Curie
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3705-3708
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3709 - A515
Neuroprotective Effect on Retinal Ganglion Cells by Transpupillary Laser
Irradiation of the Optic Nerve Head
3710 - A516
The Wnt Signaling Pathway Protects RGC-5 Cells From Pressure-Induced
Apoptosis
L. Jiang1, J. Ma2. 1Eye Center, Beijing Tongren Hospital, Beijing, China; 2Department
of Ophthalmology, Peking Union Medical College Hospital, Chinese Academy of
Medical Sciences & Peking Union Medical College, Beijing, China.
M.A. Fragoso, H. Yi, A.S. Hackam. Ophthalmology, University of Miami, Miami, FL.
Purpose: The current study demonstrated that the subthreshold transpupillary
thermotherapy (TTT) laser irradiation on optical never head protects retinal ganglion
cells (RGC) in an optic nerve crush (ONC) model.
Methods: TTT was performed in right eyes with an 810-nm diode laser aimed at the
center of the optic nerve head using the following protocol: power 60 mW, duration 60
seconds, spot size 50μm. Fluorogold was injected into bilateral superior colliculi 5 days
before sacrifice and fluorescent gold labeled RGC were counted under fluorescence
microscopy.
Results: In the ONC group, a progressive loss of RGC was observed; however, in ONC+
TTT group, RGC density was significantly higher (p < 0.05) in each corresponding
subgroup compared to the ONC group, which implied the potential neuroprotective
role of TTT. This protective effect seemed to be heat shock protein (Hsp) related,
because intraperitoneal Quercetin (an inhibitor of Hsp, 4mg/kg/d for 7 days) could
completely abolish this protective effect (p < 0.05 for each subgroup). Minimal collateral
damage of TTT on optic nerve head tissue, peripapillary RGCs and the myelin sheath
of the optic nerve were observed under transmission electron microscopy.
Conclusions: These findings suggest the subthreshold TTT is a safe and practical
approach to protect RGC. The underlying mechanism may be related TTT-induced
Hsp in the RGC.
CR: L. Jiang, None; J. Ma, None.
Support: Beijing Natural Science Foundation (7053067)
Purpose: Identifying survival factors for retinal ganglion cell (RGC) is an important
strategy in the development of novel therapies for glaucoma. Elevated intraocular
pressure (IOP) is a risk factor in glaucoma progression. Wnt signaling is an essential
pathway that regulates cell proliferation, survival and differentiation in the retina.
Our previous work showed that the Wnt pathway is upregulated during retinal
degeneration and that it protects photoreceptors from oxidative stress. In this study
we investigated whether Wnt signaling protects the retinal ganglion cell line RGC-5,
and explored possible factors involved in this neuroprotection.
Methods: Immunodetection was used to confirm expression of RGC marker genes
in the RGC-5 cell line. RGC-5 cells were subjected to elevated pressure (100 mmHg,
24 hrs) in the presence of Wnt3a, DKK1 or the appropriate control. Caspase activity
and Tunel assays were performed to quantitate apoptosis, and qPCR was used to
investigate potential mechanisms of Wnt3a activity.
Results: Expression of the RGC marker genes Thy1 and Brn3b was confirmed in the
RGC-5 cells in our culture conditions. Wnt3a reduced pressure-induced caspase
activity to 58% of control treatment (n=8). The percentage of Tunel positive cells was
significantly reduced by Wnt3a. Control treated cultures had 58% Tunel positive cells
whereas Wnt3a treated cultures had 43% (n=5, p=0.0014). Addition of Wnt inhibitor,
DKK1, in the presence of Wnt3a significantly increased the percentage of Tunel
positive cells to 52% (n=3, p<0.05). Furthermore, the Wnt3a-treated cells up-regulated
neurotrophin 3 (2.48-fold, s.e.m 0.14), bFGF (2.3-fold, s.e.m. 0.51), BDNF (1.5-fold, s.e.m.
016) and NGF (1.9 fold, s.e.m. 0.24 (n=4, p<0.05).
Conclusions: These data demonstrate that Wnt3a protected RGCs from apoptosis
induced by elevated pressure. Increased expression of neuroprotective growth factors
in the presence of Wnt3a suggests a potential mechanism of neuroprotection. These
results suggest that activation of the Wnt signaling pathway by Wnt3a could be
investigated further as a tool to develop therapeutic strategies for the prevention
of RGC death.
CR: M.A. Fragoso, None; H. Yi, None; A.S. Hackam, None.
Support: AHAF
3711 - A517
Functional and Morphological Analysis of Retinas of Sigma Receptor 1 (σR1)
Null Mice
3712 - A518
Iron and Manganese Corroles Are Neuroprotective for Serum-Deprived Retinal
Neurons
Y. Ha1A,1B, A. Saul1C,1B, E. Zorrilla2, V. Ganapathy1D,1B, S.B. Smith1A,1B. ACell Biology &
Anatomy, BVision Discovery Inst., COphthalmology, DBiochemistry & Molecular
Biology, 1Medical College of Georgia, Augusta, GA; 2Neurobiology, The Scripps
Research Institute, La Jolla, CA.
M.-M. Catrinescu1, A. Kanamori2, R. Beaubien 3, W. Chan1, Z. Gross 4, L.A. Levin 5.
1
Ophthalmology, Hospital Maisonnueve Rosemont Research, Montreal University,
QC, Canada; 2Ophthalmology, Montreal University, Montreal University, QC,
Canada; 3Centre de Recherches HMR, Ophtalmologie, Montreal University, QC,
Canada; 4Schulich Faculty of Chemistry, Technion, Israel Institute of Technology,
Israel Institute of Technology, Israel; 5Ophthalmology, Univ Montreal/Univ
Wisconsin, Montreal University, QC, Canada.
Purpose: σR1 is thought to act as a molecular chaperone at the ER membrane. Recently,
we reported robust protection against retinal ganglion cell (RGC) death when a σR1specific ligand was administered to diabetic mice (Smith et al, 2008). To understand
how σR1 mediates retinal neuroprotection, we established a colony of σR1 null (-/-)
mice to examine the retinal phenotype functionally and histologically as there have
been no reports of the consequence to retina when σR1 is not present.
Methods: A colony of σR1 wildtype (+/+), heterozygous (+/-) and homozygous (-/-)
mice were established. Neural retina and brain were subjected to RT-PCR, western
blotting (WB) and immunohistochemistry (IHC) to analyze expression of σR1. Mice
(age: 5-6 wks) were tested in scotopic/photopic conditions at a range of flash intensities
after 12 h dark adaptation. Eyes were processed for light microscopy to assess retinal
morphology.
Results: RT-PCR and WB confirmed abundant σR1 gene/protein expression in retina,
brain and other tissues of σR1 +/+ mice, but not σR1 -/- mice. IHC detected σR1 in
several retinal layers in +/+ mice, but not in σR1 -/- mice. Scotopic ERGs revealed
implicit times ranging from ~40-15 ms for a-waves, ~100-60 ms for b-waves. Responses
for +/+, +/- and -/- mice were within normal ranges (strong b-waves and OPs, strong
a-waves at high intensities), however the amplitudes in σR1 -/- mice were weaker than
+/+ or +/- mice. Morphologically, retinas of σR1 -/- mice were comparable to σR1 +/+
and +/- mice (total retinal thickness = ~ 250 μm, INL = ~35-40 µm, ~12-14 rows of PRC
nuclei, ~15 RGCs/100 μm retinal length).
Conclusion: This is the first analysis of retinas of mice lacking σR1. The data suggest
that, at least at early ages, σR1 -/- mice have retinal morphology and function similar
to wildtype. Studies are underway to assess the long-term effects of the lack of σR1
gene on retinal morphology, function and expression of ER stress genes and will set
the stage for mechanistic studies to determine how σR1 mediates neuroprotection
in diabetes.
CR: Y. Ha, None; A. Saul, None; E. Zorrilla, None; V. Ganapathy, None; S.B. Smith,
None.
Support: NIH Grant EY014560 & Medical College of Georgia Vision Discovery
Institute
Purpose: Corroles are tetrapyrrolic macrocycles that have come under increased
attention because of their unique capabilities for oxidation catalysis, reduction
catalysis, and biomedical applications. Corrole-metal complexes (metallocorroles)
can decompose a variety of reactive oxygen species (ROS), often more efficiently
than analogous metalloporphyrins. We investigated whether bipolar Fe-, Mn- and
Ga-corroles have neuroprotective effects on neurons and reduce ROS in vitro and
in vivo.
Methods: Undifferentiated and differentiated RGC-5 neuronal precursor cells were
induced to undergo apoptosis by serum deprivation for 48 hours. Differentiation
of RGC-5 cells was induced with 316 nM staurosporine. The level of cell death with
or without these metallocorroles was monitored by XTT assay and by calcein-AM/
propidium iodide assay. RGC-5 ROS were measured with hydroethidine (HEt), a
superoxide indicator. In vivo, real-time imaging using a confocal scanning laser
ophthalmoscope (CSLO) identified the production of ROS within individual rat
RGCs after optic nerve transection. Intraocular ROS was visualized by intravitreal
administration of HEt.
Results: RGC-5 cell death after serum deprivation was significantly decreased by
Fe- and Mn-corroles, but not Ga-corrole. This correlated with the ability of Fe- and
Mn-corroles but not Ga-corrole to dismutate intracellular superoxide, measured by
fluorescence after HEt administration. In vivo, intravitreal Fe- and Mn-corroles, but
not Ga-corrole decreased retinal HEt-positivity after optic nerve transection.
Conclusions: Fe- and Mn-corroles could be candidate drugs for delaying RGC death
after axonal injury in optic neuropathies via suppression of specific ROS.
CR: M.-M. Catrinescu, None; A. Kanamori, None; R. Beaubien, None; W. Chan,
None; Z. Gross, None; L.A. Levin, None.
Support: Canadian Institutes for Health Research, Canadian Foundation for
Innovation, Canadian Research Chairs program, Fonds de recherche en ophtalmologie
de l’Université de Montréal
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3709-3712
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3713 - A519
Gene Expression Profiling of the Retina After Transcorneal Electrical
Stimulation in Wildtype Brown Norway Rats
G. Willmann1A, K. Schäferhoff 1B, A. Schatz1A, M. Bonin1B, H. Enderle1A, K.U. BartzSchmidt1A, E. Zrenner1A, F. Gekeler1A. ACentre of Ophthalmology, BMedical Genetics,
1
University of Tuebingen, Tuebingen, Germany.
3714 - A520
Upregulation of Proinflammatory Genes (COX-2 and B-94) Under the Influence
of Mutant Glutamine Expansion Inhibited by NPD1 and PEDF/DHA in Human
Retinal Pigment Epithelial Cells (HRPE)
P.K. Mukherjee1A, N.G. Bazan1B. ANeuroscience Cntr/Ophthalmology, BOphthal &
Neuroscience, 1LSU Health Sciences Center, New Orleans, LA.
Purpose: To investigate the effect of transcorneal electrical stimulation (TES) on the
retina of wildtype Brown Norway (BN) rats by gene expression profiling.
Methods: TES was applied to 56 BN rats in vivo for 1h (1ms biphasic pulses at 20Hz;
current: 200 µA). Prior to microarray studies, known TES regulated neurotrophic
factors (i.e. IGF1, Bcl2, CNTF, Fgf2 and BDNF) were studied at the mRNA level by
quantitative real-time polymerase chain reaction (qPCR) at 0h, 4h, 10h, 24h, 2d, 4d and
7d post TES to determine optimal time points. Gene expression profiling was analyzed
0, 4, and 24 hours after TES. Major transcriptome-level changes were independently
validated at the mRNA level by qPCR. Electroretinograms (ERGs) were recorded for
functional analysis at the respective time points.
Results: qPCR analysis of selected neurotrophic factors revealed overall highest fold
level changes at 4h and 10h after TES. 20 BN rats were then analyzed by gene expression
profiling. In total 163 genes were differentially expressed with a p-value <0.05 and
a 1.5 fold cutoff at 4h versus 24h after TES. These results demonstrate the direct
effect of TES on the retina of wildtype BN rats and showed differential expression
of several transcription factors that may play a crucial role in neuroprotection of the
retina. Functional analysis by ERGs at the respective time points showed normal
ERG recordings.
Conclusions: TES applied to the retina of wildtype BN rats induces a variety of
transcriptome level changes. Our study may help to understand the mechanisms
underlying TES induced neuroprotection and may contribute to define more clearly
therapeutic options for patients with neurodegenerative retinal diseases.
CR: G. Willmann, Okuvision Gmbh, F; K. Schäferhoff, None; A. Schatz, Okuvision
Gmbh, F; M. Bonin, None; H. Enderle, Okuvision Gmbh, F; K.U. Bartz-Schmidt,
Okuvision Gmbh, F; E. Zrenner, Okuvision Gmbh, F; F. Gekeler, Okuvision Gmbh,
F.
Support: Okuvision Gmbh
Purpose: The mutation of the ataxin-1 protein causes polyglutamine diseases due
to glutamine expansions on the polypeptide chain. Docosahexaenoic acid (DHA)
and pigment epithelium-derived growth factor (PEDF) promote neuroprotection
through neuroprotectin D1 (NPD1) synthesis and may foster survival in stroke and
neurodegenerative diseases. Ataxin-1 mutation interacts with its normal cellular
complex, but the function of the complex is repressed due to the mutation. The function
of the complex is to up-regulate and inhibit certain gene expression. The purpose of this
study is to investigate the up-regulation of proinflammatory genes cyclooxygenase-2
(COX-2) and B-94 under the influence of mutant glutamine expansion and the effect of
NPD1 and PEDF with DHA in human retinal pigment epithelial (HRPE) cells.
Methods: HRPE cells, grown overnight, were cotransfected with 82Q polyglutamine
expansions and COX-2 promoter (830) luciferase constructs by Fugene-6 according
to the manufacturer’s protocol (Roche). A promoterless β-galactosidase construct
was used to cotransfect as transfection control. Transfected cells were harvested,
cell extracts were made, and luciferase assays were performed using luciferin as
substrate. Western blot analysis of COX-2 and B-94 was performed using respective
antibodies.
Results: Our results indicated that a transfected ataxin-1 mutant gene with 82
polyglutamine expansions in human retinal pigment epithelial (HRPE) cells induce
apoptosis in cell involving the upregulation of (COX-2) and B-94. These upregulations
were inhibited by NPD1 as well as PEDF with DHA. Western blot analysis indicated
that the ataxin-1 mutant induced proinflammatory COX-2 and proapoptotic B-94
proteins, and NDP1 and PEDF with DHA were able to inhibit the induction of these
proteins in HRPE cells.
Conclusions: The expression of proinflammatory/proapoptotic proteins COX-2 and
B-94 were reduced when the HRPE cells transfected with ataxin-1 mutant were exposed
to NPD1 or a mixture of DHA/PEDF. These neuroprotective agents enhance survival
of the HRPE cells when polyglutamine diseases are induced in HRPE cells. These
results demonstrate that survival signaling is mediated by NPD1 in an experimental
model of neurodegeneration. NPD1 may be useful in therapeutic strategies for treating
neuronal diseases like age-related macular degeneration (AMD) and stroke.
CR: P.K. Mukherjee, None; N.G. Bazan, None.
Support: NIH EY50121, EY00444
3715 - A521
Neuroprotective Effect of Ketone Bodies and Ketogenic Diet in Nmda-Induced
Rgc Damage in Rat. Possible Involvement of Kynurenic Acid?
3716 - A522
Prolonged Blockade of VEGF Receptors Does Not Damage Retinal
Photoreceptors or Ganglion Cells
T.J. Choragiewicz1,2, S. Thaler1, R. Rejdak1,2, M. Fiedorowoicz1,3, W.A. Turski4, T.
Kocki4, M. Tulidowicz-Bielak 2, E. Zrenner1, F. Schuettauf 1, T. Zarnowski2. 1Centre
for Ophthalmology, University of Tuebingen, Tuebingen, Germany; 2Tadeusz
Krwawicz Chair of Ophthalmology and Eye Hospital, Medical Unviersity, Lublin,
Poland; 3Department of Experimental Pharmacology, PAS Medical Research
Center, Warsaw, Poland; 44Departament of Toxicology, Institute of Agricultural
Medicine, Lublin, Poland.
A. Miki, K. Miki, S. Ueno, C. Berlinicke, D.M.B. Wersinger, S. Usui, B.C. Oveson, G.
Shaw, D.J. Zack, P.A. Campochiaro. Ophthalmology, Johns Hopkins Wilmer Eye Inst,
Baltimore, MD.
Purpose: To investigate neuroprotective properties of ketogenic diet and the ketone
bodies acetoacetate (ACA) and β-hydroxybutyrate (BHB) in a rat model of NMDAinduced damage of rat retinal ganglion cells (RGCs). Additionally the influence of ACA
and BHB on retinal kynurenic acid (KYNA) production after NMDA was assessed
in vitro in calf retinal slices.
Methods: ACA in a dose of 250 mg/kg body weight, BHB in a dose of 291.2 mg/kg or PBS
as control was administered intraperitoneally once a day for 21 days. Brown Norway
rats 3 weeks of age and adult rats were fed with ketogenic diet or standard diet as control
for 2 weeks prior to experiments. On day 14 animals received intravitreal NMDA (2µl,
10mM). RGC numbers were assessed 7 days thereafter using a retrolabelling method.
For HPLC measurements of KYNA production, calf retinal slices were incubated with
NMDA and different concentrations of ACA or BHB.
Results: Numbers of RGC in retinas from animals treated with ACA, BHB or fed with
ketogenic diet (only young animals) were significantly higher (48%, 41% and 25%
increases respectively, P<0.05, U Mann-Whitney) than in those treated with PBS or fed
with standard diet. However in adult rats ketogenic diet turned out to be ineffective.
De novo KYNA production in calf retinal slices was reduced by NMDA. ACA or BHB
attenuated this effect significantly (82.8% and 94.0% higher, P<0.001).
Conclusions: The ketone bodies ACA and BHB act neuroprotectively in a rat model of
NMDA-induced RGC damage. Ketogenic diet shows similar properties, but its effect
is age-related. Attenuation of NMDA induced reduction of KYNA production may be
essential for ketone bodies to exert their neuroprotective effects.
CR: T.J. Choragiewicz, None; S. Thaler, None; R. Rejdak, None; M. Fiedorowoicz,
None; W.A. Turski, None; T. Kocki, None; M. Tulidowicz-Bielak, None; E. Zrenner,
None; F. Schuettauf, None; T. Zarnowski, None.
Support: None
Purpose: A VEGF receptor kinase inhibitor, SU4312, was used to assess possible
retinal toxicity induced by antagonism of VEGF signaling.
Methods:Transgenic mice with sustained expression of VEGF in photoreceptors (rho/
VEGF mice) were used to identify a SU4312 dosing regimen that completely suppressed
the angiogenic effects of high levels of VEGF in the retina. Wild type mice were given
this dosing regimen of SU4312 for up to 12 weeks. Retinal function was assessed by
performing serial electroretinograms (ERGs) and retinal structure was assessed by
TUNEL and measurement of outer nuclear layer (ONL) thickness. Primary retinal
ganglion cell and total retinal cultures were incubated in high concentrations of
SU4312 and cell viability was assessed.
Results: Using rho/VEGF mice, we determined that periocular injection of 3 μg of
SU4312 every 5 days completely suppressed subretinal neovascularization indicating
effective blockade of VEGF signaling. Wild type mice given periocular injections of
5 μg of SU4312 every 5 days for up to 12 weeks showed normal scotopic and photopic
electroretinograms (ERGs), no TUNEL stained cells in the retina, and no reduction
in outer nuclear layer thickness. Incubation of cultured ganglion cells or total retinal
cultures containing photoreceptors with high doses of SU4312 did not reduce cell
viability.
Conclusions: We recently found that expression of a vascular endothelial growth factor
(VEGF) binding protein in the retina for 7 months completely blocked the vascular
leakage-promoting effects of VEGF, but did not cause identifiable toxicity to retinal
neurons. However, others have reported that much briefer periods of treatment with
a VEGF antagonist caused photoreceptor cell death. Our earlier work plus the data
presented here suggest that blockade of VEGF signaling in the retina for up to 12
weeks does not damage photoreceptors nor alter ERG function and should reassure
patients who are receiving frequent injections of VEGF antagonists for choroidal and
retinal vascular diseases.
CR: A. Miki, None; K. Miki, None; S. Ueno, None; C. Berlinicke, None; D.M.B.
Wersinger, None; S. Usui, None; B.C. Oveson, None; G. Shaw, None; D.J. Zack,
None; P.A. Campochiaro, None.
Support: EY12609 and core grant P30EY1765 from the NEI
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3713-3716
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3717 - A523
Inhibition of Dopamine Suppresses cGMP Accumulation in rd1 Retinal Organ
Cultures
3718 - A524
The Role of Tnfα in Photoreceptor Degeneration After Retinal Detachment;
Suppression With Dexamethasone Treatment
A.M. Richmond, J.M. Ogilvie. Biology, Saint Louis University, Saint Louis, MO.
T. Nakazawa1, M. Ryu1, T. Hisatomi2A, K. Noda2A, S. Nakao2A, M. Kayama2A, A. HafeziMoghadam2A, D. Vavvas2B, K. Nishida1, J.W. Miller2B. 1Ophthalmology, Tohoku
Univ Graduate Sch of Med, Sendai, Japan; AAngiogenesis, BOphthalmology,
2
Massachusetts Eye & Ear Infirmary, Boston, MA.
Purpose: The rd1 mouse retina is a model of early onset retinitis pigmentosa. A
mutation in the beta-subunit of the rod-specific cGMP-phosphodiesterase causes cGMP
accumulation in the retina, resulting in degeneration of the rod photoreceptors by one
month of age in vivo or in organ culture. The molecular cascade that induces apoptosis
remains poorly understood. We have previously shown that dopamine depletion blocks
the degeneration of photoreceptors in the rd1 retinal organ culture model (Neurobiol
Disease 2002, 10:33-40). Here we determine whether dopamine signaling regulates
cGMP levels in the retinal organ culture model to protect photoreceptors.
Methods: Retinas were dissected from wild type (wt) and rd1 mice at postnatal
day (P)2 and grown in organ culture in control media or in media with 6-OHDA in
order to deplete dopamine. Organ cultures were harvested under dark conditions
after 8 - 16 days in vitro. An ELISA assay was used to determine the concentration of
cGMP for each sample.
Results: The concentration of cGMP in rd1 retinal organ cultures treated with 6-OHDA
is significantly lower than in untreated control rd1 retinal organ cultures. However,
treatment of retinal organ cultures with 6-OHDA does not reduce cGMP concentration
to wt levels.
Conclusions: We have shown that dopamine depletion in rd1 retinal organ cultures
reduces cGMP accumulation, which likely contributes to photoreceptor protection.
Our results illustrate a role for dopamine signaling in regulation of cGMP, suggesting
a novel dopamine signaling pathway in differentiating rod photoreceptors.
CR: A.M. Richmond, None; J.M. Ogilvie, None.
Support: NIH Grant EY015113 (JMO), Sigma Xi (AMR)
3719 - A525
BDNF Secretion is Dependent on Optineurin Expression
A.K. Ball1A, M.T.A. Duong1B. APathology/Molecular Med HSC1R1, BPathology/Molec
Med-HSC 1R1, 1McMaster University, Hamilton, ON, Canada.
Purpose: Variants of the optineurin gene have been associated with certain forms
of glaucoma, while BDNF has been shown to be an important survival factor for
retinal ganglion cells. An interaction between optineurin, myosin VI, and Rab8
proteins suggests that optineurin may be involved in the BDNF secretory pathway.
The purpose of these experiments was to determine if BDNF secretion was dependent
on optineurin expression.
Methods: RGC-5 (retinal neuronal precursor cells) were terminally differentiated with
staurosporine for 48 hrs. Stable expression of miRNAs (miRNA.NS=non targeting;
miRNA.OPT=optineurin targeting) in RGC-5 cells was acheived for at least 2 weeks
using BLOCK-iTTM miR RNAi Select (Invitrogen). Optineurin expression in wild-type
(wt) RGC-5 cells, or RGC-5 cells transfected with control miRNA.NS, or miRNA.
OPT was determined using immunohistochemistry and western immuoblotting to
confirm knockdown only in miRNA.OPT RGC-5 cells. Each cell type was stressed
with glutamate (48 hrs; 2x10 -5M=LD50 wt-RGC-5). Some cells were pre-treated with
hrBDNF(0 to 1x10 -6 gm/ml; BioShop). BDNF secretion was measured using ELISA
using the BDNF Emax ImmunoAssay System (Promega).
Results: 48hr exposure to glutamate killed 50% wt-RGC-5 and miRNA.NS RGC-5
cells, and 99% miRNA.OPT RGC-5 cells. This result demonstrates that optineurin
expression protected RGC-5 cells from glutamate-mediated stress. Treatment with
3x10 -7 g/ml hrBDNF provided 100% protection for wt-RGC-5 and miRNA.NS RGC-5
cells and only 68% for miRNA.OPT RGC-5 cells. hrBDNF pretreatment increased BDNF
secretion (5 fold increase) in wt-RGC-5 and miRNA.NS RGC-5 cells. This increase in
BDNF secretion was greater (9 fold increase) when wt-RGC-5 and miRNA.NS RGC-5
cells were challenged with glutamate. No increase in BDNF secretion was observed
in miRNA.OPT RGC-5 cells, either treated with glutamate, or left untreated.
Conclusions: This study suggests that one mechanism of BDNF mediated
neuroprotection is through recycling of exogenous BDNF. BDNF secretion was
dependent on optineurin expression. Optineurin’s involvment in BDNF secretion
may explain why non-functional mutations of this protein have been implicated in
the onset of glaucoma.
CR: A.K. Ball, None; M.T.A. Duong, None.
Support: NSERC 171190-2008; Glaucoma Research Society of Canada
Purpose: Photoreceptor degeneration is a major cause of visual loss in various retinal
diseases. In this study, we investigated the role of anti-inflammatory treatment
with dexamethasone (DEX) in retinal detachment (RD)-induced photoreceptor
degeneration.
Methods: RD was caused by subretinal injection of hyaluronaic acid. Photoreceptor
degeneration was assessed by counting the TdT-dUTP terminal nick-end labeling
(TUNEL)-positive cells and by measuring the outer nuclear layer thickness after RD.
To understand the role of anti-inflammatory treatment, DEX (1mg/kg, IP) or nano
particle of poly (γ-glutamic acid) with DEX (NP-DEX, 0.8 mg/ul, subretinally) was
injected just after RD. As the target of anti-inflammatory treatment, the expression of
TNFα with or without DEX was examined by real-time PCR and RD was performed
in mice deficient in TNFα or its receptors (TNFRI, TNFRII, TNFRI&II).
Results: Treatment with DEX (p=0.0005) or NP-DEX (p=0.0065) significantly suppressed
RD-induced photoreceptor degeneration and the expression of TNFα. RD-induced
photoreceptor degeneration was significantly suppressed in mice deficient for TNFα
(p<0.0001), TNFRII (p=0.001), or TNFRI&II (p<0.0001). However, lack of TNFRI did
not protect from RD-induced photoreceptor degeneration (p=0.06).
Conclusions: Anti-inflammatory treatment with DEX had a neuroprotective effect
against RD-induced photoreceptor degeneration. TNFα plays a critical role in RDinduced photoreceptor degeneration. This pathway may become an important target
in the prevention of photoreceptor degeneration.
CR: T. Nakazawa, None; M. Ryu, None; T. Hisatomi, None; K. Noda, None; S.
Nakao, None; M. Kayama, None; A. Hafezi-Moghadam, None; D. Vavvas, None; K.
Nishida, None; J.W. Miller, None.
