Effect of RAGE Activation on VEGF Secretion of ARPE–19 Cells

Abstract

Purpose: : Vascular endothelial growth factor (VEGF) is an angiogenic and vascular permeability factor and plays an important role in both retinal vascular disorders and age–related macular degeneration (AMD). In this study we analyzed the involvement of receptor for advanced glycation end–products (RAGE) of ARPE–19 cells in VEGF imbalance.

Methods: : Multiple ligands of RAGE were tested on ARPE–19 cells, and VEGF secretion was detected by ELISA. Human dominant–negative RAGE (DN–RAGE) gene, subcloned into pcDNA3 Plasmid, was transfected into ARPE–19 cells with Lipofectamine²⁰⁰⁰ transfection reagent to confirm the involvement of RAGE activation in the secretion of VEGF.

Results: : Human RPE cells produced a basal amount of VEGF in normal cell culture conditions. A low level of RAGE expression was detected in ARPE–19 cells by immunoprecipitation and Western blot. RAGE ligands, including advanced glycation end–products (AGEs), S100B and amyloid–beta (Aß), could induce significantly higher secretion of VEGF, and this result was demonstrated to be RAGE–dependent by genetic manipulation of RAGE functionality. Furthermore, inhibition of NF–ΚB activation diminished the increased secretion of VEGF, demonstrating that NF–ΚB played a central role in RAGE–dependent secretion of VEGF.

Conclusions: : These findings suggest that activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF and potentially augment neovascular macular disease.

Keywords: neovascularization • signal transduction • pathobiology