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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologya major mechanism for oxidative stress-induced RPE cell death.Moreover, we have screened a collection of 2,000 FDA-approvedcompounds for molecules that inhibit oxidative stress-induced RPEnecrosis.Methods: ARPE-19 cells were treated with different concentration ofH2O2 and harvested at different time points followed by cell deathmarker staining, as well as RNA, DNA and protein analyses.Compound screening was performed using MTT assay.Results: Morphology and molecular hallmarks of dying ARPE-19cells show strong evidence of necrotic cell death induced byoxidative stress. We observed partial chromosomal condensation(pyknosis) and permeability of cell membrane by propidium iodine(PI). ARPE-19 cells treated with H2O2 did not exhibit apoptoticDNA fragmentation, consistent with our finding that ARPE-19 cellshave very low level of DFF45 protein, inhibitory subunit of DFF40main apoptotic endonuclease. RPE cell death is accompanied byincreased levels of PGAM5 and RIP3 both critical for undergoingprogrammed necrosis. Consistently, necrostatin-1, but not caspaseinhibitor zVAD, rescued oxidative stress-induced RPE cell death.Additionally our results showed that autophagic cell death is not aresult of H2O2 treatment in RPE cells. Furthermore, we screenedFDA-approved compounds and identified candidates thatsuccessfully rescued RPE cells from oxidative stress-induced celldeath.Conclusions: We provide unequivocal evidence that necrosis is amajor mechanism for oxidative stress-induced RPE cell death. Wealso identified natural compounds that can rescue oxidative stressinducedRPE cell death. Taken together, these findings provide novelinsight into the mechanism of RPE cell death, and will beinstrumental for developing novel therapeutics for dry AMD,especially GA.Commercial Relationships: Jakub Hanus, None; William C.Anderson, None; Peng Jin, None; Qinghua Liu, None; ShushengWang, UT Southwestern Medical Center (P)Support: Startup fund of Tulane University (S.W.), President’sResearch Council New Investigator Award from UT SouthwesternMedical Center (S.W.), NIH Grants EY021862 (S.W.), CareerDevelopment Award from the Research to Prevent BlindnessFoundation (S.W.).Program Number: 5002 Poster Board Number: A0131Presentation Time: 2:45 PM - 4:30 PMPhosphorylation Networks in Age-Related Macular DegenerationSrinivas R. Sripathi 1 , O'Donnell Sylvester 2 , Trevor Moser 1 , Paul S.Bernstein 3 , Folami Lamoke 4 , Manuela Bartoli 4 , Wan Jin Jahng 2 .1 Biological Sciences, Michigan Tech University, Houghton, MI;2 Retina Proteomics Laboratory, Petroleum Chemistry, AmericanUniversity of Nigeria, Yola, Nigeria; 3 Ophthalmology and VisualSciences, Moran Eye Center, University of Utah, Salt Lake City, UT;4 Ophthalmology, Georgia Health Sciences University, Augusta, GA.Purpose: Age-related macular degeneration (AMD) results from thechronic atrophy of retina and retinal pigment epithelial (RPE) cells(dry AMD) or choroidal neovascularization (wet AMD). However,the molecular mechanism underlying the progression of AMD is notwell defined yet. Light-induced oxidative stress, lipid oxidation,accumulation of soft drusen, and crystalline aggregation maycontribute to AMD pathogenesis. The aim of our phosphoproteomestudy is to identify the early biochemical events and specificphosphorylated sites in the macular, peripheral retina and RPE. Ourdata demonstrated that specific kinase/phosphatase regulatedapoptotic phosphorylation signaling in AMD progression.Methods: The senescence-associated molecular events during AMDprogression were identified by comparing the phosphoproteome ofmacular and peripheral retina, RPE from different stages of AMDcompared to age-matching control donor eyes. Phosphoproteins wereenriched by charge-based spin column chromatography and resolvedby 2D gel electrophoresis. Tryptic peptides were enriched usingGa3+/TiO2 immobilized metal ion chromatography. Elutedphosphopeptides were analyzed and confirmed by MALDI-TOF orESI/MS/MS mass spectrometry and phospho-Western blotrespectively.Results: Our preliminary data shows tyrosine phosphorylation ofbeta crystallin A3 and A4 increased but serine phosphoryaltion ofbeta crystallin decreased in RPE under stress. Our phospho-Westernanalysis reveals phosphorylation of intermediate filament vimentin(Ser) and mitochondrial heat shock protein mt Hsp70.Serine/threonine phosphatase PP2A-Cα/β (Tyr) and tubulin α1B/β2were dephosphorylated in the RPE. Further, our data shows thatprohibitin is upregulated in the retina, but down regulated