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Mark Fields, Hui Cai, Hannah Bowrey, Ernesto F Moreira, Kannan Kunchithapautham, Jie Gong, Lucian Del Priore; Bruch's Membrane Aging Reduces CD46 Expression and Enhances VEGF Secretion in RPE: Implications for Age-related Macular Degeneration . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2400.
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Age-related macular degeneration (AMD) is associated with complement activation, and the complement regulatory protein CD46 - which inhibits the alternative pathway - has been implicated in choroidal neovascularization. In AMD, basement membrane modification precedes significant cellular changes by many years. Reduction in CD46 is associated with retinal pigment epithelium (RPE) deterioration of RPE morphology in patients with advanced AMD and vascular endothelial growth factor (VEGF) expression in animal models of chroidal neovascularization. We aimed to investigate the effect of nitrate modification of extracellular matrix (ECM), in an vitro model of ECM ‘aging’, on CD46 expression and VEGF secretion in co-cultured human retinal pigment epithelium (RPE).
ARPE-19 cells plated onto one of four RPE-derived ECM conditions (untreated; nitrite modified; nitrite modified followed by washing with Triton X-100; or nitrite modified followed by washing with Triton X-100 and coated with a mixture of extracellular matrix ligands), were cultured for 7 days, and CD46 expression was analyzed by immunohistochemistry and western blot. CD46 siRNA was transfected into ARPE-19 cells, challenged with normal human serum (NHS) and VEGF levels were determined by ELISA. Finally, in the same ECM conditions as above, ARPE-19 cells were challenged with NHS and examined for expression of vascular endothelial growth factor using ELISA.
CD46 is expressed on the basolateral surface of ARPE-19 cells on RPE-derived basement membrane. Nitrate modification of ECM reduced the expression of CD46 on ARPE-19 cells (by 0.5 fold change, p = 0.003) and increased VEGF secretion in ARPE-19 cells (by 286 pg/m, p < 0.001). CD46 knockdown in ARPE-19 also increased secretion of VEGF on the basal side of ARPE-19 in culture.
These data support the hypothesis that nitrite modification of the basement membrane decreases CD46 expression, which then the release of VEGF from ARPE-19 cells. This process may play a pathological role in the development of AMD.
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