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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyAPNpII treated group was 38% less in 24 hours and 59% less in 48hours after injury compared to control. Expression of PCNA inwounded area was 20% more in APNpII treated ARPE-19 cellscompared to control group.Conclusions: RPE cells are potential target for anti-angiogenictherapy. APNpII may be used for inhibiting the VEGF and CCL2secretion by RPE cells. APNpII peptide may have therapeutic use forAMD treatment.Commercial Relationships: Puran S. Bora, None; Nalini S. Bora,None; Valeriy V. Lyzogubov, NoneSupport: Jones Eye Institute, University of Arkansas for MedicalSciencesProgram Number: 4582 Poster Board Number: A0101Presentation Time: 11:00 AM - 12:45 PMIsoform-specific interactions between human apolipoprotein Eand amyloid-beta in the retinal pigment epitheliumKimberly A. Toops, Jin Xu, Aparna Lakkaraju. Ophthalmology andVisual Sciences, University of Wisconsin - Madison, Madison, WI.Purpose: To investigate how different apolipoprotein E (ApoE)isoforms (a) influence the expression of amyloid-beta (Aβ) in theretinal pigment epithelium (RPE) and (b) preferentially interact withAβ under conditions of RPE stress. Human ApoE occurs in threeisoforms (E2, E3, E4) that vary in only two amino acids but havedifferent conformations, stabilities and interactions with lipids andproteins such as Aβ. The E4 isoform is strongly associated withincreased risk of Alzheimers’ disease (AD), but has a protectiveeffect in age-related macular degeneration (AMD). Drusen containApoE and Aβ and the RPE may be a source of both proteins.Increased intracellular cholesterol escalates neuronal Aβ productionand ApoE participates in clearing Aβ. Since RPE cells withlipofuscin store cholesterol, we investigated how lipofuscin andcholesterol storage affects Aβ expression and ApoE-Aβ interactions.Methods: Primary porcine RPE cells were transfected with GFPtaggedApoE2, E3 or E4 expressed from either a CMV or Best1promoter. Cells were treated with the lipofuscin component A2E orwith U18666A to induce cholesterol storage directly. Expression ofApoE and Aβ was measured by immunoblot in total cell lysates orafter immunoprecipitation with an ApoE antibody. ApoE and Aβcellular localization was examined by immunocytochemistry.Cholesterol was measured biochemically and by filipin staining. Liveimaging was performed using spinning disk confocal microscopy.Results: Expression of human ApoE isoforms in pig RPE induces Aβproduction. The amount of Aβ varies depending on the ApoEisoform. Pull-down assays show that the Aβ-ApoE interaction isresistant to reducing agents. Our data also suggest that lipofuscin andintracellular cholesterol affect numerous steps of the ApoE-Aβcascade including ApoE trafficking, the amount of Aβ generated inthe cell and the binding of ApoE to Aβ.Conclusions: ApoE trafficking and ApoE-Aβ interactions aremodulated by intracellular cholesterol and lipofuscin content in theRPE. In the retina, it is likely that ApoE isoforms participate inclearing Aβ from the RPE and direct it into the choroidal circulation.Thus, while there may be common pathological processes in AD andAMD, our data point to key functional differences at the cellular levelthat could help explain how the same ApoE isoform can confer riskin one disease and protection in another.Commercial Relationships: Kimberly A. Toops, None; Jin Xu,None; Aparna Lakkaraju, NoneSupport: American Health Assistance Foundation (M2009093); KarlKirchgessner Foundation; McPherson Eye Research Institute(Rebecca Meyer Brown Professorship); Research to PreventBlindness Career Development Award; Reeves Foundation forMacular Degeneration ResearchProgram Number: 4583 Poster Board Number: A0102Presentation Time: 11:00 AM - 12:45 PMEffects of the absence of both complement factor H andapolipoprotein E on VEGF expression, MMP2/9 activity andcaspase-1Laura Garcia-Garcia, Patricia Fernandez, Sergio Recalde, MaiteMoreno-Orduña, Vanessa Fernandez-Garcia, Alfredo Garcia-Layana. Ophthalmology Experimental Laboratory, ClinicaUniversidad de Navarra, Pamplona, Spain.Purpose: Apolipoprotein E deficient mouse (apoE-/-), experimentalmodel of