<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>processing. Reduced activity of α-mannosidase offers a mechanismfor the diminished expression of retinal synaptophysin in diabetes.Commercial Relationships: Alistair J. Barber, None; Travis S.D'Cruz, None; Brittany N. Weibley, NoneSupport: JDRF, ADA, PA LionsProgram Number: 272 Poster Board Number: D0221Presentation Time: 8:30 AM - 10:15 AMAutophagy in Modified LDL-induced Pericyte Loss in DiabeticRetinopathyDongxu Fu 1, 2 , Shihe Yang 1 , Mingyuan Wu 1 , Mei Du 1 , Junping Chen 1 ,Kenneth Wilson 1 , Timothy J. Lyons 1 . 1 Harold Hamm OklahomaDiabetes Ctr, Univ of Oklahoma Hlth Sci Ctr, Oklahoma City, OK;2 Department of Immunology, Harbin Medical University, Harbin,China.Purpose: We hypothesise that extravasation of plasma lipoproteinsthrough damaged blood retinal barriers and their subsequentmodification (glycation, oxidation) are more important in thepropagation of diabetic retinopathy (DR) than circulatinglipoproteins. We previously showed that extravasated, modified LDL(modeled by highly-oxidized glycated LDL, HOG-LDL) wasassociated with human retinal capillary pericyte (HRCP) apoptosisand autophagy mediated by oxidative and ER Stress. Little is knownabout role of autophagy in modified LDL-induced pericyte loss.Methods: HRCP was exposed to HOG-LDL at differentconcentrations (up to 200mg/l) and times (up to 24h); native LDL (N-LDL) was used as control. Cells were transfected with plasmid GFP-LC3, si-RNA (si-CHOP or si-JNK) ,for 24-36h or pre-treated withinhibitors of autophagy (3-methyladenine and chloroquine) andcaspase (z-VAD-fm), ER stress (4-phenyl butyric acid), or JNK(sp600125), each for 1h before LDL treatment. Autophagy,apoptosis, JNK activity, cell viability, oxidative stress and ER stresswere determined by methods including immunocytochemistry,western blots, CCK-8 assay, TUNEL assay and cellular ROSmeasurement assay. To define the relevance of the results in vivo,immunohistochemistry was performed to detect LC3 and p-JNK from(a) mouse model of DR that mimics exposure of the retina to elevatedglucose and elevated LDL levels, and (b) human subjects with andwithout diabetes and DR. Markers above were also measured byWestern blot in human retinas.Results: Compared with N-LDL, HOG-LDL induced autophagy inpericytes, pJNK played an essential role. With lower concentrationsof HOG-LDL (0-50mg/l), autophagy inhibited apoptosis promotingcell survival, but with higher concentrations (50-200mg/l), autophagyinduced autophagic death and apoptosis. In diabetic hyperlipidemicmice (vs. animals with either condition alone), retinal autophagy wasenhanced. Autophagy was also enhanced in diabetic human retina,but was not correlated with severity of DR.Conclusions: In DR, extravasated and modified plasma lipoproteinsplay an important role once blood retinal barrier (BRB) leakage isestablished, and autophagy is implicated. The findings may lead tonew treatments to inhibit the development of DR.Commercial Relationships: Dongxu Fu, None; Shihe Yang, None;Mingyuan Wu, None; Mei Du, None; Junping Chen, None;Kenneth Wilson, None; Timothy J. Lyons, NoneSupport: COBRE Program of the National Center for ResearchResources P20 RR 024215Program Number: 273 Poster Board Number: D0222Presentation Time: 8:30 AM - 10:15 AMProfiling miRNAs in a hyperglycemic and hypoinsulinemicIns2Akita mouse modelAudrey Giocanti-Auregan 1, 2 , Lisa Ahn 1 , Javier Sancho-Pelluz 3, 4 ,Stephen H. Tsang 3 , Szilard Kiss 1 , Mark Rosenblatt 1 , Thomas Tuschl 5 ,John T. Pena 1 , Donald J. D’Amico 1 . 1 Ophthalmology, Weill CornellMedical College, New York, NY; 2 Ophthalmology, Avicennehospital, Bobigny, France; 3 Bernard & Shirlee Brown GlaucomaLaboratory. Department of Ophthalmology, Columbia University,New York, NY; 4 Facultad de Medicina, Universidad Católica SanVicente Mártir, Valencia, Spain; 5 Howard Hughes Medical Institute,The Rockefeller University, New York, NY.Purpose: Diabetic retinopathy (DR) is a leading cause of blindnessworldwide involving neoangiogenesis and blood retinal barrierbreakdown. Our goal is to identify novel genetic targets involved inDR. microRNAs (miRNAs) are non-coding RNAs that regulate geneexpression and modulate several pathologic diseases. The purpose ofthis study is to characterize the in vivo perturbations of retinalmiRNAs in a hyperglycemic and hypoinsulinemic mouse model.Here, we characterize the vascular and macroscopic anatomicproperties of the Ins2Akita mouse and then profiled the miRNAabundance longitudinally.Methods: Heterozygous male Ins2Akita with a mutation in theinsulin 2 gene, were age matched with C57BL6/J mice. We analyzedvascular permeability by Evans blue extravasation, identified retinalvascular macroscopic lesions by fundus photography and fluoresceinangiography, and surveyed for functional visual impairment byelectroretinography (ERG). We measured miRNA abundance in theretina and