Biochemistry/Molecular Biology - ARVO

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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyregulators of transcription, which have a profound effect on cellfunction when altered. These observations are interesting,considering that both cholesterol and zinc have been implicated inAMD. This raises the possibility that the response of RPE-derivedcells to serum-deprivation may model some processes related toAMD.Commercial Relationships: Sanghamitra Mishra, None;Katherine M. Peterson, None; Alan E. Berger, None; Graeme J.Wistow, NoneProgram Number: 4587 Poster Board Number: A0106Presentation Time: 11:00 AM - 12:45 PMHSPA5 - a possible new contributing gene in atrophic age-relatedmacular degenerationSumana Chintalapudi, Yin H. Chan Lau, Shwetapadma Sahu, MonicaM. Jablonski. Ophthalmology, The University of Tennessee HealthScience Center, Memphis, TN.Purpose: To investigate the contribution of extracellular HSPA5 (akaGRP78) in human atrophic age-related macular degeneration usingnovel systems genetic methods, mathematical modeling andimmunostaining techniques.Methods: To determine the cellular localization and contribution ofextracellular HSPA5, single and double immunostaining in humannormal and AMD retinas were performed using well-characterizedantibodies. Quantitative trait locus (QTL) mapping was used to mapthe genomic region(s) that regulate Hspa5 in a BXD geneticreference panel. Multiple partial correlation and heatmap analyseswere performed to define the relationship between the expression ofHspa5 and all genes within the mouse genome. The top candidategenes were identified and further evaluated using SNP screening.Results: In non-AMD retinas, extracellular HSPA5 was localizedintensely around cones within the macular area; however it wasscattered in the peripheral retina. In contrast, in AMD retinas, therewere very few HSPA5 immunopositive cones, especially in themacula. In addition, the immunolabeling profiles were aberrant. Ourmapping studies in BXD mice indicate a single highly significanttrans-eQTL with an LRS of 18.4 on Chr2:169-175Mb and asuggestive trans-eQTL on Chr15:75-100Mb, both of which suggestthat other genes control Hspa5 expression either directly orindirectly. Partial correlation analyses and heatmaps confirmed theQTLs on Chrs 2 and 15. The best candidate extracellular genes inthose intervals were B4galt5, Dpm1, Sulf2, Fbln1, Glt8d3 and Pmm1.Direct correlation analyses showed that expression of Sulf2, Pmm1and Fbln1 were significantly correlated with Hspa5. Only Sulf2 had anon-synonymous SNP, which makes it our top candidate forcontrolling Hspa5 expression.Conclusions: The presence of HSPA5 in the interphotoreceptormatrix around cones in healthy human retinas and its absence inatrophic AMD retinas supports our assertion that HSPA5 may play arole in cone structural integrity in the macula. Integrated approachesusing mouse and human analyses suggest that HSPA5 may be a locusfor AMD progression and/or susceptibility. Our studies furthersuggest that Sulf2 is the best candidate gene for controlling Hspa5expression in the retina.Commercial Relationships: Sumana Chintalapudi, None; Yin H.Chan Lau, None; Shwetapadma Sahu, None; Monica M.Jablonski, 8,092,825 (P)Support: Research to Prevent BlindnessProgram Number: 4588 Poster Board Number: A0107Presentation Time: 11:00 AM - 12:45 PMQuercetin Inhibits H2O2 Stimulated PEDF and FGF2 Synthesisin hRPE CellsNandita Anand, Piyush C. Kothary, Monte A. Del Monte. Universityof Michigan, Ann Arbor, MI.Purpose: Age related macular degeneration (AMD) is a major causeof blindness. Proliferation and damage of the human retinal pigmentepithelial (hRPE) cells is involved in the pathogenesis of AMD.Oxidative stress induces in hRPE cell death and stimulates thesynthesis of angiogenic factors in hRPE cells. We studied the effectof quercetin (Q), a flavonoid that protects cells from oxidative stress,on the synthesis of the angiogenic factor, fibroblast growth factor 2(FGF2), and the anti-angiogenic factor, pigment epithelial derivedfactor (PEDF) in hRPE cells.Methods: hRPE cells were cultured from eyes obtained from theMichigan Eye Bank.Cellular proliferation in the presence ofincreasing concentrations of