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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyProgram Number: 4996 Poster Board Number: A0125Presentation Time: 2:45 PM - 4:30 PMIn-depth analysis of blood plasma proteome for discovery of agerelatedmacular degeneration biomarkersSe Joon Woo 1, 2 , Hye-Jung Kim 3 , Eui Jin Suh 3 , Ji Hyun Park 1, 2 ,Jeeyun Ahn 1, 2 , Duck Jin Hwang 1, 2 , Ji Eun Lee 3 , Kyu Hyung Park 1, 2 ,Cheolju Lee 3, 4 . 1 Ophthalmology, Seoul National University Collegeof Medicine, Seoul, Republic of Korea; 2 Ophthalmology, SeoulNational University Bundang Hospital, Seongnam, Republic ofKorea; 3 Theragnosis Research Center, Korea Institute of Science andTechnology, Seoul, Republic of Korea; 4 Biomolecular Science,University of Science and Technology, Daejeon, Republic of Korea.Purpose: To find plasma biomarkers for age-related maculardegeneration, we performed comprehensive proteomic analyses onblood plasma of AMD and age-matched healthy controlsMethods: We used a 4D protein profiling method on pooled plasmaof patient and control groups. (1) Abundant protein depletion coupledwith separation strategies - Gel-Eluted Liquid Fraction EntrapmentElectrophoresis (GELFrEE) for protein and (2) Isoelectrofocusing oftryptic peptides by OFFGEL (3) Liquid chromatography and tandemmass analsys (4) LC-MS/MS. Global functional analyses andcanonical pathway analyses were carried out using IngenuityPathway Analysis for proteins which were differentially expressed(more than 2- fold). Validation of candidate biomarkers wasperformed using ELISA and western blot on individual samples.Results: Using this profiling strategy, we identify 5042 uniquepeptides from 353 proteins, ranging over six orders of magnitude inabundance. Canonical pathways associated with AMD wereidentified for example, LXR/RXR activation, acute phase responsesignaling, complement cascades and actin cytoskeleton signaling. Todevelop potential biomarker proteins for AMD, we selected 7proteins for further verification.Conclusions: Our results implicate altered systemic pathways ofAMD and provide a new protein database for hypothesis-driven anddiscovery based studies of AMD.Commercial Relationships: Se Joon Woo, None; Hye-Jung Kim,None; Eui Jin Suh, None; Ji Hyun Park, None; Jeeyun Ahn, None;Duck Jin Hwang, None; Ji Eun Lee, None; Kyu Hyung Park,None; Cheolju Lee, NoneSupport: This study was partly supported by a grant from the KoreaHealth Technology R&D Project, Ministry of Health and Welfare,Republic of Korea (Grant No. A111161).Program Number: 4997 Poster Board Number: A0126Presentation Time: 2:45 PM - 4:30 PMPost-Transcriptional Gene Interactions in Retinal IschemiaKalina Andreeva, Maha Soliman, Nigel G. Cooper. AnatomicalSciences & Neurobiology, University of Louisville School ofMedicine, Louisville, KY.Purpose: Ischemia-Reperfusion (IR) injury has been implicated innumerous retinal disorders, the etiological expression of which can beassociated with various protein coding and micro RNA genes. Whilemuch progress has been made towards identification of genes andpathways associated with retinal disorders, the regulatorymechanisms for their coordinated expression are just beginning to beuncovered.Methods: Our laboratory has generated microarray mRNA andmiRNA data using a rat ischemic model for 3 post-ischemic timepoints (0h, 24h and 7d). We computed correlation coefficientsbetween miRNAs and their target genes in our datasets based onexpression values over the three post-ischemic time points. We thangrouped the genes and miRNAs based on their expressioncorrelations.Results: The graphic representations of changes in expression ofgenes and miRNAs showed inverse relationships, such that whengenes were elevated in expression miRNAs showed reducedexpression and vice versa. These findings support the hypothesis thatthe changes in expression of protein coding genes are in part afunction of changes in expression of their corresponding miRNAs.Conclusions: Based on preliminary data, we propose that regulatedgene expression may control different phases of the disorder.Commercial Relationships: Kalina Andreeva, None; MahaSoliman, None; Nigel G. Cooper, NoneSupport: Supported by EY017594Program Number: 4998 Poster Board Number: A0127Presentation Time: 2:45 PM - 4:30 PMAn In-Vitro Study of Wnt Pathway Modulation in Cybrid H andJ Mitochondrial Haplogroups and the Possible Implications forAge-related Macular DegenerationPayam Falatoonzadeh 1 , Grace B. Woo 1 , Nitin Udar 1 , Shari Atilano 1 ,Marilyn Chwa 1 , Miceli V. Michael 2 , S Michal Jazwinski 2 , Cristina M.Kenney 1 . 