Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologySupport: NIH R01 EY020861, RPB Challenge Grant, RPB Lew R.Wasserman Merit Award, HHMI Medical Research Fellows ProgramCommercial Relationships: Ying-Bo Shui, None; Benjamen Filas,None; Qianru Zhang, None; Shaili Sharma, None; Alyssa Panitch,Purdue Research Foundation (P); David C. Beebe, FivePrime (C),Panoptica (C), Vistakon (Johnson and Johnson) (C)Support: EY021515-01, Unrestricted grant from RPB, Core grantEY02687Program Number: 3153Presentation Time: 12:15 PM - 12:30 PMInsulin Stimulates Increased Production of Soluble Betacellulinin Retinal Pigment Epithelium CellsBailey Y. Shen, Mariya Ali, Alecia Cutler, Bela Anand-Apte.Ophthalmology, Cole Eye Institute, Cleveland Clinic, Cleveland, OH.Purpose: To determine the effects of high glucose and extracellularinsulin on insulin signaling, ADAM 10 (A Disintegrin andMetalloproteinase 10), and betacellulin (BTC) expression in retinalpigment epithelium (RPE) cells in vitro.Methods: ARPE-19 cells were incubated in low (5 mM) or high (25mM) glucose media containing varying doses of regular humaninsulin (0 pM - 100 nM). After 48 hours, cells were harvested bymechanical lysis, and western blots of cell lysates were performed tomeasure expression of insulin receptor, phosphorylated ERK-1 (amember of the insulin signaling pathway), ADAM 10, andbetacellulin. As ADAM 10 is known to cleave pro-BTC (MW ~46kDa) into soluble BTC (22 kDa) and BTC cytoplasmic remnant (~25kDa), two different types of betacellulin antibodies, one against its N-terminal ectodomain, and one against its cytoplasmic domain, wereused to study BTC cleavage.Results: Insulin concentrations greater than or equal to 10 nM causedincreased soluble BTC expression in RPE cells, while glucoseconcentrations did not have any effect on soluble BTC expression.Insulin also upregulated ADAM 10 expression, but, surprisingly, didnot increase levels of BTC cytoplasmic remnant, which would beexpected to accumulate if insulin enhanced ADAM 10-mediatedcleavage of pro-BTC into soluble BTC. Finally, insulin increasedexpression of phosphorylated ERK-1 and decreased expression ofinsulin receptor.Conclusions: Soluble betacellulin is a molecule known to increaseretinal vascular permeability, and a molecule thought to play a role inthe pathogenesis of diabetic macular edema. This study suggests thatextracellular insulin acting upon RPE cells may contribute to theincreased soluble betacellulin previously measured in retinas ofdiabetic animals and humans. Our results also raise the possibilitythat insulin-mediated increases in soluble BTC in RPE cells may beindependent of pro-BTC cleavage.Commercial Relationships: Bailey Y. Shen, None; Mariya Ali,None; Alecia Cutler, None; Bela Anand-Apte, 7 183 256 B2 (P)362 Biochemistry and Regulation of Proteins in AMDTuesday, May 07, 2013 2:45 PM-4:30 PM6A Paper SessionProgram #/Board # Range: 3651-3657Organizing Section: Biochemistry/Molecular BiologyProgram Number: 3651Presentation Time: 2:45 PM - 3:00 PMAge-Dependent Changes In Heparan Sulfate In Human Bruch’sMembrane: Implications For Age-Related Macular DegenerationTiarnan D. Keenan 1, 4 , Claire Pickford 2 , Rebecca Holley 2, 3 , Simon J.Clark 1, 4 , Catherine Merry 2 , Anthony J. Day 3 , Paul N. Bishop 1, 4 .1 Faculty of Medical and Human Sciences, University of Manchester,Manchester, United Kingdom; 2 Faculty of Engineering and PhysicalSciences, University of Manchester, Manchester, United Kingdom;3 Faculty of Life Sciences, University of Manchester, Manchester,United Kingdom; 4 Centre for Advanced Discovery and ExperimentalTherapeutics, Manchester Academic Health Sciences Centre,Manchester, United Kingdom.Purpose: Heparan sulfate (HS) plays an important role in retinalhomeostasis. It may be implicated in age-related maculardegeneration (AMD), as it is the major binding partner forcomplement factor H (CFH) in human macular Bruch’s membrane.The aim of this study was to investigate potential changes with age inthe quantity and composition of HS in the human retina.Methods: Postmortem human ocular tissue was obtained fromconsenting eye donors without known retinal disease (n=4 pairs ofyounger globes, age 26-35 years; n=6 pairs of older globes, age 71-88years). Retinal tissue was dissected, and HS was extracted andpartially purified by anion exchange chromatography. Followingheparinase digestion, HS disaccharides were labeled with AMAC andquantified according to sulfation pattern by HPLC alongsidereference standards. Separately, HS was detected byimmunohistochemistry in frozen human macular tissue sections (n=6from younger globes, age 18-34 years; n=5 from older globes, age79-89 years).Results: The quantity of HS in Bruch’s membrane was 44% lower inolder donors (p