HNE–adducts, AGEs, RAGE and VEGF in Blue Light Irradiated A2E–laden RPE.

Abstract

Abstract: : Purpose:The generation of advanced glycation end products (AGEs) and aldehyde–bearing products of lipid peroxidation is a feature of oxidative environments as is the upregulation of VEGF. We sought to determine whether these cellular changes were induced by the photooxidative events initiated by blue light irradiation of RPE cells that have accumulated the lipofuscin fluorophore, A2E. Methods: Adducts formed by lipid peroxidation products (hydroxynonenal, HNE; malondialdehyde, MDA) were detected by immunocytochemistry and immunoblotting. For the immunocytochemical detection of AGEs, antibody (clone no 6D12) to the CML epitope was used. To examine for expression of the cell surface receptor for AGE (RAGE), lysates of ARPE–19 cells were probed by western blotting with rabbit rabbit anti–RAGE IgG. VEGF mRNA was measured by real time RT–PCR with b2–microglobulin serving as an internal amplification control and secretion of VEGF in culture medium was assayed by ELISA. Results: ARPE–19 cell cultures that had accumulated A2E and were exposed to blue light were immunopositive for HNE–, MDA– and AGE–modified proteins in the zone of illumination while outside the area of exposure, only background levels were detected. On immunoblots multiple polypeptide bands exhibited immunoreactivity with anti–HNE antibody. Incubation of the cells with A2E–epoxides or A2E–bisepoxides also resulted in HNE– and AGE–specific immunofluorescence. Immunoblotting with RAGE–specific antibody, revealed that RPE cells express RAGE. In RPE cells that had accumulated A2E and were exposed to levels of illumination that do not induce cell death (430 nm; 3–7 min) VEGF mRNA was elevated several fold and secretion of VEGF into the medium over a 48 hour interval was significantly increased as compared to controls. Conclusions: Covalent modifications of proteins by AGE and aldehyde–bearing products of lipid peroxidation are thought to contribute to aging of the RPE–Bruch’s membrane complex and to the pathogenesis of age–related macular degeneration (AMD) The present results suggest that one source of these adducts may be photoxidative processes originating in the RPE cell and involving light and RPE fluorophores. Detection of the cell surface receptor RAGE connotes the presence of signaling pathways by which AGEs can modulate RPE function and upregulation of VEGF indicates that changes in gene expression can occur with blue light irradiation of A2E–laden RPE.

Keywords: age–related macular degeneration • retinal pigment epithelium • choroid: neovascularization