Density-dependent expression of FGF-2 in response to oxidative stress in RPE cells in vitro

Purpose. The purpose of this study is to demonstrate the effect of culture density on the steady state mRNA levels of fibroblast growth factor-2 (FGF-2) when retinal pigment epithelial (RPE) cells are subjected to oxidative stress in vitro. Methods. Subconfluent and confluent cultures of the established RPE cell line ARPE-19, were treated with increasing concentrations of tert-butyl hydroperoxide (tBH) or hydrogen peroxide (H₂O₂). Cell viability was measured using the WST-1 assay, and intracellular reactive oxygen intermediate (ROI) production was quantified by dichlorofluoroscein (DCF) fluorescence. Steady state changes in heme oxygenase-1 (HO-1) and FGF-2 mRNAs were measured by Northern blot analysis. Results. Confluent cultures of ARPE-19 cells were less susceptible than subconfluent cultures to the toxic effects of the chemical oxidants. The intracellular reactive oxygen intermediate production was higher in subconfluent than confluent cultres with increasing tBH concentration. At nontoxic concentrations of tBH and H₂O₂, a dose dependent increase in FGF-2 expression was seen as a function of culture density. FGF-2 mRNA expression was induced after tBH treatment in subconfluent, but not confluent cells. On the other hand, FGF-2 mRNA induction was observed after H₂O₂ treatment in confluent, but not subconfluent cultures. In contrast, no density dependent induction of HO-1 mRNA was seen after treatment with either tBH or H₂O₂. Conclusions. These results suggest that care should be taken to control for cell density in similar types of in vitro experiments.