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


METHODS
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.


RESULTS
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<0.05 vs controls). A similar, maximal increase at 5 h tBH treatment was also observed for VEGF-A and VEGF-C cellular protein levels. The secretion of VEGF-A and VEGF-C in nonpolarized ARPE showed an increase with tBH exposure. The levels of secretion of VEGF-A and -C were significantly higher in polarized monolayers and were stimulated significantly with tBH at both apical and basolateral domains. The secretion of VEGF-A increased with 150 microM of tBH treatment as a function of time (1-5 h) with maximal increases at 5 h from 410 to 2080 pg/10(6) cells on the apical and 290 to 1680 pg/10(6) cells on basolateral domains. The pattern of VEGF-C secretion was similar. VEGF-A secretion was dose-dependent for the tBH range of 50-200 microM and apical secretion tended to be higher than basolateral secretion.


CONCLUSIONS
Our data show that oxidative stress to RPE from tBH upregulates secretion of both VEGF-A and C. The secretion to the apical side was higher than that of basolateral side for VEGF-A and C. Given the role of VEGF in choroidal neovascularization, these data may be of value in understanding pathogenic mechanisms and designing antiangiogenic therapies.
Kannan
Ram
R
Department of Ophthalmology, Keck School of Medicine of the University of Southern California, Los Angeles, CA 90033, USA.
Zhang
Ning
N
Sreekumar
Parameswaran G
PG
Spee
Christine K
CK
Rodriguez
Anthony
A
Barron
Ernesto
E
Hinton
David R
DR
D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002463:Cell Membrane Permeability; D016764:Cell Polarity; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D006801:Humans; D018384:Oxidative Stress / Q000502:physiology*; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D019108:Tight Junctions / Q000187:drug effects / Q000502:physiology; D013997:Time Factors; D014018:Tissue Distribution; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D042582:Vascular Endothelial Growth Factor C / Q000378:metabolism*; D020122:tert-Butylhydroperoxide / Q000008:administration & dosage / Q000494:pharmacology
D042461:Vascular Endothelial Growth Factor A; D042582:Vascular Endothelial Growth Factor C; D020122:tert-Butylhydroperoxide