in the RPEfrom AMD donor eyes. Our study suggests that altered lipidcomposition may determine phosphorylation-dependent interactionsof target proteins that contain PH, PX, and SH domains.Conclusions: The region specific proteome changes incentral/peripheral retina and RPE from AMD donor eyes and agematchingcontrols were analyzed. Phosphorylation changes anddownstream activation may leads to the early biochemical events inAMD. Our phosphoproetomic strategy provides the detailedphosphorylation signaling,phospholipid changes, altered proteinaggregation,binding affinity and expression that include depletedlevels of anti-apoptotic, mitochondrial chaperone prohibitin in theRPE of AMD eyes.Commercial Relationships: Srinivas R. Sripathi, None; O'DonnellSylvester, None; Trevor Moser, None; Paul S. Bernstein, Kalsec(C), Kemin Health (R), Science Based Health (C), Abbott Nutrition(F), Genentech (C), DSM (R), Sequenom (R), NuSkin/Pharmanex(P), Aciont (C), Thrombogenics (C); Folami Lamoke, None;Manuela Bartoli, None; Wan Jin Jahng, NoneProgram Number: 5003 Poster Board Number: A0132Presentation Time: 2:45 PM - 4:30 PMEvaluation of RPE cell senescence as a mechanism for agerelatedmacular degeneration (AMD),Michael R. Kozlowski. Arizona College of Optometry, MidwesternUniversity, Glendale, AZ.Purpose: The purpose of this work is to assess whether or not thechanges seen in retinal pigment epithelial (RPE) cells as theyapproach cell senescence are consistent with the pathologicalprocesses that occur in age-related macular degeneration (AMD).Methods: In this study, ARPE-19 cells grown on membranecontainingmulti-well plate inserts or in cell culture flasks were usedto model the natural RPE cell layer. The cells were cultured to highpopulation doubling levels (PDL’s) and several measures of cellgrowth and function were assessed including doubling rate, viability,morphology, terminal restriction fragment (TRF) length (as ameasure of telomere erosion), trans-epithelial electrical resistance(TEER, as a measure of the quality of their tight junctions), andsenescence associated beta-galactosidase activity (SABG).Results: As ARPE-19 cells reached high population doubling levels(PDL), which is one trigger for cell senescence, they experiencedsignificant telomere erosion and displayed several senescenceassociatedchanges. First, their staining for SABG, a marker forsenescence which is related to lysosomal dysfunction, was elevated.Second, their shape became more rounded, possibly indicating achange in their adhesion characteristics. Finally, the depression oftheir TEER values by VEGF was significantly enhanced.Conclusions: The changes seen in ARPE-19 cells that had reached©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 ARVO Office at arvo@arvo.org.
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyhigh PDL’s are consistent with pathological processes that occur inAMD. For example, a change in RPE cell attachment characteristics,such as that seen in the present study, could lead to a compromise inthe transport of materials across Bruch’s membrane and, thereby, tothe development of drusen. Likewise, a change in lysosomalfunctioning, as was indicated in this study by the increased SABGstaining, could cause a buildup of improperly process outer segmentmaterials, which would also contribute to the development of drusen.In addition, increased responsiveness to VEGF in terms of looseningof tight junctions, as was indicated by the present TEER results,could be permissive to the development choroidal neovascularizationin wet AMD. These finding demonstrate that RPE cell senescencecould contribute to the occurrence of AMD.Commercial Relationships: Michael R. Kozlowski, NoneSupport: Intramural Research Stimulation GrantProgram Number: 5004 Poster Board Number: A0133Presentation Time: 2:45 PM - 4:30 PMAn In-Vitro Study of the Effect of Ultraviolet Radiation onGrowth Patterns and Gene Expression of Human ARPE-19Cybrid H and J, and their Implications with Age-related MacularDegeneration (AMD)Deepika Malik 1 , Payam Falatoonzadeh 1 , Tiffany Hsu 1, 2 , Claudio A.Ramirez 1 , Javier Cáceres del Carpio 1 , Mohamed Tarek MohamedMoustafa 1 , S Michal Jazwinski 3 , Miceli V. Michael 3 , Cristina M.Kenney 1 , Baruch D. Kuppermann 1 . 