The use of hyperoxia to induce chronic mild oxidative stress in RPE cells in vitro

Purpose: To establish a model of mild and chronic oxidative stress using hy peroxia for retinal pigment epithelial (RPE) cells in vitro. Methods: RPE340 cells and WI38 lung fibroblasts were grown in normal oxygen (20% O-2) and hyperoxia (40% O-2 or 60% O-2). After cell viability was exa mined, the levels of reactive oxygen intermediates (ROI) by flow cytometry and heme oxygenase-1 (HO-1) mRNA by northern analysis were measured as mark ers of oxidative stress in both cell types. Proliferative ability and gene expression pattern of growth factors were studied to demonstrate the phenot ypic changes induced by mild oxidative stress upon these cells. Results: While decreased by 60% O-2, 40% O-2 did not affect viability in bo th cell types, ROI production and HO-1 mRNA expression were elevated in hyp eroxia compared to controls, but were inhibited with the antioxidant dehydr o-ascorbic acid (DHA). The proliferation of cells by hyperoxia was inhibite d in both cell types. The expression of growth factors induced by hyperoxia was cell type dependent. Fibroblast growth factor-2 mRNA was unchanged in RPE cells, but was increased in fibroblasts. Transforming growth factor-bet a2 was decreased in RPE cells, but unchanged in fibroblasts. Vascular endot helial growth factor was downregulated in RPE cells, while upregulated in f ibroblasts. Connective tissue growth factor was decreased in RPE cells, but was unchanged in fibroblasts. Conclusions: The results demonstrate that hyperoxia induces mild oxidative stress which alters the phenotype of cells in a cell type specific manner.