Protein Expression Patterns of Cultured Human RPE Cells Under Hyperglycaemic Condition Investigated by Proteome Analysis

Abstract

Abstract: : Purpose: To identify differential protein expression patterns of retinal pigment epithelial (RPE) cells exposed to increased glucose concentrations equivalent to acute hyperglycemia in the diabetic patient. Methods: Human RPE cells (ARPE–19) were cultured for 4 days with normal blood glucose concentration (5.5 mM D–glucose), followed by exposure to either normal (5.5mM) or high (33mM) concentrations of D–glucose for 48 hours. Protein extracts of glucose–treated RPE cells were then subjected to comparative proteome analysis based on 2–D gel electrophoresis. Protein spots were visualized by silver staining. The differentially expressed proteins were excised and digested in–gel with trypsin, then analyzed by matrix–assisted laser desorption/ionisation time–of–flight (MALDI–TOF) mass spectrometry. Results: The expression levels of cathepsin B, glutathione peroxidase and heat shock protein 27 were increased, and that of protein disulfide isomerase decreased in high glucose treated RPE compared to normal glucose. The isoelectric point of copper/zinc–containing superoxide dismutase shifted toward acidic region in response to high glucose. Conclusions: Systematic survey of protein expression has revealed that RPE cells respond to acute, pathologically high glucose levels by the elevated expression of anti–oxidant and proteolytic enzymes.

Keywords: proteomics • retinal pigment epithelium