Support: MESCJ21659395
3720 - A526
Targeted siRNA-Mediated Knockdown of p53 Family Activators ASPP1 and
ASPP2 Delays Adult Retinal Ganglion Cell Death in vivo
A.M. Wilson1, E. Feinstein2, A. Di Polo1. 1Pathology/Cell Biology, University of
Montreal, Montreal, QC, Canada; 2Research, Quark Pharmaceuticals Inc, Ness
Ziona, Israel.
Purpose: The ASPP (Ankyrin-repeats, SH3-domain and Proline-rich-region-containing
Protein) family members, ASPP1 and ASPP2, are essential regulators of p53 activity
but their role in retinal ganglion cell (RGC) death is unknown. Here, we addressed
their function in a rat model of acute optic nerve injury (axotomy) using novel siRNAs
that selectively silence ASPP1 and ASPP2 gene expression.
Methods: RGCs were retrogradely labeled by application of Fluorogold to the rat
superior colliculus, and intraorbital optic nerve axotomy was performed one week
later. siRNA against ASPP1, ASPP2 or GFP (control) were administered by intravitreal
injection at the time of axotomy and one week after injury. The density of Fluorogoldlabeled RGCs was quantified in 12 standard retinal areas at 1 and 2 weeks postaxotomy. ASPP protein expression was examined by retinal immunohistochemistry
and western blot analysis.
Results: Endogenous, retinal ASPP1 and ASPP2 proteins were found to be primarily
expressed by RGCs, and were effectively knocked down as early as 24 hours after
intravitreal injection of targeted siRNAs. ASPP1 and ASPP2 gene silencing led to robust
protection of axotomized RGCs: ASPP2 and ASPP1 siRNA-injected eyes displayed
79% RGC survival (1,636 RGCs/mm2 ± 62, mean ± S.E.M., n=5) and 69% survival
(1,423 RGCs/mm2 ± 19, n=4), respectively, compared to 54% that survived in GFP
siRNA injected eyes (1,132±41 RGCs/mm2, n=5). Significant neuroprotection was also
observed at two weeks post-axotomy: ~26% RGC survival was achieved with either
ASPP1 or ASPP2, with respect to 6% in eyes treated with control siRNA.
Conclusions: Our data demonstrate that targeted gene silencing of ASPP1 or ASPP2
effectively delays RGC death after acute optic nerve lesion, and suggest that ASPP
proteins play an important role in the demise of these neurons following axonal
injury.
CR: A.M. Wilson, None; E. Feinstein, Quark Pharmaceuticals Inc., E; A. Di Polo,
None.
Support: This study was supported by grants from Quark Pharmaceuticals Inc. and
Canadian Institutes for Health Research (A.D.P.). A.W. is a recipient of a studentship
in excellence from the FESP, UdM
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3717-3720
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3721 - A527
The Antiapoptotic TUDCA Protects Against Mitochondrial Dysfunction, Glial
Cell Changes and Loss of the Capillary Network in the Transgenic Rat Model of
Retinitis Pigmentosa P23H
L. Fernandez-Sanchez1, G. Esquiva1, I. Pinilla2, J. Martín-Nieto1, N. Cuenca1. 1Fisiología,
Genética y Microbiología, Universidad de Alicante, Alicante, Spain; 2Servicio de
Oftalmología, Hospital Miguel Servet, Instituto Aragones de Ciencias de la Salud,
Zaragoza, Spain.
Purpose: It has been reported that TUDCA prevents the degeneration of photoreceptors
in the P23H rat retina. The aim of this study was to study the changes in the vascular
network and glial cells (astrocytes, Müller cells and microglia) in the P23H rat retina
after administration of the antiapoptotic TUDCA.
Methods: For this study homozygous P23H line 3 rats were used. The animals
(from 20 days to 4 months) were injected weekly with TUDCA (500 mg, i.p.). Vertical
retinal cryostat sections were singly or doubly immunostained for specific markers
of microglia, Müller cells and/or astrocytes. Wholemount retinas were subjected to
lectin staining to reveal the retinal vascular plexus, and an antibody to GFAP was
used to observe its relationship to astrocytes.
Results: In the P23H rat, Müller cells expressed the marker GFAP in response to
degeneration of the retina. Activation of microglia was also observed, although there
were no differences in the number of these cells in the retina of animals treated with
TUDCA compared to untreated controls. The number of astrocytes was higher in the
retina of TUDCA-treated animals, which was accompanied by minor degeneration of
retinal vessels. The morphology of astrocytes in TUDCA-treated animals was similar
to that of wild-type (Sprague-Dawley) rats, whereas in untreated P23H astrocytes
exhibited a deteriorated morphology, including changes in the Cajal’s sucker-feet.
The immunostaining for different mitochondrial markers revealed that TUDCA
prevented the loss of mitochondria in the axon terminals of rods in the OPL and in
photoreceptor inner segments.
Conclusions: This work suggests that the neuroprotective effect of TUDCA could be
useful to delay photoreceptor loss in retinitis pigmentosa and preserve the capillary
network and associated astrocytes. TUDCA is also able to avoid the mitochondrial
degeneration that occurs in the P23H rat. Therefore, TUDCA could be potentially
useful for the future treatment of retinitis pigmentosa.
CR: L. Fernandez-Sanchez, None; G. Esquiva, None; I. Pinilla, None; J. MartínNieto, None; N. Cuenca, None.
Support: MEC (BFU2006-00957/BFI, BFU2009-07793/BFI), MSyC RETICS
RD07/0062/0012, FUNDALUCE, ONCE, Generalitat Valenciana ACOMP/2009/139,
Fundación Médica Mutua Madrileña.
3723 - A529
Excess Winged Helix Transcription Factor Foxg1 in Nuc1 Astrocytes is a Possible
Cause for the Retinal Remodeling Abnormality in the Nuc1 Spontaneous
Mutant Rat
D. Sinha1, A. Klise1, S.L. Hose1, C. Zhang1, B.K. Padhi2, J.S. Zigler, Jr.1. 1Ophthalmology,
Johns Hopkins Wilmer Eye Inst, Baltimore, MD; 2Health Canada, Ottawa, ON,
Canada.
Purpose: In the neural retina, βA3/A1-crystallin is expressed only in astrocytes. The
Nuc1 phenotype results from mutation of the βA3/A1-crystallin gene. The purpose of
this study is to better understand the molecular mechanisms by which the mutation
affects the remodeling of the retina.
Methods: During development, apoptosis is associated with remodeling of the retina.
TUNEL staining, retinal morphometry and immunofluorescence studies using specific
neuronal, astrocyte and blood vessel markers were used to analyze this process.
Microarray analysis was performed using the Affymetrix system; Ingenuity pathway
analysis software identified functional groups of genes differentially regulated in Nuc1
astrocytes. In addition, Real-time PCR analysis was used to determine the expression
of certain transcription factors downstream of selected signaling pathways.
Results: Excess neurons and endothelial cells remain in the Nuc1 retina during
development, suggesting a deficiency in the apoptotic process normally associated with
remodeling. Based on microarray analysis, different functional groups of genes were
combined into networks. Major network changes observed included the neurogenesis
genes NrCam, PtprZ1, Ascl1, Olig1, Aspa, Apoe, Ntrk2, Mt3 and Fez1 (Upregulated
in Nuc1) and vessel development genes such as Cyr61, Serpine 1, Cspg4, tgfα, figf
(downregulated in Nuc1) and Apln, Ednrb, Sult1a1 and Cxcr4 (upregulated in Nuc1).
Real-time RT-PCR confirmed marked upregulation of FoxG1 in Nuc1 astrocytes.
Conclusion: Our studies suggest that the mutant protein affects several pathways
in the signaling web, such as the insulin and PI-3 kinase pathways. However, it is
tempting to speculate that the failure of the Nuc1 retina to remodel could be an
effect of FoxG1 as it has been shown earlier that FoxG1 plays an important role in the
regulation of neuronal cell apoptosis.
CR: D. Sinha, None; A. Klise, None; S.L. Hose, None; C. Zhang, None; B.K. Padhi,
None; J.S. Zigler, Jr., None.
Support: NIH Grants EY018636, EY019037, EY019037-2S1, Research to Prevent Blindness,
Helena Rubinstein Foundation (all to DS)
3722 - A528
Identification of Dkk3 Interacting Proteins and Target Genes in the Wnt
Pathway
R.E. Nakamura, H. Yi, A.S. Hackam. Ophthalmology, University of Miami, Miami,
FL.
Purpose: The Wnt pathway is a critical cellular communication pathway involved
in retinal development and degeneration. Recently, we showed that Wnt signaling
protects photoreceptors from oxidative stress by acting on Muller glia. Muller glia
secrete Dkk3, which is an anti-apoptotic Wnt pathway protein that positively modulates
Wnt signaling. The purpose of this study is to identify interacting proteins of Dkk3
and its target genes in an effort to understand how Dkk3 regulates cellular survival
in the retina.
Methods: Interactions between Dkk3 and the Wnt pathway receptors V5-Krm1,
V5-Krm2 and LRP6, and the Wnt3a ligand were performed on transfected SH-SY5Y
whole cell lysates by co-immunoprecipitation (co-IP) using anti-FLAG antibody,
followed by immunoblotting with anti-V5, anti-LRP6 and anti-Wnt3a antibodies.
Mass spectrometry (Midwest Bioservice) was used to identify novel Dkk3 interacting
proteins from co-IP complexes isolated from HEK293 stably expressing Dkk3. Primary
retinal cultures from PN8 C57/Bl6 mice were treated with Wnt3a (100 ng/ml) plus
Dkk3 (50 ng/ml) or Wnt3a (100 ng/ml) alone at 3 days in vitro, for 24 hr, and total
RNA was then harvested. cDNA was synthesized and used to test the expression of
89 Wnt signaling genes using QPCR arrays (SA Bioscience).
Results: Dkk3 interacted with Krm1 and Krm2 but not the LRP6 receptor or the Wnt3a
ligand (n=3). Mass spectrometry revealed that Dkk3 interacts with glucose response
protein 78 (GRP78), which was confirmed by co-IP (n=3) and immunolocalization.
Additional interactors found by mass spectrometry in the Dkk3-containing precipitate
included HSP70 and ATP synthetase. Finally, our preliminary data indicated that
Dkk3 upregulated multiple Wnt ligands and downregulated intracellular signaling
molecules by at least 2 fold.
Conclusions: These data suggest that Dkk3 may mediate its pro-survival pathway by
increasing Wnt ligand levels and/or by interacting with Krm receptors and GRP78,
but not by interacting with the positive regulators LRP6 and Wnt3a. Future studies
will determine whether Dkk3-mediated signaling contributes to photoreceptor
neuroprotection.
CR: R.E. Nakamura, None; H. Yi, None; A.S. Hackam, None.
Support: Karl Kirchgessner Foundation NEI core grant P30EY014801 RPB Career Dev
Award RPB Unrestricted grant (BPEI) 1R01EY017837
3724 - A530
Culture of Rat Müller Cells in Alginate Capsules for Sustained Release of
Brain-Derived Neurotrophic Factor
M. Hirabayashi, M. Nakatani, Y. Shinohara, H. Mori, N. Asai, S. Nishimura.
Bioengineering Institute, Nidek Co Ltd, Gamagori, Japan.
Purpose: The brain-derived neurotrophic factor (BDNF), among other neurotrophic
factors, is known for its preventive effect against retinal degeneration. We have
previously reported that the immortalized rat Müller cell line, TR-MUL, act protectively
on rat retinal ganglion cells through the secretion of humoral factors (ARVO 2009). In
this study, we modified the TR-MUL for stable expression of BDNF and attempted to
encapsulate the modified cells with future in-vivo manipulation in view.
Methods: To obtain a cell line that stably expresses BDNF (the BD-TM4 cell line),
TR-MUL cells were transfected with a plasmid that contains the BDNF gene. The
BD-TM4 cells were encapsulated in 0.5% alginate at a density of 50,000 cells/capsule.
Dehydrogenase activity in encapsulated cells and BDNF levels released from the
capsules were quantified at days 3, 7, 14, and 21 after encapsulation by WST-8 assay
and ELISA respectively. Immunostaining for glutamine synthetase (GS), a marker for
the Müller cell, was performed to evaluate cell functionality.
Results: The encapsulated Müller cells, which initially appeared as round-shaped
and isolated, gradually started to aggregate to form inhomogeneous regions.
Dehydrogenase activity in encapsulated cells was significantly higher at day 21 than
at day 3. The capsules continuously secreted BDNF throughout 21 days at the range of
3.8-2.5 pg/day/capsule. The cells had remained immunoreactive for GS at day 21.
Conclusions: These results demonstrate that alginate-encapsulated BDNF-secreting
Müller cells maintain its viability and functionality at least for 21days.
CR: M. Hirabayashi, Nidek Co.,Ltd., E; M. Nakatani, Nidek Co.,Ltd., E; Y. Shinohara,
Nidek Co.,Ltd., E; H. Mori, Nidek Co.,Ltd., E; N. Asai, Nidek Co.,Ltd., E; S. Nishimura,
Nidek Co.,Ltd., E.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3721-3724
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3725 - A531
Molecular Basis for Rapid Corticosteroid Effects: Isolation of Plasma Membrane
Glucocorticoid Receptor from Retinal Glia
3726 - A532
Sequential Increase of Müller Glia in a Non-Intraocular-Pressure Related
Glaucoma Model
G. Hoppe, A. Sears, J.E. Sears. Cole Eye Institute, Cleveland Clinic, Cleveland, OH.
V.D. Stahl1A, S.C. Joachim1A, O.W. Gramlich1A, P. Laspas1A, N. Pfeiffer1B, F.H. Grus1B.
A
Experimental Ophthalmology, BOphthalmology, 1University of Mainz, Mainz,
Germany.
Purpose: To identify the alternative pathway of for anti-inflammatory and antiangiogenic effects of glucocorticoids (GC). GC reduce neovascularization and
resolve retinal edema through one of the two distinct mechanisms, the “classic” GC
nuclear receptor or an unconventional membrane-associated receptor that initiates
transcription-independent cell response. We hypothesize that this non-genomic GC
receptor in retinal glia is responsible for rapid destabilization of key mRNA of the
inflammatory and angiogenic factors.
Methods: Isolation of GC-binding proteins from human Müller cells (MIO-M1)
was achieved by using cortisol tagged with biotin and a photocrosslinker. Cellular
membrane fractions were obtained by sequential extraction into non-denaturing
detergent cocktails. Following magnetic streptavidin affinity purification and
separation by SDS-PAGE, GC-bound proteins were detected by anti-biotin antibody
and identified by mass spectrometry and bioinformatics.
Results: Optimization of crosslinking/extraction/purification protocol yielded
consistent protein band patterns on 1D PAGE, yet biotinylated cortisol was competed
out by an excess of unlabeled ligand from only two bands, 65 kDa and 250 kDa. To
further reduce the complexity of protein mixtures we performed 2D PAGE, which was
able to resolve only the 65 kDa spot that was consequently identified as cytoskeletonassociated protein 4 (CKAP4). CKAP4 is a single-pass protein found in endoplasmic
reticulum and plasma membranes and is ubiquitously expressed in many tissues.
CKAP4 is a receptor for tissue plasminogen activator, a receptor for antiproliferative
factor and a receptor for surfactant protein. The sole previously-identified binding
partner of CKAP4, ASCC2 is implicated multiple nuclear receptor pathways including
NF-kB, estrogen receptor, and AP1.
Conclusions: A GC-specific biotin-photocrosslinker helped to identify novel cortisolbinding membrane protein implicated in inflammatory and stress pathways. This
discovery opens the possibility of developing therapies for inflammation, vascular
permeability and neovascularization that provide the benefit of GC action without
the debilitating side effects of prolonged steroid use.
CR: G. Hoppe, None; A. Sears, None; J.E. Sears, None.
Support: Research to Prevent Blindness Challenge Grant, Cleveland Clinic Product
Development Fund, Knights Templar Eye Foundation.
3727 - A533
Treatment With Salmeterol Restores Insulin Signaling in Retinal Müller Cells
R.J. Walker, J.J. Steinle. Ophthalmology, Univ of Tennessee - Memphis, Memphis,
TN.
Purpose: To determine the regulation of insulin signaling by beta-adrenergic receptor
subtypes on Müller cells in culture.
Methods: rMC-1 cells were cultured in high glucose (25mM) conditions until the cells
reached 80% confluency. After becoming appropriately confluent, cells were serumstarved for 18-24 hours to eliminate any effects of insulin from the FBS. Following
starvation, cells were treated with 10μM isoproterenol for 1, 6, 12, and 24 hours,
followed by Western blot and ELISA analysis. For investigation of dominant receptor
subtype in Müller cells, cells were treated with either 10uM xamoterol (selective
beta-1-adrenergic agonist) or 10uM salmeterol (selective beta-2-adrenergic receptor
agonist) to stimulate beta-adrenergic receptors without presence of antagonist. We then
compared the effects of each of these drugs cleaved caspase 3 levels, phosphorylation
of Akt, and insulin receptor to previous results with isoproterenol (a non-selective
beta-adrenergic receptor agonist).
Results: Treatment of cells with 10uM isoproterenol significantly increased the
phosphorylation activity of insulin receptor beta and Akt, while significantly
decreasing caspase 3 levels at 24 hours. Treatment with a beta-1-adrenergic receptor
agonist, xamoterol, significantly decreased caspase 3 levels following 1 and 6 hours
of treatment. Treatment with a beta-2-adrenergic receptor agonist, salmeterol,
significantly decreased cleaved caspase 3 levels at all timepoints. Both xamoterol
and salmeterol significantly increased phosphorylation of insulin receptor beta in
cells cultured in high glucose. Phosphorylation of Akt, an anti-apoptotic factor, was
also significantly increased following treatment with isoproterenol and salmeterol,
but salmeterol produced a stronger response than isoproterenol.
Conclusions: These results indicate that change in glucose environment reduces
insulin signaling in cultured rat Muller but treatment with a non-selective- or
selective beta- adrenergic receptor agonist can restore normal signaling. These
results also suggest that treatment with a beta-2 adrenergic receptor agonist can
restore insulin signaling for longer periods of time vs. a non-selective- or beta-1adrenergic receptor selective drug. Overall, these results are the first to report that
beta-adrenergic receptors (specifically beta-2-adrenergic receptors) can regulate
insulin signaling in hyperglycemic Müller cells, which may offer a new avenue for
therapeutic development.
CR: R.J. Walker, None; J.J. Steinle, None.
Support: CDA JDRF 2-2006-114, JDRF Translational Award 17-2008-1044, William and
Mary Greve Special Scholars Award RPB, P30EY013080 (PI: Dianna Johnson), RPB
(PI:Barrett Haik), NIH 1F31 EY19240
Purpose: A method for automated glial fibrillary acidic protein (GFAP) analysis after
immunohistology was developed using eyes of our established Experimental Autoimmune
Glaucoma animal model (EAG). This model is non-intraocular pressure dependant. Slow
progressive retinal ganglion cell (RGC) loss is a result of immunizing rats with ocular
antigens. Intent of this study was to analyze the behavior of Müller glia with software based
detection shortly after immunization.
Methods: During the EAG-study IOP was measured regularly. After removal of rat’s eyes
RGC density was examined. For GFAP-immunofluorescence staining cross-sectioned eyes
taken 8, 12 and 22 days after immunization with optic nerve homogenate (ONH) and equal
controls (Co) were used (n=5 per group). 2 central areas, near optic nerve head, and 2
peripheral areas of each retina were photographed. GFAP was quantified using 2 computer
based methods: Method A was based on area measurements with respective GFAP intensity
after threshold setting, to detect Müller cell expansion. Method B was based on different
intensities of GFAP immunofluorescence, as initial point for comparison. Integrated density
was acquired. Both groups at all time-points were compared using t-test.
Results: IOP stayed stable throughout the EAG-study (P=0.9). A significant RGC loss was
detectable at 22 days (P=0.001). Examining method A, the mean area for both ONH and
control group increased from 8 (% of GFAP positive area: ONH=7.1%±2.3, Co=5%±3.1) to
14 (ONH=7.8%±4.6, Co=5.5%±1.2) to 22 days (ONH=8.5%±3.1, Co=6%±3). However, the
immunized group ascended significantly higher each time (P<0.05). Analyzing method
B, an increase in integrated density of GFAP could be detected in time. A first significant
raise could be ascertained for the 22 day time-point with an integrated density of 218.4±53
for ONH and 173.1±53.2 for Co (P=0.01). Data reveals a reactive gliosis after immunization.
At first swelling of Müller glia was observed. Later in process, as RGCs die off, Müller glia
produce more GFAP-filament per area.
Conclusions: As EAG is a glaucoma model with slow progressive RGC loss, the common glia
scoring systems, for example ranging from 0 to 3, were not detailed enough to detect discrete
changes. A software based analyzing method, to examine even discrete increase in expression
of GFAP, was developed and applied. Analyzing the sequential increase of Müller glia was
only possible with the combination of methods A and B (area plus intensity of GFAP). This
analytical method could also be useful for other animal models in the future.
CR: V.D. Stahl, None; S.C. Joachim, None; O.W. Gramlich, None; P. Laspas, None; N.
Pfeiffer, None; F.H. Grus, None.
Support: Deutsche Forschungsgemeinschaft (DFG JO-886/1-1), Gertrud Kusen
Foundation
3728 - A534
Green Tea (Camellia Sinensis) Attenuates Oxidative Stress And The Activation
Of Nitric Oxide Synthase Isoforms In The Retina Of Diabetic Spontaneously
Hypertensive Rats.
J.M. Lopes De Faria, K.C. Silva, M.A.B. Morales, J.B. Lopes de Faria. Research
Department, Laboratory of Pathophysiology, Faculty of Medical Sciences,
UNICAMP, Brazil.
Purpose/Aim of the study: The role of oxidative stress is pivotal in the pathogenesis
of diabetic retinopathy (DR). Green tea (GT), or its major polyphenolic compound,
has been shown to have potent antioxidant activity. In this study, we investigated
the possible effects of GT on the early markers of diabetic retinopathy (DR) through
oxidative/nitrosative mains.
Materials and Methods: Diabetes was experimentally induced in spontaneously
hypertensive rats (SHR) with twelve-week-old by streptozotocin. Control rats received
only vehicle (citrate buffer). The diabetic SHR (DM-SHR) groups were assigned to
receive or not receive, oral GT (13.3 g/L) in drinking water.
Results: After 12 weeks, the retinal glial reaction, demonstrated by a local increase
in glial fibrillary acidic protein (GFAP) levels were increased in DM-SHR group
compared with control rats (p=0.0003). Retinal oxidative stress analyzed by
immunohistochemistry for 8-hydroxy-2’-deoxyguanosine (8-OHdG) and nitrotyrosine
(NT) levels, were greater in diabetic than in nondiabetic rats (p<0.0001 for 8-OHdG
and p=0.04 for NT). The phosphorylated isoforms of neuronal (nNOS) and endothelial
nitric oxide synthase (eNOS) were also increased in retina of diabetic SHR rats
compared with control (p<0.05 for nNOS and p=0.02 for eNOS). The treatment with
GT reestablished all of the above-mentioned parameters.
Conclusions: GT reestablished the redox state and reduced the activation of nitric
oxide synthase isoforms, preventing the early diabetic retinal change. The possible
mechanisms by which GT ameliorates the redox status are under investigation.
CR: J.M. Lopes De Faria, None; K.C. Silva, None; M.A.B. Morales, None; J.B. Lopes
de Faria, None.
Support: FAPESP grant 08/540687, CNPQ, FAEPEX
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3725-3728
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3729 - A535
Alpha-Crystallin Neuroprotective Function in the Retina and its Regulation
During Diabetes
3730 - A536
Activated Protein C Rescues Photoreceptor and Retinal Pigment Epithelial Cells
From Ischemia-Induced Cell Death
P.E. Fort, M.K. Losiewicz, T.W. Gardner. Ophthalmology, Penn State Univ College of
Medicine, Hershey, PA.
T. Ueda, T. Yamamoto, N. Matsumura, Z.J. Du, M. Kamei. Osaka University, Oosaka,
Japan.
Purpose: To investigate the role and regulation of the crystallin proteins in the retinal
cells in relation with stress such as diabetic retinopathy. We previously showed that
several proteins of the crystallin protein superfamily are expressed in the retina and
over-expressed during diabetes. We also demonstrated that their regulation is sensitive
to insulin but their basal function as well as the function of their upregulation during
diabetes remained unclear.
Methods: iTRAQ method was used to characterize the proteome modifications in the
retinas of diabetic mice and rats compared to their age-matched controls. Changes
were confimed using western-blot and immunohistochemistry analysis, respectively,
to determine levels of protein expression and cellular localization. Protein interactions
were assessed using co-immunoprecipitation methods followed by western-blots
analysis. Cell death was assessed using a Cell Death DNA fragmentation Elisa assay
or a caspases-3/7 activity assay.
Results: We demonstrated that the diabetes-induced crystallin upregulation was
among the largest and the most conserved changes observed during diabetes since it is
conserved across different models, strains and species. We showed that the interaction
between alpha-crystallins and pro-apoptotic proteins observed in retinal tissue from
non-diabetic animals was destabilized by diabetes and correlates with increased
cell death. We also demonstrated that retinal neuronal cells over-expressing alphacrystallin proteins were protected from diabetes related stress induced cell death. We
showed that this was related to decreased caspases-dependant apoptosis.
Conclusions: This study shows for the first time that crystallin upregulation is one of
the major and most conserved changes observed during diabetes. It also demonstrated
that the normal function of alpha-crystallin in retinal cells is to prevent cell-death
through interaction and inhibition of pro-apoptotic proteins. This study suggest that
control of the function of alpha-crystallin could be a new therapeutic alternative for
the prevention of retinal cell death during diabetes and potentially any retinal cell
degeneration related disease.
CR: P.E. Fort, None; M.K. Losiewicz, None; T.W. Gardner, None.
Support: JDRF, JDRF Diabetic Retinopathy Center
Purpose: Activated protein C (APC) has been demonstrated to reduce cell death
associated with ischemia in the brain, lung, and kidney. The purpose of this study is
to examine the ability of APC to rescue hypoxia-induced retinal cell death.
Methods: Cultured mice retinal photoreceptor cells (661w) and human retinal pigment
epithelial cells (ARPE-19) were placed in a hypoxic chamber. Immediately before
placing in the ischemic condition, various concentrations of APC (0.3, 3 and 30 ug/
ml for 661w and 3µg/ml for ARPE-19) were added to the culture medium. After
incubation for 4, 8 or 12 hours, cells were subjected to the MTT assay to determine
the number of viable cells.
Results: When 661w and ARPE-19 cells were cultured in a hypoxic chamber, viable
cells were decreased in a time-dependent manner (91.4, 78.3 and 62.7% compared to
the baseline (0 hour)) and (82.8, 49.8 and 46.9%) at 4, 8 and 12 hours. In contrast, the
viability of cells treated with APC were, for 661w cells, (0.3 ug/ml; 94.9, 93.7 and
83.5%), (3 ug/ml; 96.3, 97.9 and 90.0%), (30 ug/ml; 92.3, 103.3 and 95.7%) at 4, 8, and 12
hours, and for ARPE-19 cells, (3 ug/ml; 99.8, 100.8 and 102.0%), at 4, 8, and 12 hours.
APC significantly protected 661w and ARPE-19 cells from hypoxia-induced cell death,
and the level of cytoprotection did not differ between the doses tested.
Conclusions: APC reduced the cytotoxicity to retinal cells induced by hypoxia in
vitro. APC therefore is a promising candidate molecule for protecting the retina from
ischemic retinal diseases.
CR: T. Ueda, None; T. Yamamoto, None; N. Matsumura, None; Z.J. Du, None; M.
Kamei, None.
