Stimulation of apical and basolateral vascular endothelial growthfactor-A and vascular endothelial growth factor-C secretion by oxidativestress in polarized retinal pigment epithelial cells

[摘要] Purpose: To investigate whether oxidative stress modulates vascularendothelial growth factor (VEGF)-A and VEGF-C expression and polarizedsecretion in a human retinal pigment epithelium cell line (ARPE-19).Methods: Long-term culture of ARPE-19 cells in Dulbecco's modifiedEagle medium (DMEM)/F12 containing 1% fetal bovine serum (FBS) ontranswell filters (12 mm or 6 mm, pore size 0.4 μm) was performed toproduce polarized retinal pigment epithelium (RPE) monolayers. Theintegrity of polarized monolayer was established by measurement oftransepithelial resistance (TER) and presence of tight junctionsassessed by zonula occludens (ZO-1) and occludin expression and apicalNa/K ATPase localization. Paracellular permeability was studied usingradiolabeled mannitol. Confluent cells were treated with tertiary butylhydrogen peroxide (tBH) for varying durations (0-5 h) and doses (50-200μM). VEGF-A and -C expression was evaluated by western blot andquantitative RT-PCR, while secretion to the apical and basolateralsurfaces was quantitated by ELISA.Results: Polarity of ARPE-19 cells was verified by the localizationof tight junction proteins, ZO-1 and its binding partner occludin byconfocal microscopy as well as by localization of Na,K-ATPase at theapical surface. The TER in confluent ARPE-19 cells averaged 48.7±2.1Ω. cm2 and tBH treatment (0-5 h) did not alter TERsignificantly (46.9±1.9 Ω. cm2; p>0.05 versuscontrols) or ZO-1 expression. Whole cell mRNA in nonpolarized ARPE-19increased with tBH at 5 h both for VEGF-A and VEGF-C and the increasewas significant (p<0.05 vs controls). A similar, maximal increase at5 h tBH treatment was also observed for VEGF-A and VEGF-C cellularprotein levels. The secretion of VEGF-A and VEGF-C in nonpolarized ARPEshowed an increase with tBH exposure. The levels of secretion of VEGF-Aand -C were significantly higher in polarized monolayers and werestimulated significantly with tBH at both apical and basolateraldomains. The secretion of VEGF-A increased with 150 μM of tBHtreatment as a function of time (1-5 h) with maximal increases at 5 hfrom 410 to 2080 pg/106 cells on the apical and 290 to 1680pg/106 cells on basolateral domains. The pattern of VEGF-Csecretion was similar. VEGF-A secretion was dose-dependent for the tBHrange of 50-200 μM and apical secretion tended to be higher thanbasolateral secretion.Conclusions: Our data show that oxidative stress to RPE from tBHupregulates secretion of both VEGF-A and C. The secretion to the apicalside was higher than that of basolateral side for VEGF-A and C. Giventhe role of VEGF in choroidal neovascularization, these data may be ofvalue in understanding pathogenic mechanisms and designingantiangiogenic therapies.

[学科分类] 生物化学/生物物理