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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyBiostatistique, Bordeaux, France; 5 INSERM, ISPED, CentreINSERM U897-Epidemiologie-Biostatistique, Bordeaux, France;6 Unité de soutien méthodologique à la recherche clinique etépidémiologie (USMR), Pôle de Santé Publique, CHU Bordeaux,Bordeaux, France.Purpose: To compare macular pigment optical density (MPOD)measured by two techniques (autofluorescence, reflectometry) inhealthy subjects at high risk for age-related macular degeneration(AMD).Methods: The Limpia Study is a double-blind, placebo controlled,prospective randomized clinical trial performed in 120 subjects withat least one parent affected by neovascular AMD. To be included,subjects had to be aged 40-70 years, have best-corrected visual acuity(BCVA) greater than 20/25, be free of late AMD and other major eyeconditions (severe glaucoma, high myopia, severe retinal disease,cataract surgery…). Subjects having used supplements containinglutein and/or zeaxanthin in the preceding year were not included.MPOD was measured using two methods: the two-wavelengthautofluorescence method with a modified scanning laserophthalmoscope (Heidelberg Retinal Analyzer (HRA), Heidelberg,Germany) and a method based on reflectometry (Visucam200 MPD,Carl ZeissMeditec, Germany).Results: At baseline, mean MPOD within 0.51°, measured with themodified HRA method, was 0.5 ± 0.2. Maximal MPOD, measuredwith Visucam, was 0.4 ± 0.1. Parameters from the modified HRA(optical density within 0.5°, 1°, 2° and 6°) and from the Visucammethod (volume, area, mean and maximum optical density) wereweakly correlated. The best correlations were observed for MPODwithin 2° and 6° with volume from Visucam (r=0.21 and r=0.22,respectively), and mean MPOD from Visucam (r=0.21 and r=0.18).Conclusions: The two methods propose different parameters toevaluate macular pigment, which overall correlated weakly in thispopulation of middle-aged healthy subjects at high risk for AMD.Further research is needed to characterize the differences between themethods and identify the best parameters and techniques to measuremacular pigment.Commercial Relationships: Catherine P. Garcher, Alcon (C),Allergan (C), Baush and Lomb (C), Bayer Pharma (C), Novartis (C),Laboratoire Théa (C); Marie-Noelle Delyfer, Thea Laboratories (F);Marie B. Rougier, THEA (C), Bausch&Lomb (C), Allergan (C),Kemin (C); Hélène Savel, None; Geneviève Chêne, None; CecileDelcourt, Laboratoires Théa (F), Novartis (C), Bausch+Lomb (C);Jean-Francois Korobelnik, Alcon (C), Allergan (C), Bayer (C), CarlZeiss Meditec (C), Novartis (C), Thea (F)Support: Théa, Carl Zeiss MeditecClinical Trial: NCT01269697Program Number: 3782 Poster Board Number: A0121Presentation Time: 2:45 PM - 4:30 PMSerial Assessment of Macular Pigment Distribution ProfilesObtained Using Minimum Motion Photometry: 10 to 15-YearFollow-UpJack D. Moreland 1 , Anthony G. Robson 2, 3 , Daniel Pauleikhoff 4 ,Frederik J. van Kuijk 5 . 1 Life Sciences, Keele University, Keele,United Kingdom; 2 Electrophysiology, Moorfields Eye Hospital,London, United Kingdom; 3 Institute of Ophthalmology, UniversityCollege London, London, United Kingdom; 4 St Franziskus Hospital,Muenster, Germany; 5 Life Sciences, Keele University, Keele, UnitedKingdom.Purpose: To monitor macular pigment (MP) distribution profilesover periods of more than a decade in order to assess the long-termstability of MP optical density values at different retinal locations.Methods: Multiple MP spatial distribution profiles were obtainedusing a motion photometer in 4 healthy subjects (A-D) monitoredover periods of 15.6, 14.7, 11.7 and 10.7 years respectively. A squarewave grating (460nm and 580nm) was moved at constant horizontalvelocity (26 deg/sec and/or 37 deg/sec) within 2 circular fields(radius 0.45° and 1.1°) and 11 annular segments (maximum radius7.5°). The radiance of the 580nm stimulus was adjusted to minimizethe perceived motion. Optical density (OD) was computed at eachlocation relative to the most eccentric area from log (Rref/R), whereRref is the mean radiance setting for the most eccentric locations andR is the radiance setting at any location. The shape of the meanspatial distribution profiles for each of the 4 subjects (average of 46,42, 12 and 10 profiles respectively) were characterised in detail.Results: Mean peak MPOD values obtained with a circular field(radius = 0.45°) were 0.89 (A), 0.76 (B), 0.45 (C) and 0.17 (D). Theshapes of the mean MP profiles varied in a way representative