The effect of oxidative stress on retinal pigment epithelial cell migration

Abstract

This study sought to determine if the migration and adhesion of retinal pigment epithelial (RPE) cell could be modulated by oxidative stress induced by t-butyl hydroperoxide (TBH). Intracellular RPE reactive oxygen species (ROS) was labeled by Carboxy-H(2)DCFDA staining. Cell migration was measured by a modified Boyden Chamber Assay. After RPE cells were treated with different concentrations of TBH (1-200 micromol/L) for one hour, chemotaxis toward PDGF (30 ng/ml) was measured after additional 5 hours incubation. The actin cytoskeleton was evaluated by phalloidin (F-actin) staining. Focal adhesion kinase (FAK) and vinculin expression were evaluated using immunofluorescence staining. The expression of MAP Kinase and FAK were studied by western blot. Cell survival and attachment of treated RPE cells were determined using MTT assay. ROS production was elevated in the RPE cells pretreated with TBH. The PDGF-independent RPE cell migration was enhanced by TBH at low concentration up to 30 micromol/L, and the PDGF-induced RPE cell migration was significantly inhibited by TBH at concentration higher than 50 micromol/L. F-actin was aggregated in the periphery of the RPE cells after treatment with TBH at a dose higher than 100 micromol/L. FAK and vinculin expression in RPE cells decreased and their distribution were changed after the treatment of higher concentrations of TBH. MAP Kinase level was up-regulated in response to 10-200 micromol/L TBH exposure. The cell attachment was declined upon addition of TBH above 50 micromol/L. Oxidant stress induced by TBH can inhibit or promote RPE cell migration.