Purpose: The importance of VEGF in stimulating neovascular age-related macular degeneration (AMD) is well-recognized, but the initiating factors that induce local upregulation of VEGF remain unclear. The current study was conducted to test the hypothesis that activation of RAGE (receptor for advanced glycation end products [AGEs]) by its ligands, including AGEs, amyloid-beta peptide (Abeta), and S100B/calgranulins, some of which are known components of drusen and Bruch's membrane deposits, modulate secretion of VEGF by retinal pigment epithelial (RPE) cells.
Methods: ARPE-19 cells were used for all experiments. The cells were transfected with constructs encoding a signal transduction mutant of human RAGE to assess the RAGE-dependence of intracellular signaling. VEGF secretion and gene expression were assessed by ELISA and quantitative real-time PCR. SDS-PAGE and size exclusion chromatography were performed to analyze the structural changes of S100B after oxidation of its thiol groups under denaturing and nondenaturing conditions, respectively. NF-kappaB activation was assessed via electrophoretic mobility shift assay (EMSA). The impact of the NF-kappaB inhibition was assessed by using parthenolide.
Results: ARPE-19 cells basally secreted VEGF under normal cell culture conditions. Immobilized ligands of RAGE increased VEGF secretion in a RAGE-dependent manner. In contrast, soluble AGE-BSA, fresh Abeta, and S100B were less effective in increasing VEGF secretion. Studies with Abeta demonstrated that oligomeric and surface-immobilized forms of Abeta, but not soluble monomeric forms of Abeta, were effective upregulators of VEGF secretion via RAGE. Oxidation of S100B's thiol groups resulted in the formation of oligomers that displayed distinct RAGE biological activity compared with the simple dimeric form. RAGE-mediated upregulation of VEGF secretion by ARPE-19 cells was largely dependent on NF-kappaB, as indicated by studies with parthenolide.
Conclusions: Immobilized or oligomerized ligands for RAGE induce RPE cells to increase VEGF secretion. NF-kappaB plays a central role in RAGE-dependent RPE secretion of VEGF. In AMD, activation of the RAGE axis in RPE cells may contribute to upregulation of VEGF, potentially inciting or propagating neovascular macular disease.