Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyResults: The Affymetrx array analyses showed that compared to Hcybrids, the L cybrids and J cybrids had 5.7 fold and 3.5 fold lowerexpression levels of IL-33 but the K cybrids were similar (1.7 folddifference). As determined by Q-PCR, the gene expression levels ofIL-33 for cybrids J, K, and L were measured relative to theexpression in H cybrid. An analysis of the data showed decreasedexpression of the IL-33 gene in J cybrids (0.46 fold, p=0.004) and Lcybrids (0.30 fold, p=0.002), but similar expression levels of IL-33 inthe K cybrids and H cybrids (1.35 fold, p=0.40).Conclusions: This study demonstrates that expression of the IL-33gene varies in cybrids that have identical nuclei but differenthaplogroups defined by specific mtDNA variants. This is significantbecause it suggests that the mtDNA variants may mediate IL-33, apro-inflammatory cytokine that may be important in the pathogenesisof AMD.Commercial Relationships: Thomas A. Vo, None; Marilyn Chwa,None; Shari Atilano, None; Deepika Malik, None; Claudio A.Ramirez, None; Javier Cáceres del Carpio, 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); Cristina M. Kenney, NoneSupport: Discovery Eye Foundation, Guenther Foundation,Beckman Macular Research Initiative, Polly and Michael SmithFoundation, Max Factor Family Foundation, Skirball Foundation,Lincy Foundation, Iris and B. Gerald Cantor Foundation,Unrestricted grant from Research to Prevent Blindness, grant fromNational Institute on Aging (AG006168)Program Number: 4989 Poster Board Number: A0118Presentation Time: 2:45 PM - 4:30 PMResveratrol Protects Human Retinal Pigment Epithelial Cellsfrom Inflammatory InsultsR K. Kutty 1 , Chandra N. Nagineni 1 , William Samuel 1 , Todd Duncan 1 ,Camasamudram Vijayasarathy 2 , Cynthia Jaworski 1 , T. MichaelRedmond 1 . 1 National Eye Institute, National Institutes of Health,Bethesda, MD; 2 National Institute on Deafness and OtherCommunication Disorders, National Institutes of Health, Bethesda,MD.Purpose: Inflammatory responses of retinal pigment epithelium(RPE) are implicated in the pathogenesis of age-related maculardegeneration (AMD). RPE cells in culture respond to theproinflammatory cytokines IFN-γ, TNF-α and IL-1β by increasingthe expression of chemokines and cytokines. Resveratrol, a naturallyoccurring polyphenol, is a well characterized anti-inflammatoryantioxidant. The purpose of the present study is to investigatewhether resveratrol can modulate the effect of IFN-γ, TNF-α and IL-1β on RPE cells.Methods: Confluent cultures of ARPE-19 cells were treated with aproinflammatory cytokine mixture containing IFN-γ, TNF-α and IL-1β in a serum free medium for various time intervals. Total RNAfraction was then isolated and real-time PCR analysis of geneexpression was performed using TaqMan reagents (AppliedBiosystems) with GAPDH as an endogenous control. The activationof nuclear factor-κB (NFκB) was examined by western immunoblotanalysis of cell extracts using anti-phospho-NFκB p65 (Ser536)antibody.Results: Pretreatment of ARPE-19 cells with resveratrol (50 µM)effectively blocked the induction of CCL5, CXCL9, CSF2 andNOS2A (inducible form of nitric oxide synthase) by theproinflammatory cytokines (IFN-γ + TNF-α + IL-1β). Resveratrolalso prevented the decrease in the expression of HMOX1 (hemeoxygenase-1) in cells exposed to proinflammatory cytokines.Immunoblot analysis showed that the phospo-NFκB p65 increased incells in response to the treatment with proinflammatory cytokines.This increase in phosphorylation was considerably reduced in thepresence of resveratrol.Conclusions: Resveratrol attenuated the inflammatory response ofthe RPE cells in culture by its ability to block the activation of NFκBsignaling pathway by the proinflammatory cytokines. Thus,resveratrol, due to its ability to protect RPE from inflammatoryinsults, may have therapeutic potential as a nutraceutical in arrestingthe development of ocular degenerative diseases such as AMD.Commercial Relationships: R K. Kutty, None; Chandra N.Nagineni, None; William Samuel, None; Todd Duncan, None;Camasamudram Vijayasarathy, None; Cynthia Jaworski, None;T. Michael Redmond, NoneSupport: Intramural Research Program of the National Eye Institute,National Institutes of HealthProgram Number: 4990 Poster Board Number: A0119Presentation Time: 2:45 PM - 4:30 PMBone morphogenetic protein 4 induces ID1 and ID3 transcriptionfactors in trabecular meshwork cellsAvani A. Mody, Robert J. Wordinger, Abbot F. Clark. Visual sicence/NTERI, UNTHSC, Fort Worth, TX.Purpose: Bone morphogenetic protein4 (BMP4) attenuatestransforming growth factor β2 (TGFβ2) mediated responses intrabecular meshwork (TM) cells. However, The overall regulatorymechanisms of this inhibition remain unclear. BMP4 regulatesvarious cellular processes by induction of transcription factor knownas inhibitors of DNA binding protein (ID1, ID3) which bind andsuppress other transcription factors. The purpose of the current studyis to determine whether BMP4 will induce ID1 and ID3 expression incultured human TM cells. This study will aid in understandingwhether BMP4 transcriptionally suppresses TGFβ-2 responses in TMcells via induction of IDs.Methods: Transformed ( GTM-3) and primary TM cells were treatedwith different doses of recombinant BMP-4 for various time points(0-48 hr) to study ID1/ID3 induction. Quantitative RT- PCR andwestern immunoblotting were used to study mRNA and proteinexpression of ID1 and ID3.Results: BMP-4 significantly induces ID1 and ID3 m RNA levels inTM cells (p