<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>Results: The results show that only the fraction corresponding to2.5% BSA contained most cells displaying PROX-1 and Islet-1positive immunoreactivities with a typical HC morphology. Based onan accurate morphological analysis, a number of cells in this fractionresembled H1- and H3-types of HCs. Strikingly, Opn4ximmunoreactivitywas observed in cultures from both the 2.5 and 3 %BSA gradient phases. It is noteworthy that the 3% phase containscells that express the neuronal filament of 200 KDa (NF200) anddisplay longer processes that morphologically resemble RGCs. Byflow cytometry we found a 30% of cells from the wholedisaggregated retina and 80% of cells from the 2.5% of BSA gradientthat were positively labeled for Prox-1 respectively. Afterimmunopurification, cultures contained only OPN4x (+) HCs whichwere also found to express the Gq mRNA.Conclusions: In conclusion, by means of this method we selectivelyobtained primary cultures highly enriched in HCs expressing nonvisualphotoreceptor components.Commercial Relationships: Mario E. Guido, None; Luis P.Morera, None; Nicolás Díaz, NoneSupport: ANPCyT-FONCyT PICT Bicentenario 2010 Nr. 647,PICT, CONICET, SeCyT-UNC, and MinCyT of Córdoba.Program Number: 2008 Poster Board Number: D0039Presentation Time: 11:00 AM - 12:45 PMEffect of prostaglandin E2 on collagen gel contraction in mouseretinal pigment epithelium cellsTomoko Orita, Kazuhiro Kimura, Koh-hei Sonoda. Ophthalmology,Yamaguchi University, Ube, Japan.Purpose: An epithelial to mesenchymal transition of retinal pimentepithelium (RPE) cells plays an important role in the formation ofmembrane in proliferative vitreoretinopathy (PVR) or scar tissue inage-related macular degeneration. Growth factors and inflammatorycytokines are involved in this process. Meanwhile prostaglandin E2(PGE2) has a variety of strong physiological effects and is concernedwith inflammation. Some articles reported that the level of PGE2 invitreous body increases in PVR patients. In this study, weinvestigated that effect of PGE2 on fibrillary twitch of RPE cells byusing collagen gel contraction model.Methods: Mouse RPE cells were prepared and incubated in threedimensional type 1 collagen gel culture system with transforminggrowth factor (TGF)-β2 (1ng/ml) in the absence or presence of PGE2(0-10μM). The expression of α-smooth muscle actin and matrixmetalloproteinase (MMP)-3 as well as the phosphorylation of Smad2and paxillin were examined by immunoblot analysis.Results: TGF-β2 significantly caused a gel contraction in RPE cells,oherwise PGE2 inhibited this effect of TGF-β2 in does dependentmanner. The expression of α-smooth muscle actin as well as thephosphorylation of Smad2 and paxillin induced by TGF-β2 was alsoreduced by PGE2. In contrast, TGF-β2 have no effect on theexpression of MMP-3.Conclusions: PGE2 inhibited the RPE cell mediated collagen gelcontraction. This indicates that PGE2 may inhibit the contraction ofproliferative membrane in progressive vitreoretinal diseases.Commercial Relationships: Tomoko Orita, None; KazuhiroKimura, None; Koh-hei Sonoda, NoneProgram Number: 2009 Poster Board Number: D0040Presentation Time: 11:00 AM - 12:45 PMAge-dependent Biochemical Changes in the Retina ofGlutaredoxin 2 (Grx2) Knockout MiceHongli Wu 1 , Marjorie F. Lou 1, 2 . 1 VBS, University of Nebraska-Lincoln, Lincoln, NE; 2 Redox <strong>Biology</strong> Center, University ofNebraska-Lincoln, Lincoln, NE.Purpose: Glutaredoxin 2 (Grx2) is a mitochondrial isozyme ofthioltransferase in the oxidoreductase family that is a key regulator ofredox homeostasis in the cells through dethiolation of proteinglutathionemixed disulfides (PSSG). Previously, we have found thatGrx2 gene knockout (KO) mouse developed cataract faster duringaging, and that the lens epithelial cells were more sensitive tooxidative stress-induced apoptosis. In this study, we investigated thepresence of Grx2 in the mouse retina, and the potential biochemicalchanges in the mouse retina with Grx2 KO during aging.Methods: Grx2 gene KO mouse model was used to study the agedependentchanges in the retina. Neruo-retina tissues from 1, 7, and16 months old Grx2 KO mice were surgically removed. Five pairs ofthe retina tissues were pooled, homogenized with a glass-to-glasshomogenizer. The mitochondrial fraction was isolated followingpublished procedure. Retina from age-matched wild type (WT)littermates were processed in parallel and used as the control. Thelevels of glutathione (GSH) and protein thiols (PSH) were eachquantified by DTNB colorimetric method. PSSG were analyzed usingWestern blot with GSH-specific antibody. Mitochondrial complex I,complex IV activity and ATP levels was each measured usingmicroplate assay kit.Results: Grx2 was found in the neuro-retinal tissues with higherprotein level and enzyme activity than that of the lens from the sameanimal. Grx2 was absent in the retina of the Grx2 KO model. Withincreasing age, both WT and Grx2 KO groups showed a gradualdecrease in GSH and PSH levels, but this alteration was moreexaggerated in the Grx2 KO group. Glutathionylated protein or PSSGaccumulation in the retina was more prominent in the Grx2 KO micethan that of the WT controls. Remarkably, in