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ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologynumbers were similar to each other in the H vs J cybrids(1.34±0.0228 vs 1.36±0.017, p=0.59).Conclusions: The OCR/ECAR ratio confirms that the H cybrids haveincreased OXPHOS while the J cybrids use glycolysis. This findingis supported by lower expression levels of the mtDNA encoded genesin complex I (MT-ND1, MT-ND2, MT-ND3, MT-ND4/ND4L), IV(MT-CO2, MT-CO3) and V (MT-ATP6). Our findings show thatmtDNA variants can mediate energy production pathways andmtRNA expression which in turn may alter the signaling pathwaysand stress-response patterns of the cells.Commercial Relationships: Cristina M. Kenney, None; MarilynChwa, None; Shari Atilano, None; Payam Falatoonzadeh, None;Deepika Malik, None; Claudio A. Ramirez, None; S MichalJazwinski, None; Miceli V. Michael, 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: Supported by Discovery Eye Foundation, GuentherFoundation, Beckman Macular Research Initiative, Polly andMichael Smith Foundation, Max Factor Family Foundation, SkirballFoundation, Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on AgingProgram Number: 5006 Poster Board Number: A0135Presentation Time: 2:45 PM - 4:30 PMThe serine protease HTRA1 is a potential regulator of theinflammatory cytokine GDF15Chloe M. Stanton 1 , Elod Kortvely 2 , Caroline Hayward 1 , MariusUeffing 2, 3 , Alan F. Wright 1 . 1 MRC Human Genetics Unit, MRCIGMM, University of Edinburgh, Edinburgh, United Kingdom;2 Centre for Ophthalmology, Institute for Opthalmic ResearchTubingen, Tubingen, Germany; 3 Research Unit of Protein Science,Helmholtz Zentrum Munchen, Munich, Germany.Purpose: An AMD risk locus on chr10q26 spans two genes -ARMS2 and HTRA1 - and controversy exists as to whether one orboth genes contribute to AMD. This study was performed toinvestigate the protein interaction network of the serine proteaseHTRA1 (high-temperature requirement A-1) and to elucidate apossible role in AMD pathogenesis.Methods: Potential interacting partners of HTRA1 were identified ina yeast two-hybrid screen performed using a placental cDNA library.Protein interactions were subsequently verified using recombinantproteins in pull-down assays. Proteins found to interact directly withHTRA1 were subjected to protease activity assays to identifysubstrates of the protease. Plasma GDF15 was measured by ELISA,and gene expression determined by RT-PCR.Results: Growth differentiation factor 15 (GDF15), a divergentmember of the TGFβ superfamily, with a proposed role in mediatinginflammation, interacts directly with HTRA1 in vitro. ProGDF15 is asubstrate for the HTRA1 protease in vitro. GDF15 and HTRA1 areexpressed at very low levels by monocyte-like cells, and theirexpression is highly upregulated in activated macrophages. PlasmaGDF15 is significantly elevated in AMD cases carrying thechromosome 10 risk haplotype, relative to disease-free controls, or toAMD cases not carrying the chromosome 10 risk haplotype.Conclusions: ProGDF15 is a substrate for the HTRA1 protease invitro, suggesting a regulatory role for the protease on thisinflammatory cytokine. Given the previously described role ofGDF15, the substantial genetic risk for AMD associated with thechromosome 10 risk haplotype, and the involvement of macrophagesin AMD, GDF15 may contribute towards pathological inflammationobserved in the disease. Further work to understand the geneticcontribution to regulating GDF15 expression in the generalpopulation, and in disease, and to determine the biologicalsignificance of the interaction between HTRA1 and GDF15 is ongoing.Commercial Relationships: Chloe M. Stanton, None; ElodKortvely, None; Caroline Hayward, None; Marius Ueffing, None;Alan F. Wright, NoneSupport: UK Medical Research CouncilProgram Number: 5007 Poster Board Number: A0136Presentation Time: 2:45 PM - 4:30 PMRole of complement in altered RPE function and depositformation in Efemp1 mutant mice: A primary cell culture modelRosario Fernandez-Godino, Eric A. Pierce, Donita Garland.Ophthalmology, Ocular Genomics Institute. Mass Eye and EarInfirmary. Harvard Medical School, Boston, MA.Purpose: The R345W mutation in the Efemp1 gene causes theinherited macular degeneration Doyne Honeycomb RetinalDystrophy/ Malattia Leventinese (DHRD/ML). Gene targetedEfemp1-R345W mice develop sub-RPE basal deposits and vacuolesin the RPE. In previous studies using Efemp1-R345W : complementC3 null mice we found that the complement system had a critical rolein the formation of basal laminar deposits in the mutant mice. Thepurpose of this study was to use a primary RPE cell culture system toinvestigate