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Ernesto F Moreira, Hui Cai, Tongalp H Tezel, Mark Fields, Lucian V Del Priore; Reengineering Human Bruch’s Membrane Increases Retinal Pigment Epithelium Phagocytosis Using an Integrin Receptor Pathway. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2343.
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© ARVO (1962-2015); The Authors (2016-present)
We have shown previously that Bruch’s membrane (BM) aging decreases retinal pigment epithelium (RPE) phagocytosis of rod outer segments (ROS) and that reengineering BM increases RPE repopulation and attachment to BM. Herein we investigated the effects of reengineering aging BM on RPE phagocytosis.
Explants of aged BM (donors > 55 years) were reengineered by cleaning with Triton X-100 and/or coating with extracellular matrix (ECM) ligands: laminin, fibronectin and vitronectin. ARPE-19 cells were plated onto BM and cultured to confluence (14 days). In parallel, RPE-derived ECM (RPE-ECM) modified by nitration, and untreated controls, were subjected to similar treatments. Bovine ROS were purified by sucrose gradient centrifugation, labeled with fluorescein isothiocyanate (FITC), fed to the RPE, and incubated at 37oC for 16 hours. For phagocytosis kinetics studies, ROS were removed and analyzed every 2 hours (for 12 hours). Phagocytosis pathway was assayed with 50µg/ml of αV-β5 blocking antibody for 2 hours. Image acquisition and quantification was performed by fluorescence microscopy and Image J.
Cleaning aged BM with a detergent did not increase the uptake of ROS, but a combination of cleaning and re-surfacing with extracellular matrix proteins significantly increased RPE phagocytosis to levels similar to RPE on young BM (Old vs. Old+Det.+ECM: 54.9±6.2 vs. 83.5±6.5; P < 0.05. (N=10)). Similar effects were observed on nitrite-modified RPE-ECM subjected to the same treatments. Time course analysis revealed a statistically significant difference in ROS internalization starting at 3-4 hours suggesting that ROS receptor binding and internalization may be affected in nitrite-treated samples. Challenging RPE phagocytosis by the addition of αV-β5 blocking antibody showed a reduced cellular fluorescence intensity of 70-95% as compared to samples devoid of antibody.
These results suggest that the detrimental effects of aging Bruch’s membrane on RPE phagocytosis can be reversed by “rejuvenation” of the surface with combined detergent cleaning and coating with extracellular matrix ligands. Aging and nitration may affect phagocytosis by altering the expression or integrity of the αV-β5 surface receptor as well as the internalization process.
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