Support: None
3731 - A537
Treatment of Laser-Induced Retinal Injury and Visual Loss Using Sustained
Release of Intra-Vitreal Neurotrophic Growth Factors
3732 - A538
Constituents of Bile, Bilirubin and TUDCA, Protect Against Retinal
Degeneration
H. Kecova1, R.H. Kardon2,3, E. Lavik4, S.A. Park1, S. Jacobson1, K. Hamouche1, E. Alward1,
M.M. Harper1,3, S.D. Grozdanic1. 1Veterinary Clinical Sciences, Iowa State University,
Ames, IA; 2Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA;
3
Veterans Administration Medical Center, Iowa City, IA; 4College of Engineering,
Case Western University, Cleveland, OH.
B.C. Oveson1A, S.F. Hackett1A, T. Iwase1A, S. Lee1A, T.W. Sedlak1B, S.H. Snyder1B, P.A.
Campochiaro1A, J.U. Sung1A. AOphthalmology, BNeuroscience, 1Johns Hopkins
University Wilmer Eye Inst, Baltimore, MD.
Purpose: To develop a novel therapeutic approach to laser retinal damage using
intravitreal injection of microspheres with sustained release of glial cell-derived
neurotrophic factor (GDNF).
Methods: Laboratory beagles (n=20) with experimentally induced laser retinal damage
were divided into 3 groups: group 1 received intravitreal injection of GDNF-PLGA
spheres (with degradation and release kinetics of 3 months) 1 hour after damage
induction (GDNF1), group 2 received empty spheres (E2) and group 3 received laser
damage but no additional treatment (LT3). Functional (pattern and ISCEV ERG)
recordings were done at 14, 30, 90 and 180 days post laser exposure.
Results: Ninety days after laser exposure, significant decrease in scotopic a-wave
amplitude was present in dogs which received empty microspheres (mean ± SEM:
86.51 ± 3.97µV, p=0.0044, unpaired t-test) and dogs which received laser damage
only (56.95 ± 3.77µV, p<0.0001), but not in GDNF treated dogs (113.8 ± 11.03µV,
p=0.5041) when compared to healthy controls (123.3 ± 8.49µV). However, 180d post
laser injury, a-wave amplitude significantly decreased in GDNF-treated group
(48.4 ± 7.27µV, p<0.0001) when compared to control values and was not significantly
different when compared to values in dogs, which received empty spheres
(45.63±4.39µV, p=0.9455) or dogs with laser damage only (42.75±5.58µV, p=0.671).
Almost identical trend of changes has been observed with inner retina function
(b-wave amplitudes and oscillatory potentials).
Conclusion: Intravitreal injection of microspheres with sustained release of GDNF
protected retinal function in laser-damaged eyes for 3 months. At six months post laser
injury, when GDNF was no longer present in the eyes, functional deficits continued to
progress, which is suggestive of progressive loss of retinal function even months after
initial laser damage in an environment depleted of neurotrophic support.
CR: H. Kecova, None; R.H. Kardon, None; E. Lavik, None; S.A. Park, None; S.
Jacobson, None; K. Hamouche, None; E. Alward, None; M.M. Harper, None; S.D.
Grozdanic, None.
Support: Department of Defense (DOD) Peer Reviewed Medical Research Program
(PRMRP PR064674), and Iowa City VA Center for Prevention and Treatment of Vision
Loss
Purpose: To assess the effects of bilirubin, a physiologic cytoprotectant, and
tauroursodeoxycholic acid (TUDCA) in the rd10+/+ mice and mice with light-induced
retinal degeneration.
Methods: Subcutaneous injections of bilirubin or TUDCA were administered to rd10+/+
mice every three days from postnatal day (P) 6 to P49. Retinal function was assessed
by scotopic and photopic electroretinograms (ERGs) at P30 and P50. Photoreceptor
cell survival was assessed by measuring cell density of peanut-agglutinin-stained
retinal whole mounts and outer nuclear layer (ONL) thickness. Albino BALB/c mice
were injected 24 hours and 1 hour before exposure to 8 hours of light at an intensity
of 5,000 lux. Photoreceptor survival was assessed by scotopic and photopic ERGs
taken 24 hours and 7 days after light exposure.
Results: At P30, bilirubin- (n=10) and TUDCA-treated rd10 +/+ mice (n=6) showed
higher (P<0.02) peak mean scotopic ERG (µV) a-wave amplitudes (76+4 and 76+2,
respectively) compared to vehicle-treated littermates (52+4 and 45+5). Mean scotopic
b-wave amplitudes for bilirubin (377+33) and TUDCA (367+30) groups were also higher
(P<0.05) compared to vehicle-treated littermates (193+45 and 267+28, respectively).
Mean photopic b-wave amplitudes for bilirubin- (242+19) and TUDCA-treated mice
(254+22) were greater (P<0.01) than those in vehicle-treated littermates (148+23 and
131+19). ONL thickness measurements were greater (P<0.05) at three out of the six
measurement locations in the bilirubin group, and at four out of six locations in the
TUDCA group. At P50, bilirubin- (n=9) and TUDCA-treated rd10+/+ mice (n=8) showed
higher (P<0.05) peak mean photopic b-wave amplitudes (55+8 and 83+15) compared to
untreated littermates (26+3). Mean cone cell density (cones/0.0529 mm 2) was greater
for bilirubin-treated rd10+/+ (215+30), and TUDCA-treated rd10+/+ (278+21) compared to
untreated rd10+/+ (93+13). Light-exposed BALB/c mice treated with bilirubin or TUDCA
showed significant preservation of ERG function compared to untreated mice.
Conclusions: Treatment with bilirubin or TUDCA effectively slowed photoreceptor
degeneration in rd10+/+ mice through P50 and reduced light-induced photoreceptor
death in BALB/c mice.
CR: B.C. Oveson, None; S.F. Hackett, None; T. Iwase, None; S. Lee, None; T.W.
Sedlak, None; S.H. Snyder, None; P.A. Campochiaro, None; J.U. Sung, None.
Support: NIH Grant EY015025-03, FFB Grant C-NP-0707-0419-JHU05, Knights Templar
Eye Foundation
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3729-3732
Tuesday, May 4, 3:45 PM - 5:30 PM Hall B/C Poster Session Program Number/Board # Range: 3689 - 3735 / A495 - A541
381. Neuroprotection
Organizing Section: RC
3733 - A539
The Effect of Müller Cell Reactivity on Photoreceptor Cell Survival
3734 - A540
Overexpression of Norrin in the RPE of Transgenic Mice Protects Against LightInduced Photoreceptor Damage
S.M. Shah, T. McDaniel, D.A. DiLoreto, Jr.. Flaum Eye Institute, University of
Rochester Medical Center, Rochester, NY.
Purpose: To investigate the role that Müller cell reactivity plays on photoreceptor
cell survival in culture.
Methods: Experiment 1: Four separate sets of Müller cell cultures were prepared
from the central and peripheral retinas of the 3 m (n=3) and 24 m (n=3) old Fischer
344 rats. These were co-cultured with photoreceptor cells from 3 month old Fishcer
344 rats. After 10 days, photoreceptor cell viability was assessed in the co-cultures.
Experiment 2: Four separate sets of Müller cell cultures were prepared from the
central and peripheral retinas of the 3 m (n=3) and 24 m (n=3) old Fischer 344 rats.
Conditioned medium from each of these cultures was added to 5 day old photoreceptor
cell cultures of 3 m old Fischer 344 retinas and cell survival was assessed after 5 more
days. From previous studies based on glial fibrillary acidic protein expression, Müller
cell reactivity was designated in the following fashion: 3m central = normal; 3 m old
peripheral = mildly reactive; 24 m old central = moderately reactive; and 24 m old
peripheral retina severely reactive.
Results: For experiment 1, photoreceptor cells co-cultured with Müller cells from 3 m
old peripheral retinas showed greater survival rates (p < 0.019) than all other groups.
For experiment 2, the only statistical difference seen was a higher photoreceptor cell
survival rate for those cultured with conditioned media from 24 month old Müller
cells versus those from 3 m old Müller cells (p = 0.041).
Conclusions: Previous studies have shown that Müller cell reactivity increases in the
area of photoreceptor cell degeneration and may actually precede it in the Fischer
344 rat. From our experiments, Müller cells seem to play a role in photoreceptor cell
survival due to the secretion of unknown factors related to their reactive state and
also possibly to a mechanism involving cell-to-cell contact.
CR: S.M. Shah, None; T. McDaniel, None; D.A. DiLoreto, Jr., None.
Support: Research to Prevent Blindness
B.M. Braunger1, A. Ohlmann1, S.C. Beck 2, G. Huber2, N. Tanimoto2, Y. Yang3, M. Bösl4, A.
Cvekl3, M.W. Seeliger2, E.R. Tamm1. 1University of Regensburg, Institute of Human
Anatomy and Embryology, Regensburg, Germany; 2University of Tübingen,
Division of Ocular Neurodegeneration, Institute for Ophthalmic Research, Centre
for Ophthalmology, Regensburg, Germany; 3Ophth & Vis Sci & Genetics, Albert
Einstein Coll of Medicine, Bronx, NY; 4Max Planck Institute of Neurobiology,
Martinsried, Germany.
Purpose: Norrin is a secreted protein that controls capillary formation in the developing
retina and acts via the Wnt/ß-catenin pathway. We tested if Norrin has neuroprotective effects
on photoreceptors that are independent from its effects on vascular development.
Methods: Transgenic Rpe65-Norrin mice that express norrin under the control of the
Rpe65 promoter were developed and analyzed by Northern/Western blotting, and by
immunohistochemistry. Wnt/ß-catenin signaling was investigated by analyzing ß-catenin,
and by LacZ staining of mixed Rpe65-Norrin/TOP-Gal Wnt reporter mice. Apoptotic cell
death was analyzed by TUNEL-labeling and histone ELISA. Outer nuclear layer thickness
was measured on semithin sections. Retinal structure and function were tracked in vivo by
SLO, OCT and ERG. Rhodopsin regeneration after bleaching was determined by measuring
rhodopsin levels at different time points in darkness.
Results: Retinae of Rpe65-Norrin mice showed a distinct increase in the amounts of Norrin,
but otherwise expressed no obvious phenotype. Levels of retinal ß-catenin were higher, as
was the intensity of LacZ staining in Rpe65-Norrin/TOP-Gal mice indicating activation
of the Wnt/ß-catenin pathway. 30 hours after light-induced damage (white light, 5000 lux
for 1 h), retinae of Rpe65-Norrin mice showed significantly fewer TUNEL-positive cells as
compared to wild-type littermates, an effect that was supported by histone ELISA. 7 and 14
days after damage, the outer nuclear layer was significantly thicker in Rpe65-Norrin mice
than in controls, indicating an increase in photoreceptor survival. By ERG, retinal function
was significantly better in Rpe65-Norrin mice as compared to wild-type littermates. The
effects on photoreceptor survival could be partially blocked by adding Dickkopf-1, an
inhibitor of the Wnt/ß-catenin pathway. Metabolic rhodopsin regeneration was delayed in
Rpe65-Norrin mice when compared to wild-type littermates.
Conclusion: Transgenic overexpression of Norrin via the RPE protects photoreceptors from
light-induced apoptotic cell death, indicating a neuroprotective role of Norrin. This effect
might is mediated, at least partially, through the Wnt/ß-catenin pathway, and may act via
an inhibition of metabolic rhodopsin regeneration.
CR: B.M. Braunger, None; A. Ohlmann, None; S.C. Beck, None; G. Huber, None; N.
Tanimoto, None; Y. Yang, None; M. Bösl, None; A. Cvekl, None; M.W. Seeliger, None; E.R.
Tamm, None.
Support: Supported by DFG Research Unit (Forschergruppe) FOR1075 (TP7 to ERT), Se837/5-1
& 6-1, and BMBF FKZ 0314106.
3735 - A541
The Effect of Valproic Acid in Mouse Models of RP
S. Kaushal1, S.M. Noorwez1, R. Tzekov1, D. Huang2, Y. Li2, R. Wen2. 1Department of
Ophthalmology, University of Massachusetts Medical School, Worcester, MA;
2
Bascom Palmer Eye Institute, University of Miami, Miami, FL.
Purpose: To determine the effect of valproic acid (VPA) on the amounts of folded
rhodopsin in a P23H mouse model and assess VPA’s neuroprotective effect in the
S334ter-3 rat model.
Method: Heterozygous P23H opsin mice and S334ter-3 rats were produced by
previously published methods. VPA (200 mg/kg) or a placebo was administered
daily (i.p.) to P23H mice, littermate controls, or C57/B6 mice every other day for two
weeks. The mice were dark-adapted overnight and their eyes were then harvested in
dim red light. The total folded rhodopsin was purified from each eye and quantified
by UV/visible spectroscopy using our previously published methods. Separately, in
the S334ter rats, either VPA (300 mg/ml) or a placebo was administered via i.p. daily,
starting at PD 9. Eyes were collected at PD 21, embedded in an Epon/Araldite mixture,
and sectioned at 1 µm thickness to display the entire retina along the vertical meridian.
Retinal sections were examined by light microscopy and evaluated independently
by three observers.
Results. When compared to the retinas receiving a placebo, the P23H mice treated with
VPA had an approximately 30-40% increase in the amounts of folded rhodopsin, which
was statistically significant. There was no substantial effect of VPA on the littermate
controls or the C57/B6 mice. In the retinas of the S334ter rats treated with VPA, the
outer nuclear layer (ONL) had 2-3 rows of photoreceptor nuclei in the superior retina.
In contrast, animals treated with PBS had only 1 row of nuclei in the ONL of the same
retinal region. Separate quantitative data showed that the ONL thickness in treated
animals was significantly higher than the controls.
Conclusion. Systemically delivered VPA in P23H mice resulted in increased folded
rhodopsin levels and in modest but significant preservation of photoreceptors in
the S334ter-3 rats.
CR: S. Kaushal, None; S.M. Noorwez, None; R. Tzekov, None; D. Huang, None; Y.
Li, None; R. Wen, None.
Support: Retina Research and Education Fund, NIH grant EY-018586, JEK grant 08KN09, Hope for Vision, Foundation Fighting Blindness, NIH center grant P30-EY014801,
and Research to Prevent Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3733-3735
Wednesday, May 5, 8:30 AM - 10:15 AM Floridian A Paper Session Program Number Range: 3823 - 3829
405. Advances in Therapeutic Targets for Diabetic Retinopathy
Organizing Section: RC
3823 - 8:30AM
Impaired Balance of NGF/proNGF Causes Retinal Neuronal/Vascular Injury via
Activation of RhoA
3824 - 8:45AM
Inhibition of Plasminogen Activator Inhibitor-1 (PAI-1) Corrects Diabetic
Endothelial Progenitor Cells (EPC) Dysfunction
A.B. El-Remessy1, M.M. Al-Gayyar1, M.A. Abdelsaid1, S. Matragoon1, B.A. Pillai1,
J.J. Nussbaum2. 1Clinical and Experimental Therapeutics, University of Georgia,
Augusta, GA; 2Ophthalmology, Medical College of Georgia, Augusta, GA.
M.B. Grant1A, S. Hazra1A, A.D. Bhatwadekar1A, S. Bartelmez2, P. Higgins3, M.E. Boulton1B.
Pharmacology and Therapeutics, BAnatomy and Cell Biology, 1University
of Florida, Gainesville, FL; 2Beta Stem Therapeutics, Inc, San Francisco, CA;
3
Pharmacology, Albany Medical College, Albany, NY.
A
Purpose: We have previously shown positive correlation between breakdown of blood
retina barrier (BRB), neuronal death and accumulation of the proform of nerve growth
factor (proNGF) in diabetic rat retinas. The purpose of this study is to elucidate the
causal role of proNGF in mediating glial activation, BRB breakdown and neuronal
death and to characterize its downstream signal. In addition, levels of NGF and
proNGF were assessed in human samples from non-diabetic, diabetic patients (DR)
or proliferative diabetic retinopathy (PDR) patients.
Methods: Overexpression of the proNGF in SD rats was achieved by intravitreal
injection of the GFP-conjugated plasmid of cleavage resistant proNGF construct (5μg)
that was successfully incorporated into rat retina by electroporation. Additional groups
received either proNGF/the specific Rho kinase inhibitor Y27632 (100 nmoles/eye) or
GFP-conjugated plasmid only. Neuronal cell death was determined by TUNEL assay
and ganglion cell (GC) count. BRB was assessed by extravasation of BSA-fluoresciene.
Expression of NGF, proNGF and P38MAPK were determined by Western-Blot.
Activation of RhoA was determined by pull down assay.
Results: Significant accumulation of proNGF (3- and 5-fold) and reduction of NGF
levels (35% or 65%) were observed in samples from DR and PDR-patients, respectively,
compared to samples from non-diabetic. Overexpression of proNGF in rat retina
mimicked diabetes action by inducing BRB breakdown (2-fold), retinal neuronal
cell death (5-fold) and reducing GC count (25%). ProNGF caused glial activation as
indicated by GFAP staining and activation of RhoA/p38 MAPK pathway. Treatment
of proNGF expressing animals with Y27632 blocked activation of RhoA/p38MAPK
pathway and preserved BRB and restored GC in rat retina.
Conclusions: Diabetes-induced peroxynitrite alters the homeostasis of NGF in the
retina by decreasing NGF maturation, increasing proNGF accumulation which
activates the common pathway RhoA/p38MAPK leading to retinal neurodegeneration
and vascular permeability. Similar to diabetes, overexpression of proNGF in normal rat
retina caused vascular and neuronal injury that was reversed by Rho kinase inhibitor.
Thus, inhibiting RhoA may be effective therapeutic targets in DR.
CR: A.B. El-Remessy, None; M.M. Al-Gayyar, None; M.A. Abdelsaid, None; S.
Matragoon, None; B.A. Pillai, None; J.J. Nussbaum, None.
Support: SDG from AHA and CDA from JDRF
3825 - 9:00AM
Leukocytes Regulate Retinal Capillary Degeneration in Diabetes Via
Generation of Leukotrienes
R.A. Gubitosi-Klug, R. Talahalli. Pediatrics, Rainbow Babies &Childs Hosp/CWRU,
Cleveland, OH.
Purpose: Degeneration of retinal capillaries in diabetes may result from a chronic
inflammatory process that damages the endothelium. Leukocytes participate in
inflammatory responses through the generation of potent pro-inflammatory mediators
such as the leukotrienes. We investigated the role of the leukocyte and leukotriene
generation in inducing retinal inflammation and retinal capillary degeneration in
the diabetic mouse.
Methods: We generated chimeric mice which lacked the ability to generate leukotrienes
by transplanting 5-lipoxygenase deficient (5LO-/-) bone marrow cells into non-diabetic,
wild-type (WT) mice{5LO-/- → ND.WT} and into streptozotocin-induced diabetic, wildtype mice {5LO-/- → SD.WT}. Comparisons were made to 1) chimeric mice using the
reverse strategy of transplanting WT bone marrow cells into 5-lipoxygenase deficient
mice{WT→ND.5LO -/- and WT →SD.5LO-/-} and to 2) non-chimeric, standard nondiabetic and diabetic WT mice. In vitro experiments were carried out with mouse
leukocytes from diabetic and non-diabetic WT and 5LO-/- mice co-cultured with
mouse retinal microvascular endothelial cells.
Results: Diabetes-induced leukocyte adherence and retinal superoxide generation
were significantly reduced in retinas from the leukotriene deficient mice {5LO-/- →
SD.WT}, compared to both{WT →SD.5LO-/-} and non-chimeric, diabetic WT mice.
(p<0.05) These findings correlated with retinal histology where a significant decrease
in the capillary degeneration and pericyte loss was seen in {5LO-/- → SD.WT}compared
to both{WT →SD.5LO-/-} and diabetic WT mice. (p<0.05) Immunostaining of paraffin
sections of retinas from chimeras indicated a significant decrease in the diabetesinduced increase in expression of NF-kB p65, ICAM-1, MCP-1 and TNF-α in retinas
from chimeras containing 5LO-/- deficient bone marrow cells. Co-cultures of peripheral
blood leukocytes from diabetic WT mice with retinal microvascular endothelial cells
resulted in increased endothelial cell death compared to endothelial cells co-cultured
with non-diabetic WT leukocytes. In contrast, retinal microvascular endothelial cell
death was not increased when cells were cultured with diabetic 5LO-/- leukocytes.
Under co-culture conditions, diabetic WT leukocytes induced a translocation of NF-kB
p65 to the nucleus of mouse retinal microvascular endothelial cells, which was not
detected in co-cultures with diabetic 5LO-/- leukocytes.
Conclusions: In vivo and in vitro data suggest the involvement of leukocytes and
leukotrienes in the regulation of inflammation and capillary degeneration in diabetic
retinopathy.
CR: R.A. Gubitosi-Klug, None; R. Talahalli, None.
Support: NEI KO8EY016833
Purpose: CD34+ EPCs from diabetics demonstrate reduced vascular reparative function
and cellular senescence which may contribute to the vasodegenerative phase of
diabetic retinopathy. Diabetic EPC dysfunction (reduced proliferation, migration
and bioavailable NO) is associated with elevated TGF-β1 expression by these cells
which can be corrected by transient inhibition of TGF-β using phosphorodiamidate
morpholino oligomers (TGF-β1-PMO). We asked whether the cytostatic effects of
TGF-β requires PAI-1 in diabetic EPCs and assessed whether the reparative ability of
diabetic CD34+ cells is improved by transient blockade of PAI-1.
Method: Peripheral blood CD34+ cells from diabetics (n=10) were treated ex vivo
with TGF-β1-PMO and were analyzed for PAI-1 expression. CD34+ cells were stabily
infected with retroviral constructs expressing PAI-1 shRNA or nonfunctional shRNA.
To assess knockdown efficiency we determined mRNA levels of PAI-1. Effect on EPC
function was assessed by cell proliferation (cell number and colony forming ability),
cell migration (Boyden chamber assay) and NO levels (DAF-FM fluorescence). SMAD2
phosphorylation was assessed by western blot.
Results: Treatment of diabetic cells with TGF-β1-PMO resulted in a 80% reduction in
PAI-1 mRNA levels. PAI-1 levels were suppressed by more than 90% in cells expressing
retroviral constructs of PAI-1 shRNA. Retroviral treatment of cells with the PAI-1
shRNA resulted in a 2-fold increase in cell numbers at day 3 (p<0.05) and a 6-fold
increase in colony formation by day 12 (p<0.001) compared to cells treated with
nonfunctional shRNA. SDF-1-induced migration of PAI-1 shRNA treated cells was
enhanced compared to control treated cells (p<0.05). In the diabetic shPAI-1 treated
CD34+ cells, NO production was restored to non-diabetic levels. TGF-β1 treatment
of nondiabetic cells resulted in growth inhibition while knockdown of PAI-1 using
viral PAI-1 shRNA in these cells resulted in cell proliferation. SMAD activation by
TGF-β1 (200 pM) was not affected by PAI-1 knockdown in either diabetic or controls.
Conclusion: Our results suggest that the cytostatic activity of TGF β1 in diabetic CD34+
cells requires PAI-1 since knockdown PAI-1 resulted in bypass of the growth inhibition
by TGF-β1. Blockade of PAI-1 may offer a promising therapeutic strategy for restoring
vascular reparative function in senescent diabetic CD34+ cells.
CR: M.B. Grant, None; S. Hazra, None; A.D. Bhatwadekar, None; S. Bartelmez, Beta
Stem Therapeutics, Inc., E; P. Higgins, None; M.E. Boulton, None.
Support: NIH grants 2RO1 EY012601-08 , 2RO1 EY007739-17, R01 EY018358
3826 - 9:15AM
TNFα is Essential for Progressive BRB Breakdown in Diabetic Retinopathy and
Oxygen-Induced Ischemic Retinopathy and It Promotes Apoptosis in Retinal
Vessels and Neurons
S.A. Vinores1, H. Huang1, J. Gandhi1, X. Zhong1, W. Xiao2. 1Ophthalmology, Johns
Hopkins Wilmer Eye Inst, Baltimore, MD; 2Cancer Comprehensive Center, Ohio
State Univ, Columbus, OH.
Purpose: Blood-retinal barrier (BRB) breakdown in diabetic retinopathy (DR) and
other ocular disorders is associated with inflammation and retinal leukostasis is
almost totally suppressed in the absence of TNFα. This study was conducted to
determine whether TNFα is critical for BRB breakdown in DR and other ischemic
retinopathies and for apoptosis of retinal vascular cells and neurons resulting from
oxidative stress and in DR.
Methods: Diabetes was induced in TNFα(KO) mice with streptozotocin (STZ). Ins2Akita
mice were crossed with TNFα(KO) mice to provide a genetic model of diabetes, devoid
of TNFα. Oxygen-induced ischemic retinopathy (OIR) was induced by placing P7
mice in 75% oxygen for 5 days and removing them to room air for 5 days. The BRB
was assessed using a quantitative assay with 3H-mannitol as a tracer. Apoptosis was
evaluated with TUNEL and activated caspase-3 staining.
Results: The absence of TNFα did not influence DR-associated BRB breakdown at
6 weeks, but it completely prevented it at 3 months in TNFα(KO) X Ins2Akita mice
and at 6 months in STZ-diabetic TNFα(KO) mice and it significantly reduced BRB
breakdown in OIR. An absence of TNFα also significantly reduced apoptosis after 3
months of STZ-induced diabetes and protected retinal vascular cells in the inner and
outer plexiform layers, photoreceptors, retinal ganglion cells, and retinal neurons in
the inner nuclear layer from oxidative stress-induced apoptosis, which is associated
with age-related macular degeneration (AMD).
Conclusions: TNFα is critical for late, but not early BRB breakdown in DR and it
promotes BRB breakdown in OIR. This suggests that TNFα-induced inflammation is
not responsible for the early BRB breakdown that occurs in DR, but it may be essential
for the later BRB breakdown and apoptosis in the retina as the disease progresses. TNFα
blockade also protects retinal vascular cells and neurons from oxidative stress. The
results suggest that TNFα inhibition could provide benefit in DR and other ischemic
retinopathies as well as in AMD.
CR: S.A. Vinores, None; H. Huang, None; J. Gandhi, None; X. Zhong, None; W.
Xiao, None.
Support: NIH Grant EY017164 and an unrestricted grant from Research to Prevent
Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3823-3826
Wednesday, May 5, 8:30 AM - 10:15 AM Floridian A Paper Session Program Number Range: 3823 - 3829
405. Advances in Therapeutic Targets for Diabetic Retinopathy
Organizing Section: RC
3827 - 9:30AM
IL-6 Is a Mediator of Retinal Neovascularization During Ischemic Retinopathy
M.A. Rojas, Sr.1A, W. Zhang1A,1B, Z. Xu1A, S.K. Virmani1A, A. Patel1A, N.T. Tsai1A, S.E.
Brooks1A, R.W. Caldwell1B, R.B. Caldwell1A,2. AVascular Biology Center, BPharmacology
and Toxicology, 1Medical College of Georgia, Augusta, GA; 2VA Medical Center,
Augusta, GA.
Purpose: Retinal neovascularization is a major cause of blindness in proliferative diabetic
retinopathy and retinopathy of prematurity. Interleukin-6 (IL-6) has been found to be
increased in vitreous samples from patients with proliferative diabetic retinopathy, but
the specific role of IL-6 in retinal neovascularization is not known. The goal of this study
was to directly test whether IL-6 expression is required for retinal neovascularization
during ischemic retinopathy.
Methods: Oxygen-induced ischemic retinopathy (OIR) was induced by maintaining
neonatal mice in 70% oxygen from postnatal day (P)7 to P12 and room air from P12 to
P17. Control mice were kept in room air. C57BL/6 wild type (WT) or IL-6 +/- or IL6-/mice were used in this study. Retinas were prepared for analysis of mRNA for IL-6, IL-6
receptor, and VEGF by quantitative RT-PCR. Superoxide was assayed by dihydroethidium
(DHE) labeling and quantified by the Metamorph Image System. The retinal vasculature
was examined by fluorescence microscopy after labeling retinal wholemounts with
isolectin-B4. Morphometric data were quantified using NIH imageJ.
