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C.K. Spee, N. Zhang, R. Kannan, S.J. Ryan, D.R. Hinton; Effect of Oxidative Stress on the Polarity of Secretion of VEGF–A and VEGF–C in RPE . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1607.
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Purpose: The aim of the present study was to determine a) if VEGF–A and VEGF–C exhibit polarity in secretion in ARPE–19 cells and b) whether oxidative stress causes selective changes in secretion. Methods: ARPE–19 cells were grown in DMEM containing 1% FBS on transwell filters (0.4µm) for four months. Transepithelial resistance (TER) was measured using a Voltohmmeter and presence of tight junctions was assessed by staining with a monoclonal antibody for ZO–1. Cells were treated with tert. butyl hydrogen peroxide (TBH) in both apical and basolateral media. The TBH treatment protocol included varying times (0–5h) and doses (50–200µM). VEGF–A and –C secretion was quantitated by ELISA. Cell viability for the entire duration of the experiment was monitored and remained unchanged. Results: ZO–1 expression confirmed the presence of intercellular tight junctions in RPE. The TER remained unchanged during TBH treatment and averaged 32.7 ± 2.3 ohms/cm2. VEGF–A and –C secretion was markedly stimulated in polarized ARPE–19 cells as compared to non–polarized cells. Unstressed RPE secreted VEGF–A and VEGF–C at both apical and basolateral surfaces. The secretion of VEGF–A increased with 150µM of TBH treatment as a function of time (1–5h) with maximal increases at 5 hours (from 402 to 1921 pg/106 cells on the apical and 269 to 1507 pg/106 cells for basolateral). A similar trend was observed for VEGF–C secretion. VEGF–A secretion was dose–dependent for the TBH range of 50–200µM with apical secretion greater than basolateral secretion. However, VEGF–C did not exhibit a concentration–dependent increase in apical or basolateral secretion. Conclusions: VEGF is secreted at both apical and basolateral surfaces in normal RPE. Our data showed that oxidative stress to RPE resulted in selective increase in apical secretion of VEGF–A. Thus, oxidative stress promotes VEGF secretion from RPE in vitro and may play a role in the development of choroidal neovascularization.
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