of anormal population and were best described by the sum of 2 Gaussianfunctions, accounting for at least 99% of the variance. Linearregression through serial data points (circular field; radius 1.1°) gavegradients of 0.0093 (A), 0.0012 (B), -0.0003 (C) and -0.0024 (D) peryear. Maximum gradients at 4 eccentricities at or between 0.82 and2.31 degrees were 0.0075 (A), -0.0070 (B), 0.0026 (C) and 0.0049(D) per year.Conclusions: Widely different MP distribution profiles in healthysubjects demonstrate a high degree of stability over periods of up to15 years. The findings provide an assessment of normal stability thatis pertinent to studies that aim to monitor MP in disease.Commercial Relationships: Jack D. Moreland, None; Anthony G.Robson, None; Daniel Pauleikhoff, None; Frederik J. van Kuijk,NoneSupport: Foundation Fighting Blindness and NIHR MoorfieldsBiomedical Research Centre (AGR). Research to Prevent Blindnessunrestricted grant to University of Minnesota (EVK)Program Number: 3783 Poster Board Number: A0122Presentation Time: 2:45 PM - 4:30 PMEffects of Zeaxanthin on Constitutive and TPA-InducedSecretion of VEGF by Human Retinal Pigment Epithelial CellsIn VitroDan-Ning Hu, Richard B. Rosen, Anthony Sclafani, Steven A.McCormick. Pathology & Ophthalmology, New York Eye & EarInfirmary, New York, NY.Purpose: TPA (12-0-tetradecanoyl phorbol 13-acetate) is a proteinkinase C activator, which can stimulate the secretion of VEGF byretinal pigment epithelial (RPE) cells. The purposes of this studywere to investigate the effects of zeaxanthin on the constitutive andTPA-induced secretion of VEGF by human RPE cells in vitro.Methods: Human RPE cells (ARPE19) were seeded into 12-wellplates and cultured with F12 medium with 10% serum. After 24hours, culture medium was replaced by F12 medium without serum.Zeaxanthine at various concentrations (1, 10 and 100 µM) was added.TPA (100 ng/ml) was added 1 hour later. After 24 hours, conditionedmedium was collected and the amount of VEGF was measured usinga sandwich enzyme-linked immunosorbent assay kit (R & DSystems). All tests were performed in triplicate. A primary culture ofRPE cells isolated from a donor eye was also tested.Results: VEGF could be detected in the conditioned medium fromcultured ARPE cells (70.7±5.5 pg/ml). TPA significantly stimulatedthe secretion of VEGF (149.3±12.1 pg/ml, p
ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologylevels in conditioned medium from cells cultured with TPA andzeaxanthin (100 µM) was 93.3 ± 7.8 pg/ml, which was significantlylower than that in cells cultured with TPA alone (p < 0.01) but stillhigher than that in cells cultured without TPA (0.05 > p > 0.01).Study in primary culture of cell line obtained a similar result.Conclusions: Zeaxanthin only slightly inhibits constitutive secretionof VEGF by human RPE cells in vitro but significantly reduced TPAinducedsecretion of VEGF by RPE cells in a dose-dependentmanner.Commercial Relationships: Dan-Ning Hu, Zeovision (F); RichardB. Rosen, Opko-OTI (C), Optos (C), Clarity (C), OD-OS (C),Topcon (R), Zeavision (F), Genetech (F), Optovue (C); AnthonySclafani, None; Steven A. McCormick, NoneSupport: Supported by the Pathology Research Fund, New York Eyeand Ear Infirmary and the Research Fund from ZeaVision.401 Biochemistry of GlaucomaWednesday, May 08, 2013 8:30 AM-10:15 AM6A Paper SessionProgram #/Board # Range: 4002-4008Organizing Section: Biochemistry/Molecular BiologyContributing Section(s): Biochemistry/Molecular BiologyProgram Number: 4002Presentation Time: 8:30 AM - 8:45 AMMyocilin affects differentiation of oligodendrocytes and regulatesmyelination of the optic nerve via Lingo-1/RhoA signalingStanislav I. Tomarev 1 , Heung Sun Kwon 1 , Naoki Nakaya 1 , Mones S.Abu-Asab 2 . 1 SRGCB, LRCMB, National Eye Institute, Bethesda,MD; 2 Histopathology Core Facility, National Eye Institute, Bethesda,MD.Purpose: To investigate the functions of the glaucoma-associatedprotein, myocilin, in the optic nerve.Methods: Optic nerve astrocytes and oligodendrocytes were isolatedfrom postnatal day 6 (P6) or P7 mice. Optic nerves were examined byelectron microscopy. Binding of myocilin to the Lingo-1/NgR1complex was assessed by co-immunoprecipitation from the opticnerve and by binding assays using myocilin-alkaline phosphatasefusion proteins. Conductivity of the optic nerve was evaluated by avisual evoked potential test. Changes in gene expression weremeasured by RNA sequencing.Results: Myocilin was first detected in optic nerve astrocytes at P6and its level increased with