Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular Biologyfrom Atg5flox/flox; nestin-Cre mice, as well as a clear reduction intheir visual function.Conclusions: We have identified a primary malfunction ofmacroautophagy in the aging retina that may contribute to the agerelatedretinopathies and reduction of visual function.Commercial Relationships: Enrique J. de la Rosa, ProRetinaTherapeutics, S.L. (F), ProRetina Therapeutics, S.L. (I), ProRetinaTherapeutics, S.L. (C), US2010/0042 A1 (P); Natalia Rodriguez-Muela, None; Hiroshi Koga, None; Lucia Garcia-Ledo, None;Pedro de la Villa, ProRetina Therapeutics, S.L. (I), US2010/0042 A1(P); Ana M. Cuervo, None; Patricia Boya, US2010/0042 A1 (P),ProRetina Therapeutics, S.L. (I)Support: SAF-2010-21879 to EJdlR and PdlV, CONSOLIDERCSD2010-000454 to EJdlR and PB, NIH-AG031782 and AG038072to AMC, SAF-2009-08086 to PBProgram Number: 4577 Poster Board Number: A0096Presentation Time: 11:00 AM - 12:45 PMHigh glucose activates ChREBP-mediated HIF-1α and VEGFexpression in human RPE cells under normoxiaMin-Lee Chang, Chung-Jung Chiu, Fu Shang, Allen Taylor. HNRCATufts University, Boston, MA.Purpose: Because retina-damaging angiogenesis is controlled byVEGF and people with higher glucose intakes are more susceptible toretinal complications that may be due to increased VEGF, it is crucialto elucidate relations between glucose exposure and VEGFexpression. We aimed to determine if a carbohydrate responseelement binding protein (ChREBP) plays a role in the transcriptionalup-regulation of hypoxia-inducible factor-1α (HIF-1α) anddownstream vascular endothelial growth factor (VEGF) expression inretinal pigment epithelial (RPE) cells exposed to normoxia and highglucose.Methods: ARPE19 cells were exposed to 5.6, 11, 17, 25 and 30 mMglucose for 48h in serum-free culture media under normoxic (21%O2) conditions. Protein and mRNA expression of indicated geneswere determined by immunoblot analyses and real-time RT-PCR,respectively. An enzyme-linked immunosorbent assay (ELISA) wasused to detect the concentrations of VEGF in the media.Immunofluorescence (IF) and chromatin immunoprecipitation (ChIP)for ChREBP were used to demonstrate nuclear translocation andHIF-1α gene promoter association, respectively.Results: Immunoblot analyses showed that HIF-1α levels werepositively related to levels of glucose exposure between 5.6-25 mMin the RPE cells, indicating the induction and stabilization of HIF-1αby elevated glucose under normoxic conditions. Human lensepithelial cells and HeLa cells did not respond to high glucose,implying that this phenomenon is cell type-specific. Real-time RT-PCR for HIF-1α and VEGF and ELISA for VEGF indicated that highglucose is associated with elevated production of HIF-1α-inducedVEGF, an established inducer of neovascularization, in the RPE cells.IF analyses showed that, although ChREBP was equally expressedunder both low (5.6 mM) and high (25 mM) glucose conditions, itappeared more in the nuclear region than in the cytosol of the RPEcells after the high glucose treatment. ChIP analyses suggested aHIF-1α gene promoter association with ChREBP under the highglucose condition. These results imply that RPE cells use cytosolicChREBP as a glucose sensor to up-regulate HIF-1α expression.Conclusions: These results suggest a high glucose-induced,ChREBP-mediated, and normoxic HIF activation that may bepartially responsible for the neovascularization in both diabetic andage-related retinopathy.Commercial Relationships: Min-Lee Chang, None; Chung-JungChiu, None; Fu Shang, None; Allen Taylor, NoneSupport: NEI R01 EY021826, EY021212, EY013250, Ross AgingInitiative, and USDA agreements 1950-5100-060-01A.Program Number: 4578 Poster Board Number: A0097Presentation Time: 11:00 AM - 12:45 PMAMD-associated silent polymorphisms in HtrA1 ameliorate IGF-1 antagonismSarah Melissa P. Jacobo 1, 2 , Margaret M. DeAngelis 3, 4 , AndriusKazlauskas 1, 2 . 1 Ophthalmology, Harvard Medical School, Boston,MA; 2 The Schepens Eye Research Institute, Boston, MA;3 Ophthalmology and Visual Sciences, University of Utah, Salt LakeCity, UT; 4 John A. Moran Eye Center, Salt Lake City, UT.Purpose: Single nucleotide polymorphisms (SNPs) within atranscript’s coding region that do not change the amino acid sequenceof the protein product are intuitively assumed to have no effect onprotein properties and function. The synonymous SNPs rs1049331and rs2293870 within the exon 1 of HTRA1 are associated withincreased risk of neovascular age-related macular degeneration(NvAMD), and convert the common codons for Ala34 and Gly36 toleast frequently used codons. The purpose of this project was toassess the impact of these SNPs on HtraA1’s properties andfunctions.Methods: To evaluate the effect of optimum-to-rare codonconversion on the rate of protein synthesis, synthetic mRNAtemplates encoding HtrA1+SNPs or WT HtrA1 were used in in vitrotranslation. We evaluated the effect of the synonymous SNPs onprotein structure by partial proteolysis and immunoprecipitation witha conformation-sensitive antibody. The expression level of HtrA1 inneovascular AMD (NvAMD) patients homozygotic for rs1049331and rs2293870 were compared to age-matched unaffected controls.Given that rs1049331 and rs2293870 lie within HtrA1’s Mac25domain, we evaluated the ability of HtrA1 to influence IGF-1signaling in endothelial cells.Results: We report that this common-to-rare codon conversionreduced mRNA translation rate, increased susceptibility to partialproteolysis, and reduced recognition by an antibody raised against theregion of HtrA1 that includes exon 1. Exon 1 encodes a Mac25domain, which is structurally very similar to proteins that bindinsulin-like growth factor-1 (IGF-1). The NvAMD-associated SNPsreduced HtrA1’s ability to antagonize IGF-1-stimulated signalingevents, and to suppress IGF-1-dependent tube formation of primaryhuman choroidal endothelial cells.Conclusions: These observations demonstrate the potential ofsynonymous SNPs within HTRA1 to impact its previouslyunappreciated ability to titrate IGF-1, and suggest that the loss of thiscapacity contributes to pathogenesis of NvAMD.Commercial Relationships: Sarah Melissa P. Jacobo, None;Margaret M. DeAngelis, None; Andrius Kazlauskas, NoneSupport: NIH Grant EY014458Program Number: 4579 Poster Board Number: A0098Presentation Time: 11:00 AM - 12:45 PMDifferences in Reactive Oxygen Species (ROS) Production andExpression of Genes from the Complement and InflammatoryPathways Between H and K Haplogroup CybridsJavier Cáceres del Carpio 1 , Mohamed Tarek 1 , Claudio A. Ramirez 1 ,Payam Falatoonzadeh 1 , Marilyn Chwa 1 , Deepika Malik 1 , S MichalJazwinski 2 , Miceli V. Michael 2 , Baruch D. Kuppermann 1 , Cristina M.Kenney 1 . 1 Gavin Herbert Eye Institute, University of CaliforniaIrvine, Irvine, CA; 2 Tulane Center for Aging, Tulane University,New Orleans, LA.Purpose: Age-related Macular Degeneration (AMD) is the leadingcause of blindness in people older than 50 in developed countries.©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.