1 Ophthalmology, Gavin HerbertEye Institute, Irvine, CA; 2 Warren Alpert Medical School, BrownUniversity, Providence, RI; 3 Tulane Center for Aging, TulaneUniversity, New Orleans, LA.Purpose: Studies have shown that mitochondrial DNA (mtDNA)plays an important role in the aging process. Various haplogroups ofmtDNA have been classified based on variable combinations ofsingle nucleotide polymorphisms (SNPs). Cybrids (cytoplasmichybrids) have identical nuclei but mitochondria of differenthaplogroups. Although both haplogroups H and J representEuropean-Caucasian populations, individuals with haplogroup J havea higher susceptibility to develop AMD and H is protective. The aimof this study was to evaluate the effect of UV radiation (a knownenvironmental risk factor for AMD) on cell growth patterns and geneexpression in various cellular pathways in H and J cybrids.Methods: ARPE-19 cells were depleted of mitochondria (Rho0) andfused with platelets isolated from haplogroup H or J subjects to createindividual cybrids. Cybrids were treated with 10mJ of UV radiation.Growth patterns of cybrids H and J were studied over 14 days in bothUV-treated and untreated groups. RNA extraction and cDNAsynthesis were done at 0, 72 and 120 hours, after exposure. Variouspro-apoptotic genes RARA (Retinoic Acid Receptor Alpha), BBC3(BLC2 Binding Protein) and BLC2L13 (BCL2 like 13) (both BBC3and BCL2L13 induce mitochondrial outer membranepermeabilization and caspase activation); pro-inflammatory geneIL33 (Interleukin-33); and angiogenic gene TGF-A (TransformingGrowth Factor-Alpha) were studied using Q-PCR.Results: A stepladder growth pattern was seen in UV-treated groups,which was significantly different than control groups. J cybridsshowed more growth than H cybrids at all studied time points. At 120hours, both UV-treated cybrids H and J showed increase in geneexpression levels for pro-apoptotic genes RARA, BBC3 andBCL2L13, but cybrid H showed a greater increase. UV-treated cybridJ showed higher expression of pro-inflammatory gene IL-33 andangiogenic gene TGF-A as compared to UV-treated cybrid H.Table 1: Fold differences relative to untreated cybrid H (0 hours)Conclusions: Our study shows that SNP variations in mtDNA mayalter nuclear gene expression and cellular responses to stress, andcontribute to differential genetic risk factors for AMD.Fold differences relative to Untreated Cybrid H (0 Hours)Commercial Relationships: Deepika Malik, None; PayamFalatoonzadeh, None; Tiffany Hsu, None; Claudio A. Ramirez,None; Javier Cáceres del Carpio, None; Mohamed TarekMohamed Moustafa, None; S Michal Jazwinski, None; Miceli V.Michael, None; Cristina M. Kenney, None; Baruch D.Kuppermann, Alimera (C), Allegro (C), Allergan (C), Genentech(C), Glaukos (C), GSK (F), Novagali (C), Novartis (C), Ophthotech(C), Pfizer (C), Regeneron (C), Santen (C), SecondSight (C), Teva(C), ThromboGenics (C)Support: Discovery Eye Foundation, Guenther Foundation,Beckman Macular Research Initiative, Polly and Michael SmithFoundation, Max Factor Family Foundation, Skirball Foundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 5005 Poster Board Number: A0134Presentation Time: 2:45 PM - 4:30 PMCybrids with Different mtDNA Haplogroups Show DifferentialExpression of Respiratory Complex GenesCristina M. Kenney 1 , Marilyn Chwa 1 , Shari Atilano 1 , PayamFalatoonzadeh 1 , Deepika Malik 1 , Claudio A. Ramirez 1 , S MichalJazwinski 2 , Miceli V. Michael 2 , Baruch D. Kuppermann 1 .1 Ophthalmology, Gavin Herbert Eye Inst, UC Irvine, Irvine, CA;2 Tulane Center for Aging, Tulane University, New Orleans, LA.Purpose: Mitochondrial DNA (mtDNA) haplogroups representdifferent human populations and are defined by accumulation ofspecific SNPs. The J haplogroup is high risk for age-related maculardegeneration (AMD) while the H haplogroup is protective for AMD.Cybrids (cytoplasmic hybrids) are an excellent model to studymitochondrial functions because the cells have identical nuclei andvary only in the mtDNA content. This study was designed to analyzethe oxygen consumption rates (OCR representing OXPHOS) andextracellular acidification rates (ECAR representing glycolysis) andthe expression of mtDNA encoded genes that are associated with theelectron transport chain complexes I, III, IV, and V.Methods: Cybrids were created by fusing Rho0 ARPE-19 cells(depleted of mitochondria) with platelets isolated from haplogroup Hor J subjects (n=3 each). The Seahorse FX24 extracellular fluxanalyzer was used to measure OCR and ECAR in H vs J cybrids.These measurements allow for evaluation of cellular metabolicactivity, mitochondrial function, and energy expenditure. Q-PCR wasperformed on H cybrids vs J cybrids using primers for the mtDNAgenes of complexes I, III, IV, and V. mtDNA copy numbers weremeasured by comparing nDNA:mtDNA ratios (18S:MT-ND2).Results: The J cybrids had a significantly lower OCR/ECAR ratiothan the H cybrids (14±2.7 vs 24 ±2.5, p
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