Results: Levels of IL-6, IL-6 receptor and VEGF mRNA were markedly and progressively
increased in the retinas of OIR mice as compared with the controls (P<0.05). Associated
with the increases in IL-6 signaling components, the retina from OIR mice displayed
increased superoxide formation as determined by DHE labeling (P<0.05). This increase
was blocked by IL-6 knockout (P<0.05). The specificity of the staining was verified by
quenching superoxide with PEG-SOD or inhibition of NADPH oxidase with apocynin.
To address the role of IL-6 in ischemic neovascularization, areas of neovascular tufts
and vascular dropout were determined in IL-6 +/- and IL-6 -/- littermates. There was no
difference in the vascular dropout area at P12. However, at P17 the neovascular tuft area
was decreased by ~50% in IL-6 -/- mice as compared IL-6 +/- mice (P<0.01). In contrast,
physiological revascularization was increased by IL-6ko. The capillary-free area was
reduced by 20.2% in IL-6 -/- as compared with IL-6 +/- mice (P<0.001).
Conclusions: Lack of IL6 prevents oxidative stress and retinal neovascularization while
enhancing physiological revascularization during ischemic retinopathy. This result
indicates that IL6 is critically involved in the progress of retinal neovascularization
during ischemic retinopathy.
CR: M.A. Rojas, Sr., None; W. Zhang, None; Z. Xu, None; S.K. Virmani, None; A. Patel,
None; N.T. Tsai, None; S.E. Brooks, None; R.W. Caldwell, None; R.B. Caldwell, None.
Support: JDRF 10-2009-575, NIH Grants EY11766, EY04618, HL70215, VA Merit Award.
3828 - 9:45AM
Thioredoxin Interacting Protein (TXNIP) Mediates Inflammation, Fibrosis and
Gliosis in Early Diabetic Retinopathy
P.L. Singh1A, L. Perrone1B, T.S. Devi1B. AAnatomy/Cell Biology and Ophthalmology,
B
Department of Anatomy and Cell Biology, 1Wayne State Univ Sch of Med, Detroit,
MI.
Purpose: We have recently shown that TXNIP induces histone remodeling and
inflammatory gene expression in retinal endothelial cells under diabetic conditions.
Here, we investigate whether TXNIP mediates inflammation, fibrosis and glial cell
reactivity in early diabetic retinopathy.
Methods: Diabetes was induced in Sprague Dawley rats by Streptozotocin and
maintained for 4 weeks. TXNIP was down-regulated by intravitreal injection of
promoter-targeted siRNAs using cell penetrating peptides as cargo carriers, and
retinal inflammation and fibrosis were determined.
Results: Messenger RNA and protein levels of TXNIP are significantly increased in
the diabetic retina when compared with normal rat retina. The increase in TXNIP
is associated with enhanced levels of its downstream targets such as inflammatory
cyclooxygenase 2 (Cox-2) and sclerotic fibronectin (FN). Furthermore, we observed that
4 weeks of diabetes induction in rats causes retinal muller cell gliosis (GFAP induction)
and neuronal injury (synapsin 1 down-regulation and caspase-3 activation). TXNIP
gene silencing blunts Cox-2, FN, and GFAP expression in the retina of diabetic rats.
Conclusions: The results demonstrate that TXNIP is a mediator of inflammation,
fibrosis and gliosis in early diabetic retinopathy.
CR: P.L. Singh, None; L. Perrone, None; T.S. Devi, None.
Support: JDRF International
3829 - 10:00AM
The Chemokine Ccl2/MCP-1: A Possible Therapeutic Target in Diabetic
Retinopathy
A. Das1A, S. Rangasamy1B, J. Maestas1B, P. McGuire1B. AMSC10-5610 Surgery, BDept of
Cell Biology and Physiology, 1Univ of New Mexico Sch of Med, Albuquerque, NM.
Purpose: One of the hallmarks of diabetic retinopathy is the alteration of the bloodretinal barrier resulting in macular edema. Although the VEGF has been identified
as an important mediator of this process, other molecules may also play a role. In this
study, we examined the angiogenesis related gene expression in retinas of diabetic
rats. Furthermore, we investigated in depth one of the most upregulated proteins, Ccl2
(MCP-1), and the molecular mechanisms of the actions of this molecule.
Methods: Diabetes was induced in Sprague-Dawley rats by a single intraperitoneal
injection of streptozotocin. Human retinal microvascular endothelial cells (HREC)
were grown to confluence, and treated with high glucose (30.5 mM) for 5 days. RNA
was extracted from the rat retina and HREC. The expression pattern of angiogenesis
related gene was analyzed using PCR based arrays from SA Biosciences.
Results: The rat angiogenesis array revealed that the Ccl2 gene was significantly
up-regulated in the retinas of rats with 4 weeks (p=0.005) and 8 weeks (p=0.02) of
diabetes by more than 20 fold over the non-diabetic group. We found that the Ccl2
is most prominently up-regulated compared to other genes. We also found that Ccl2
gene was significantly increased (p<0.01) by more than two fold in HRECs treated
with high glucose for 5 days compared to low glucose treated cells. Furthermore,
angiopoetin-2 (Ang-2), an important vasopermeability factor, significantly up-regulated
the expression of Ccl2 in the HRECs.
Conclusions: Hyperglycemia induces the expression of Ccl2 in the retina of diabetic
rats at early stages as well as in isolated human retinal endothelial cells. We also find
that Ang-2 upregulated the Ccl2 expression indicating a cross talk between Ccl2
and Ang-2. An understanding of the role of Ccl2 during the early stages of diabetic
retinopathy may help us to elucidate the molecular mechanisms and possibly lead to
the development of novel therapeutic targets for this disease.
CR: A. Das, None; S. Rangasamy, None; J. Maestas, None; P. McGuire, None.
Support: NIH EY12604
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
3827-3829
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4028 - A421
The Zebrafish Patched2 Mutant as a Model for the Study of Basal Cell Naevus
Syndrome (BCNS)-Related Ocular Pathologies
4029 - A422
Histopathology of the Retina in an Eye Donation From a Patient With an RPE65
Mutation
J. Bibliowicz, J.M. Gross. Molecular Cell and Developmental Biology, The University
of Texas at Austin, Austin, TX.
M.E. Rayborn1, V.L. Bonilha1, Y. Li1, G.H. Grossman1, J.G. Hollyfield1, E.L. Berson2.
1
Ophthalmology, Cole Eye Institute/Cleveland Clinic Lerner College of Medicine,
Cleveland, OH; 2Ophthalmology, The Berman-Gund Laboratory, Harvard Medical
School, MEEI, Boston, MA.
Purpose: Müller glia are retina-specific glia that normally maintain neuronal health
and retinal organization, and are known to become ‘reactive’ upon exposure to
growth factors or in response to retinal injury. Our previous characterization of the
embryonic zebrafish patched2 mutant retina revealed localized upregulation of GFAP,
a marker for ‘reactive’ glia, as well as morphological abnormalities at the vitreo-retinal
interface, where Müller glial endfeet terminate. Here, we extend our analysis of the
patched2 mutant retina to later developmental stages to investigate whether Müller
glia homeostasis and retinal organization are perturbed when Hedgehog pathway
activity is upregulated.
Methods: Histological and immunohistochemical analyses were utilized to analyze
retinal architecture and Müller glial reactivity in juvenile patched2 mutants and their
heterozygous and homozygous siblings.
Results: Analysis of the juvenile patched2 mutant retina revealed upregulation of
GFAP, a marker for ‘reactive’ glia, throughout the central retina. Ectopic proliferation
and rosette formation were detected in the central retina and these abnormalities
were spatially associated with displaced Müller glia. Current research efforts are
focused on determining whether Müller glia reactivity may play a role in the onset
of these retinal abnormalities, and/or whether Müller glia are displaced in response
to these defects.
Conclusions: patched2 deficiency in zebrafish results in phenotypes that are similar to
the ocular abnormalities observed in human patients suffering from Basal Cell Naevus
Syndrome (BCNS), a disorder that has been linked to mutations in the human PTCH
gene (the orthologue of the zebrafish patched2), and further support the utility of the
patched2 mutant line as a model for the study of BCNS-related ocular pathologies.
CR: J. Bibliowicz, None; J.M. Gross, None.
Support: NIH RO1 EY18005
Purpose: RPE65 is an enzyme exclusively expressed in the retinal pigment epithelium
(RPE) that converts all-trans retinol to the 11-cis form. Mutations in the RPE65 gene
are found in some patients with Leber congenital amaurosis. We evaluated the
histopathology of a donor eye from a patient with a homozygous missense change
Ala132Thr in the RPE65 gene.
Methods: Autopsy eyes were obtained from a 56 year old woman who died from
metastatic breast cancer. The eyes were fixed in 4% paraformaldehyde and 0.5%
glutaraldehyde in phosphate buffer within 13.5 hours postmortem. Small areas
from the fundus periphery were processed for electron microscopy and indirect
immunofluorescence, using monoclonal antibodies to rhodopsin (mAB B630N) and
cone arrestin (mAB 7G6). The content of autofluorescent material was analyzed. The
donor eye was compared to a matched normal eye.
Results: The patient had night deficiency and decreased side vision since childhood.
At age 51 she had hand motions OD and 20/200 OS, a central island of vision with far
peripheral crescents OU, and peripheral bone spicule pigmentation OU. Cone ERGs
were barely detectable. Histologic findings revealed a highly disorganized retina
with indistinct layers in each quadrant. The RPE layer displayed thinning in some
regions of the periphery. Rods were virtually absent in the affected retina. Cones
were present in the macula, but were mostly absent from the retinal periphery. Cone
synapses were not observed. Autofluorescent material was greatly reduced in the
RPE in all areas studied.
Conclusions: This patient with an RPE65 mutation showed an absence of rods and
cones in the periphery. In the macula, cone photoreceptors were present, albeit highly
disorganized. The lack of autofluorescent material in the RPE suggested that these
cells had not been functional in the photoreceptor outer segment renewal process
for an extended period of time.
CR: M.E. Rayborn, None; V.L. Bonilha, None; Y. Li, None; G.H. Grossman, None; J.G.
Hollyfield, None; E.L. Berson, None.
Support: Supported by Histopathology Grant and Research Center Grants from The
Foundation Fighting Blindness; Research to Prevent Blindness Unrestricted Grants;
and the National Eye Institute (NIH).
4030 - A423
Identification and Distribution of Prominin-Like Molecules in the Retina of
Lower Vertebrate Model Organisms
4031 - A424
Endoplasmic Reticulum Stress Precedes Photoreceptor Degeneration in Ccl2 -/-/
Cx2cr1-/- Mice
J. Jaszai, D. Corbeil. Tissue Engineering Laboratories, University of Technology,
Dresden, Germany.
M.K. Ertel1, K.G. Sheets2A, Y. Zhou2A, C.-C. Chan 3, J. Tuo 4, W.C. Gordon2B, N.G. Bazan2C.
1
Neuroscience Center of Excellence, LSU Health Sciences Center - New Orleans,
New Orleans, LA; ANeuroscience Center, BOphthalmology & Neuroscience
Center, COphthal & Neuroscience, 2LSU Health Sciences Center, New Orleans, LA;
3
Immunopathol Section, Lab of Immunology, National Eye Institute/NIH, Bethesda,
MD; 4Laboratory of Immunology, National Eye Institute/NIH, Rockville, MD.
Purpose: Mutations in the human PROM1 gene, encoding for the pentaspan
transmembrane glycoprotein prominin-1, have been previously linked with autosomal
recessive progressive retinal degeneration [retinitis pigmentosa] mainly affecting rods
and peripheral vision (Maw et al. Hum Mol Genet, 2000; Zhang et al. Hum Genet, 2007).
A missense mutation of the gene causes dominant macular degeneration, affecting
mainly cones and central vision (Yang et al. J Clin Invest, 2008). The features of human
progressive retinal degeneration can be phenocopied by a murine prominin-1 gene
knockout model (Zacchigna et al. J Neurosci, 2009). Given that the major cell classes
and laminar anatomical organization of the vertebrate retina appears to be remarkably
conserved during evolution, the retina of lower vertebrate species, e.g. zebrafish (D.
rerio), axolotl (A. mexicanum) and chick (G. gallus) offers unique model systems for
disentangling problems associated with retinal morphogenesis, degeneration and
regeneration. To establish a comparative framework for future functional studies
we have isolated and analyzed the retinal distribution of prominin-like genes from
lower vertebrate species (fish, axolotl, chick).
Methods: Prominin-like genes were cloned from zebrafish, axolotl and chick and their
expression was investigated by in situ hybridization in the retina.
Results: In the analyzed species an evolutionarily conserved expression of prominin1-like molecules was found in photoreceptor cells. Interestingly, the zebrafish genome
encodes for two related prominin-1-like genes (co-orthologues), i.e. prominin-like
1a and 1b, showing co-incident expression in photoreceptors and unique, nested
expression profile in the inner nuclear layer harbouring among others slowly dividing/
quiescent potential stem cells.
Conclusions: Our analysis revealed that not only protein sequences of lower vertebrate
prominins show high similarity to their mammalian (mouse, human) counterpart, but
also their expression features. Furthermore, our findings suggest a lineage specific
sub-functionalization of zebrafish prominin-1 co-orthologues, beside the potential
functional redundancy at overlapping sites within the photoreceptor cells. These
data will be discussed.
CR: J. Jaszai, None; D. Corbeil, None.
Support: None
Purpose: The endoplasmic reticulum (ER) is a sensor of homeostatic disruptions that
provides quality control to prevent unfolded or misfolded protein transit to the Golgi,
channeling them to degradation. Retinal degenerations and other perturbations that
accumulate misfolded proteins in the ER lumen trigger ER stress, which is expressed
as the unfolded protein response in an attempt to cope with this abnormal buildup. In
retinal degenerative diseases, ER stress markers are up-regulated. Consequently, in this
study, we examined whether ER stress markers demonstrated increased expression in the
Ccl2-/-/Cx3cr1-/- (DKO) mouse model of age-related macular degeneration (AMD).
Methods: Eyes from 2 and 18 month DKO mice and age matched C57/Bl6 controls were
fixed, cryo-sectioned at 20 micron thickness, and immunohistochemically examined
for ER stress markers (e.g., GRP78-BiP). Typical immunolocalization methods were
employed. Confocal microscopy was used to visualize the location of this marker in
histological sections.
Results: Retinal sections from 18 month DKO mice, immunolabeled, demonstrated
localization and increased expression of the ER stress marker GRP78-BiP. Inferior
portions of these retinas, the areas that exhibited more profound pathological changes,
demonstrated significant photoreceptor loss. The superior retina maintained a greater
degree of structural integrity, but expressed higher levels of the ER stress marker GRP78BiP. In 2 month DKO mice, retinal damage was already apparent in the inferior retina
with focal disorganization in the outer nuclear layer. As early as 2 months, these mice
showed increased levels of ER stress. Retinal sections of 2 month controls showed little
ER stress; older controls demonstrated increased labeling. However, control labeling
was less than that observed in mutants.
Conclusions: Retinas of these mice contained varying, focal pathological abnormalities.
In areas where retinal architecture was relatively conserved, increased expression of ER
stress markers was found. Expression of the ER stress marker precedes structural damage
in RPE, photoreceptors, and inner retina, implicating ER stress as an important event
that precedes photoreceptor loss in the DKO mice. This may be an amenable therapeutic
target to explore for AMD.
CR: M.K. Ertel, None; K.G. Sheets, None; Y. Zhou, None; C.-C. Chan, None; J. Tuo,
None; W.C. Gordon, None; N.G. Bazan, None.
Support: NIH/NEI R01 EY005121
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4028-4031
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4032 - A425
The Influence of MicroRNAs on Nr2e3 Associated Retinal Disease in the rd7
Mouse Model
4033 - A426
Erg Responses in the Dystrophin Dp71-Null Mice
A.S. Jelcick1, J. Reinecke1, Y. Yuan1, N.B. Haider2. 1Genetics Cell Biology and Anatomy,
University of Nebraska Medical Center, Omaha, NE; 2Genetics, Cell Biology,
Anatomy, Univ of Nebraska Medical Ctr, Omaha, NE.
D. Cia1, M. Simonutti2, A. Sene2, R. Benard2, M. Roux2, M. Doly1, J. Sahel2, A. Rendon2.
1
Laboratory of Sensory Biophysics, School of Medicine - University of Clermont
1, Clermont-Ferrand, France; 2Inserm U-968 - UMR S 968, University of Pierre et
Marie Curie – Paris 6, Paris, France.
Purpose: The variability of expression genes can contribute to the observed phenotype
both a positive and negative manner. Micro RNAs (miRNA) influence gene expression
by negative regulation. The purpose of this study is to examine the effect of microRNAs
on Nr2e3 associated retinal disease. Nr2e3 is a crucial factor for proper photoreceptor
development and function. Lack of Nr2e3 in patients is associated with enhanced
S-cone syndrome and retinitis pigmentosa and in a superfluous production of blue
opsin expressing cone cells and a slow progressive retinal degeneration as observed
in the rd7 mouse model. We examine the influence of miRNAs on Nr2e3 associated
retinal degeneration by comprehensive gene expression profile in the developing
and mature rd7 retina.
Methods: microRNA was collected from embryonic (E) 18, P2, P6, P14, and P30 B6
(control) and rd7 retinas. microRNA samples were hybridized to mirVana miRNA
chips. Initial normalization and analysis of miRNA expression data was performed
using BRB ArrayTools. Subsequent analysis to determine statistically significant
miRNA’s was performed using Stanford Array Tool’s SAM software. microRNA gene
expression differences were confirmed by quantitative real time PCR.
Results: Initial filtering, normalization and organization of miRNA’s expressed
revealed 1981 miRNA’s to be expressed across B6 and rd7 samples. A subset of over
200 miRNA’s were entered into the SAM analysis software and statistically significant
miRNA’s found to be differentially expressed across time points were also evaluated.
Of the genes confirmed to be differentially expressed, it was observed that the target
genes belonged to multiple families with multiple functions.
Conclusions: Differential expression of microRNAs were observed in B6 versus
rd7 retinas and thus may contribute to retinal degeneration in these mice. Further
pathway analysis will shed additional light on the possible functions of the genes
regulated by these miRNAs, but initial analysis shows their involvement in regulation
of genes encoding f-box proteins, nuclear receptors, and genes encoding for various
types of ECM related fibers.
CR: A.S. Jelcick, None; J. Reinecke, None; Y. Yuan, None; N.B. Haider, None.
Support: Grant P20-RRO18788-03, NIH Grant EY017653
Purpose: The dystrophin Dp71 is the most abundant Duchenne Muscular Dystrophy
(DMD) gene product expressed in the retina. This protein is localized in the Müller
glial cells (MGC) and regulates the retinal homeostasis by clustering Kir4.1 and AQP4
channels. It is known that 80% of DMD patients and the mdx3cv mouse strain with a
mutation affecting all the DMD gene products show an increasing implicit time and
a high attenuation of the b-wave amplitude. The aim of this study was to determine
whether, solely the absence of Dp71 affects the electroretinogram (ERG) response.
Methods: Ganzfeld ERGs were obtained from dark adapted Dp-71 null mice and
wild-type littermates. Scotopic flash ERGs were recorded at different light intensities.
Amplitudes and implicit times were measured for a-wave and b-wave. The b-wave
sensitivity curves were fitted to calculate the saturated b-wave amplitude (Vmax)
and the retinal sensitivity parameter (k). Oscillatory potentials (OP) were isolated by
filtering the ERG responses. Photopic flash ERGs were recorded after 10 minutes of
light adaptation. Photopic Flicker ERGs were obtained with light flashes.
Results: The scotopic ERGs recorded from Dp-71 null mice showed normal a-wave
amplitudes, but revealed a reduction in the b-wave amplitudes at the highest light
intensities. Consequently, the b/a-wave amplitude ratio was smaller in Dp71-null mice
compared with littermates. The Vmax value was reduced by 25%, but no change was
found in the k parameter. Implicit times of the a-wave and b-wave were not different
between Dp71-null mice and wild-types. The OP amplitudes and implicit times showed
no difference between the two strains. No change was observed in the photopic ERG
and flicker responses in Dp71-null mice compared with littermates.
Conclusions: The absence of dystrophin Dp71 is associated with a reduction in
b-wave amplitude of the dark-adapted ERG. This result suggests that Dp71 together
with all the DMD gene products expressed in mouse retina may contribute to normal
retinal electrophysiology.
CR: D. Cia, None; M. Simonutti, None; A. Sene, None; R. Benard, None; M. Roux,
None; M. Doly, None; J. Sahel, None; A. Rendon, None.
Support: Institut National de la Santé Et de la Recherche Médicale (INSERM),
Association Française contre les Myopathies (AFM)
4034 - A427
Receptor Interacting Protein 1 Kinase is an Essential Mediator of Programmed
Photoreceptor Necrosis After Retinal Detachment
4035 - A428
βA3/a1-Crystallin is Required for Cellular Integrity of Astrocytes
Y. Murakami1, G. Trichonas1, A. Thanos1, Y. Morizane1, S. Jardeleza1, T. Hisatomi2, E.S.
Gragoudas1, J.W. Miller1, D. Vavvas1. 1Department of Ophthalmology, Massachusettes
Eye and Ear Infirmary, Boston, MA; 2Department of Ophthalmology, Graduate
School of Medical Sciences, Kyushu University, Fukuoka, Japan.
Purpose: To investigate the role of receptor interacting protein 1 (RIP1) kinase during
photoreceptor death after retinal detachment (RD).
Methods: RD was created in the eyes of Brown Norway rats by subretinal injection of
viscoelastic (Provisc). The eyes without RD were used as a control. Expression levels
of RIP family members were assessed by quantitative real-time PCR. Treated eyes
received Z-VAD (pan-caspase inhibitor) and/or necrostatin-1 (RIP1 kinase inhibitor)
injected subretinally at the time of RD induction. Photoreceptor death was evaluated
by transmission electron microscopy and changes of outer nuclear layer thickness
at 3 days after RD. Oxidative stress in the retina was assessed by ELISA for carbonyl
adducts on proteins. Cellular localization of apoptosis-inducing factor (AIF) was
assessed by immunostaining.
Results: RIP3 expression, a key activator of RIP1 kinase, increased over 10-fold in the
retina 3 days after RD. Caspase inhibition by Z-VAD failed to prevent photoreceptor
loss after RD; however, ultrastructural analysis revealed that the form of photoreceptor
death changed from apoptosis to necrosis. These necrotic changes observed in Z-VADtreated retina were substantially prevented by combined treatment with necrostatin-1
and Z-VAD, along with a reduction in oxidative stress and the nuclear translocation
of AIF.
Conclusions: These findings indicate that RIP1 kinase plays an essential role after
RD to induce programmed necrosis and that simultaneous inhibition of RIP1 kinase
and caspases may be a novel therapeutic strategy to prevent photoreceptor death
after RD.
CR: Y. Murakami, None; G. Trichonas, None; A. Thanos, None; Y. Morizane, None; S.
Jardeleza, None; T. Hisatomi, None; E.S. Gragoudas, None; J.W. Miller, None; D.
Vavvas, None.
Support: Bacardi, Research to Prevent Blindness, Lions, Onassis, Fight For Sight,
Harvard Ophthalmology Department Support, NIH Core Grant for Vision Research
to MEEI
S.L. Hose, C. Zhang, R. Grebe, G.A. Lutty, J.S. Zigler, Jr., D. Sinha. Ophthalmology,
Johns Hopkins Wilmer Eye Inst, Baltimore, MD.
Purpose: To determine the effect of the Nuc1 mutation in βA3/A1-crystallin on
astrocytes.
Methods: The Nuc1 spontaneous mutant rat (Molecular & Cellular Neuroscience,
2008) and age-matched Sprague-Dawley wildtype rats were used in these studies.
Astrocytes were cultured from brains of Wildtype and Nuc1 neonatal rats. Expression
of autophagy proteins in the astrocytes was assessed by immunohistochemistry
and western blotting. Structural changes in the astrocytes including the assembly of
intermediate filaments were evaluated using electron microscopy.
Results: In the retina and the brain, βA3/A1-crystallin is expressed only in astrocytes.
Our studies with the Nuc1 mutant astrocytes indicate that βA3/A1-crystallin plays an
important role in the assembly of intermediate filaments (Ifs) in that large aggregates of
Ifs are observed in the cytoplasm. The mutation also causes nuclear membrane fragility,
change in nuclear shape and altered disposition of chromatin. In Nuc1 astrocytes, the
nucleolus is larger, singular and consistently located at the periphery of the nucleus
rather than in the more central location typical of normal astrocytes. In addition,
unique perinuclear vacuolar structures containing amorphous and electron-dense
material are present in the mutant astrocytes. These structures appear to be part of an
autophagy process. The activation of autophagy is supported by an increase in Beclin
1 in Nuc1 astrocytes compared to normal during postnatal development. Beclin 1 is
required for initiation of autophagosome formation during autophagy.
Conclusion: Our studies with Nuc1 suggest that βA3/A1-crystallin may have an
important role in maintaining the cellular integrity of astrocytes. Our studies also
indicate that autophagy, the only known process in eukaryotic cells for degrading
cellular organelles and recycling them to ensure cell survival, is severely affected in
astrocytes with the βA3/A1-crystallin gene mutation, Nuc1.
CR: S.L. Hose, None; C. Zhang, None; R. Grebe, None; G.A. Lutty, None; J.S. Zigler,
Jr., None; D. Sinha, None.
Support: NIH Grants EY018636, EY019037 and EY019037-2S1 (to DS), EY09357 (to
GAL), EY01765 (to WEI), Research to Prevent Blindness (to DS), Helena Rubinstein
Foundation (to DS)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4032-4035
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4036 - A429
Connexin Modulation During Neurodegeneration of the Chick Retina
4037 - A430
Elucidation of Protein Interaction Network for Tubby-Like Protein 1
V. Paschon, G. S. V. Higa1, T. Barbosa1, P.S. Akamine2, D.E. Hamassaki2, L.R.G. Britto1
& A.H. Kihara1. Institute of Bioquemistry, University of Sao Paulo, Sao Paulo,
Brazil.
G.S. Alvarado, N. Caberoy, W. Li. Ophthalmology, Bascom Palmer Eye Inst.-Univ. of
Miami, Miami, FL.
Purpose: Intercellular communication mediated by connexin (Cx) channels in the gap
junctions (GJ) plays an important role in a variety of tissues allowing the exchange of
small molecules up to 1kDa and ions. Accruing evidence indicates that gap junctions
are involved in neurodegeneration after injury. The passage of calcium ions through
the GJs plays an important role in neuronal survival/death, due to its cytoxicity in
high concentrations. However, studies using GJ pharmacological blockers and KO
animal models endowed apparently contradictory results in relation to the role of
coupling in neuroprotection. In this work, we analyzed the expression of Cx in a
focal retinal lesion induced by mechanical trauma (Fig.1). Remarkably, this model
allowed spatial and temporal de[[Unsupported Character - &#64257;]]nition of the
lesion with high reproducibility, permitting visualization of the focus, penumbra
and adjacent areas.
Methods: In the retina, Cx36 is abundantly expressed and plays essential roles in the
visual signaling. Combining techniques such as real time PCR, western blot and IHC,
we assessed the spatiotemporal expression of Cx in this experimental condition.
Results: We analyzed Cx36 and Cx43 expression pattern during retinal degeneration.
Cx36 expression steady state levels mildly changed during the degenerative process,
whereas Cx43 exhibited modifications in the phosphorylation status, protein levels
and gene expression. The functional role of cell coupling was assessed by employing
GJ blockers and openers, combined with methods for evaluating cellular death/
viability (TUNEL, Fluoro-Jade and lactate dehydrogenase, LDH). Indeed, changes in
cell coupling affected secondary cell loss. CBX, a broad-spectrum GJ blocker, reduced
LDH release after 4 hours in a concentration-dependent manner. Quinine, a Cx36- and
Cx50- specific blocker, reduced significantly the LDH released after 1, 2 and 4 hours
in a concentration-dependent manner.
