Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyPurpose: Purpose: Pericyte degeneration is an early event in diabeticretinopathy (DR) and plays an important role in retinal vascularleakage. Fenofibrate, a Peroxisome Proliferator-Activated Receptor α(PPARα) agonist, has been shown to have robust protective effectsagainst DR. This study is to evaluate the efficacy and molecularmechanism of Fenofibrate on pericyte loss in DR.Methods: Methods: Fenofibrate was administrated to streptozotocin(STZ)-induced diabetic Wistar rats at dose of 160 mg/kg/day for 2months. Periodic acid schiff (PAS) and hematoxylin staining wereperformed to quantify pericyte loss and acellular capillaries in retinadigestion samples. Palmitate, a saturated fatty acid, was used as adiabetic stressor in cultured primary human retinal capillary pericytes(HRCP). Cell viability was quantified by MTT assay, and apoptoticcells were measured by TUNEL assay . Intracellular ROS levels weremeasured by CM-H2DCFHDA. Western blot analysis was employedto measure changes of protein levels.Results: Results: Fenofibrate significantly decreased retinal pericyteloss and acellular capillaries in diabetic rat retina. Fenofibrateimproved HRCP viability and protected the cells from palmitateinducedapoptosis. Fenofibrate decreased palmitate-induced ROSproduction and down-regulated NOX4 expression. Fenofibratesuppressed NF-kB activation and prevented the down-regulation ofplatelet-derived growth factor-B (PDGF-B) expression in HRCP afterpalmitate treatment. Furthermore, over-expression of PPARα hadantioxidant and anti-inflammation effects in pericytes, similar toFenofibrate.Conclusions: Conclusions: Fenofibrate has therapeutic potential onretinal pericyte loss in DR and the effect is, at least in part, throughactivating PPARα.Commercial Relationships: Lexi Ding, None; Rui Cheng, None;Yang Hu, None; Jian-Xing Ma, NoneSupport: NIH grants EY018659, EY012231, EY019309 andP20GM104934Program Number: 280 Poster Board Number: D0229Presentation Time: 8:30 AM - 10:15 AMIGF-1R EXPRESSION IN AN OXYGEN-INDUCEDRETINOPATHY (OIR) MODELMaria C. Sanchez 1 , Valeria E. Lorenc 1 , Luna D. Jose 2 , Gustavo A.Chiabrando 1 . 1 CIBICI-Dpto de Bioquimica Clinica, Fac de CienciasQuimicas UNC, Cordoba, Argentina; 2 Clinica de Ojos Romagosa-Funadacion Ver, Cordoba, Argentina.Purpose: Various eye diseases, including proliferative diabeticretinopathy (PDR) and retinopathy of prematurity (ROP), are theresult of a pathological neovascularization. Different animal modelssuch as oxygen-induced retinopathy (OIR) have been developed inorder to understand the cellular and molecular mechanisms of thesediseases. One of the growth factors involved in both physiologicaland pathological neovascularization is the Insulin-like growth factor-1 (IGF-1). Previously, we have demonstrated the IGF-1 effect onretinal cell migration via IGF-1R and the expression of this receptorin retina of animals with and without OIR treatment (ARVO Meeting2012). Herein, we examine the IGF-1R expression, tissue distributionand cellular localization in relationship with the retinal cell death.Methods: Neonatal C57BL/6J mice were subjected to 75% oxygenfrom postnatal day 7 (P7) to P12 and then returned to room air forfive days. Control mice were exposed to room air from birth untilP17. Retinal blood vessel patterns were visualized by GSA labeling.Retinas from animals with and without OIR treatment were analyzedfor IGF-1R expression and tissue distribution at selected time points(P3, P12, P15, P18, P21 and P27). The IGF-1R localization wasexamined using cell-type specific markers (GFAP, GS, Brn3a, PKCalpha and calbindin among other) by immunofluorescence andconfocal microscopy. In order to identify apoptotic cells in the sameretinas, TUNEL assay (Roche) was used.Results: In retinas without OIR we first observed IGF-1R expressionin endothelial cells which was confirmed by using the GSA. Thisreceptor was also expressed at level of ganglion cells as well as in theend feet of Muller glial cells. In addition, we visualized staining forIGF-1R in inner and outer nuclear layers. When the IGF-1Rexpression in the OIR model was analyzed, we observed a change inthe distribution due to an alteration in the structure of the neuralretina. Considering that this model produces Muller glial cellsactivation and retinal cell death, then the expression of GFAP wasanalyzed. Increased GFAP expression was detected in Müller cells inOIR retinas from P15. Finally, in agreement with GFAP expression,TUNEL possitive cells in the retinas were also observed at P15.Conclusions: The IGF-1R distribution and localization in retinaswere modified after OIR treatment coinciding with the onset ofGFAP expression and the retinal cell death.Commercial Relationships: Maria C. Sanchez, None; Valeria E.Lorenc, None; Luna D. Jose, None; Gustavo A. Chiabrando, NoneSupport: FONCyT, CONICET, SeCyT-UNC.Program Number: 281 Poster Board Number: D0230Presentation Time: 8:30 AM - 10:15 AMPhotoreceptors are a main generator of superoxide in retinas ofdiabetic miceTimothy S. Kern 1, 2 , Yunpeng Du 1 , Krzysztof Palczewski 3 , Bruce A.Berkowitz 4 . 1 Medicine, Case Western Reserve University, Cleveland,OH; 2 Stokes Veterans Administration Hospital, Cleveland, OH;3 Pharmacology, Case Western Reserve University, Cleveland, OH;4 Anatomy/Cell Biol, Wayne State Univ Sch of Med, Detroit, MI.Purpose: Prior work by us and others has provided strong evidencethat oxidative stress and inflammatory processes play important rolesin the development of the vascular lesions of early diabeticretinopathy. Oxidative stress is known to regulate expression of proinflammatoryproteins, thus making it important to understand thesource of the oxidative stress. We have investigated the contributionof photoreceptors to the retinal oxidative stress and induction ofproinflammatory ICAM-1 in diabetes.Methods: Retinal oxidative stress was assessed histologically usingdichlorofluorescein staining, and quantitatively using luciginenluminescence in C57Bl/6J mice (nondiabetic and diabetic for 2 mos).Two models that caused degeneration of photoreceptors (a geneticmodel (rhodopsin knockout) and a chemically-induced degenerationof photoreceptors (iodoacetic acid)) were studied after 2 mosdiabetes.Results: In the diabetic mice, dichlorofluorescein stain indicated thatthe majority of oxidative stress was localized in the photoreceptorlayer. Both models of photoreceptor degeneration resulted inessentially total obliteration of photoreceptors. The diabetes-inducedincrease in superoxide observed in wildtype C57Bl/6J mice wassignificantly inhibited in both of the diabetic models lackingphotoreceptors. The diabetes-induced induction of ICAM-1 (used asa marker of inflammation) was significantly inhibited in diabeticanimals missing photoreceptors (rhodopsin knockout).Conclusions: These findings demonstrate a critical role ofphotoreceptors in the diabetes-induced oxidative stress in the retina,and presumably in the pathogenesis of other lesions of diabeticretinopathy.Commercial Relationships: Timothy S. Kern, Bausch & Lomb (F),PamLab (F); Yunpeng Du, None; Krzysztof Palczewski, QLT Inc(F), Polgenix Inc (E), Visum Inc (P), Amegen Inc (F); Bruce A.Berkowitz, NoneSupport: EY00300 and VA Merit grant©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.