choroid/retinal pigmented epithelium (RPE) at several timepoints using multiplexed libraries of Ins2Akita and wild-type mice at1 and 3 months of age. We identified cell-type specificity of miRNAsin fresh frozen retina tissue sections using a modified in situhybridization (ISH) procedure.Results: Morphological changes and fluorescein angiogramabnormalities were identified in the retinas of Ins2Akita at 1 and 3months. We observed substantially increased vascular permeability inthe Ins2Akita retina at 3 months. However, no functional changeswere observed by ERG for up to 3 months of age. miRNA profileswere generated for Ins2Akita and control mice (1 and 3 months),which showed substantial down regulation of several miRNAsincluding miR-126 and miR-143 in the retina and choroid/RPE. ISHstudies showed cell-type specific staining in several retinal layers inwild-type retina tissue sections.Conclusions: We further characterized the Ins2Akita mouse modelof hyperglycemia and hypoinsulinemia. We found that despitesubstantial macroscopic morphological changes in the fundus andincrease in retinal vascular permeability of Ins2Akita mice, nofunctional change was yet detectable by ERG at 3 months. SeveralmiRNAs are deregulated in the Ins2Akita mouse and severalmiRNAs are cell type specific. The miRNAs identified in our screenoffer potential targets for genetic modulation of hyperglycemicinduced pathology in the eye.Commercial Relationships: Audrey Giocanti-Auregan, None;Lisa Ahn, None; Javier Sancho-Pelluz, None; Stephen H. Tsang,None; Szilard Kiss, Alcon (F), Alimera (F), Alimera (C), Alimera(R), Allergan (F), Allergan (C), Allergan (R), Genentech (F),Genentech (C), Genentech (R), Regeneron (F), Regeneron (C),Regeneron (R), Optos (F), Optos (C), Optos (R), Eytech (C),Merge/OIS (C), Merge/OIS (I); Mark Rosenblatt, None; ThomasTuschl, The Rockefeller University (P), MIT (P), max planckinstitute (P), alnylam pharmaceuticals (C); John T. Pena, TheRockefeller University (P); Donald J. D’Amico, Ophthotech, Inc (I),OptiMedica, Inc (I), Neurotech, Inc (I), Genentech, Inc (C), LuxBiosciences, Inc (C)Support: NIH CTSC grant, Margaret M. Dyson Vision Research andInstitute Research to Prevent Blindness.©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 <strong>ARVO</strong> Office at arvo@arvo.org.
<strong>ARVO</strong> 2013 Annual Meeting Abstracts by Scientific Section/Group - <strong>Biochemistry</strong>/<strong>Molecular</strong> <strong>Biology</strong>Program Number: 274 Poster Board Number: D0223Presentation Time: 8:30 AM - 10:15 AMInfluence of Proliferative Diabetic Retinopathy Aqueous inCausing Defective CD34 cellsSankarathi Balaiya 1 , Maria B. Grant 2 , K V Chalam 1 .1 Ophthalmology, Univ of Florida College of Med, Jacksonville, FL;2 Pharmacology and Therapeutics, University of Florida College ofMedicine, Gainesville, FL.Purpose: Proliferative diabetic retinopathy (PDR), a majorcomplication of diabetes induces retinal microvascular endothelialdysfunction lead to decreased retinal perfusion, hypoxia andsubsequent induction of angiogenic factors. Endothelial progenitorcells, a subpopulation of the circulating mononuclear cells arerecruited to sites requiring vascular repair and can contribute to therepair and viability of the vasculature. However, in diabetes,dysfunctional EPCs may not repair this injury and promotedevelopment of acellular capillaries and sustained retinal ischemia. Inthis study, we investigated whether angiogenic cytokines present inaqueous of PDR subjects might affect the reparative nature of CD34cells.Methods: Aqueous fluid was obtained from individuals with PDRundergoing pars plana vitrectomies in accordance with Institutionalreview board at the University of Florida, Jacksonville. Aqueousfluid from healthy individuals who underwent cataract extractionwithout any ocular diseases served as controls. Mobilized healthyhuman CD34 cells were maintained in an undifferentiated state andtreated with 1% and 5% concentration of PDR and control aqueousfluid. The effect of PDR aqueous on CD34 cells were analyzed usingtrypan blue exclusion assay on day 4 and day 7. In addition,migration chamber assay was performed on aqueous enriched CD34cells (10,000 cells); conditioned medium was analyzed withnanoelectrospray ionization mass spectrometry (nESI-MS).Results: In presence of aqueous, CD34 cells showed increasedproliferation in a time-dependent manner. On day 4, they did notshowed increased proliferation compared to control (12.8x106 Vs20x106), whereas proliferation increased on day 7 (3.73x106 Vs3.06x106). Pretreated CD34 cells with varying concentration ofaqueous fluid from PDR patients did not cause any increase inmigratory response compared to aqueous fluid from healthy patients.However, the migratory response was significantly reduced to 12.2%in comparison to control after treatment with 5% concentration ofaqueous fluid from PDR patients (p
Change language