Conclusions: To summarize, Cx expression in chick retina was modulated during
degenerative processes. In addition, control of GJ channel permeability may take part
in reliable neuroprotection strategies.
CR: V. Paschon, None.
Support: FAPESP
4038 - A431
Absence of Neuronal Transmission Occurs Prior to the Development of Schisis
in a New Mouse Model for X-Linked Juvenile Retinoschisis (Rs1 E4E5)
Y. Ueki1, M.H. Elliott2, L.L. Molday3A, R.S. Molday3B, J.D. Ash2. 1Neuroscience,
University of Oklahoma Health Sciences Center, Oklahoma City, OK;
2
Ophthalmology, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK;
A
Biochemistry & Molecular Biology, BBiochemistry/Molecular Biology, 3University
of British Columbia, Vancouver, BC, Canada.
Purpose: Mutations in retinoschisin (Rs1), a gene encoding retinoschisin (Rs1), cause
a progressive retinal disorder called X-linked juvenile retinoschisis (XLRS) often in
young males. Hallmarks of the disease are the splitting of the inner retina (schisis)
and reduced retinal function. We have identified a mouse line with a new spontaneous
mutation in retinoschisin gene (Rs1E4E5). The purpose was to characterize the mutation
and to investigate the role of Rs1 in retinal development and/or maintenance of
retinal function.
Methods: Western blots and immunohistochemistry were performed to detect Rs1
protein and synaptic marker expression in developing wildtype and Rs1E4E5 retinas.
The mutant cDNA was amplified by PCR and sequenced to find potential mutations.
Based on the cDNA clones, primers were designed to amplify the RS1 mutant allele
in the mouse genome. Morphology and function of the retina were analyzed by
histology and electroretinography (ERG).
Results: mRNA and DNA analyses revealed a duplication of Rs1 exons 4 and 5, most
likely the result of unequal recombination between two X chromosomes. The mRNA
from the Rs1E4E5 allele is expressed, but affected males (Rs1E4E5/ Y) and homozygous
females (Rs1E4E5/ E4E5) have no detectable Rs1 protein expression in the retina. Rs1E4E5/
Y
or Rs1E4E5/ E4E5 mice have typical inner retinal schisis and no detectable inner retinal
function at any age. Our data suggest that synapses develop, but signal transmission
to the inner retina does not occur. Data also suggest that schisis occurs subsequent
to failure of neuronal transmission.
Conclusions: Analyses showed that this Rs1E4E5 line is another useful model for
studying progressive retinal degeneration that occurs in human XLRS. Data suggest
that the C-terminus of RS1 is necessary for proper folding and expression in vivo. Our
data also suggest that schisis is not likely the cause of transmission defects. Rather, cyst
formation and schisis occurs subsequent to the absence of neuronal transmission.
CR: Y. Ueki, None; M.H. Elliott, None; L.L. Molday, None; R.S. Molday, None; J.D.
Ash, None.
Support: R01 EY016459, EY02422, P20 RR017703, P30 EY012190, Foundation Fighting
Blindness, and Research to Prevent Blindness
Purpose: Mutations in tubby-like protein 1 (Tulp1) cause progressive retinitis
pigmentosa. However, delineation of its pathological mechanisms is hindered by its
poorly defined protein interaction networks. The purpose of this study is to elucidate
its binding partners by a newly developed technology of functional proteomics.
Methods: Tulp1-binding proteins were identified by open reading frame (ORF) phage
display technology as the following. ORF phage display cDNA library of mouse eye
was incubated with immobilized Tulp1 protein on ELISA plates, washed, eluted,
amplified and reselected. A total of three rounds of affinity selection were performed.
Enriched individual phage clones were re-verified for their binding activity to Tulp1
and identified by sequencing. Their binding specificity to Tulp1 and other proteins in
the same family were analyzed. Identified Tulp1-binding proteins were independently
validated by yeast two-hybrid system or protein pull-down assay.
Results: Several new Tulp1-binding proteins were efficiently identified by ORF phage
display technology, including acidic nuclear phosphoprotein 32 A (Anp32a), cyclic
nucleotide gated channel beta 1 (Cng1), cell division cycle 2-like 1 (cdc2l1), PRP38 premRNA processing factor 38B (Prpf38b), DnaJ (Hsp40) homolog A1 (Cnaja1) and D4
zinc and double PHD fingers 3 (Dpf3). They exhibited different binding specificities
to Tulp1 and other proteins in the same family. Furthermore, their interactions with
Tulp1 were independently verified.
Conclusions: These data revealed that Tulp1 has multiple binding partners and
may play multiple intracellular and extracellular roles. The elucidation of these new
binding partners will facilitate the delineation of their functional roles and disease
mechanisms. This study also demonstrates that ORF phage display is a powerful
technology of functional proteomics with efficiency, sensitivity and convenience for
re-verification and analysis of their binding specificity.
CR: G.S. Alvarado, None; N. Caberoy, None; W. Li, None.
Support: NIH Grant R01EY016211 and P30EY014801
4039 - A432
Restoring CNGB Expression With Cre Recombinase in a CNGB -/-Mouse Line
T. Wang1, F.A. Concepcion2, H. Moaven1, J. Chen1. 1Zilkha Neurogenetic Institute, Keck
School of Medicine, University of Southern California, Los Angeles, CA; 2Baylor
College of Medicine, Houston, TX.
Purpose: Retinitis pigmentosa (RP) is a form of retinal degeneration that affects
nearly 1 in 3000 people globally. For all retinal rescue strategies, retinal remodeling
as a consequence of degeneration constitutes a major challenge, and the potential
of functional recovery after such remodeling events is an important question to
address. The CNGB-/- mice is a good RP model exhibiting similar retinal degeneration
patterns as human RP. Here, we have generated a CNGB -/- mouse line that is capable
of CNGB gene reactivation upon Cre-mediated recombination. These mice will allow
for a systematic study of the extent of vision recovery following defined periods of
retinal degeneration.
Methods: The Loxp-flanked neomycin resistance (LBL) cassette was introduced into
the endogenous CNGB1 locus by homologous recombination. The targeting vector
was engineered to introduce a mutation within the CNGB1 gene that deletes its
calmodulin binding site. Importantly, the insertion of the targeting vector precludes
CNGB gene expression, and expression of CNGB1ΔCaM can be achieved by excision
of the LBL cassette by the Cre recombinase. Mice that expressed CNGB1ΔCaM were
created following germline excision of the LBL cassette. Visual function was assessed
by electroretinogram. The retinal structures at different ages were investigated by
light and electron microscopy as well as by immunocytochemistry.
Results: The removal of LBL cassette led to the expression of the CNGB1ΔCaM protein.
The CNGB1ΔCaM protein expressed at the same level as WT CNGB1, and is located at
the rod outer segment plasma membrane. The dark-adapted CNGB1ΔCaM mice showed
normal flash sensitivities and light adaption, while the CNGB -/- mice had little or no
measurable rod response. Similar to WT, the CNGB1ΔCaM mice maintained normal
retinal structure up to 12 month. In contrast, retinas from CNGB1-/- mice show a
moderate rate of degeneration which was complete by 3 months.
Conclusions: Our data showed that the regulated activation of the CNGB1ΔCaM protein
is feasible, and the CNGB1ΔCaM protein can form a functional CNG channel at the
rod outer segment and restore normal light responses. Therefore, the CNGB-/- mice
can be conditionally activated to express the functional channel. This will allow for
the systematic study of the extent of functional recovery following different stages
of retinal degeneration. The conditional activation of CNGB1ΔCaM will be achieved
by crossing the CNGB -/- mice with transgenic mice that express a ligand-inducible
ERT2CreERT2 construct in the rod photoreceptors
CR: T. Wang, None; F.A. Concepcion, None; H. Moaven, None; J. Chen, None.
Support: EY12703
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4036-4039
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4040 - A433
Comparative in vitro Secretion of TGF-β1 and Proteinases by wt and rd1 Mouse
Retinal Explants
4041 - A434
Two Novel CRX Mutant Proteins Causing Autosomal Dominant Leber
Congenital Amaurosis (LCA) Interact Differently With NRL
S. Ahuja1A, P. Ahuja-Jensen1A, A. Caffe1A, M. Abrahamson2, P.A. Ekstrom1B, T. van Veen1A.
A
Ophthalmology, Clinical Sciences, Lund, BOphthalmology, Clinical Sci Lund,
1
Lund University, Lund, Sweden; 2Clinical Chemistry and Pharmacology, Lund
University Hospital, Lund, Sweden.
E. Boobalan1A, L. Nichols, II1A, R.P. Alur1A, Y.V. Sergeev1A, R.C. Caruso1A, E.M.
Stone2, A. Swaroop1B, M.A. Johnson 3, B.P. Brooks1A. AOphthalmic Genetics & Visual
Function Branch, BN-NRL, 1National Eye Institute/NIH/DHHS, Bethesda, MD;
2
Ophthalmology and Visual Sciences, HHMI, University of Iowa, Iowa City, IA;
3
Ophthalmology and Visual Science, University of Maryland School of Medicine,
Baltimore, MD.
Purpose: Due to Pde6brd1 gene mutation in β subunit of phosphodiesterase, rd1 mouse
retina shows degeneration homologous to a form of Retinitis Pigmentosa. The secretion
of TGF-β1, matrix metalloproteinases (MMPs) and cathepsins and their endogenous
inhibitors TIMPs and cystatin C by wt and retinal degeneration (rd1) mouse retinas
was compared to understand the mechanism of retinal degeneration.
Methods: Retinal explants from postnatal day 2 (PN2) and PN7 wt and rd1 mice were
cultured and the retinal conditioned medium (RCM) was collected at PN12 and PN21
and analyzed. TGF- β1, MMPs / TIMPs, and cathepsins / cystatin C secreted into the
RCM were determined by ELISA.
Results: In vitro secretion of TGF-β1 into RCM was low and activities of total proteinases,
MMP-9 and cathepsin B were persistently higher in case of rd1 retinal explants and this
was due to relatively lower levels of TIMP-1 and cystatin C, respectively. Whereas, in
case of wt explants, in vitro secretion of TGF-β1 was higher along with transient increase
in the secretion of MMPs and cathepsins relative to their endogenous inhibitors.
Conclusion: In wt explants higher in vitro levels of TGF-β1 may transiently increase
the secretion of MMPs and cathepsins which activate TGF-β1 and remodel the ECM.
In rd1 retina consistent increases in the expression of proteinases relative to their
inhibitors could degrade TGF-β1, ECM and possibly lead to retinal degeneration.
Therapeutic treatment with individual proteinase inhibitors and a combination(s) of
inhibitor(s) needs investigation.
CR: S. Ahuja, None; P. Ahuja-Jensen, None; A. Caffe, None; M. Abrahamson,
None; P.A. Ekstrom, None; T. van Veen, None.
Support: Dutch Retina Foundation, KMA (Sweden), Stiftelsen Synfrämjandets
Forskningsfond (Sweden); Stiftelsen för Synskadade i f d Malmöhus Län (Sweden),
FFB, VRM
4042 - A435
Mutant Rhodopsin (P23H) Forms Aggregates in the Rod Outer Segment
Purpose: To report the clinical and molecular phenotype associated with 2 novel, de
novo, CRX mutations causing dominant LCA.
Method: Patients underwent detailed ophthalmic examination, full field ERGs and
retinal imaging. Mutations were detected using standard PCR and DNA sequencing.
The transactivation capacity, protein expression, and subcellular localization of wildtype (WT) and mutant CRX proteins were analyzed in vitro, both in the presence and
absence of Neural Retina Lucine Zipper (NRL).
Results: Both patients showed early macular degenerative changes and unrecordable
ERGs. One patient showed retinal thinning at 2.5 years of age, with possible
preservation of some photoreceptors by OCT. We found two novel CRX mutations,
c.G264T (p.K88N) and c.413delT (p.Il38fs48), which reduced transactivation to 10%
& 30% of WT activity, respectively. Both mutants are expressed in vitro at levels
comparable to WT protein, and their proteins show cytoplasmic localization as well
as nuclear subcellular localization observed in WT and mutants transfected cells. NRL
increased luciferase activity by 5 fold & 30 fold respectively, when expressed alone
or with wild type CRX. However, activation was decreased to only 2- and 9- fold,
respectively, when mutant constructs were coexpressed with NRL. Unlike WT or
c.413delT constructs, coexpression of mutant c.G264T with NRL drastically reduced
amounts of both the proteins.
Conclusion: Both mutations significantly reduce baseline CRX activity and are
partially mislocalized. The c.G264T mutant acts in a dominant-negative-like fashion
with NRL, whereas the c.413delT mutant allows NRL to exert its normal baseline
activity. Although some photoreceptors may be present in patients early in disease,
a simple gene-replacement strategy in dominant CRX-LCA is unlikely to address the
pathophysiology of this condition.
CR: E. Boobalan, None; L. Nichols, II, None; R.P. Alur, None; Y.V. Sergeev, None; R.C.
Caruso, None; E.M. Stone, None; A. Swaroop, None; M.A. Johnson, None; B.P.
Brooks, None.
Support: NIH intramural program
4043 - A436
Dogs Heterozygous for a PDE6A and a PDE6B Null Mutation Do Not Develop
Retinal Degeneration
A. Adekeye, M. Haeri, J. Chen, B.E. Knox. Biochem & Molecular Bio and Ophthal,
SUNY Upstate Medical University, Syracuse, NY.
Purpose:Mutations in rhodopsin cause inherited retinal degenerative diseases. We
characterized the oligomeric state of the P23H rhodopsin mutation in transgenic
rods and mammalian cells.
Methods:We generated transgenic Xenopus rods that express opsin (Rho-EGFP)
or P23H mutant (RhoP23H-EGFP) cDNAs with EGFP fused to its carboxyl terminus.
Retinal explants were examined for EGFP expression using quantitative confocal
microscopy of live rods. Parallel experiments were performed in transiently transfected
mammalian cells. SDS-PAGE was used to study potential oligomeric forms of the
fusion proteins.
Results:Rho-EGFP was observed primarily in the rod outer segment. Rods expressing
RhoP23H-EGFP exhibited an abnormal fluorescent protein distribution. Fluorescence was
observed within the inner segment and in small focal regions in both inner and outer
segments. The outer segment protein foci were immobile. When retinal extracts from
Rho-EGFP expressing tadpoles were examined by western blot analysis, Rho-EGFP was
detected as monomers, homodimers and heterodimers with endogenous rhodopsin. By
contrast, retinal extracts from tadpoles expressing RhoP23H-EGFP primarily contained
homodimers. Minor amounts of monomers were observed, but unlike Rho-EGFP,
dimers with endogenous rhodopsin were undetectable. The aggregation behavior is
currently under biochemical analysis.
Conclusions:RhoP23H-EGFP, but not Rho-EGFP, forms aggregates in the rod outer
segment. Although both mutant and wild type proteins were able to form detergent
resistant dimers, RhoP23H-EGFP selectively formed homodimers but not heterodimers
with endogenous rhodopsin. Our results suggest that the P23H mutation may render
rhodopsin susceptible to form self-aggregates, which may be cytotoxic. Future studies
will determine if mutant rhodopsin forms aggregates in other species.
CR: A. Adekeye, None; M. Haeri, None; J. Chen, None; B.E. Knox, None.
Support: National Eye Institute/NIH (EY011256, EY012975); Fight For Sight;
Unrestricted grant from Research to Prevent Blindness.
K.E. Pierce, Jr.1, J.T. Bartoe1, G.M. Acland2, S.M. Petersen-Jones1. 1Small Animal Clinical
Sci, Michigan State University, East Lansing, MI; 2James A Baker Institute, Cornell
University, Ithaca, NY.
Purpose: To determine if dogs heterozygous for both the PDE6A mutation that causes
rod cone dysplasia type 3 and also for the PDE6B mutation that causes rod cone
dysplasia type 1 have rod dysfunction or develop a retinal degeneration.
Methods: A PDE6A -/- bitch was bred to a PDE6B -/- male to produce PDE6A+/PDE6B+/- puppies. Three PDE6A+/- /PDE6B+/- puppies were born. These were
monitored by complete ophthalmic examinations, fundus photography, and
electroretinography for 32 months. Full-field dark-adapted ERG intensity:response
series and rod-mediated flicker responses were recorded followed by light adaptation
and a light-adapted intensity:response series and cone flicker responses.
Results: No ophthalmoscopic evidence of retinal degeneration was observed over
the time period. No significant abnormalities in rod-mediated ERGs were detected
suggesting that the dogs had normal rod function. The ERG amplitudes remained
comparable to those of wild-type dogs over the study period.
Conclusion: Dogs heterozygous for both the previously described PDE6A and the
PDE6B null mutations do not appear to have abnormal retinal function and over the
first 32 months of life show no evidence of retinal degeneration.
CR: K.E. Pierce, Jr., None; J.T. Bartoe, None; G.M. Acland, None; S.M. PetersenJones, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4040-4043
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4044 - A437
Peripheral Retinal Vascular Leakage in Retinitis Pigmentosa Evaluated With
Optos Widefield Fluorescein Angiography
4045 - A438
Computational Molecular Phenotyping and Excitation Mapping in a Human
Patient With Retinitis Pigmentosa
K.V. Miller, A.W. Eller, T.R. Friberg. Ophthalmology, University of Pittsburgh
Medical Center, Pittsburgh, PA.
B.W. Jones1, R.E. Marc2. 1Ophthalmology, Moran Eye Center, Salt Lake City, UT;
2
Ophthalmology-Sch of Med, Univ of Utah/Moran Eye Center, Salt Lake City, UT.
Purpose: Analysis of the retinal vasculature in patients with retinitis pigmentosa (RP)
utilizing the Optos P200A (Dunfermline, Scotland) widefield fluorescein angiographic
(FA) system.
Methods: Patients with the diagnosis of RP were identified through a search of billing
records. Medical records were then reviewed to identify patients with RP who had
been evaluated with Optos widefield FA and Cirrus OCT. Images were analyzed for
cystic changes or retinal thinning on OCT and correlated with the FA for leakage in
the macula, posterior pole, and peripheral retina.
Results: Sixteen patients were identified with RP who had undergone Optos
widefield FA, Cirrus OCT, and clinical examination. No patients were found to
have peripheral retinal exudates (Coats’-like response) on examination. Ten of 16
patients (63%) had FA and OCT findings consistent with cystoid macular edema
(CME). Five patients (31%) showed peripheral vascular leakage on widefield FA.
Of these five, three were identified as having CME. One patient had a history
of telangiectasis and had been treated with laser. The vascular leakage was
predominant in the temporal periphery and was associated with capillary
nonperfusion. There was no evidence of retinal neovascularization in any patient.
Discussion: Coats’-like response has been reported in RP. However, the peripheral
retinal vasculature has not been previously studied in RP patients without
telangiectasis or exudates. The advent of the Optos widefield FA has made this
possible. Abnormal peripheral retinal vasculature in RP may represent a forme-fruste
of Coats’-response, which may be more common than previously reported. The cause
of this vascular damage is unknown, and may or may not reflect an inflammatory
etiology. Interestingly, the peripheral vascular leakage was associated with CME in
some (3/5) but not all cases. The high percentage (31%) of patients with peripheral
vascular leakage in this study may be related to selection bias, as those patients with
a history of CME were more likely referred for testing.
Conclusions: A significant percentage of RP patients may have peripheral retinal
vascular leakage identified with Optos widefield FA. Further studies are needed to
identify the prevalence, mechanism, treatment, and effect on prognosis.
CR: K.V. Miller, None; A.W. Eller, None; T.R. Friberg, Research support, F; Advisory
board, C.
Support: None
Purpose: Evaluation of animals models of human retinitis pigmentosa have been
extensively documented. However, substantive documentation of human samples
of retinitis pigmentosa have not been introduced into the literature. Our goal was to
assess the state and condition of the late stage human retina with retinitis pigmentosa
and evaluate the dependance of retinal remodeling on cone survival.
Methods: Samples from human subjects with retinitis pigmentosa were collected
post-mortem through the Utah Lions Eye Bank and incubated with 1-amino-4guanidobutane (AGB). Retinal fragments were incubated 10 minutes at 35 degrees
C in oxygenated Ames-Hepes medium with 5mM AGB with and without iGluR
agonists (KA 50uM, NMDA 1mM), followed by conventional fixation in buffered
aldehydes and embedding in epoxy resins. Tissues were sectioned at 200nm followed
by classification with computational molecular phenotyping (CMP) using an array
of small and macromolecular signatures (aspartate, glutamate, glycine, glutamine,
glutathione, GABA, taurine, CRALBP, GS, rhodopsin, LWS1 cone opsin, tyrosine
hydroxylase).
Results: As in animal models of retinitis pigmentosa and most notably the P347L
transgenic rabbit, progressive rod-specific degeneration leads to complete elimination
of rods and rod signaling. There is extensive survival of substantially altered cones.
However, the presence of cones prevents the onset of radical remodeling in the retina,
also preserving iGluR mediated signaling to surviving horizontal and bipolar cells.
Those surviving bipolar and horizontal cells demonstrate neurite sprouting and
potential bipolar cell and retinal reprogramming through upregulation of iGluR
expression.
Conclusions: Disease progression observed in the human condition shows that animal
models of retinal degeneration recapitulate the human condition. Cone mediated
preservation of bipolar cell signaling, retinal reprogramming and retinal remodeling
seen in the human retinitis pigmentosa retina are all duplicated in animal models.
CR: B.W. Jones, None; R.E. Marc, Signature Immunologics, E.
Support: NIH Grants EY02576 (RM), EY015128 (RM), EY014800 Vision Core (RM),
Research to Prevent Blindness CDA (BWJ)
4046 - A439
Whole-Genome Association Study Identifies the Canine Cone-Rod Dystrophy
2 Locus
4047 - A440
RPGR Deficiency Results in Retinal Degeneration in Zebrafish
G.M. Acland1A, J.G. Mezey1, A.R. Boyko1, C. Gao1, W. Wang1, C.D. Bustamante1, G.D.
Aguirre2, O. Goldstein1. AJames A Baker Institute, 1Cornell University, Ithaca, NY;
2
School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA.
Canine Cone-Rod dystrophy 2 (crd2) is an early onset, hereditary retinal degeneration
identified in the American Pit Bull Terrier dog breed. It is characterized by initial
severe day-blindness, progressing to total blindness by 1 year. A colony segregating
the disease has been established.
Purpose: To identify the locus responsible for Cone-Rod Dystrophy 2 by whole-genome
association study (WGAS).
Methods: DNA was extracted from 14 crd2-affected cases and 13 unaffected related
controls using standard protocols. A WGAS was undertaken using the Affymetrix
Version 2, Canine SNP chip following the standard protocol. Genotypes were called
using “MAGIC” algorithm and tested for association using Fisher’s Exact test. A
Bonferroni correction for multiple tests set the significance threshold at -Log10(p)
> 6.39.
Results: Two potentially associated loci were identified, although neither passed
the significance threshold. The lower of the 2 peaks yielded a signal of 4.17. Linkage
analysis to crd2 in pedigrees showed no co-segregation, suggesting a false positive
hit. The second locus on CFA33 included 5 SNPs with a signal of 6.13, approaching
the Bonferroni threshold. Linkage analysis showed significant co-segregation.
Haplotype analysis identified a 2.7 Mb homozygosity block- the presumed crd2
Linkage Disequilibrium interval. Candidate genes evaluation is now in progress.
Conclusions: Carefully selected case and control groups for a WGAS, with as few
as 13-14 dogs per group, successfully identified a region on CFA33 that potentially
harbors the crd2 gene. Combined WGAS and Linkage analysis can exclude false
positives and confirm true positive association hits even if genome wide significance
thresholds are not achieved.
CR: G.M. Acland, Optigen LLC, I; Optigen LLC, C; J.G. Mezey, None; A.R. Boyko,
None; C. Gao, None; W. Wang, None; C.D. Bustamante, None; G.D. Aguirre, Optigen
LLC, I; Optigen LLC, C; O. Goldstein, None.
Support: NIH Grants EY006855, -13132,-17549, GM082910; Foundation Fighting
Blindness; Morris Animal Foundation; Van Sloun Fund
X. Shu1, Z. Zeng2, P. Gautier1, A. Lennon1, M. Gakovic1, E. Patton1, A. Wright1. 1Medical
and Developmental Genetics, MRC Human Genetics Unit, Edinburgh, United
Kingdom; 2Cancer Research Centre, University of Edinburgh, Edinburgh, United
Kingdom.
Purpose: X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of
human retinal degeneration, as determined by age-of-onset and progression, and
accounts for 6-20% of all RP cases. The RP GTPase Regulator (RPGR) gene is mutated
in 70-80% of XLRP patients. Over 290 mutations in RPGR have been identified, which
can give rise to central or peripheral retinal dystrophies. The function of RPGR is
unclear although the N-terminal half of RPGR (exons 2-11) is structurally similar to
the regulator of chromosome condensation (RCC1). Here we use zebrafish as a model
to investigate RPGR function.
Methods: Zebrafish RPGR orthologues were predicted by bioinformatic analysis and
confirmed by reverse transcriptase polymerase chain reaction (RT-PCR). Zebrafish
RPGR expression patterns during development and in adult tissues were examined
by RT-PCR and whole-mount in situ hybridisation, while the localisation was studied
by immunostaining. Knock-down of zebrafish RPGR was carried out by morpholino
injections. The phenotype of the RPGR deficient morphants was analysed using
standardized procedures.
Results: RPGR is duplicated in zebrafish. RT-PCR analysis showed ZFRPGR1mRNA
was readily detected from the13-somite stage (15hpf) and throughout development
up to 5dpf. ZFRPGR2 was detected at the time of fertilization and persisted during
gastrulation and through the tailbud and larval stages. In situ hybridisation showed
that both ZFRPGR1 and ZFRPGR2 are expressed in central retina, brain and neural
tube. ZFRPGR2 knockdown results in a severely abnormal eye phenotype that
is rescued by the wildtype human RPGRORF15 isoform. Morphants display retinal
dysplasia and extensive apoptosis in the central retina.
Conclusions: We identified and examined the expression of zebrafish RPGR. We
generated a vertebrate model of RPGR insufficiency using antisense methodology
in zebrafish, which showed abnormal retinal development and retinal degeneration
due to extensive apoptosis. Human RPGRORF15 can rescue the zebrafish RPGR-deficient
phenotype.
CR: X. Shu, None; Z. Zeng, None; P. Gautier, None; A. Lennon, None; M. Gakovic,
None; E. Patton, None; A. Wright, None.
Support: Medical Research Council, BRPS GR558, UK Fight for Sight, EVI-GENORET
FP6 512-036, AICR 07-0421,
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4044-4047
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4048 - A441
Impairment of Pupillary Light Responses in Murine Models of Retinal
Degeneration
4049 - A442
Retinal Changes in the R6/1 Transgenic Mouse Model of Huntington’s Disease
E.L. Fletcher1, A. Hussain Batcha1, U. Greferath1, K.A. Vessey1, J. Nithianantharajah2,
A. Hannan2. 1Dept Anatomy/Cell Biology, University of Melbourne, Parkville,
Australia; 2The Howard Florey Institute, The University of Melbourne, Parkville,
Australia.
M.C. Canver1, K.E. Revere1, G.-S. Ying1, A.C. Canver2, J. Bennett1, D.C. Chung1. 1F.M.
Kirby Center for Molecular Ophthalmology, Scheie Eye Institute, University
of Pennsylvania, Philadelphia, PA; 2School of Biomedical Engineering, Drexel
University College of Medicine, Philadelphia, PA.
Purpose: To establish normative reference values for pupillary light response (PLR)
parameters during disease progression in comparison with those of selected murine
models of retinal degeneration.