1 Ophthalmology, Gavin Herbert Eye Inst., UC Irvine,Irvine, CA; 2 Tulane Center for Aging, Tulane University, NewOrleans, LA.Purpose: The wet form of age-related macular degeneration (AMD)can be associated with increased VEGF, vascular cell proliferation,and induced choroidal neovascularization. Mitochondrial DNA(mtDNA) haplogroups are defined by accumulation of SNPs thatrepresent different geographic origins of populations. Individualswith haplogroup H mtDNA are lower risk for AMD, whilehaplogroup J individuals have higher predisposition. The Wntpathway and β-Catenin, one of its downstream proteins, arecommonly associated with cell proliferation, and studies havefocused on its role in angiogenesis related to cancer and possiblyAMD. Our present study uses a mitochondrial haplogroupcytoplasmic hybrid (cybrid) model to assess the effects of mtDNAvariations on the Wnt pathway.Methods: Mitochondria-deficient (Rho0) human ARPE-19 cellswere fused with platelets from individuals with mtDNA haplogroupsH (n=3) or J (n=3) to establish cybrid cell lines. RNA was extractedfrom each cybrid and cDNA synthesized. Q-PCR was performedusing primers for various genes associated with the Wnt pathway andanalyzed for changes in gene expression levels of J cybrids relative toH cybrids.Results: Gene expression levels were significantly decreased in Jcybrids relative to H cybrids for inhibitors of the Wnt pathway,including SFRP1 (0.29 fold, p=0.0001), DKK3 (0.46 fold, p=0.004),and RARA1 (0.57 fold, p=0.007). There was also a relative increasein expression of GSK3 (1.6 fold, p=0.005). Expression levels of othermembers of the Wnt pathway, including CSNK1E, WNT9A, LRP1,KREMEN1, MDM2, and TGFβ were not found to be significantlydifferent in the J versus the H cybrids.Conclusions: SFRP1 and DKK3 inhibit the binding and interactionof the Wnt ligand with cell surface receptors. Decreased expressionof these genes in J cybrids could potentially lead to more Wnt ligandbinding and increased signal transduction within the cell. This wouldinclude recruitment of increased GSK3A away from the β-catenindegradation complex. Also, decreased RARA1 levels, a nuclearinhibitor of β-catenin, would allow for more β-catenin driventranscription. These observations correlate with faster cybrid Jgrowth patterns in culture, and may also have implications for howdifferent haplogroups might modulate the Wnt pathway and possiblyinfluence AMD.©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 BiologyCommercial Relationships: Payam Falatoonzadeh, None; GraceB. Woo, None; Nitin Udar, Illumina (E); Shari Atilano, None;Marilyn Chwa, None; Miceli V. Michael, None; S MichalJazwinski, None; Cristina M. Kenney, NoneSupport: Supported by Discovery Eye Foundation, GuentherFoundation, Beckman Macular Research Initiative, Polly andMichael Smith foundation, Max Factor Family Foundation, SkirballFoudnation, Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 4999 Poster Board Number: A0128Presentation Time: 2:45 PM - 4:30 PMExpression of VEGF-C, VEGF-D and their Cognate Receptors inExperimental Choroidal Neovascularization and Clinical AMDKameran Lashkari 1 , Jie Ma 1 , Gianna Teague 1 , Jorge G. Arroyo 2 .1 Schepens Eye Research Institute/ Massachusetts Eye & Ear, HarvardMed School, Boston, MA; 2 Beth Israel Deaconess Medical Center,Harvard Med School, Boston, MA.Purpose: VEGF-C has been implicated in tumor-associated andcorneal angiogenesis through its effects on lymphangiogenesis andactivation of VEGFR3. However, recent evidence implicates VEGF-C and D in pathological angiogenesis in absence of lymphaticinvolvement, possibly through activation of VEGFR2. The goal ofthis study is to define the role of VEGF-C and its cognate receptors inretinal angiogenesis including neovascular AMD and relateddisorder.Methods: The levels of VEGF-C and D, and soluble VEGFreceptors, R1, R2 and R3 were measured in vitreous samples ofsubjects with various vitreoretinal conditions using combination ofLuminex and Elisa assays. The mouse model of laser-inducedchoroidal neovascularization (CNV) was used to detect theexpression of VEGF-C, D and receptors, R2 and R3 using imagingand immunohistochemistry. Expression of these factors was alsoexamined in donor eyes with various stages of AMD.Results: Both VEGF-C and D and soluble (s) R1, R2 and R3 weredetectable in the human vitreous. sR3 levels averaged 200 pg/ml incontrol subjects, and one log order higher than sR2 levels. Therespective concentrations of VEGF-C and D positively correlatedwith age and were affected by retinal diseases including AMD.VEGF-C and its cognate receptors were detected within cellularcomponents of laser-induced CNV membranes. A similar expressionpattern was seen in clinical