Effects of concentration of amyloid (A) on viability of cultured retinal pigment epithelial cells

BackgroundAmyloid beta (A) is a constituent of drusen that is a common sign of age-related macular degeneration (AMD). The purpose of this study was to investigate the effect of A on human retinal pigment epithelial (RPE) cells in culture.MethodsCells from a human RPE cell line (ARPE-19) were exposed to 0 to 25M of A 1-40 for 48h, and the number of living cells was determined by WST-8 cleavage. Replicative DNA synthesis was measured by the incorporation of 5-bromo-2-deoxyuridine. The cell death pathway was investigated by the WST-8 cleavage assay after the addition of caspase-9 inhibitor, an anti-apoptotic factor. Real-time qRT-PCR was performed using A-exposed cellular RNA to determine the level of vascular endothelial growth factor (VEGF)-A and pigment epithelium derived factor (PEDF). To determine the effect of receptor-for-advanced glycation end products (RAGE), the siRNA for RAGE was inserted into ARPE-19 treated with A, and the levels of expression of VEGF-A and PEDF were determined.ResultsThe number of living ARPE-19 cells was increased by exposure to 5M A but was decreased by exposure to 25M of A. Replicative DNA synthesis by ARPE-19 cells exposed to 25M of A was significantly decreased indicating that 25M of A inhibited cell proliferation. Real-time RT-PCR showed that the level of the mRNA of PEDF was increased by exposure to 5M A, and the levels of the mRNAs of PEDF and VEGF-A were also increased by exposure to 25M A. The addition of an inhibitor of caspase-9 blocked the decrease the number of ARPE-19 cells exposed to 25M A. Exposure to si-RAGE attenuated the increase of VEGF-A and PEDF mRNA expression in ARPE-19 exposed to A.ConclusionsExposure of ARPE-19 cells to low concentrations of A increases the level of PEDF which then inhibits the apoptosis of ARPE-19 cells leading to RPE cell proliferation. Exposure to high concentrations of A induces RPE cell death and enhances the expression of the mRNA of VEGF-A in RPE cells. The A-RAGE pathway may lead to the expression VEGF-A and PEDF in RPE cells. These results suggest that A is strongly related to the pathogenesis of choroidal neovascularization.