Methods: Cohorts of wildtype (C57Bl6) and retinal degeneration (nrl-/-, rd10-/-) mice
underwent PLR testing, utilizing the Neuroptics Pupillometer (San Clemente, CA,
USA). Mice were tested at postnatal day 21 (P21), 35(P35), 49(P49) and if indicated
77(P77). Mice were dark-adapted for >12 hours prior to testing and exposed to three
light pulses at 10 second intervals at three intensities: 4.6, 37.5, 300 μW/cm 2, with left
and right eyes exposed in alternating fashion. Both direct and consensual responses
were measured. A custom algorithm for automated analysis of the raw pupillometric
data was formulated and facilitated the extraction of twelve physiologically relevant
parameters: (1) baseline diameter, (2) minimum amplitude, (3) response amplitude,
(4) re-dilation amplitude, (5) percent constriction, (6) response time, (7) re-dilation
time, (8) average constriction velocity, (9) average re-dilation velocity, (10) maximum
constriction velocity, (11) maximum re-dilation velocity, and (12) onset latency.
Normative parameters for each mouse strain were established as a function of age for
all 12 parameters. Within each mouse strain, each of the 12 parameters was compared
for each intensity using the GENMOD procedure to analyze changes over time.
Results: C57Bl6 wildtype strain had robust PLR responses at all light intensities
and the PLRs did not exhibit significant changes over the test period. In contrast,
rd10-/- mice exhibited significant impairment of PLRs between 3 and 7 weeks. PLRs
also diminished in the nrl-/- mice over time, especially at lower light intensities. The
timecourse of PLR changes in the retinal degeneration mice correlated with changes
in the outer nuclear cell layer (ONL) thickness over time.
Conclusions: Pupillometric measures are non-invasive, rapid, and quantitative and can
be used as outcome measures in assessments of treatment efficacy of pharmacologic
and gene based therapies in mice.
CR: M.C. Canver, None; K.E. Revere, None; G.-S. Ying, None; A.C. Canver, None; J.
Bennett, None; D.C. Chung, None.
Support: NIH RO1 EY10820, K08 EY017024, Hope for Vision Foundation, Ira
Rosenmertz/Fidelity Charitable Trust, FFB Center grant C-GT-0607-0390-UPA02,
RPB, and the Paul and Evanina Mackall Foundation Trust
4050 - A443
Age-Related Dendrite Pruning in the Adult Brown Norway (BN) Rat Retina
L. Kisiswa1, J. Albon1, M.A. Wride2, J.E. Morgan 3. 1School of Optometry and Vision
Sciences, Cardiff University, Cardiff, United Kingdom; 2Zoology Department,
University of Dublin Trinity College, Dublin, Ireland; 3Ophthalmology, University
Hospital of Wales, Cardiff, United Kingdom.
Purpose: To determine the extent in which age-related dendritic pruning occurs in
the adult rat retina.
Methods: Whole-mount retinal explants from 6 (young) and 24-52 weeks (mature)
old BN rats were prepared and RGCs were diolisticaly (DiI) labelled using BioRad
gene gun and Hoeschst 33342. Changes in RGC dendritic complexity were assessed
utilising Sholl analysis. RGC cell density was determined in the whole mounts while
outer and inner nuclear layer (ONL and INL) cell number was assessed in retinal
slices stained with Hoeschst 33342
Results: RGC dendrite branching was significantly reduced (p<0.0002) in 24-52
compared to 6 weeks old RGCs, although dendrite length remained unchanged. There
was marginal RGC loss (7%) in mature compared to young retinae. RGC density was
significantly reduced (p<0.001) (26%) in the central (1mm from optic disc), but not in
the peripheral (2-3mm from the optic disc) retina of 24-52 compared to 6 weeks old
BN retina. Overall retinal area was significantly increased (p<0.001) (23%) in mature
compared to younger BN retina. A significant reduction in the INL and ONL cell
numbers were also significantly reduced (p<0.001 and p<0.001 respectively (25%
and 20% respectively)) occurred in mature retina although there was no evidence
of thinning of the layers.
Conclusions: Age-related reduction in RGC dendritic complexity implies that dendritic
pruning occurs during adulthood. Developmental retinal expansion that occurs during
the period studied, which accounts, in part for the reduction in RGC density, suggests
that cell loss is marginal. Age-related dendritic modifications should be considered
when determining the effects of disease models in RGC morphology.
CR: L. Kisiswa, None; J. Albon, None; M.A. Wride, None; J.E. Morgan, None.
Support: National Eye Research Centre (NERC), UK, Grant RCOP256
Purpose: Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by a
CAG repeat expansion, resulting in an expanded polyglutamine tract in the huntingtin
protein. While altered visual function has been reported in HD patients and mouse
models, analysis at the level of the retina has not been thoroughly addressed. The aim
of this study was to characterize in detail changes in retinal structure and function
in the R6/1 transgenic mouse model of HD.
Methods: C57Bl6 (wildtype, WT) and R6/1 (HD) mice from 7 to 20 weeks of age were
used in this study. Retinal function of WT (N=9) and HD mice (N=11) at 13-14 weeks
of age was measured using a twin flash electroretinogram paradigm. Retinae from
all age groups were aldehyde fixed and either resin embedded and Nissl stained,
or sectioned at 12μm on a cryostat and processed for indirect immunofluorescence.
Using antibodies targeted against specific cell types, and synaptic markers, changes
in photoreceptors and inner retinal neurons were examined. Cell death was assessed
by quantification of TUNEL positive cells.
Results: Electrophysiological findings revealed significantly reduced cone and variable
rod responses in HD mice compared with WT. HD mice showed a progressive decrease
in cone opsin immunolabeling, but not peanut agglutinin lectin suggesting a defect
in cone-opsin expression, rather than a loss of cone photoreceptors. A small number
of photoreceptor nuclei displayed TUNEL labelling from 13 weeks of age and an
increase in GFAP immunolabelling of Müller cell processes was detected suggesting
the presence of retinal stress. Interestingly, from 13 weeks of age, there was evidence
of ectopic synapses within the distal ONL that were immunoreactive for the ribbon
markers RIBEYE, mGluR6 and PSD-95. Extension of rod and cone bipolar cells, towards
ectopic synapses was also apparent.
Conclusions: These results suggest that with progression of the disease, HD mice
display decreased cone opsin expression and cone dysfunction. This is accompanied
by retinal stress and remodeling of bipolar cells. This data suggest that the retina
is affected structurally as well as functionally in addition to neurodegeneration in
Huntington’s disease.
CR: E.L. Fletcher, None; A. Hussain Batcha, None; U. Greferath, None; K.A. Vessey,
None; J. Nithianantharajah, None; A. Hannan, None.
Support: NH&MRC #566814; Retina Australia
4051 - A444
A Detailed Analysis of the Transient Photoreceptor Degeneration in the ConeOnly Nrl-Knockout Mouse Retina
K. Ranganath1A, J. Roger1A, A. Hiriyanna1A, S. Veleri1A, R. Cojocaru1A, L. Zhao1B, W.T.
Wong1B, R. Bush2, A. Swaroop1A. ANeurobiology Neurodegeneration & Repair
Laboratory, BUnit on Neuron-Glia Interactions in Retinal Disease, 1National Eye
Institute, NIH/Bethesda, MD; 2Section on Translational Research-Retina & Macular
Degeneration, National Institute of Deafness and Communication Disorders, NIH/
Bethesda, MD.
Purpose: NRL (Neural Retina Leucine Zipper) is a bZIP transcription factor necessary
and sufficient for rod photoreceptor differentiation. The deletion of Nrl in mice results
in a cone-only retina, with complete transformation of rod precursors to cones. Nrl
knockout (Nrl-/-) mice are widely used due to their distinctive phenotype; however
the detailed onset and pattern of cone degeneration has not been reported. This study
aims to evaluate age-associated changes in the Nrl-/- retina and possible use of these
mice as a model for cone degeneration.
Methods: The morphology of the aging retina of Nrl-/- mice was evaluated by histology,
immunohistochemistry, and TUNEL assays. Cone function was assessed by ERG.
Microarray analysis using Affymetrix was performed using retina from 1M, 2M,
4M, 6M, and 10M old Nrl-/- mice.
Results: As Nrl-/- mice age, the thickness of the ONL decreases with maximum cell
death occurring between 2-4 M. The ERG response drops in the same timeframe
but levels out after 4M; subsequently the ONL thins to a 4-nuclei thickness and the
characteristic rosettes disappear. Between 2-4M retinal microglia also migrate to
the subretinal space and attain an activated morphology. After 4M, TUNEL staining
disappears and retinal microglia return to the inner retina where they recover their
resting morphology. Müller cells are activated at all ages examined, as shown by GFAP
immunoreactivity. At 10M the remaining cones express S-opsin and M-opsin, contain
shortened outer segments, and display a preserved scotopic ERG. Microarray analysis
done across multiple stages of degeneration identified a set of misregulated genes
early on, and will help elucidate possible mechanisms of cone cell death.
Conclusions: These findings provide a detailed assessment of retinal changes in Nrl/mice; cone cell death occurs rapidly between 2-4M after which the retina stabilizes.
The retinal degeneration (RD) observed in Nrl-/- mice is unusual in that degeneration
does not continue indefinitely as in other RD lines. The Nrl-/- retina is potentially
useful for understanding mechanisms underlying cone degeneration and for exploring
therapeutic strategies that sustain or replace cone loss in human disease.
CR: K. Ranganath, None; J. Roger, None; A. Hiriyanna, None; S. Veleri, None; R.
Cojocaru, None; L. Zhao, None; W.T. Wong, None; R. Bush, None; A. Swaroop,
None.
Support: NIH/NEI Intramural funding
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4048-4051
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4052 - A445
Environmental Enrichment Delays Photoreceptor Degeneration in a Mouse
Model of Retinitis Pigmentosa
4053 - A446
Assessment of Paramacular Sensitivity in Patients With Retinitis Pigmentosa
E. Strettoi1, I. Barone1, E. Novellli2. 1CNR Neuroscience Institute, Pisa, Italy; 2GB BIetti
Foundation for Ophthalmology, Rome, Italy.
D. Shimizu1, T. Takeshi2, A. Hagiwara2, A. Tawada2, S. Yamamoto2. 1Chiba university
hospital, Chiba-city,chu-o-ku, Japan; 2Chiba University Hospital, Chiba-City,Chuo-ku, Japan.
Purpose: Enviromental enrichment (EE) is a combination of complex innimate and
social stimuli in which animals are reared in large groups and cages with toys, tunnels,
nesting material and running wheels that are changed frequently. EE increases brain
plasticity, accelerates neural development and slows down neuronal degeneration,
partly through increased production of trophic factors. We tested the hypothesis that
early exposure to EE could slow down photoreceptor degeneration in a mouse model
of Retinitis Pigmentosa (RP).
Methods: Pregnant rd10 mutant mice and their litters were exposed to EE. Retinal
structure of the offspring was assessed at P24, P45 and P60. Controls were age-matched
rd10 mice raised in standard (ST) conditions. Survival of photoreceptors was estimated
measuring ONL thickness on retinal sections after fluorescent nuclear staining and
confocal microscopy. Pycnotic (condensed) nuclei from degenerating photoreceptors
were counted in confocal images of the ONL of retinal whole mounts. For cone counts,
retinas from age-matched EE and ST mice were stained with Red-Green Opsin and
Blue-Opsin antibodies. Cone-rich and cone-poor areas were imaged by means of
fluorescence digital pictures of retinal whole mounts and used to trace cone isodensity
curves. Cone densities within each curve were estimated separately by automatically
counting cones on high resolution confocal images. Local cone densities were then
assigned to corresponding isodensity areas on retinal maps using a contour plot
graph. The total number of cones/retina was then estimated.
Results: rd10 mice born in EE conditions open their eyes 3 days earlier than ST
counterparts. The number of pycnotic nuclei from dying photoreceptors in the ONL
is lower in EE than in ST mice at P24 and P45. The number of surviving cones, on the
contrary, is higher in retina of EE mice. An abrupt decrease in cone number occurs
in the rd10 retina between P45 and P60 but it is strongly reduced by EE. At P60, an
average of 80,000 intact cones, with elongated outer segments, persist in EE retinas,
against only 40,000 dystrophic cones remaining in ST retinas. A higher number of
rods, with outer segments still expressing the light-sensitive channel, is preserved
as late as P45. Synaptic terminals of photoreceptors in the OPL of EE retinas are also
maintained.
Conclusions: Exposure of rd10 mutant mice to EE decreases photoreceptor degeneration
and promotes remarkable preservation of rods and cones up to 2 months of age.
EE might represent a non invasive approach to enhance photoreceptor survival in
inherited photoreceptor degeneration.
CR: E. Strettoi, None; I. Barone, None; E. Novellli, None.
Support: RSTL CNR fund; R01 EY12654
Purpose: We have reported that the paramacular sensitivity (PMS) determined by
microperimetry-1 (MP-1, Nedek, Japan) was significantly correlated with the visual
acuity in patients with retinitis pigmentosa (RP). The purpose of this study was to
determine whether PMS can be used to assess the visual condition in RP patients.
Methods: Thirty eyes of 15 typical RP patients (mean age, 51 years; range, 26 to 62
years) who visited Chiba University Hospital from January to May in 2009 were
studied. The PMS was calculated as the mean sensitivity of the 16 sites in the central
2 to 10 degrees of the MP-1. We compared the PMS to the visual acuity, the sensitivity
in the central 2 degrees of the MP-1, results of the 10 -2 program of the Humphrey
field analyzer (HFA), and results of Goldmann perimetry (GP).
Results: The PMS was strongly correlated (correlation ratio >0.85) to the other visual
functions. For eyes with a PMS <10 dB, the mean deviations of the HFA were not
altered significantly. For eyes with a PMS <20 dB, there was considerable variation
in the PMS in the central 2 degrees.
Conclusions: The PMS can be used to assess the visual status of RP patients. This is
important because RP patients can often have good visual acuity even if other visual
functions are severely impaired. The PMS of the MP-1 may measure visual functions
that are not related to the visual acuity.
CR: D. Shimizu, None; T. Takeshi, None; A. Hagiwara, None; A. Tawada, None; S.
Yamamoto, None.
Support: None
4054 - A447
Absolute Autofluorescence (AbsAF) in Stargardt Disease (STGD)
4055 - A448
Clinical and Functional Findings in a Large Sample of Children Affected by
Joubert Syndrome
T.R. Burke1, S.H. Tsang1, J.P. Greenberg1, T. Duncker1, J.R. Sparrow1, G.R. Barile1, F.C.
Delori2, T. Smith1. 1Ophthalmology, Columbia University, New York, NY; 2Schepens
Eye Research Institute, Harvard Medical School, Boston, MA.
Purpose: To demonstrate AbsAF levels in patients with STGD can correlate with in vivo
lipofuscin levels.
Methods: AbsAF measurements were done on 22 patients with STGD (ages 10 to 67 yrs,
37 eyes) using the HRA2 (Heidelberg Engineering Inc., Vista, CA) modified by insertion
of a fluorescence reference chip in a retinal image plane (Fig 1). Correction was made for
refractive error and optical media density from normative data.
Results: Median AbsAF values in the central 3000 micron diameter region as well as superior
and temporal macular regions were compared to the data from age matched controls.
The internal fluorescence reference (horizontal strip) is at the top of each image. The AbsAF
images are generated by Matlab using the formula in Fig 2 - SC is the refractive error correction
and (T-1om,488T-1om,emi) is the optical media density correction. The zero (GL0) is calculated from
darkest pixels, usually at the optic disc. The reference gray level (GLR) is measured from the
original AF images. Two tailed t-test was used for analysis.
Results: Excluding 10 eyes of 6 patients where atrophy or poor centration precluded imaging
of focal macular hyperfluorescence, background levels of AbsAF were found to be significantly
elevated relative to controls (Table 1, Chart 1) and levels 400% greater were detected in flecks
compared with background AbsAF in STGD patients (mean fleck: 159.7, mean background:
94.5, p<0.003), while areas in the transition zone from hyper to hypofluorescence (atrophy)
demonstrate levels double the highest background levels in STGD.
Conclusions: Absolute AF measurements in STGD can shed
quantitative light on the disease process which is not possible
with previous in vivo imaging techniques. This will not only
help with monitoring disease progression, but will be a
useful marker of individual mutation effect and of response
to interventional therapies in the future. Our patients
demonstrate a markedly increased level of AbsAF in youth,
but tapering with age as atrophy of the pigment epithelium
ensues. Flecks and transition zones show the highest
AbsAF.
CR: T.R. Burke, None; S.H. Tsang, None; J.P. Greenberg,
None; T. Duncker, None; J.R. Sparrow, None; G.R. Barile,
None; F.C. Delori, None; T. Smith, None.
Support: New York Community Trust, NEI R01 EY015520, R01EY018213 (SHT), Foundation
Fighting Blindness, Bernard Becker-Association of University Professors in OphthalmologyResearch to Prevent Blindness Award
G. Ruberto1, S. Signotini2, C. Bertone1, M. Suzani1, M. Antonini2, P.E. Bianchi1. 1Clinica
Oculistica, IRCCS Policlinico San Matteo, Pavia, Italy; 2Department of Child
Neurology and Psychiatry, IRCCS C. Mondino Institute of Neurology,Pavia, Pavia,
Italy.
Purpose: Depict clinical and functional results of children affected by Joubert
Syndrome.
Methods: Joubert syndrome (JS) is a rare genetic disorder that affect the cerebellum.
Some of these babies are at the same time affected by syndromic retinal degeneration,
associated or not with mental retardation . In 26 children (mean age 7,2) diagnosed as
JS affected by Mrn image of molar tooth sign a whole clinical and functional set of tests
were performed. All subjects were examined by mean of complete ophthalmologic
visit ( inclusive of visual acuity (VA) with Teller acuity cards or optotype, slit lamp and
fundus) and short Electroretinonagraphy ( ERG) examination. ERG was performed
in awake condition with a single registering electrode positioned at nasion. After 15
minutes dark adaptation mixed cone-rod response was registered and after 10 minutes
light adaptation 30 hz response was recorded. Statistical analysis of mean values and
“p” regarding ERG “a” and “b” waves amplitudes and latencies were done. VA and
ERG data were matched with 35 normal subjects of similar mean age.
Results: We found funduscopic retinal degeneration in 9 subject affected by JS . Mean
VA was 4,02. Significant differences in ERG between JS children versus normals we
found in “b” wave amplitude (16,02±10,1 versus 33,25±, “p” <0,03) and latencies (53,71±
8,4 versus 39,1±4,8, “p”<0,06). No other statistically significant differences were found
, even tough a worse trend in JS children.
Conclusions: An interdisciplinary neuro-visual approach is worthwhile in order to
a correct diagnosis and rehabilitation program in this rare disorder.
CR: G. Ruberto, None; S. Signotini, None; C. Bertone, None; M. Suzani, None; M.
Antonini, None; P.E. Bianchi, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4052-4055
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4056 - A449
A Novel Mechanism for Retinal Degeneration: Photoreceptor Membrane and
Structure Destabilization Caused by Rhodopsin Aggregation
4057 - A450
Impact of Rod Loss on the Retinal Cone Population in the ELOVL4 Mouse
Model of Stargardt-Like Dystrophy (STGD3)
M. Haeri, P.D. Calvert, B.E. Knox. Biochemistry & Molecular Biology and
Ophthalmology, SUNY Upstate Medical University, Syracuse, NY.
M.-A. Filion1A, E. Brown1A, F.P. Gaillard2, Y. Sauve1B. AOphthalmology, BDept of
Ophthalmology, 1University of Alberta, Edmonton, AB, Canada; 2IPBC, University
Poitiers, Poitiers, France.
Purpose: RhoP23H is the most common rhodopsin mutation in humans. Current
animal models that express RhoP23H in rods exhibit retinal degeneration. In cultured
mammalian cells, RhoP23H was found in aggresomes containing chaperones (Saliba et
al. 2002, J. Cell. Sci. 115, 2907) suggesting this could also be a cellular fate of RhoP23H in
retina. Thus, we examined rods for potential RhoP23H oligomers or protein aggregates
by quantitative live cell imaging.
Methods: We generated transgenic Xenopus that express opsin (Rho-EGFP) or P23H
mutant (RhoP23H-EGFP) cDNAs with EGFP fused to its carboxyl terminus. We utilized
high-resolution live cell imaging and fluorescence recovery after photobleaching to
reveal the localization and dynamics of RhoP23H-EGFP within the photoreceptor.
Results: Live imaging of transgenic rods showed that RhoP23H-EGFP appears in
concentrated fluorescent foci both in inner and outer segments. Based on dynamic
FRAP analysis, we found that the outer segment foci were immobile aggregates.
RhoP23H-EGFP inner segment foci were mobile, with fluorescence recovery rates
after photobleaching that were similar to non-aggregated Rho-EGFP. The immobile
fluorescent foci were more abundant toward the distal end of the outer segment,
suggesting a time-dependence for foci formation. Fluorescent foci were more abundant
in rods with high RhoP23H-EGFP expression levels, and were commonly found in disk
incisures in contrast to Rho-EGFP. Rods co-expressing both RhoP23H-EGFP and RhomCherry showed exclusion of Rho-mCherry from immobile fluorescent foci. Retina
from transgenic Xenopus expressing RhoP23H-EGFP showed abnormal vesicotubular
structures in rod outer segment membranes in electron micrographs, which were not
observed in transgenic Xenopus expressing Rho-EGFP.
Conclusion: We have shown, for the first time, that RhoP23H-EGFP forms aggregates in
the outer segment of transgenic rods. In addition, we observed abnormal vesicotubular
structures in the outer segment membranes of these animals. These results suggest a
new mechanism for destabilizing the rod: mutant protein aggregation. Formation of
such protein aggregates has been shown in a number of neurodegenerative diseases
such as Alzheimer’s disease. Further studies will address the nature of these aggregates
and their efficacy to trigger photoreceptors degeneration.
CR: M. Haeri, None; P.D. Calvert, None; B.E. Knox, None.
Support: National Eye Institute/NIH (EY011256, EY012975, EY018421); Fight For Sight;
Unrestricted grant from Research to Prevent Blindness
4058 - A451
The Metabolic Response of Müller Glial Cells to Photoreceptor Degeneration
F.R. Vazquez-Chona1, W.D. Ferrell2, B.W. Jones2, R.E. Marc3. 1Ophthalmology, Univ of
Utah, Salt Lake City, UT; 2Ophthalmology, Moran Eye Center, Salt Lake City, UT;
3
Ophthalmology-Sch of Med, Univ of Utah/Moran Eye Center, Salt Lake City, UT.
Purpose: To test the hypothesis that glial cells provide neuroprotection, we
are visualizing the metabolic response of Müller glial cells to photoreceptor
degeneration.
Methods: Visualizing and quantifying the metabolic states of activated glia is possible
through the integration of high resolution, N-dimensional metabolic profiling
(Computational Molecular Phenotyping, CMP), electron microscopy, classic glial
proteome profiling, and proliferation assays. We are using the classic light-induced
retinal damage model in albino mice to characterize the glial response to photoreceptor
degeneration.
Results: CMP is capable of visualizing thousands of Müller cells and photoreceptors
covering large stretches of retina while resolving the metabolic response of individual
Müller cells and photoreceptors, preserving all histological context. We found that glial
processes surrounding stressed photoreceptors exhibit changes in metabolic envelopes,
displaying altered metabolic signals for glutamate metabolism, osmoregulation,
anti-oxidation and retinoid metabolism. These metabolic profiles may reveal altered
metabolic stabilities, altered metabolic programming or biochemical response profiles
indicative of cell stress.
Conclusions: This work is aimed at investigating the metabolic relationships between
Müller cells and photoreceptors in retinal stress situations. Metabolic networks are
likely complex and we propose that the metabolic response of activated Müller cells
assists metabolically stressed or challenged photoreceptors. The power of CMP to
integrate various levels of cell regulation (metabolism, energetics and proteomics)
with high spatial resolution is paving the way to discover the molecular glial
transformations that confer neuroprotection.
CR: F.R. Vazquez-Chona, None; W.D. Ferrell, None; B.W. Jones, None; R.E. Marc,
Signature Immunologics, E, P.
Support: NIH EY02576, EY015128 and EY014800 (RM); RPB Career Development
Award (BWJ); and FFS, IRRF and KTEF (FVC)
Purpose: To examine whether the initial loss of rods in STGD3 affects the cone mosaic
and opsin expression prior to detectable cone loss
Methods: We relied on an ELOVL4 transgenic mouse [line TG1-2, Kuny et al. IOVS
(2009), in press] to model STGD3. Firstly, the cone population was quantified on retinal
cross sections stained with DAPI and a cone-specific marker (gamma transducin).
Secondly, retinas of 12 month old ELOVL4/TG1-2 and wild type (WT) littermates were
flatmounted and immunolabeled for M- and S-opsins. Potential differences between
groups in M-cone mosaic and M-opsin expression across the retina were estimated
using Voronoi domain and fluorescence imaging analysis, respectively.
Results: In WT mice, there is a higher density of photoreceptors in the center than in
the periphery. The total number of photoreceptors does not vary with age. In ELOVL4/
TG1-2 retinas, there is a progressive loss of photoreceptors beginning by 6 months
of age. Near complete photoreceptor loss is achieved at 24 months. At 12 months of
age, approximately 50% of the photoreceptors are lost. However, regardless of the
area investigated, the cone density remains similar to that found in WTat this time
point. The initial photoreceptor loss in transgenic mice is therefore mostly due to rods.
Voronoi analysis of the M-cone population further shows no significant variation in
mean area and regularity index values between WT and transgenic mice. Finally,
the dorso-ventral gradient of M-opsin expression seen in WT is still preserved in
ELOVL4/TG1-2 mice. This is in striking contrast with older (24 months) transgenic
mice where only sparse, severely distorted cones (confined to a single plane along
the outermost periphery of the degenerating retina) can be seen.
Conclusions: Despite an important initial rod loss, the pattern and the phenotype of
the M-cones look normal in 12 months old ELOVL4/TG1-2 mice. Studies at later time
points will reveal when cones start degenerating.
CR: M.-A. Filion, None; E. Brown, None; F.P. Gaillard, None; Y. Sauve, None.
Support: CIHR, AHFMR
4059 - A452
Müller Cells Play a Key Role in Maintaining a Functional Retina After Stem
Cell Transplantation in Rcs Rats
Z.Q. Yin, C. Tian, T. Zhao. Southwest Eye Hospital, Southwest Hospital, Chongqing,
China.
Purpose: Retinal degeneration is a leading cause of blindness worldwide, and
currently there is no practical clinical treatment. Stem cell-based therapy offers a
novel therapeutic approach for the treatment of retinal degeneration. This study
was to evaluate whether Müller cells are potential progenitor cells in a chronic
retinal degeneration model (RCS rats) and in rats with subretinal stem cell (rSC)
transplants.
Methods: The morphology of Müller cell in a RCS rats and in rats with rSC transplants
was showed by immunohischemistry with anti-bodies to Chx10 (progenitor
marker), vimentin (Müller cell marker) and opsin (photoreceptor cell marker).The
function of retina in a RCS rats and in rats with rSC transplants was evaluated by
electroretinaogram.
Results: We found that some cells in retina co-expressed anti-bodies to Chx10 and
vimentin in a RCS rats and in rats with rSC transplants. The number of Chx10vimentin positive cells (dedifferentiated Müller cells) significantly increased after rSC
transplantation in the graft region (temporal retina) as well as nasal retina. In addition,
some Müller cells co-express photoreceptor cell markers. Electroretinaogram analysis
showed that retinal function was significantly prolonged after transplants.
Conclusions: Müller cells play a key role in restoring/maintaining retinal function
in cell based treatments of degeneration. Investigation of the relationship between
Müller cell dedifferentiation and functional recovery may lead to new treatments for
retinal diseases such as retinitis pigmentosa.
CR: Z.Q. Yin, None; C. Tian, None; T. Zhao, None.
Support: National Basic Research Program of China(2007CB512203)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4056-4059
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4060 - A453
Cyclic GMP, PDE6β and CREB Regulation in P23H-1 and S334ter-3 Mutant
Rhodopsin Transgenic Rats in the Course of Retinal Degeneration
4061 - A454
Does Activation of Mislocalized Opsin Cause Rod Cell Sprouting?