specimens of AMD.Conclusions: Based on these examinations, VEGF-C, D andsVEGFRs are detectable in the human vitreous, positively associatedwith age, and affected by disease states such as AMD. VEGF-C andD were detected within experimental and clinical CNV membranes.VEGF-C and D may participate in pathological angiogenic processessuch as neovascular AMD in absence of any observedlymphangiogenesis.Commercial Relationships: Kameran Lashkari, CircadianTechnologies (F), Regeneron (R); Jie Ma, None; Gianna Teague,None; Jorge G. Arroyo, NoneSupport: Circadian TechnologiesProgram Number: 5000 Poster Board Number: A0129Presentation Time: 2:45 PM - 4:30 PMMitochondrial DNA Damage Increases with Age-related MacularDegeneration in the RPE but not Neural RetinaMARCIA R. TERLUK 1 , Lauren M. Soukup 2 , Sandra R. Montezuma 1 ,Deborah A. Ferrington 1 . 1 Ophthalmology and Visual Neurosciences,University of Minnesota, Minneapolis, MN; 2 Biology, University ofSt. Thomas, St. Paul, MN.Purpose: Reports of increased mitochondrial DNA (mtDNA)damage in the retinal pigment epithelium (RPE) from human donorswith age-related macular degeneration (AMD) provide compellingevidence supporting the hypothesis that mitochondrial dysfunctionplays a prominent role in AMD pathology (Karunadharma et al.,2010; Lin et al., 2011). However, it is still not clear whether thisdamage is limited to the RPE or if it also involves the neural retina.The purpose of this study was to determine if (1) mtDNA damageincreases in the neural retina with AMD, and (2) the extent ofmtDNA damage in the RPE correlates with the extent of damage inthe neural retina in individual donors.Methods: Human donor eyes were obtained from the MinnesotaLions Eye Bank, Minneapolis, MN. Genomic DNA was isolated froma 5 mm trephine punch of the macular region of the RPE and neuralretina from human donor eyes categorized into four progressivestages of AMD (MGS 1-4, Olsen and Feng, 2003). MitochondrialDNA damage was assessed in 32 donors (n=8/stage) using the longextension polymerase chain reaction. In this assay, decreasedamplification reflects increased DNA damage. The amplified productwas quantified using Pico Green fluorescent dye. Statistical analysisincluded one way ANOVA and Tukey’s post-hoc tests to determine ifthere was a significant increase in mtDNA damage with AMDprogression. Pearson Product-Moment Correlation analysis wasperformed comparing the extent of damage in the RPE and retina foreach donor to determine if there was an association between damagein these two ocular regions.Results: There was no significant change in mtDNA damage in themacula of the neural retina when comparing age-matched controlsand donors with AMD (p=0.29). Furthermore, comparison of mtDNAdamage in the RPE and retina from individual donors showed nocorrelation (Pearson’s r=0.03; p>0.10) between the extent of mtDNAdamage in these two macular tissues.Conclusions: Collectively our data indicates that mtDNA damagethat increases with AMD is limited to the RPE and not the retina.These results suggest that targeting treatments that protect themitochondria in the RPE may be the most efficacious therapeuticstrategy for AMD.Commercial Relationships: MARCIA R. TERLUK, None;Lauren M. Soukup, None; Sandra R. Montezuma, None; DeborahA. Ferrington, NoneSupport: Unrestricted grant to the Department of OVNS fromResearch to Prevent Blindness; Beckman Initiative for MacularResearch, Minnesota Lions Club.Program Number: 5001 Poster Board Number: A0130Presentation Time: 2:45 PM - 4:30 PMOxidative Stress-Induced Necrosis in RPE CellsJakub Hanus 1 , William C. Anderson 1 , Peng Jin 3 , Qinghua Liu 4 ,Shusheng Wang 1, 2 . 1 Cell and Molecular Biology, Tulane University,New Orleans, LA; 2 Ophthalmology, Tulane University, New Orleans,LA; 3 Human Genetics, Emory University, Atlanta, GA;4 Biochemistry, University of Texas Southwestern Medical Center,Dallas, TX.Purpose: Atrophy of retinal pigment epithelial (RPE) cells andphotoreceptors in the macula is characteristic of geographic atrophy(GA), an advanced state of dry age-related macular degeneration(AMD). RPE cells are subjected to chronic oxidative stress.Currently, the mechanism of oxidative stress-induced RPE cell deathis controversial, with most pointing to a dominant role for apoptosis.However, apoptosis does not induce inflammatory response, which isnot consistent with chronic inflammation observed in dry AMD. Weset out to clarify the mechanism of oxidative stress-induced RPE celldeath using cultured RPE cells. We test the hypothesis that necrosis is©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.
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