Abstract
Abstract: :
Purpose: The receptor for advanced glycation end (AGE) products is upregulated in RPE cells adjacent to histopathology and AGE product deposition in aging and AMD maculas. We propose that AGE products induce RAGE–mediated RPE cellular activation responsible for altered expression of genes regulating matrix organization, cytokines, growth factors and inducers of apoptosis. In order to directly examine AGE product induced RPE cellular activation we will treat differentiated ARPE–19 cell cultures with AGE products and another RAGE ligand, S100B, to examine altered expression of genes which are implicated in AMD pathogenesis as well as shown to be regulated by RAGE in other aging disorders. Methods: ARPE–19 cells are differentiated on matrigel and 1% FCS for 2–3 weeks. AGE products are produced by previously published protocols, using glycolaldehyde treated BSA. ARPE–19 cells are treated with AGE products and micromolar concentrations of S100B for 1–4 days. After treatments, media is collected and cells are removed from the matrigel by dispase (2.4U/ml). Protein or total RNA is extracted from cell pellets and examined by western blot or real time RT–PCR. Primers were generated and antibodies used to examine matrix organizing proteins (MMPs, TIMPS), cytokines (MCP1,PAI–1), growth factors (TGFß, PDGF, VEGF) and apoptosis regulators (caspase 3, bcl2). Apoptosis was measured with the TUNEL assay. Results: We found that AGE products and S100B inhibit matrix organizing proteins MMP2 & MMP9 and upregulate VEGF, MCP1, and PAI–1 mRNA levels. AGE products and micromolar S100B induce apoptosis as measured with the TUNEL assay. Conclusions: AGE products induce RAGE–mediated alterations in expression of proteins which have been strongly implicated in AMD pathogenesis. These and future studies will confirm a molecular mechanism for AGE product induced receptor– mediated RPE cell activation in AMD pathogenesis.
Keywords: age-related macular degeneration • retinal pigment epithelium • gene/expression