age. Myocilin was also detected inastrocytes but not oligodendrocytes that were isolated and culturedfrom the optic nerve. Addition of purified myocilin to immatureoligodendrocytes stimulated their differentiation and increasedmRNAs encoding myelin-associated proteins (MBP, MAG andMOG) when compared with untreated oligodendrocytes. MBP levelsin the optic nerve of Myoc null mice were lower than in wild-typelittermates at early postnatal stages (P5-P21). Electron microscopyexamination of the optic nerve showed that myelin thickness wasreduced and the microvilli at the nodes of Ranvier were disorganizedin P13 Myoc null mice when compared with wild-type littermates.These defects in the myelination of the optic nerve were accompaniedby a reduction of RhoA activity. Myocilin regulates RhoA activitythrough interaction with the Lingo-1/NgR1 complex. Changes in themyelination of the optic nerve led to a delayed latency and reducedamplitude of the visually evoked potential in adult Myoc null micewhen compared to wild-type littermates. The levels of multiplemRNAs in the optic nerves of adult mice were different from wildtype,Myoc null and transgenic mice expressing mutated humanmyocilin (Tyr437His).Conclusions: Our data suggest that myocilin regulates myelination ofthe optic nerve via the Lingo-1/RhoA signaling pathway.Commercial Relationships: Stanislav I. Tomarev, None; HeungSun Kwon, None; Naoki Nakaya, None; Mones S. Abu-Asab,NoneProgram Number: 4003Presentation Time: 8:45 AM - 9:00 AMGenomewide DNA methylation analysis of human trabecularmeshwork cells with dexamethasone stimulationAkira Matsuda, Nobuyuki Ebihara, Akira Murakami. Department ofOphthalmology, Juntendo Univ School of Med, Tokyo, Japan.Purpose: To investigate the change of DNA methylation statusduring glucocorticoid exposure using cultured human trabecularmeshwork cells.Methods: Human trabecular meshwork (TM) cells established fromglaucoma patients (GTM) and normal control (NTM) were culturedwith and without 100nM dexamethasone (DEX) for 14 days. Genomewide methylation analysis was carried out using Illumina 450Kmethylation analysis. Gene expression analysis was also carried outwith Agilent 44K whole human genome array using cultured TMcells. The effect of DNA methylation in response to DEX treatmentsfor gene expression was verified by DNA methyltransferase inhibitor(5-aza-2’-deoxycytidine) treatment and subsequent realtime PCRanalysis.Results: We found 174/297 cytosine-phosphate-guanine (CpG) sitesshowing significant demethylation and 102/184 CpG sites showingsignificant methylation by 2 week-DEX treatment in GTM/NTM,respectively. We found 23 sets of demethylated-CpG sites after DEXexposure with significant changes of corresponding gene expression,and 15 sets methylated-CpG sites after DEX exposure withsignificant changes of corresponding gene expression. Among themwe further evaluated the expression of FKBP5, ZBTB16 and DUSP1.ZBTB16 gene-body methylation was observed after DEX treatment,accompanied with increased gene expression. 5-aza-2’-deoxycytidine-treatment of TM cells abolished the increase ofZBTB16 mRNA expression. DNA-demethylation of FKBP5promoter region was observed with increased FKBP5 geneexpression by DEX treatment. Promoter region of DUSP1 generegion is highly methylated in NTM cells. Significant DNAdemethylationand increase of DUSP1 gene expression was observedafter DEX treatment in NTM cells but not in GTM cells.Conclusions: DEX treatments induce the alteration of DNAmethylation status in human TM cells. Epigenetic modification couldaffect TM gene expression profile.Commercial Relationships: Akira Matsuda, None; NobuyukiEbihara, None; Akira Murakami, SEED(Japan) JP4855782 (P),SEED(Japan) JP5132958 (P)Support: Grants-in-Aid from MEXT Japan (No. 24592652).Program Number: 4004Presentation Time: 9:00 AM - 9:15 AMStabilization of Myocilin with Small Molecules Prevents ItsAggregationRaquel L. Lieberman, Shannon E. Hill, Rebecca K. Donegan,Katherine C. Turnage. School of Chemistry & Biochemistry, GeorgiaInstitute of Technology, Atlanta, GA.Purpose: The vast majority of mutations in the myocilin geneleading to glaucoma are located within the 30 kDa C-terminalolfactomedin (OLF) domain. Disease-causing myocilin variantsaggregate within human trabecular meshwork (TM) cells. Thisaccumulation taxes the cells and leads to TM cell death, resulting inincreased intraocular pressure, and a hastening of glaucoma-©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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