fetal bovine serum (FBS) was measuredby 3[H] thymidine incorporation. hRPE cell viability was measuredin the presence of increasing concentrations of FBS and H2O2 by thetrypan blue exclusion method (T).The effect of (Q) on hRPE cellviability was also measured in presence of H2O2 by (T).The effect ofvarious concentrations of H2O2 on intracellular synthesis of PEDFand FGF2 in presence and absence of (Q) was measured byimmunoprecipitating 14C-methionine-PEDF (14C Met-PEDF) and14C-methionine-FGF2 (14C Met-FGF2) and immunocytochemical(IC) analysis.Results: FBS (0-10%) stimulated cell proliferation and cell viabilityin cultured hRPE cells. H2O2 (0-0.5mM) decreased cell viability (T)in a dose dependent manner. The addition of (Q) (50μM) to 0.5mMH2O2 treated cells increased hRPE cell viability(130,000 ± 48,989vs. 40,625± 28,252, n=8, cells±SEM, p
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyphenotype of Tg and age-matched wild-type (Wt) mice in 18- to 24-month-old mice. Real time PCR, immunohistology, transmissionelectron microscopy and fluorescein angiography (FA) were used toanalyze the phenotype of this transgenic mouse model and agematchedcontrols.Results: This BMP4 over-expressing mouse accumulated remarkableamounts of oxidized lipid in RPE and on Bruch’s membrane (BM).This suggests that over-expressed BMP4 induces oxidative stress ofRPE which might lead to subsequent AMD phenotypes, includingRPE atrophy and thickened BM. RPE in Tg mice showed decreasedheight, hypo- or hyper-pigmentation, disorganized basal infoldingsand cellular lipid vesicles. Some Tg mice eventually developedchoroidal neovascularization (CNV) with fluorescein leakage on FA,isolectin B4 positive staining on flat mounted posterior eye cups andCNV-like lesions by histology. These changes were not seen in aged-matched controls.Conclusions: The BMP4 over-expressing mice mimic thepathological process of AMD and may be a good model for studyingAMD. Our work suggests that a dysregulated growth factor, BMP4,may be involved in the pathogenesis of AMD.Commercial Relationships: Danhong Zhu, None; Jing Xu, None;Xuemei Deng, None; Jamie Hsiung, None; Stephen J. Ryan,Alergan (S); David R. Hinton, RPT (I), RPT (P)Support: EY03040Program Number: 4590 Poster Board Number: A0109Presentation Time: 11:00 AM - 12:45 PMVitamin D Status and Subretinal Fibrosis in Neovascular Age-Related Macular DegenerationAmardeep Singh 1, 2 , Mads K. Falk 1, 2 , Yousif Subhi 1, 2 , Torben L.Sørensen 1, 2 . 1 Ophthalmology, Copenhagen University HospitalRoskilde, Roskilde, Denmark; 2 University of Copenhagen,Copenhagen, Denmark.Purpose: Vitamin D has been shown to have anti-fibrotic propertiesand deficient levels of circulating 25-hydroxyvitamin D have beenassociated with fibrogenesis in several extra-ocular tissues. Evidencelinking vitamin D deficiency to age-related macular degeneration(AMD) is inconsistent. Thus, we sought to investigate 25-hydroxyvitamin D levels across clinical subgroups of AMD andwhether the presence of subretinal fibrosis in neovascular AMD atdiagnosis is associated with lower levels of circulating vitamin D.Methods: Plasma samples were collected from 202 participantsacross 4 groups: controls (n=73), early AMD (n=22), geographicatrophy (n=12) and neovascular AMD (n=95). All participants weresubjected to a structured interview and retinal examination wasperformed using stereoscopic funduscopy on mydriatic eyes, digitalcolor fundoscopy, autofluorescence imaging, spectral-domain opticalcoherence tomography and fluorescein and indocyanin greenangiography in patients suspected of having neovascular age-relatedmacular degeneration. Clinical grading was performed using theClinical Age-Related Maculopathy Grading System. Venous bloodwas obtained for measurement of 25-hydroxyvitamin D2 and D3 inplasma using liquid chromatography-tandem mass spectrometry.Genomic DNA was extracted from leukocytes and genotyping wasperformed for single nucleotide polymorphisms in the vitamin Dmetabolism.Results: Patients with subretinal fibrosis in addition to neovascularAMD had significantly lower levels of circulating 25-hydroxyvitaminD compared to patients without subretinal fibrosis (47.2 versus 75.6nmol/L, p
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