B. Arango-Gonzalez, J. Kaur, G. Eske, F. Paquet-Durand, E. Zrenner. Division of
Experimental Ophthalmology, Centre for Ophthalmology, Tuebingen, Germany.
J. Wang, E. Townes-Anderson. Neurology and Neurosciences, UMDNJ- New Jersey
Medical School, Newark, NJ.
Purpose: Our focus lies on using two rhodopsin mutant rats as models for RP to
delineate the sequence of events that lead to retinal degeneration. In the present study
we examined the synthesis of Cyclic GMP (cGMP), and expression of phosphodiesterase
6 ß (PDE6ß) and cAMP response element binding protein (CREB) in P23H-1 and
S334ter-3 Mutant Rhodopsin Transgenic Rats.
Methods: Retinas of P23H-1, S334ter-3 and CD rats were collected at different
developmental ages (PN0 - PN30). Retinas were examined by immunohistochemistry
on cryo- and paraffin- sections using specific antibodies against cGMP, PDE6ß, CREB
and phosphoCREB.
Results: Using a well validated antibody directed against cGMP (Tanaka et al. 1997),
in both transgenic retinas, particularly in the S334ter-3 mutants, we observed an
increased cGMP immunoreactivity in photoreceptor cell bodies, processes and
segments compared to their age matched controls. Early in the postnatal development
(PN12), immunostaining showed only a weak labelling in the ONL of wild type
retinas. This staining disappeared after PN20. PDE6ß immunostaining was limited
to the OS of photoreceptors in CD as well as in mutant rats. However, OS length in
S334ter-3, was reduced as the degeneration progressed. CREB expression was found
in the cells localized in the INL and GCL of CD retinas on all analysed ages. On the
other hand, in P23H-1 or S334ter-3 retinas, the absence of CREB staining was clear.
PhosphoCREB expression was observed on all cell layers in the retina in wild type
animals, being more intense in the inner retina. In both mutant models, a decrease
in the staining intensity in the inner retina and in the number of stained cells in the
ONL was found.
Conclusions: Our results suggest a seemingly critical role of cGMP and CREB in
photoreceptor degeneration mechanisms in rhodopsin transgenic rats. Further
experiments are required to establish the cause of cGMP up-regulation and its
contribution to photoreceptor degeneration.
CR: B. Arango-Gonzalez, None; J. Kaur, None; G. Eske, None; F. Paquet-Durand,
None; E. Zrenner, None.
Support: Tistou und Charlotte Kerstan Stiftung; Transgenic animals were kindly
provided by Dr. M. M. LaVail (UCSF, San Francisco, CA)
Purpose: In retinal disease, such as retinitis pigmentosa, and after reattachment of
detached retina, rod photoreceptor cells sprout neurites. We have demonstrated that
activity of adenylyl cyclase (AC) contributes to sprouting by isolated salamander rod
cells. We also reported that β-ionone, an opsin agonist, can stimulate AC activity
through G protein in isolated rod cells in which opsin occurs along the inner segment
and cell body (Alfinito and Townes-Anderson, 2002). In this study, we have tested
whether β-ionone can induce sprouting by rod cells in cell culture and retinal
tissue.
Methods: For cell culture, retinal cells were isolated from adult tiger salamanders,
cultured for 3 days, and stained for rod opsin and phosphorylated cAMP-dependent
transcription factor (pCREB), to analyze for neuritic growth and CREB activity
respectively. For tissue culture, eyeballs with cornea, lens and iris removed, were
cultured for 7 days, sectioned at 35 μm, and stained for opsin and propidium iodide
to determine rod cell sprouting.
Results: β-ionone (2.5 μM) increased the length of the longest process of rod but not
cone cells after 3 days culture in vitro. For comparison, Forskolin (FSK, 10 μM), an AC
agonist, increased the longest process length of both rod and cone cells. Densitometry
analysis showed that β-ionone increased pCREB immunolabeling of rod cells whereas
FSK increased that of both rod and cone cells. Frozen sections from retinal tissue
cultured with DMSO (0.05%) or β-ionone (2.5 μM) were examined with confocal
microscopy. Rod cells appeared normal after 7 days, but opsin was present on all
plasma membranes. Along regions of retina detached from the retinal pigment
epithelium, especially in the β-ionone treated group, rod photoreceptors sprouted
neurites into the inner nuclear layer.
Conclusion: β-ionone was able to increase the neurite outgrowth of isolated rods cell
in culture, most likely through the pCREB signaling pathway stimulated by AC. Rod
cell sprouting, similar to that observed in disease, was stimulated in intact retina
by retinal detachment, which causes mislocalization of opsin, and opsin activation.
This in vitro system of intact retina may provide a new way to study the mechanism
behind such sprouting.
CR: J. Wang, None; E. Townes-Anderson, None.
Support: NIH Grant EY012031 and the F. M. Kirby Foundation
4062 - A455
Histologic Study of Autopsy Eyes From a Male With X-Linked Stationary Night
Blindness With Myopia and a Novel Mutation in the NYX Gene, Leu202Pro
4063 - A456
Characterization of Photoreceptor Loss and Inner Retinal Remodeling in a
Canine Model of Cone-Rod Dystrophy (CRD2)
M. Adamian, T.L. McGee, E.L. Berson. Berman-Gund Laboratory, Massachusetts Eye
& Ear Inf., Harvard Medical School, Boston, MA.
E.M. Scott1, G.M. Acland2, G.D. Aguirre1, W.A. Beltran1. 1Section of Ophthalmology,
University of Pennsylvania School of Veterinary Medicine, Philadelphia, PA;
2
James A Baker Institute, Cornell University, Ithaca, NY.
Purpose: To identify the gene defect causing X-linked congenital stationary night
blindness (CSNB) with myopia in a male whose ocular findings and electroretinograms
(ERGs) were previously described (Hill, Arbel, and Berson, AJO 78:127-136, 1974) and
to define the histopathologic retinal changes in his autopsy eyes.
Methods: Autopsy eyes were obtained from an 87 year-old male with CSNB and
high myopia who died of congestive heart failure. Leukocyte DNA from this patient
was screened for mutations in the NYX gene since mutations in the NYX gene have
been associated with this condition. His right eye was fixed 24 hours after death in
2.5% glutaraldehyde and 1% formaldehyde for ultrastructural studies and the left
eye in buffered 4% formaldehyde for immunocytochemistry. Autopsy eyes from an
84 year-old unaffected male with an identical post-mortem interval were similarly
prepared for histopathologic comparison.
Results: DNA sequencing revealed a novel Leu202Pro mutation of the NYX gene.
Light microscopic and ultrastructural examination of the midperiphery of his right
eye showed a disorganized, vacuolated outer plexiform layer and loss of some cells
in the inner nuclear layer. Cell loss was seen in the outer nuclear layer in this region
also. The macula appeared unaffected. Cone and rod inner and outer segments
appeared normal. These findings correlate well with previously reported ERGs by
Hill et. al. which showed preservation of the rod a-wave and loss of the rod b-wave.
Immunocytochemical studies are in progress to further define the observed inner
retinal pathology.
Conclusions: To our knowledge this is the first histopatholgic report of a male with
X-linked CSNB with myopia with a Leu202Pro missense mutation in the NYX gene.
We show that previous ERGs recorded from this patient are consistent with the current
histologic abnormalities seen in the inner retinal layers with relative preservation
of the outer retinal layer.
CR: M. Adamian, None; T.L. McGee, None; E.L. Berson, None.
Support: Foundation Fighting Blindness, Owings Mills, MD
Purpose: Inherited retinal degenerations (RD) are a frequent cause of blindness
sharing a common feature, the degeneration and death of photoreceptor cells. Cone-rod
dystrophies (CRD) are a subset of diseases in which cones initially show clinical signs
of dysfunction and disease, followed by rods secondarily. This study characterized
the kinetics of photoreceptor loss, and evaluated the progression of photoreceptor
degeneration and inner retinal remodeling in an early onset canine model of conerod dystrophy, (CRD2).
Methods: The retinas from four affected and three age-matched non-affected carrier
dogs were embedded in OCT, and sectioned at 7 or 10 µm for H&E staining, TUNEL
assays and/or immunohistochemistry. The sections extended from the optic nerve to
the ora serrata along the superior, inferior, nasal and temporal meridians.
Results: Abnormal morphology of photoreceptors was recognized at 6 weeks of age,
the earliest time point examined. Cone outer segments were scarce at 6 weeks and
mostly absent by 14 weeks of age. Cone inner segments were stunted and broadened
at 6 weeks, and scarcely present by 31 weeks. The proportion of dying photoreceptors,
particularly rods, was highest at 6 weeks and reduced by 50% between 14 and 42 weeks
of age. Following the early loss of cone outer segments, a decline in the density of
both short and medium/long wavelength cone somatas was observed later between
31 and 42 weeks of age. In addition to rod and cone opsin mislocalization, there was
also retraction of ON (rod and cone) bipolar cell dendrites in the affected retinas.
Conclusions: CRD2 is an early onset disease characterized by abnormal cone and rod
maturation and function followed by the progressive death of rods and secondary
structural degeneration of cones. Although both cones and rods are affected, the cone
functional and structural abnormalities are more severe. The results suggest a potential
time window for cone-directed therapy within the first 31 weeks of life.
CR: E.M. Scott, None; G.M. Acland, None; G.D. Aguirre, None; W.A. Beltran,
None.
Support: EY13132, EY06855, EY17549, FFB grants, Fight for Sight Nowak Family Grant,
Univ Pennsylvania Research Foundation, NIH-Merck/Merial Fellowship, NIH T35
RR07065, Hope for Vision, Van Sloun Fund.
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4060-4063
Wednesday, May 5, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 4028 - 4066 / A421 - A459
418. Retinal Degeneration Genes/Structure and Function
Organizing Section: RC
4064 - A457
Expression Analyses of TTC21B Mutants in mIMCD3 and Rat Retinal
Photoreceptors
4065 - A458
Identification and Characterization of a Novel Whirlin Isoform as a RPGR
ORF15 Binding Protein
Q. Zhang1, Q. Liu1, E. Davis2, N. Katsanis2, E. Pierce1. 1FM Kirby Center for Molecular
Ophthalm, Univ of Pennsylvania, School of Medicine, Philadelphia, PA;
2
Ophthalmology, Duke University, Durham, NC.
R. Wright, D. Hong. Veterinary Pathobiology, Texas A&M University, College
Station, TX.
Purpose: Mutations in cilia genes cause syndromic disorders. Common clinical
features in these ciliopathies include renal cystic disease, retinitis pigmentosa. We
recently found that mutations in the novel cilia protein TTC21B cause Meckel and Jeune
syndromes and nephronophthisis. We performed analyses of the locations of multiple
mutant TTC21B proteins in cultured renal cells and the retina in vivo to investigate the
mechanisms by which the mutations cause cilia dysfunction and disease.
Methods: Mutant and wt cDNAs were cloned into an entry vector, and moved to aV5tag destination vector. The plasmids were transfected into mIMCD3 cells and neonatal
rat retinas using lipofection and in vivo electroporation, respectively. The location
of TTC21B in mIMCD3 and retinal cells were determined by expressing V5-epitope
tagged mutants. The cellular location of the wt and mutant proteins was evaluated by
immunoflourecence using anti-V5 Ab. The location of the proteins in photoreceptor
sensory cilia was evaluated in 3D reconstructions of confocal image stacks obtained
from immunostained vibratome sections of transfected retina. The levels of expression
of the mutant proteins were evaluated using Western blotting.
Results: The wt TTC21B protein localized in the transition zone of mIMCD3 and rat
photoreceptor. Seventeen missense mutations were identified in patients with cilia
disorders. The locations of all 17 mutant proteins were evaluated in mIMCD3 cells.
The location of 6 mutants was evaluated in rat retinas in vivo. Five of the mutants were
mislocalized in both the mIMCD3 and photoreceptor cells in vivo. Three mutants were
grossly mislocalized in the photoreceptor cell bodies. Two were partially mislocalized,
but retained some transition zone localization. Seven mutations resulted in reduced
levels of protein.
Conclusions: The results suggest that a subset of TTC21B mutations cause disease
by resulting in mislocalization of the mutant in ciliated cells. An additional group
of mutations may cause disease by reduced protein level. Evaluation of the ability of
mutant proteins to localize correctly in specific cell structures can be a useful method
for assessing the pathogenicity of potentially disease-causing sequence variants.
CR: Q. Zhang, None; Q. Liu, None; E. Davis, None; N. Katsanis, None; E. Pierce,
None.
Support: NIH EY12910, FFB, RPB, FM Kirby Foundation, and Rosanne Silbermann
Foundation
Purpose: Mutations in the retinitis pigmentosa GTPase regulator (RPGR) are a frequent
cause of inherited retinal degeneration. The RPGR gene undergoes complex alternative
splicing and encodes constitutive and ORF15 variants. The constitutive transcripts
are widely expressed and contain nineteen exons (RPGRex1-19), while the RPGRORF15
variants are preferentially expressed in the retina and contain exons 1-14 plus a large,
alternatively spliced exon 15. A large number of disease causing mutations in the
ORF15 exon suggest that this isoform is essential for the viability of photoreceptors.
This study was designed to further elucidate the function of the RPGR ORF15 isoform
by identifying potential interacting partners.
Methods: Mouse retinal cDNA was used in a yeast two-hybrid screen with the
C-terminus of RPGR ORF15. RT-PCR and immunoblot analysis were used to confirm
the presence of selected candidate partners in the retina. Results obtained in the yeast
two-hybrid screen were confirmed by pull-down assays using recombinant protein
and transfected cell lines, and immunohistochemistry was used to determine the
cellular localization.
Results: Our yeast two-hybrid screen identified a novel variant of whirlin, a PDZ
scaffold protein expressed in cochlear hair cells and retinal photoreceptors, as a protein
interacting with RPGR ORF15. This short, N-terminal whirlin isoform contains 322
amino acid residues resulting in a full PDZ1 domain and truncated PDZ2 domain.
Polyclonal antibodies against the N-terminus of whirlin confirmed expression of
this novel isoform in both the retina and cochlea, and immunohistochemistry using
antibodies against the N-terminal and C-terminal ends of full-length whirlin showed
isoform specific localization within photoreceptors. Whirlin isoforms detected by the
N-terminal antibody localized to the root and connecting cilium while those detected
by the C-terminal antibody localized in the outer segment and connecting cilium.
Conclusions: These findings indicate that whirlin binds RPGR ORF15 and the
N-terminal isoform may be required for photoreceptor function. Mutations in the
N-terminus of whirlin disrupt the structure of the PDZ domain and result in Usher
syndrome type 2 (USH2D), a hereditary disorder characterized by severe hearing loss
and retinitis pigmentosa. The interaction between RPGR ORF15 and whirlin provides
a potential mechanism for the retinal degeneration observed in USH2D.
CR: R. Wright, None; D. Hong, None.
Support: NIH Grant EY14188
4066 - A459
Functional Changes Correlate With Structural Changes in North Carolina
Macular Dystrophy
C.M. Cutler Peck, S. Grover. Ophthalmology, University of Florida, Jacksonville, FL.
Purpose: North Carolina macular dystrophy (NCMD) is one of the rarer forms of
juvenile-onset macular dystrophies. One-third of these patients initially present
as having a coloboma-like macular lesion with good retinal function. The aim of
this study was to describe the structural findings, as obtained by spectral-domain
optical coherence tomography (SD-OCT) and correlate with the retinal function, as
measured by visual acuity, visual fields and microperimetry (MP-1) in two young
siblings with NCMD.
Methods: The proband, a 16-year old girl and a 14-year old brother underwent a
complete ophthalmological examination and were clinically diagnosed to have NCMD.
Visual acuity, visual field testing, SD-OCT (Spectralis), fundus autofluorescence (FAF),
color fundus photography and microperimetry were performed on both siblings.
Results: Both siblings with NCMD had a normal-appearing anterior segment.
However, the 16-year old sibling showed a large, bilateral, coloboma-like macular
lesion whereas the 14-year old brother showed a relatively smaller macular lesion with
no evidence of a coloboma. This was consistent with slightly reduced visual acuity,
retinal pigment epithelium (RPE) changes with coloboma-like structure on SD-OCT
and hypoautofluorescent areas on FAF in the older sister as compared to normal visual
acuity, milder RPE changes on SD-OCT and normal FAF in the younger brother. The
retinal functional changes were also consistent with structural changes - MP-1 showed
increased thresholds in the older sister with normal thresholds in the younger brother.
It was interesting to note that although the macular lesions were large and there was
RPE involvement, the photoreceptor cell layer looked intact on SD-OCT.
Conclusions: Well-preserved retinal function as measured by microperimetry
correlates with the relatively spared photoreceptor layers as shown by Spectralis
SD-OCT and provides an insight into the pathophysiology of a rare condition like
North Carolina macular dystrophy.
CR: C.M. Cutler Peck, None; S. Grover, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4064-4066
Wednesday, May 5, 1:45 PM - 3:30 PM Floridian A Paper Session Program Number Range: 4304 - 4309
442. Retinal Development and Regeneration
Organizing Section: RC
4304 - 1:45PM
Role of Epigenetics (DNA Methylation) in RPE and Photoreceptor Development
I.O. Nasonkin1, D. Hambright1, K. Lazo1, R. Fariss2, R. Rachel1, K. Bharti 3, M. Jamrich4,
R. Jaenisch 5, A. Swaroop1. 1N-NRL, National Eye Institute, Bethesda, MD; 2NNRL,
Biological Imaging Core, National Eye Institute, Bethesda, MD; 3NINDS, NIH,
Bethesda, MD; 4Molecular and Cellular Biology, Baylor College of Medicine,
Houston, TX; 5Whitehead Institute for Biomedical Research, MIT, Cambridge, MA.
4305 - 2:00PM
Transcriptional Regulation of Eph Receptor Expression by DNA Methylation in
Embryonic Retinal Cells and Müller-Derived Retinal Stem Cells
T.D. Petkova1, G.M. Seigel2, D.C. Otteson1. 1College of Optometry, University of
Houston, Houston, TX; 2Center for Hearing and Deafness, University of Buffalo,
SUNY, Buffalo, NY.
Purpose: Differential DNA methylation takes place during organogenesis in cells
acquiring different cell fates, and is conducted in all mammalian species (including
humans and mice) by three DNA methyltransferases. Dnmt3a and Dnmt3b are
responsible for de novo methylation of DNA, while Dnmt1 maintains the newly
established methylation patterns in differentiating cells. DNA methylation mostly
leads to stable silencing of gene expression. We hypothesize that differentiation
and/or maturation of neural retina (nr) and retinal pigment epithelium (RPE) are
influenced by DNA methylation.
Methods: The Dnmt1-floxed mice were mated to Rx-Cre mice to generate conditional
loss of Dnmt1 expression in developing nr and RPE. Histological changes were
evaluated by light and electron microscopy. Immunolocalization studies were
performed on retinal sections using cell-specific antibodies to photoreceptors, RPE
and Muller cells, and second order neurons.
Results: Severe morphological changes were observed in the retina and RPE of Dnmt1
knockout animals. These pathologic changes included lack of photoreceptor outer
segment elongation, retinal folding and pseudorosette formation, and abnormalities
in RPE development and maturation. Inner segments and rhodopsin and recoverin
were present in Dnmt1 2lox, Rx-Cre mutant retinas. Retina-RPE adhesion in mutants
was affected during early postnatal development. Multiple localized RPE defects such
as lack of organized regular hexagonal cell structure were observed in flatmount
RPE preparations of newborn mutant pups (P0.5) and even in e16.5 embryos stained
with phalloidin. PNA and cone arrestin staining of P10.5 and P15.5 mutant retinas
demonstrated significant underrepresentation of cones.
Conclusions: Our studies provide the first evidence of Dnmt-mediated epigenetic
mechanisms guiding the development of RPE and photoreceptors.
CR: I.O. Nasonkin, None; D. Hambright, None; K. Lazo, None; R. Fariss, None; R.
Rachel, None; K. Bharti, None; M. Jamrich, None; R. Jaenisch, None; A. Swaroop,
None.
Support: NEI/NIH
Purpose: Understanding gene regulation in the retina during development and in
retinal stem cells will be key for successful optic nerve regeneration. EPH receptor
signaling functions in axon guidance, formation of retinotopic maps and maintaining
stem cell characteristics. We hypothesize that differential DNA methylation of CpG
islands regulates Eph promoter activity in retinal development and retinal stem
cells.
Methods: Genomic DNA and RNA were isolated from mouse Müller glia-derived
neurospheres (ImM10 cell line) and embryonic retina (E17). DNA was bisulfite
converted (ZymoGold), CpG islands were PCR amplified, subcloned and sequenced.
Gene expression was analyzed by quantitative RT-PCR. Statistical analysis used
SPSS. EphA5 promoter activity, with and without in vitro promoter methylation, was
assayed by dual luciferase assays in R28 cells.
Results: In Müller-derived neurospheres, EphA5 and EphB1 promoters were heavily
methylated (48%-67%) and no mRNA expression was detected. The EphB2 promoter was
minimally methylated (0.5%) and mRNA expression was detected (mean Ct=29.5). In E17
retina, CpG methylation of EphA5 and EphB1 was 1.2% and both genes were expressed
(mean Ct=21.7, EphA5; 20.1, EphB1). Pearson correlation of CpG methylation vs. mean
Ct across all samples was 0.876 (p=0.026), R square=0.767. Total CpG methylation by
SssI decreased EphA5 and EphB1-luc promoter activity by 97% and 87% respectively
(p<0.0001, T-test). Partial methylation using HhaI decreased EphA5 promoter activity
by 20%, while partial methylation using HpaII increased activity by 35%.
Conclusions: The inverse relationship between promoter methylation and mRNA
expression in Müller glia-derived retinal stem cells and embryonic retina supports
a role for CpG methylation in regulating Eph promoter activity. Differential effects
of partial methylation on EphA5 promoter activity suggests site specificity of CpG
methylation.
CR: T.D. Petkova, None; G.M. Seigel, None; D.C. Otteson, None.
Support: FFS Summer Fellowship, Borish Ezell Fellowship (TDP); TR Lee Award for
National Glaucoma Research AHAF, Glaucoma Research Foundation (DCO); NIH
EY07551 (core UHCO); NIH CA127061, EY016662, RPB (GMS)
4306 - 2:15PM
mTOR Activity Restores Retinal Ganglion Cell Dendritic Arbors After Axonal
Injury in vivo
4307 - 2:30PM
Sphingosine-1-Phosphate Has a Key Role in the Crosstalk and Development of
Retina Glial and Neuronal Cells
B.J. Morquette1A, P.P. Roux1B,2, A.R. McKinney3, A. Di Polo1B. APathology and Cell
biology, BPathology and Cell Biology, 1University of Montreal, Montreal, QC,
Canada; 2Institute for Research in Immunology and Cancer, Montreal, QC, Canada;
3
Department of Pharmacology/Therapeutics, McGill University, Montreal, QC,
Canada.
N.P. Rotstein1A, C.E. Abrahan1B, L.E. Politi1B. AUns-conicet, BUns-Conicet, 1Instituto de
Investigaciones Bioquimicas, Bahia Blanca, Argentina.
Purpose: Dendrites are major determinants of how retinal neurons integrate and
process incoming information. We previously showed that retinal ganglion cells (RGCs)
undergo a significant reduction of dendritic arbors soon after axonal injury. However,
the molecular mechanisms that underlie injury-induced dendritic remodeling are
poorly defined. Here, we investigated the role of the mTOR (mammalian target of
rapamycin) pathway in RGC dendritic structure after acute optic nerve lesion.
Methods: Adult transgenic mice carrying the yellow fluorescent protein (YFP) gene
under control of the Thy-1 promoter, which allows visualization of RGC dendrites,
were subjected to optic nerve axotomy. Retinal mTOR activity was manipulated
using two approaches: i) intraocular injection of siRNA against the mTOR repressor
REDD2, therefore increasing mTOR activity; and ii) intraperitoneal administration
of rapamycin, an mTOR inhibitor. Three days after lesion, prior to the onset of RGC
death, retinal whole-mounts were prepared and YFP-positive RGC dendritic trees were
reconstructed from images obtained by confocal microscopy using Imaris software
(Bitplane). mTOR activity in RGCs was examined by immunohistochemistry using
an antibody against phospho-S6, a downstream target of mTOR.
Results: Our data demonstrate that optic nerve axotomy leads to marked
downregulation of mTOR activity in RGCs, which correlated with dendritic
shrinkage at 3 days after injury. Treatment with siREDD2 stimulated mTOR activity
in axotomized RGCs and promoted a significant increase in total dendritic length
and surface (n=10) compared to retinas treated with control siRNA (n=10). Scholl
analysis revealed a marked increase in the complexity of dendritic arbors from RGCs
with increased mTOR activity. Administration of rapamycin completely blocked the
effect of siREDD2 on dendritic growth and branching confirming that this response
occurred via mTOR stimulation.
Conclusions: Our study reveals a novel role for the mTOR pathway in the maintenance
of dendritic structure in adult, injured RGCs.
CR: B.J. Morquette, None; P.P. Roux, None; A.R. McKinney, None; A. Di Polo,
None.
Support: Fond de Recherche en Santé du Québec (FRSQ).
Purpose. We have shown that Müller glial cells in coculture with retinal neurons
preserve the proliferative potential of neuronal progenitors and protect neurons from
oxidative stress, which in turn enhances glial proliferation. We have also identified
the sphingolipid sphingosine-1-phosphate (S1P) as a crucial mediator in proliferation,
survival and differentiation of photoreceptors. We have now investigated the roles
played by S1P in neuroglial development.
Methods. Pure glial cultures and neuroglial cocultures prepared from rat retinas
were treated with or without S1P. The effect of S1P on proliferation was evaluated by
Br-deoxyuridine (BrdU) or [3H]thymidine uptake, and on neuronal migration and
axonal growth and orientation by fluorescence and confocal microscopy, after staining
cells with rhodamine-phalloidin and acetylated tubulin. Cocultures were treated with
BML-241, a S1P receptor 3 (S1P3) antagonist or with a sphingosine kinase 1 (SphK1)
inhibitor to inhibit S1P synthesis, and oxidative stress was then induced with the
oxidant paraquat (PQ). Apoptosis was then determined by TUNEL labeling.
Results. Addition of S1P increased [3H]thymidine and BrdU uptake in pure glial
cultures and augmented BrdU uptake in neuroblasts in coculture. Inhibiting S1P
synthesis or adding BML-241 before treating neuroglial cocultures with PQ blocked
glial protection from oxidative stress-induced apoptosis in photoreceptors. S1P
addition also regulated neuronal differentiation and organization in neuroglial
cocultures. S1P promoted the aggregation and rearrangement of neurons in coculture,
leading to the formation of conspicuous round aggregates mainly comprised of
neurons; thick bundles of axons connected these aggregates, forming an extensive
network that was absent in controls. Concurrently, S1P increased the expression of
N-CAM, a cell adhesion molecule.
Conclusions. Our results suggest that S1P promotes proliferation of glial cells. S1P
might also be among the key factors released by glial cells to prevent neuronal
apoptosis, stimulate neuroblast proliferation and participate in neuronal migration
and axonal outgrowth and orientation.
CR: N.P. Rotstein, None; C.E. Abrahan, None; L.E. Politi, None.
Support: FONCyT PICT 06 711; CONICET Grant PIP 112-200801-02105; Universidad
Nacional del Sur, Argentina
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4304-4307
Wednesday, May 5, 1:45 PM - 3:30 PM Floridian A Paper Session Program Number Range: 4304 - 4309
442. Retinal Development and Regeneration
Organizing Section: RC
4308 - 2:45PM
Rabac1 is Differentially Localized in Developing rd1 Compared to Wild Type
Retina
V.M. Maggio1, A.M. Richmond2, J.G. Martak1, S.C.E. Hoge1, A.Y. Chowdhury1, J.M.
