Glycated serum albumin induces chemokine gene expression in human retinal pigment epithelial cells

Abstract

Chronic hyperglycemia is thought to be important in the development of diabetic neovascularization but the mechanisms involved remain poorly understood. Interleukinâ 8 (ILâ 8) is a leukocyte chemokine and activating agent with angiogenic properties that is present in diabetic vitreous and may play a role in diabetic vasculopathy. We studied ILâ 8 and monocyte chemotactic proteinâ 1 (MCPâ 1) production by human retinal pigment epithelial (hRPE) cells exposed to glycated human serum albumin (GHSA). Enzymeâ linked immunoassay GHSA (500 μg/mL)â treated hRPE cells secreted levels of ILâ 8 and MCPâ 1 detectable within 4 h and reached 26.0 ± 1.3 and 42.2 ± 0.4 ng/106 cells/mL after 24 h, respectively. Induction of ILâ 8 and MCPâ 1 by GHSA at concentrations ranging from 62.5 to 3,000 μg/mL exhibited doseâ dependent kinetics. The GHSAâ induced chemokine secretion by hRPE was almost completely inhibited by actinomycin D and cycloheximide, suggesting that de novo mRNA and protein synthesis are necessary for the GHSAâ induced ILâ 8 and MCPâ 1 production. Northern blot analysis of GHSAâ induced hRPE ILâ 8 and MCPâ 1 mRNA expression corresponded to the timeâ and doseâ dependent increases measured by enzymeâ linked immunosorbent assay. High concentrations of glucose (20 mM; 360 mg/dl) increased GHSAâ induced hRPE ILâ 8 and MCPâ 1 secretion, whereas added insulin (0.5 ng/mL) inhibited ILâ 8 but not MCPâ 1 protein secretion and mRNA expression. GHSA also induced hRPE to secrete GROâ α, RANâ TES, and NAPâ 2 chemokines. GHSA induction of hRPE chemokines further suggests a role for the hRPE in leukocyte infiltration, vascular injury, and neovascularization. J. Leukoc. Biol. 60: 405â 414; 1996.