comparison with theWT control, the retina in Grx2 gene KO mice showed extensive loss(50%) in complex I and complex IV enzyme activities. The ATP poolin the same Grx2 null retina was correspondingly suppressed to lessthan 30% as that of the WT control.Conclusions: Grx2 was found in the retina and deletion of its geneaffected the redox homeostasis and compromised the mitochondrialfunction. It is likely that mitochondrial dethiolase Grx2 may play animportant role in regulating retinal function.Commercial Relationships: Hongli Wu, None; Marjorie F. Lou,NoneSupport: NIH Grant EY010595Program Number: 2010 Poster Board Number: D0041Presentation Time: 11:00 AM - 12:45 PMRole of Hypoxia-inducible Factor 1 Alpha in Hydrogen PeroxideinducedHuman Retinal Pigment Epithelial Cell DeathPiyush C. Kothary, Patrick Lee, Neil B. Parikh, Sara Abraham,Monte A. Del Monte. Ophthalmology, Univ of Michigan-Kellogg EyeCtr, Ann Arbor, MI.Purpose: <strong>Molecular</strong> biological investigations led the development ofanti-vascular endothelial growth factor (VEGF) therapy for choroidalneovascularization (CNV). Since anti-VEGF therapy has not beeneffective in all patients, we investigated the mechanism of action ofVEGF: whether there is a role for hypoxia-inducible Factor 1 Alphain the VEGF pathway in our cultured human retinal pigmentepithelial (hRPE) cell model.Methods: Human RPE cells were cultured from normal eyesobtained from the Michigan Eye Bank. Cellular proliferation in thepresence of increasing concentrations of FBS was measured by 3(H)thymidine incorporation (3H-thy) and cell viability by the trypan blueexclusion method. Intracellular synthesis of caspase 3, an apoptosiscell death marker, HIF 1 alpha, a regulatory subunit of hypoxiainduciblefactor 1 and VEGF were quantitated and localized byimmunoprecipitation and immunohistochemistry. Statistical analysis©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the <strong>ARVO</strong> Office at arvo@arvo.org.
<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>was done by Student t-test with p≤0.05 considered to be significant.Results: Fetal bovine serum stimulated hRPE cell proliferation andH2O2 exposure inhibited proliferation of viable hRPE cells also in ain a dose dependent manner. H2O2 (0.5 mM) exposure decreasedhRPE cell number by 209.6% (5.70±0.73 vs 2.72±0.35, (Viable cellsx 10,000±SEM, n=4, p≤0.05)) and increased 14C-caspase-3 and 14C-HIF1 alpha synthesis in a dose dependent manner. H2O2 (0.5 mM)exposure increased 14C-caspase synthesis by 209.4% (1022.44±432vs. 2141.26± 666.31, n=4, p≤0.05)) and HIF1 alpha by 211.1%(197.62±43.44 vs. 417.20± 21.34, n=6, p≤0.05)). H2O2 (0.5 mM)exposure also increased 14C-VEGF synthesis by 130.0% (613.43.26±143.49 vs.793.68 ±124.02, n=4, p≤0.05)). Phase contrast microscopyconfirmed oxidative damage hRPE cell morphology following H2O2exposure. Immunohistochemical analysis confirmed and localizedincreased expression of HIF1 alpha and VEGF expression inpresence of H2O2 when compared with controls.Conclusions: Our data suggest that H2O2 may stimulateangiogenesis in CNV by stimulating HIF1 alpha as well as VEGF,suggesting anti-HIF1 alpha targeted therapy may provide a novelapproach to treating CNV.Commercial Relationships: Piyush C. Kothary, None; PatrickLee, None; Neil B. Parikh, None; Sara Abraham, None; Monte A.Del Monte, NoneSupport: Supported by the Skillman Foundation EndowmentMADM.Program Number: 2011 Poster Board Number: D0042Presentation Time: 11:00 AM - 12:45 PMExtremely brief light-preconditioning in pigmented mouse modelof light-induced retinal degeneration (LIRD)Priscila P. Cunha, Micah A. Chrenek, Jana T. Sellers, Jeffrey H.Boatright. Ophthalmology, Emory University School of Medicine,Atlanta, GA.Purpose: Preconditioning of albino mice using moderate intensitycyclic light may be confounded due to shortened photoreceptor outersegments and reduced rhodopsin expression such that the retinas areless sensitive to light. We hypothesize that preconditioning can beinduced in pigmented mice with brief, sub-toxic light exposure. Herewe describe the parameters used to induce such preconditioning.Methods: Sixty-four male 129sv mice were obtained from CharlesRiver (100 days old) and kept in a 12:12 light: dark cycle. Lightpreconditioningwas done using the following procedure: mice weretreated with 1% atropine eye drops at 9:00AM and maintained in thedark for 1 hour to allow the pupils to dilate. Non-toxic light exposureconsisted of 5k lux of white light for four hours using a LED lamp.Light-preconditioning was done either 1 or 3 days before LIRD.LIRD was done according to the following procedure: at 9:00AMmice were treated with 1% atropine eye drops and maintained in thedark for 1 hour to allow the pupils to dilate. Toxic light exposureconsisted of 40-50k lux of white light for four hours using a LEDlamp. Visual acuity was measured using OptoMetry optokinetictracking system (OKT). Retinal function was assessed using ERGs:scotopic a- and b-waves were measured using 5 flash intensities(0.0039-24.9 cd s/m^2).Results: We found that intense white light causes light damage in129sv mice. This can reliably be measured with either OKT (82% ofdim controls, P
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