Stimulation of apical and basolateral VEGF-A and VEGF-C secretion by oxidative stress in polarized retinal pigment epithelial cells

To investigate whether oxidative stress modulates vascular endothelial growth factor (VEGF)-A and VEGF-C expression and polarized secretion in a human retinal pigment epithelium cell line (ARPE-19). Long-term culture of ARPE-19 cells in Dulbecco's modified Eagle medium (DMEM)/F12 containing 1% fetal bovine serum (FBS) on transwell filters (12 mm or 6 mm, pore size 0.4 microm) was performed to produce polarized retinal pigment epithelium (RPE) monolayers. The integrity of polarized monolayer was established by measurement of transepithelial resistance (TER) and presence of tight junctions assessed by zonula occludens (ZO-1) and occludin expression and apical Na/K ATPase localization. Paracellular permeability was studied using radiolabeled mannitol. Confluent cells were treated with tertiary butyl hydrogen peroxide (tBH) for varying durations (0-5 h) and doses (50-200 microM). VEGF-A and -C expression was evaluated by western blot and quantitative RT-PCR, while secretion to the apical and basolateral surfaces was quantitated by ELISA. Polarity of ARPE-19 cells was verified by the localization of tight junction proteins, ZO-1 and its binding partner occludin by confocal microscopy as well as by localization of Na,K-ATPase at the apical surface. The TER in confluent ARPE-19 cells averaged 48.7+/-2.1 Omega. cm(2) and tBH treatment (0-5 h) did not alter TER significantly (46.9+/-1.9 Omega. cm(2); p>0.05 versus controls) or ZO-1 expression. Whole cell mRNA in nonpolarized ARPE-19 increased with tBH at 5 h both for VEGF-A and VEGF-C and the increase was significant (p