the mechanisms involved in the pathogenesis of basaldeposit formation in the Efemp1-R345W mutant mice and the role ofthe complement system in this process.Methods: Primary RPE cells were cultured from Efemp1-R345Wknockin, C3 null, and homozygous Efemp1-R345W:C3 null mice.The RPE cells, from 2.5 months old mice, were grown on apermeable support under polarizing conditions. mRNA expressionlevels of complement genes and Efemp1 were quantified by qRT-PCR. Protein expression in the RPE was determined by Western blotanalysis and immunofluorescence using confocal microscopy.Proteins secreted from both the apical and basal surfaces werequantified by Western blot analyses.Results: Results of qRT-PCR analysis reproducibly showed that RPEcells from each of the mouse strains express complement C3 mRNAand protein. The C3 null mice used for these studies express aninactive form of C3. Moreover, the major complement components ofthe classical pathway, C1q, C2, C4 as well as Cfh of the alternativepathway were also expressed in the RPE of each mouse strain. Theexpression of Efemp1 mRNA was significantly higher in Efemp1-R345W knockin mice compared to wild type (Wt)(ANOVA,p=0.0005). However, this overexpression was repressed inthe Efemp1-R345W:C3 null mice (p=0.0005).Conclusions: The primary RPE cell culture provides an excellentapproach to investigate the early effects of the Efemp1 mutation onRPE protein expression and also on the effects of alternations in thecomplement system on RPE function. The results suggested a criticalrole of C3 in the pathogenesis of the R345W mutation in DHRD/ML.They also suggest that the mutant EFEMP1 protein might cause localcomplement activation in the RPE, which in turn leads to RPE celldysfunction and an altered pattern of proteins secreted to Bruch’smembrane.Commercial Relationships: Rosario Fernandez-Godino, None;Eric A. Pierce, None; Donita Garland, NoneSupport: Ocular Genomics InstituteProgram Number: 5008 Poster Board Number: A0137Presentation Time: 2:45 PM - 4:30 PM©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 BiologyQuantifying progression of disease in aging mouse and humanRPEJohn M. Nickerson 1 , Xin Qi 2 , Tingting Jiang 3 , Yuwei Cheng 3 , Jing M.Zhang 3 , Micah A. Chrenek 1 , Alia Rashid 1 , Shagun K. Arora 1 , Hans E.Grossniklaus 1 , Yi Jiang 2 . 1 Ophthalmology, Emory Univ, Atlanta, GA;2 Mathematics, Georgia State University, Atlanta, GA; 3 Statistics,Yale University, New Haven, CT.Purpose: RPE sheet patterns change with age and progression indiseased RPE. We developed quantitative predictors of age andgenotype from the RPE sheet, and built a model of how the RPEsheet changes in disease. Using functional principal componentanalysis (FPCA) and multidimensional scaling (MDS), we classifiedRPE sheets into major categories of genotypes and age groups.Methods: Over 130 mice of many different ages and genotypes wereused, including WT C57BL/6J and mutant rd10, rpe65 KO, rd12,IRBP KO, and tvrm148, which were congenic with C57BL/6J. Over16 human cadaveric eyes were tested. ~10,000 cells per eye wereanalyzed. Borders of RPE cells were identified with anti-ZO-1 orphalloidin. Thresholding was by Ridler-Calvard adaptive method.RPE segmentation and analysis with 23 metrics were done withCellProfiler. Four classification methods were used after FPCA andMDS.Results: We compared the morphology of RPE cells from eyesacross various mouse strains and normal human and AMD eyes ofdifferent ages. Disrupted RPE had distinctive characteristics withgreat variability in size and shape of cells. In old RPE, there weregaps, atrophy, and interruptions in the sheet with significant showthroughof the choroid. Joint aspect ratio and cell area providedprincipal components predictive of age (< or > 70 days) and genotype(WT or rd10) in mice with high accuracy (>98%). With furtherdivision of mice into four age groups and spatial locations, PCA andMDS showed that no single metric was significant as a signature. Butspatial information revealed regional changes. At >P180 days, therewas extensive deformation and a subpopulation of large cells. Amathematical model of RPE cell death recapitulated changes in vivoin AMD: Normal RPE progressed to abnormal only when multiplelocal clusters of RPE cells were killed. The death of single cells orsparse clusters of RPE cells were handled effectively by stretchingthe sheet. Preliminary analysis on human normal RPE showed noage-related trends or changes in morphology.Conclusions: Morphometrics readily discriminates young vs old,normal vs mutant RPE sheets. Classification accuracy was high anddepended on metric and location of the RPE