genetic hypercholesterolemia, shows retinal proteinexpression patron alterations. Complement factor H (CFH) isinvolved in inflammatory process associated to retinal degenerations.We aimed to investigate the effect of the absence of CFH and apoEgenes in mouse protein expression in the retina.Methods: Wild type (WT), CFH-/-/apoE-/- (DKO) and CFH+/-/apoE+/- (DH) mice were used and divided in groups of age (youngeror older than 12 months) having 3-5 animals per group (all mice werein C57BL6/J background). Western blot analysis for vascularendothelial growth factor (VEGF) expression, zymography for matrixmetalloproteinase (MMP) activity and immunohistochemistrytechniques for caspase-1 detection were assessed. For comparisonsafter a significant ANOVA or Kruskal-Wallis among groups, posthoctests or U Mann-Whitney were applied (SPSS v.15.0).Results: VEGF expression was significantly lower in DH groupcompared with WT and DK (p
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynegative regulator of alternative pathway complement activation),native properdin (P2 dimer, P3 trimer, and P4 tetramer; a positiveregulator of the same pathway), and complement C3b binding toCEP-BSA.Methods: SPR experiments were performed using a Biacore 3000.CEP-BSA with pyrrole to protein (P/P) ratio 7 (as measured by theEhrlich assay) and BSA were immobilized on CM5 chips by aminecoupling. The human CFH, C3b, P2, P3, or P4, or recombinant CFH(rCFH) fragments diluted in HBS-P buffer were injected over thechips, and the sensorgrams analyzed. rCFH fragments includedcomplement control protein (CCP) repeat domains 1-3, 5-7, 6-8, 7-9,17-19 (with 6XHis tag), as well as CCPs 18-20 and 19-20.Results: CFH, native properdin forms, and C3b bound to CEP-BSAwhile they didn’t bind to the reference surface where BSA wasimmobilized. Kinetic rate constants were determined usingBiaevaluation software (V4.0.1) by the global-fit method. The datashowed affinity values (KD) in the range 10 -6 M (C3b) to 10 -7 M(P3). CFH appears to have the highest association-rate constant (ka),while the native properdins have the slowest dissociation kinetics(kd). The rCFH fragments CCPs 5-7, 6-8, 18-20 and 19-20 bound toCEP-BSA P/P 7, with affinity values in the range of 1.4 X 10 -6 M to4.4 X 10 -6 M. The other recombinants did not bind.Conclusions: CFH likely binds to CEP moieties through its “heparinbinding”domains, CCPs 18-20 and 6-8. Properdin, a positivelychargedmultimer, avidly bound to the CEP moieties. Therefore, thenegative charges of CEP are probably responsible for attractionthrough electrostatic interactions. As the CEP-BSA was bound byboth positive and negative regulators of the complement pathway, abalance may be necessary for allowing complement activation(promoted by properdin), while regulating the extent of activation (byCFH) to a level that facilitates opsonization and removal of lipidoxidation products, but not tissue damage.Commercial Relationships: Lisa Kuttner-Kondo, None; VivianaP. Ferreira, None; Claudio Cortes, None; Satya P. Yadav, None;Joe G. Hollyfield, NoneSupport: Research to Prevent Blindness Unrestricted Grant & NIHR01EY014240-10 (JGH); NIH RR016789-01A1 (SPY);AHA12GRNT10240003 (VPF)Program Number: 4585 Poster Board Number: A0104Presentation Time: 11:00 AM - 12:45 PMThe aging retina: Macula-less rat and macula-bearing monkeyretinae exhibit common age-related changes in their retinalproteinsMichael R. Boehm 1, 2 , Sonja Mertsch 1 , Harutyun Melkonyan 1 , SolonThanos 1 . 1 Institute for Experimental Ophthalmology, School ofMedicine, WWU Muenster, Muenster, Germany; 2 CurrentAffiliation: Department of Ophthalmology, University of MuensterMedical Center, Muenster, Germany.Purpose: The visual consequences of age-related alterations in theneural retina are well documented, but little is known about theirmolecular bases. We performed a comparative proteomic analysis ofthe retinae in marmosets and rats, in order to identify proteins forwhich the expression profiles in retinas are altered with age.Methods: Protein profiles were compared in the newborn, juvenile,middle-aged, and aged retinae using two-dimensional gelelectrophoresis. Seven proteins exhibited changes in contentthroughout the