Thiol Regulation of Vascular Endothelial Growth Factors A and C Secretion by Retinal Pigment Epithelial Cells

Abstract

Abstract: : Purpose: To investigate the role of glutathione (GSH) in VEGF–A and VEGF–C secretion by retinal pigment epithelial (RPE) cells. Methods: Oxidative stress was induced by GSH depletion in early passage human fetal RPE cells using dl–buthionine sulfoximine (BSO; 500 µM) in 1% fetal bovine serum. Secretion of VEGF–A and C, along with cellular redox ratios (GSH/GSSG), was quantified as a function of time (0–24hr) by specific ELISA assays in control, BSO–treated, and BSO plus GSH monoethylester (GSH–MEE; 5mM)–treated cells. Results: BSO treatment caused a time–dependent increase in VEGF–A secretion, from undetectable at 0 hr to 491.8pg/ml and 968.6pg/ml at 12 and 24 hrs, respectively, and an increase in VEGF–C secretion from undetectable at 0 hr to 3543.8pg/ml and 4898.4pg/ml at 12 and 24 hrs, respectively. The maximal increase in secretion (24hr) was five–fold higher for VEGF–C than for VEGF–A. BSO treatment also caused a significant decrease in the cellular GSH/GSSG ratio. Pre–incubation of RPE with 5 mM GSH–MEE significantly inhibited the BSO–induced increase in VEGF–A and VEGF–C secretions, and was associated with restoration of the cellular GSH level. Conclusions: BSO treatment induced the up–regulation of VEGF–A and VEGF–C secretion. Pretreatment of RPE with GSH–MEE strongly inhibits VEGF secretion, suggesting that GSH may play a critical role in this process.

Keywords: retinal pigment epithelium • oxidation/oxidative or free radical damage