Ogilvie2. 1Biology, Saint Louis University, St. Louis, MO; 2Biology, Saint Louis
University, Saint Louis, MO.
Purpose: The rd1 mouse is a well studied model of retinal degeneration. During early
postnatal development and prior to degeneration (postnatal days P4-P8), pathology
consistent with defects in vesicular trafficking has been reported (Blanks et al., 1974;
Caley et al., 1972; Sanyal and Bal, 1973). In microarray analysis of wild-type (wt) and
rd1 retinas at P2-P8, Rab acceptor 1 (Rabac1) was the only identified gene, other than
the mutant PDE6b, to be downregulated at all time points (Ogilvie et al., 2006). Rabac1
acts as a GDI dissociation factor to facilitate transfer of cytosolic GDP-bound Rab to
the membrane during vesicular trafficking. Here, we investigate the distribution of
Rabac1 protein in postnatal wt and rd1 mouse retinas.
Methods: Eyecups from wt and rd1 age-matched littermates were harvested at
developmental ages between birth and P21. Tissue was fixed, cryoprotected and
processed for immunohistochemistry using an antibody against Rabac1 and double
labeled with the cis-Golgi marker, GM130. Images were obtained using a Zeiss LSM
510 Meta confocal microscope.
Results: In wt retinas at all ages examined, Rabac1-like immunoreactivity (LIR) is
associated with Golgi and the perinuclear region of most inner retinal cells. Punctate
labeling is also seen throughout both plexiform layers. A similar pattern of Rabac1LIR is seen in the rd1 inner retina at all ages. At P21, intense Rabac1-LIR is observed
in photoreceptor inner segments (IS), where Golgi membranes reside, and in outer
segments with no staining in the outer nuclear layer (ONL). The P10 wt retina shows
intense Rabac1-LIR in developing IS with the Golgi neatly aligned at the IS margin.
Some Rabac1-LIR is also seen in the ONL associated with Golgi. Mislocalization of the
Golgi and Rabac1-LIR is observed in the rd1 retina at P10. Prior to P10, few differences
were observed in the staining pattern between wt and rd1 retinas, although less
Rabac1-LIR is observed in the rd1 retina.
Conclusions: Together, our data suggests that a decrease in Rabac1 expression may
correlate with disruption of Golgi and vesicular trafficking prior to photoreceptor
cell death in rd1 mouse retina.
CR: V.M. Maggio, None; A.M. Richmond, None; J.G. Martak, None; S.C.E. Hoge,
None; A.Y. Chowdhury, None; J.M. Ogilvie, None.
Support: Saint Louis University
4309 - 3:00PM
Optimizing Retinal Regeneration: A Role for Sonic Hedgehog
D.L. Stenkamp, T. Sherpa, S.S. Hunter. Biological Sciences, University of Idaho,
Moscow, ID.
Purpose:Teleost fish can regenerate their retinas after damage. We demonstrated
recovery of visual function in zebrafish subjected to widespread retinal damage
inflicted by intraocular injection of ouabain (Sherpa et al., 2008; Dev Neurobiol 68:16681). Functional recovery required 98 days, matching the time of optic nerve head
(ONH) restoration. Later recovery times were characterized by overproduction of cells
in the retinal ganglion cell (RGC) layer and ONH hypertrophy. Here we determine
whether a selective lesion to inner retinal layers will result in faster visual recovery
and fewer histological errors, and identify the molecular basis of any differences in
regeneration following broad vs. selective retinal damage.
Methods:Zebrafish were injected intraocularly with high or low doses of ouabain,
creating widespread retinal damage, or damage limited to the inner retinal layers,
respectively (Fimbel et al., 2007; J Neurosci 27:1712-24). Visual function was monitored
by a place-preference assay. Radial sections of the retina were used to measure the
ONH, or were stained with anti-HuC/D antibodies to facilitate counting of cells in
the RGC layer and assessment of laminar pattern. QRT-PCR was used to measure the
expression of selected genes in both lesion models as a function of recovery time.
Results:Following selective retinal damage, recovery of visual function was observed
at 58 days post-lesion, matching the time of ONH restoration (60 days; Fimbel et al.,
2007). Eyes evaluated at later recovery times did not show ONH hypertrophy, did
not contain supernumerary cells in the GCL, and the lamination errors seen at early
recovery times became virtually nonexistent. In contrast, visual recovery following
broad retinal damage was slower (Sherpa et al., 2008), and was accompanied by
increasing retinal disorganization as a function of recovery time. In both damage
models, several genes related to RGC development (Brn3b, ath5, fgf8) were significantly
downregulated immediately following the lesion, then expression returned to normal,
adult levels. However, although the sonic hedgehog (shh) gene was downregulated in
both models, expression returned to normal, adult levels only in the selective lesion
model, remaining at reduced levels in the broad lesion model.
Conclusions:Regeneration of RGCs in a manner that restores vision is optimized by the
presence of other, undamaged retinal cells. Delays in visual recovery, overproduction
of RGCs, retinal disorganization, and overgrowth of the ONH are correlated with
errors in shh expression during retinal regeneration. An experiment to assess retinal
regeneration in conditions of reduced shh signaling is underway.
CR: D.L. Stenkamp, None; T. Sherpa, None; S.S. Hunter, None.
Support: NIH R01 EY012146, NIH P20 RR016454, UI Multicultural Award
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4308-4309
Wednesday, May 5, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 4458 - 4487 / A391 - A420
451. Oxygen-induced Retinopathy
Organizing Section: RC
4458 - A391
Early Anti-Angiogenic Intervention in the Immature, Neovascular Retina: A
Cautionary Tale
4459 - A392
Hyperoxia Modulates the Physiology of the Developing Retinal
Microvasculature
T.L. Favazza1, I.Y. Benador1, Y.A. Tsabur1, A.B. Fulton1,2, R.M. Hansen1,2, J.D. Akula1,2.
1
Ophthalmology, Children’s Hospital Boston, Boston, MA; 2Ophthalmology,
Harvard Medical School, Boston, MA.
D.M. Wu1A, D.G. Puro1B. AOphthalmology and Visual Sciences, BOphthalmology,
1
Univ of Michigan-Kellogg Eye Ctr, Ann Arbor, MI.
Purpose: Angiogenic growth factors (e.g., VEGF) are also neurotrophic factors (SaintGeniez et al., PLoS One, 2008). Thus, we and others have urged considerable caution in
the use of such molecules, although promising in age-related macular degeneration and
diabetic retinopathy, for prevention of retinopathy of prematurity (ROP). We tested a
proprietary, anti-angiogenic molecule in two rat models of ROP and studied functional
and vascular outcomes. This drug has been shown to reduce neovascularization (NV)
in a choroidal NV model and have low neural toxicity in the mature murine eye.
Methods: Retinopathy-inducing oxygen exposures were delivered to Sprague-Dawley
rat pups from postnatal day (P) 0-14 (“50/10 model”, n=4) or from P7-14 (“75 model”,
n=4). Room-air-reared controls (n=4) were also studied. Drug or vehicle were injected
in contralateral eyes, in a masked fashion, in each rat. Activity in photoreceptor and
postreceptor neurons was derived from binocular ERG recordings. Retinae were
then flatmounted, and clock hours of NV and percent of the retinal surface lacking
capillary perfusion (avascular retina, AR) were quantified. Data were analyzed by
two-factor (group, eye) repeated-measures analysis of variance.
Results: Significant attenuation in ERG response amplitudes, both receptor (RmP3,
P<0.001), postreceptor (RmP2, P=0.04), and oscillatory potentials (Em, P=0.02) was
observed. Retinal sensitivity, Sm, was also significantly (P<0.001) negatively impacted
by drug. The negative impact of drug on retinal function was found in both ROP (50/10,
75) and control retinae. The drug did not significantly reduce either NV (P=0.45) or
AR (P=0.47) in ROP retinae.
Conclusion: Anti-angiogenic drugs safe in the mature eye may not be safe for the
immature retina. Indeed, since neural and vascular dysfunction are associated (Akula
et. al., IOVS, 2007; MolVis, 2008), even improvements in vascular outcomes are not
assured, as increased damage to retinal neurons may offset the presumed benefit of
early anti-angiogenic interventions.
CR: T.L. Favazza, None; I.Y. Benador, None; Y.A. Tsabur, None; A.B. Fulton,
None; R.M. Hansen, None; J.D. Akula, None.
Support: Massachusetts Lions Eye Research Fund, Pearle Vision Foundation, Scholl
Foundation, March of Dimes, NIH Grant RC1 EY020308
4460 - A393
Examining a Role for Melanin in Retinal Ion Regulation and
Neovascularization in Experimental ROP
Purpose: In retinopathy of prematurity (ROP), fluctuating oxygen levels may reversibly
alter blood flow prior to the onset of permanent structural changes. To test the hypothesis
that ion channels play a role in the response of the developing retinal microvasculature to
hyperoxia, we performed the first patch-clamp study of developing retinal microvessels.
Methods: A tissue print method was used to isolate retinal microvascular complexes
from postnatal (P2-P6) rats. In freshly isolated microvessels, ionic conductances were
monitored via perforated-patch pipette sealed onto a microvascular cell; simultaneous
time-lapse photography allowed us to correlate changes in electrophysiological activity
with vascular tone. An oxygen-enriched environment was created by bubbling a mixture
of 95% O2/ 5% CO2 into the perfusate.
Results: We found that hyperoxia caused the membrane potential of cells in postnatal
retinal microvessels to increase by 7.8 ± 2.2 mV, p<0.005. This hyperoxia-induced
hyperpolarization was due to the inhibition of a conductance whose reversal potential was
near 0 mV. Hyperoxia also reversibly inhibited spontaneously occurring depolarizations,
which were often observed in the postnatal microvasculature. Associated with these
responses, microvascular relaxation and lumen dilation were detected in many
of the monitored complexes. Because hyperpolarizing ion channels in adult retinal
microvessels are redox-sensitive, we assessed the possibility that oxidants mediated
the electrophysiological responses to hyperoxia. While we found that the oxidant, H2O2
(10-20 μM), also induced hyperpolarization in the immature retinal microvasculature,
this voltage increase was larger (45 mV ± 2.9 in H2O2, p <0.001) than that induced by
hyperoxia and was due to the activation of a current whose reversal potential was near
EK, (i.e., -103 mV) and with a sensitivity to the K ATP channel blocker glibenclamide. Thus
H2O2 did not mimic the microvascular response to hyperoxia.
Conclusions: In the microvasculature of the postnatal rat, hyperoxia causes membrane
potentials to increase, spontaneous depolarizations to cease, microvascular cells to relax,
and vessel lumens to dilate. The hyperoxia-induced hyperpolarization is due to the
inhibition of a non-specific cation conductance by an oxidant-independent mechanism.
Our demonstration that ion channels play a role in the response of the immature retinal
microvasculature to hyperoxia supports the idea that modification of ion channel activity
is a possible strategy for altering events early in the course of ROP.
CR: D.M. Wu, None; D.G. Puro, None.
Support: DMW was supported by a grant from the Knights Templar Eye Foundation
4461 - A394
Role of Mip-1β in Mouse Model of Oxygen-Induced Retinopathy
K. Ishikawa. Ophthalmology, Kyushu University, Fukuoka, Japan.
B.A. Berkowitz1A, U.B. Kompella2, D. Bissig1B, C. Durairaj2, R. Roberts1B. AAnatomy/
Cell Biol & Ophthal, BAnatomy/Cell Biol, 1Wayne State Univ Sch of Med, Detroit,
MI; 2Pharmaceutical Sciences & Ophthalmology, University of Colorado Denver,
Aurora, CO.
Purpose: Coordinated retinal ion flux is critical for proper vision, whereas retinal ion
dysregulation is linked with neovascularization (NV). We tested the hypothesis that
the ion buffer melanin in RPE/choroid, influences intraretinal uptake of manganese,
a biomarker of regulation of ions like calcium, in control rats, as well as NV severity
in retinopathy of prematurity (ROP) rats.
Methods: In dark adapted, awake and freely moving control rats (Brown Norway
(BN, postnatal day 50 (P50)), Long Evans (LE, P20, P50), and Sprague Dawley (SD, P14,
P20, P50)), and experimental rats (LE rats 6 and 36 days after 50/10 oxygen exposure,
and SD rats 0, 6, and 36 days after 50/10) central retinal ion regulation was measured
using manganese-enhanced MRI (MEMRI, MnCl2 ip). In select groups, RPE/choroid
melanin content, NV incidence and severity, and visual performance (optokinetic
tracking) were evaluated.
Results: In P50 controls, manganese uptake in RPE/choroid correlated with melanin
content (BN > LE > SD); uptake in ONL and inner retina (BN > SD >> LE) did not. In
experimental ROP, NV severity (LE > SD ~ BN) did not correlate with melanin levels.
50/10 LE, but not SD, pups had supernormal inner and outer retinal uptake through
P50. Supernormal intraretinal uptake in LE rats was associated with subnormal
visual performance.
Conclusion: As expected, melanin content and manganese uptake were linked in
RPE/choroid. Melanin levels were not correlated with ion regulation in the rest of
the retina, or with NV. NV severity and visual performance impairment were closely
associated with apparent intraretinal depolarization.
CR: B.A. Berkowitz, None; U.B. Kompella, None; D. Bissig, None; C. Durairaj,
None; R. Roberts, None.
Support: EY018109 (BAB); JDRF (BAB); AMDCC/MMPC (BAB); RPB (Kresge Eye
Institute); and EY017533 (UBK)
Purpose:We previously reported that the gene expression level of Macrophage
inflammatory protein-1β(MIP-1β), a member of CC chemokine subfamily which may
play a role in the recruitment of macrophages, was significantly up-regulated in the
ischemic retinas of mouse model of oxygen induced retinopathy using gene microarray
analyses. The aim of this study was to examine expression time course, localization
and function of MIP-1β in the model.
Methods: C57BL/6N pups were placed in a 75% oxygen environment on postnatal
day 7 for 5 days and then returned to room air. Retinas were removed from mice
at 0,3,6,12 hours (h),1,2,3,and 5 days after relative ischemia. Total RNA and protein
were extracted from each retina, and the expression levels of MIP-1β were measured
by real-time quantative PCR and ELISA method. The localization was examined
by immunohistochemstry and laser capture microdissection. The number of F4/80
positive cells and mRNA expression levels of F4/80 were measured in the P17 whole
retinas after administration of the neutralizing antibodies against MIP-1β.
Results: MIP-1β mRNA levels increased at 6 h and maximal at 12 h after ischemia.
MIP-1β protein levels were also increased at 12 h and maximal at 2 days after ischemia.
Immunohistochemistry demonstrated that MIP-1β was localized in the ganglion cell
layer and the inner nuclear layer. Administration of the neutralizing antibodies against
MIP-1β decreased levels of F4/80 mRNA expression and the number of infiltrating
F4/80 positive cells(p<0.05, n=8).
Conclusions: MIP-1β was expressed in the hypoxic inner retina in the mouse model
of oxygen induced retinopathy. Our data suggest that MIP-1β may play a role in the
inflammation induced by the ischemic retinopathy.
CR: K. Ishikawa, None.
Support: None
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4458-4461
Wednesday, May 5, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 4458 - 4487 / A391 - A420
451. Oxygen-induced Retinopathy
Organizing Section: RC
4462 - A395
The Role of AMP-Dependent Kinase a1 Isoform (AMPKa1) in Retinopathy of
Prematurity
4463 - A396
Choroidal Vasculature Degeneration in Oxygen Induced Retinopathy: The Role
of 15deoxy-Δ12, 14 -Prostagladin J2
X. Koufomichali1, N.M. Krah2, G. Trichonas3, A. Manola3, A. Thanos3, Y. Morizane3, E.
Gragoudas3, D. Vavvas3. 1Ophthalmology, Massachusetts Eye and Ear Infirmary,
HARVARD MEDICAL SCHOOL, Boston, MA; 2Ophthalmology, Children’s
Hospital, Harvard Medical School, Boston, MA; 3Ophthalmology, Massachusetts
Eye and Ear Infirmary, Harvard Medical School, Boston, MA.
Z. Shao1, A.L. Dorfman2, P. Sapieha3, P. Lachapelle2, S. Chemtob4. 1Pharmacology and
Therapeutics, McGill University, Montreal, QC, Canada; 2Ophthalmology, McGill
U-Montreal Childrens Hosp, Montreal, QC, Canada; 3Ophthalmology, Harvard
Medical School, Boston, MA; 4Pediatrics & Pharmacology, Research Ctr/Hosp Ste
Justine, Montreal, QC, Canada.
Purpose: Retinopathy of Prematurity (ROP) is a retinal vasoproliferative disease,
which, despite the recent advancement in treatment, remains among the leading
causes of visual impairment in childhood that will lead to a lifetime of incapacity.
It features cessation of vessel growth that leads to a stressed, hypoxic peripheral
retina and subsequent retinal neovascularization. Increased energy expenditure
and decreased energy supply depletes ATP and increases AMP levels leading to
activation of the energy sensor protein AMP-dependent kinase (AMPK). Previously
it has been shown that AMPK activation leads to increased levels of HIF1a, VEGF
and eNOS; molecules important in ROP pathogenesis. We study the role of AMPK in
this angiogenic disease by examining the effect of its absence in the model of Oxygen
Induced Retinopathy (OIR).
Methods: Seven days after birth (postnatal day 7, P7), litters of a1 isoform KO
(AMPKa1-/-) and WT mice were exposed to 75% oxygen for 5 days until P12.
Immediately after exposure, they were transferred to room air for 5 days until P17.
On P17 and under deep anesthesia, eyes were enucleated, retinas were isolated and
stained for Isolectin B4-FITC.The retinas were whole-mounted and quantification of
retinal vaso-obliteration and neovascularization was performed.
Results: In two independent experiments, whole-mount analysis in WT and a1 isoform
KO mice revealed a 30% to 130% more vaso-obliteration in the a1 knockout mice (p<0.05)
but no statistically significant difference in neovascularization.
Conclusions: AMPKa1 does not appear to be a major factor in the pathogenesis of
oxygen induced retinopathy. Further investigation will be contacted with the use of
CNS specific double a1/a2 isoform KO mice.
CR: X. Koufomichali, None; N.M. Krah, None; G. Trichonas, None; A. Manola,
None; A. Thanos, None; Y. Morizane, None; E. Gragoudas, None; D. Vavvas, None.
Support: Research to Prevent Blindness , LIONS , NIH Core Grant for Vision Research
to MEEI
Purpose: To investigate the contribution of choroidal vascular involution to the
retinal functional changes associated with retinopathy of prematurity (ROP) and
determine the involvement of 15deoxy-Δ12, 14-Prostagladin J2 (15d-PGJ2), a potent
anti-inflammatory and important anti-angiogenic factor, in this process.
Methods: Two different rat models of oxygen induced retinopathy (OIR; 80% O2
or 50%/10% (24hr cycle) from postnatal day (P) 1-P14) were used to mimic ROP.
Retinal function was assessed by multifocal electroretinogram (mfERG) at P60 and
compared to controls raised in room air (21% O2). Structural changes were assessed
in open sections and drifts in protein expression patterns were determined by
immunohistochemistry on cryo-sagittal sections. Choroidal vascular density and
morphology was determined by eye corrosion casts examined by scanning electron
microscopy (SEM) while vessel leakage was assessed following intra-cardiac injections
of Evans blue. Levels of 15d-PGJ2 were measured by ELISA immunoassay and 15d-PGJ2
was delivered intravitreally to room air animals to investigate the in vivo cytotoxic
effect of this compound on choroidal vasculature.
Results: Pronounced and predominantly central choroidal vascular involution
(choroidal vascular thickness and choriocapillary vessel density) was detected in both
models of OIR animals and was accompanied by increased choroidal vessel leakage.
This translated to a significant loss of function largely restricted to the central retina
(when compared to the periphery) as determined by mfERG. Relevantly, 15d-PGJ2
levels increased dramatically during the first week of hyperoxic exposure coinciding
with the initial apoptotic nuclei in the choroid. Confirmation of the involvement of
15d-PGJ2 in choroidal involution was obtained after its intraocular administration to
normal animals compromised choroidal vasculature in an analogous manner to OIR.
Conclusions: The involvement of choroidal decay in the progression of ROP has to
date not been addressed. Our data demonstrates that central choroidal involution is
associated with the cardinal retinal dysfunction associated with ROP and suggests that
effective future therapeutic strategies should take this vascular bed into consideration.
CR: Z. Shao, None; A.L. Dorfman, None; P. Sapieha, None; P. Lachapelle, None; S.
Chemtob, None.
Support: CIHR Grant 178871
4464 - A397
Caspase-14: A Novel Molecule With a Possible Role in Ischemic Retinopathy
4465 - A398
Genetic Deletion of Aldose Reductase Protects the Neonatal Mouse Retina From
Oxygen-Induced Retinopathy
M.A. Al-Shabrawey1A,2, R. Mussell1B, A. Tawfik1B, S. Hsu1B. AOral Biology, Anatomy,
Ophthalmology and Vision Discovery Institute, BOral Biology and Anatomy,
1
Medical College of Georgia, Augusta, GA; 2Ophthalmology, King Saud University,
Riyadh, Saudi Arabia.
Purpose: Caspase14 expression was found in tissues involved in barrier function
such as epidermis, choroid plexus, hair follicles, retinal pigment epithelium, and
thymic Hassall’s bodies, and keratinized oral epithelium,. The function of caspase14 is
relatively unexplored. Unlike other caspases, it is not involved in the apoptotic caspase
cascade, but is associated with terminal differentiation of normal human epidermal
keratinocytes and barrier formation. We have recently, shown reduction in the size
of tumor made of human salivary gland cells (HSG) transfected with caspase14 and
this was associated with significant reduction in tumor angiogenesis. The purpose
of this study was to characterize the expression of caspase14 in normal retina and in
retina with pathological neovascularization.
Methods: Experimental retinal neovascularization was developed by incubating
a group of mice at postnatal day 7 (P7) in high oxygen (75%) for 5days, followed
by 5 days in room air (normoxia). Mice were then sacrificed on P17 and caspase14
expression was tested in retina sections and homogenates using immunofluorescence
and Western blotting respectively.
Results: Caspase14 is localized in retinal pigment epithelium and vasculature in
normal retina. However, ischemic retinopathy is associated with a marked reduction
in retinal expression of caspase14.
Conclusion: This finding suggests a regulatory role for caspase14 in retinal
neovascularization. Further investigations are required to identify the specific role
of caspase-14 in ischemic retinopathy.
CR: M.A. Al-Shabrawey, None; R. Mussell, None; A. Tawfik, None; S. Hsu, None.
Support: AHA00104 and PSRP (MCG)
A.C. Lo1A,1B, Z.-J. Fu1A, S.-Y. Li1A, D. Wong1A,1B, S.K. Chung1C,1B. AEye Institute, BResearch
Center of Heart, Brain, Hormone and Healthy Aging, CAnatomy, 1University of
Hong Kong, Hong Kong, Hong Kong.
Purpose: Retinopathy of prematurity (ROP) is a common retinal disease that occurs in
premature babies with initial vessel loss followed by vessel proliferation. Oxidative stress
and retinal dysfunction has been reported. We previously showed that genetic deletion or
pharmacological inhibition of aldose reductase (AR), a rate limiting enzyme in polyol pathway,
prevented RGC loss and oxidative stress after retinal ischemia/reperfusion injury (Cheung
et al Exp Eye Res 2007 85:608). Here, we assessed the effects of aldose reductase deletion on
retinal injury induced by hyperoxic exposure using a mouse model of ROP.
Methods: Seven-day-old pups (P7) were exposed to 75% oxygen for 5 days and then
returned to room air environment for 5 days. Vessel architecture and neuronal responses
after hyperoxia were examined and compared between wild-type and AR-deficient retinae.
Immunohistochemistry for immunoglobulin G (IgG), calretinin, glial fibrillary acidic protein
(GFAP), nitrotyrosine (NT), inducible nitric oxide synthase (iNOS) were performed.
Results: At P12 (immediately after hyperoxia), AR-deficient retina displayed significantly
smaller central avascular area (57.9±1.1% vs. 64.3±1.4% in wild-type retina, p<0.003). There was
significant reduction in thickness of central INL and entire IPL in wild-type retina but not in
AR-deficient retina (p<0.05). GFAP immunoreactivity was increased and the intensity was
stronger in AR-deficient retina, but that for calretinin in wild-type and AR-deficient retinae
was similar. At P17 (5 days after hyperoxia), central avascular area persisted in wild-type
retina (24.8±1.7%). Again, AR-deficient retina showed a smaller area (16.1±1.2%, p<0.0005).
Interestingly, blood vessels along GCL of wild-type retina displayed IgG extravasation after
hyperoxia. Yet, this was much reduced in AR-deficient retina. There was absence of calretinin
staining in INL together with seriously distorted strata in IPL of wild-type retina but not
AR-deficient retina. Increased intense GFAP staining was seen in central-middle retina of
wild-type but not AR-deficient mice. Increased NT immunoreactivity was found in INL of
wild-type retina but this increase was less apparent in AR-deficient retina. Similar increase
in iNOS immunostaining was observed in wild-type and AR-deficient retina.
Conclusion: Our observations indicated that AR deletion reduced avascular area, IgG
leakage, amacrine cell loss, and NT induction upon hyperoxia, suggesting a protective role
of AR deficiency in ROP.
CR: A.C. Lo, None; Z.-J. Fu, None; S.-Y. Li, None; D. Wong, None; S.K. Chung, None.
Support: University Development Fund and Seed Funding for Basic Research, The University
of Hong Kong
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at pubs@arvo.org. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
4462-4465
Wednesday, May 5, 1:45 PM - 3:30 PM Hall B/C Poster Session Program Number/Board # Range: 4458 - 4487 / A391 - A420
451. Oxygen-induced Retinopathy
Organizing Section: RC
4467 - A400
Modulation of Vascular Endothelial Growth Factor and Pigment EpithelialDerived Factor Signaling by Alternating Periods of Hyperoxia and Hypoxia in a
Relevant Model of Retinopathy of Prematurity
4466 - A399
FAD286, an Aldosterone Synthase Inhibitor, Reduces Retinal Vasculopathy
K.J. Binger, D. Deliyanti, G. Tan, A.G. Miller, J.L. Wilkinson-Berka. Immunology,
Monash University, Prahran, Australia.
Purpose: Recently we identified that aldosterone has potent angiogenic and
inflammatory actions in the retina, and that these occur via the mineralocorticoid
receptor. The aim of this study was to determine if direct inhibition of aldosterone
synthase with FAD286 reduces retinal vascular disease.
Methods: Oxygen-induced retinopathy (OIR) was induced in Sprague Dawley rats with
exposure to 80% oxygen (22hrs/day) from postnatal days (P) 0 to 11, followed by 7 days
in room air. Sham rats were always in room air. FAD286 (30mg/kg/day, sc) or its control
vehicle tartaric acid (10mg/kg/day, sc) were administered from P12 to P18 to either
OIR or sham rats (N=8 to 10 rats/group). Pathological angiogenesis in the inner retina
and vitreous was assessed in paraffin sections, and by counting neovascular tufts in
isolectin stained retinal wholemounts. The under-developed avascular region of the
peripheral retina that develops in OIR was quantitated using ImageJ software (NIH).
Vascular endothelial growth factor (VEGF) and intercellular adhesion molecule-1
(ICAM-1) were measured in retina by real time PCR. The expression of NOX4, a
subunit of NADPH oxidase that is implicated in aldosterone-mediated events, was
also evaluated in retina with real-time PCR.
Results: In OIR, pathological angiogenesis (22.43±1.56 blood vessels/field) was
increased compared to shams (12.91±0.38, P<0.01), and was normalised with FAD286
(14.29±0.58, P<0.01). Retinal neovascular tufts were increased 7.8-f