life of rats, and 21 proteins that exhibited age-relatedcontent changes in marmosets were identified by matrix-assisteddesorption ionization-time-of-flight mass spectrometry. Western-blot(WB), quantitative reverse-transcriptase PCR (qRT-PCR), andimmunohistochemistry (IHC) analyses of selected proteins and theirmRNA were employed to determine whether changes identified byproteomics were verifiable at the cellular and molecular levels.Results: We found four proteins common to both species [Parkinsondisease (autosomal recessive, early onset) 7/DJ-1, stathmin,peroxiredoxin, and β-synuclein] whose contents were regulated withage. WB, IHC, and qRT-PCR analyses confirmed these findings. Theproteins were localized in certain layers and subsets of cells withinthe retinae of both species. Alterations in the expressions of theseproteins between the localization of the macular and retinal peripherywere analyzed with IHC. Detection of found proteins in the adulthuman retina were confirmed with IHC.Conclusions: This study is the first to provide evidence that theaging retina is physiologically characterized by specific changes in itsproteome. These changes occur in key functional pathways during theprocessing of visual signals and may be involved in the developmentof age-related pathologies, such as age-related macular degeneration.Commercial Relationships: Michael R. Boehm, None; SonjaMertsch, None; Harutyun Melkonyan, None; Solon Thanos, NoneSupport: Supported by Interdisciplinary Centre for Clinical Researchof Muenster (IZKF) grants to MRRB and ST (Tha3/002/09)Program Number: 4586 Poster Board Number: A0105Presentation Time: 11:00 AM - 12:45 PMGene expression profile of Retinal Pigment Epithelium derivedARPE-19 cells under serum starvationSanghamitra Mishra 1 , Katherine M. Peterson 1 , Alan E. Berger 2 ,Graeme J. Wistow 1 . 1 Section for Molecular Structure and FunctionalGenomics, National Eye Institute, Bethesda, MD; 2 JHBMC LoweFamily Genomics Core, Johns Hopkins University - School ofMedicine, Baltimore, MD.Purpose: Age Related Macular Degeneration (AMD) is associatedwith formation of subretinal deposits (drusen) and damage to Bruch’smembrane (BM) basal to the retinal pigment epithelium (RPE). Thiscould lead to serum deprivation of overlying RPE. Here we studiedgene expression in the human RPE derived cell line, ARPE-19,during a seven day time course of serum starvation.Methods: Gene expression levels in serum starved ARPE-19 cellswere measured for 0, 1, 3, 5, and 7 days using cDNA microarrays.Standard statistical tools and pathway analysis software were used toidentify regulated genes and altered pathways. Differentiallyexpressed genes and pathways, identified from microarray analysis,and the effect of supplements were confirmed by qPCR, western blotanalysis and confocal microscopy.Results: Differentially expressed genes at days 1, 3, 5, and 7 wereidentified. The most notable changes in gene expression were upregulationof cholesterol and lipid gene pathways and downregulationof methallothionein genes. This was confirmed by qPCRand western blotting. Supplementing LDL reversed the change incholesterol pathway genes. Uptake of LDL in the serum starved cellswas also tested. Cells at days 3, 5 and 7 of serum starvation showedincreasing uptake of LDL-Dy549, whereas no uptake was seen atDay 0. The labeled LDL uptake was abolished in the presence ofunlabeled LDL. Downregulation of metallothionein genes along withupregulation of zinc binding genes is consistent with a reduction inthe intracellular levels of bio-available zinc. Zinc supplementationreversed expression changes of the metallothionein and zinctransportergenes.Conclusions: Serum starved ARPE-19 cells showed significantchanges in expression patterns of cholesterol and zinc-relatedpathway genes. Cholesterol synthesis enzymes were upregulated andcells showed increased uptake of exogenous cholesterol. Thissuggests that serum deprivation initiates a cellular program requiringcholesterol in RPE cells. The cells also have an increasedrequirement of zinc. Zinc-binding proteins frequently function as©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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