cell. Tissue-stretchingrobustly maintains layer integrity. However, death of multiple localclusters of RPE cells is overwhelming and results in highly variableappearance and eventually frank holes.Commercial Relationships: John M. Nickerson, None; Xin Qi,None; Tingting Jiang, None; Yuwei Cheng, None; Jing M. Zhang,None; Micah A. Chrenek, None; Alia Rashid, None; Shagun K.Arora, None; Hans E. Grossniklaus, None; Yi Jiang, NoneSupport: RPB; NIH (R01EY016470, P30EY06360, TL1TR000456,UL1TR000454)Program Number: 5009 Poster Board Number: A0138Presentation Time: 2:45 PM - 4:30 PMZinc Staining of sub-RPE Deposits in Murine Models of RetinalDegenerationRebecca J. Kapphahn 1 , Heidi Roehrich 2 , Deborah A. Ferrington 1 ,Frederik J. van Kuijk 1 . 1 Ophthalmology, University of Minnesota,Minneapolis, MN; 2 Histology Core for Vision Research, Universtiyof Minnesota, Minneapolis, MN.Purpose: One of the hallmarks of age-related macular degeneration(AMD) is the accumulation of sub-retinal pigment epithelial (RPE)deposits, including drusen and basal laminar deposits, in Bruch’smembrane. We have previously shown that these sub-RPE depositscontain high concentrations of zinc in human donors with AMD andthat these zinc-enriched deposits were especially prevalent in themacula (Lengyel et al, 2007). Since sub-RPE deposits may contributeto AMD pathology, zinc-containing sub-RPE deposits may be animportant characteristic for animal models used to investigate AMDmechanisms or test therapeutic interventions. The purpose of thisstudy was to determine if zinc-enriched sub-RPE deposits are presentin animal models of retinal degeneration.Methods: The presence of zinc-enriched sub-RPE deposits wasassessed in multiple murine models that exhibit retinal degeneration(sod1-/-, mice carrying the rd8 mutation, old C57BL6). In order tovisualize sub-RPE zinc deposits, the anterior segment was removedfrom enucleated globes. The retina and RPE were then removedusing zinc-free PBS and the unfixed eye cup was flat mounted on aglass slide without a cover slip. A Leica DM4000B microscope wasused to visualize the autofluorescence associated with the optic nerve.Exposure time was then decreased to determine the exposureparameters where autoflourescence was no longer visible. Theseparameters were used to capture all subsequent images. Membranepermeable fluorescent zinc stain, Zinpyr-1 (Strem Chemicals,Newburyport, MA) (10uM diluted in PBS) was then applied to sub-RPE flat mounts and incubated for 5 min. Excess Zinpyr-1 wasremoved by rinsing with zinc-free PBS and the sections were coverslipped. The fluorescence associated with zinc deposits wasimmediately visualized.Results: High levels of zinc-containing sub-RPE deposits wereobserved in sod1-/- mice, mice carrying the rd8 mutation, and old (15months) but not young (3 months) C57BL6 mice. The presence ofhigh levels of zinc-enriched sub-RPE deposits in the sod1-/- mouse,proposed as a model for AMD, further validates its use to investigateAMD disease pathology and test interventions.Conclusions: Membrane permeable fluorescent zinc stainingprovides a quick assessment of sub-RPE deposits that can be easilyquantified. Staining of sub-RPE deposits helps to validate this AMDassociatedphenotype in animal models used in AMD research.Commercial Relationships: Rebecca J. Kapphahn, None; HeidiRoehrich, None; Deborah A. Ferrington, None; Frederik J. vanKuijk, NoneSupport: : Unrestricted grant from Research to Prevent Blindness tothe Department of Ophthalmology and Visual Neurosciences,Beckman Initiative for Macular Research, Minnesota Lions Club,NIH (P30-EY00374).Program Number: 5010 Poster Board Number: A0139Presentation Time: 2:45 PM - 4:30 PMVEGF, VEGF-C, and VEGF-D Increases in Aqueous HumorSamples in a LPS Induced Model of Uveitis in New ZealandWhite RabbitsDouglas Decker, Jeffery E. Burr, Kelly Lester, Vicki Zaworski, StevenJ. Weber. Biochemistry, PharmOptima, Portage, MI.Purpose: To determine the levels of VEGF, VEGF-C, and VEGF-Dfrom aqueous humor samples in an E.coli lipopolysaccharide (LPS)induced model of uveitis in rabbits.Methods: Uveitis was induced by intravitreal (IVT) injection of LPSinto the right eye(OD) of New Zealand Rabbits (NZW). The left eye(OS) received an IVT injection of vehicle. At 24 hours post injectionrabbits were evaluated by an ophthalmic exam and the aqueoushumor (AH) collected for cell counts and protein. VEGF, VEGF-C,and VEGF-D were quantitated in AH using a Meso Scale DrugDiscovery (MSD) electrochemiluminescent ELISA.Results: Ophthalmic exams of LPS treated rabbit eyes (OD)©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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