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Dan-Ning Hu, Richard Rosen, Anthony Sclafani, Steven McCormick; Effects of Zeaxanthin on Constitutive and TPA-Induced Secretion of VEGF by Human Retinal Pigment Epithelial Cells In Vitro. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3783.
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TPA (12-0-tetradecanoyl phorbol 13-acetate) is a protein kinase C activator, which can stimulate the secretion of VEGF by retinal pigment epithelial (RPE) cells. The purposes of this study were to investigate the effects of zeaxanthin on the constitutive and TPA-induced secretion of VEGF by human RPE cells in vitro.
Human RPE cells (ARPE19) were seeded into 12-well plates and cultured with F12 medium with 10% serum. After 24 hours, culture medium was replaced by F12 medium without serum. Zeaxanthine at various concentrations (1, 10 and 100 µM) was added. TPA (100 ng/ml) was added 1 hour later. After 24 hours, conditioned medium was collected and the amount of VEGF was measured using a sandwich enzyme-linked immunosorbent assay kit (R & D Systems). All tests were performed in triplicate. A primary culture of RPE cells isolated from a donor eye was also tested.
VEGF could be detected in the conditioned medium from cultured ARPE cells (70.7±5.5 pg/ml). TPA significantly stimulated the secretion of VEGF (149.3±12.1 pg/ml, p<0.01 as compared with cells cultured without TPA). Zeaxanthin slightly decreased the constitutive secretion of VEGF only at 100 µM concentration (55.7±5.5 pg/ml; p<0.05 as compared with cells cultured without zeaxanthin). Zeaxanthin significantly inhibited TPA-induced secretion of VEGF by RPE cells in a dose-dependent manner. VEGF levels in conditioned medium from cells cultured with TPA and zeaxanthin (100 µM) was 93.3 ± 7.8 pg/ml, which was significantly lower than that in cells cultured with TPA alone (p < 0.01) but still higher than that in cells cultured without TPA (0.05 > p > 0.01). Study in primary culture of cell line obtained a similar result.
Zeaxanthin only slightly inhibits constitutive secretion of VEGF by human RPE cells in vitro but significantly reduced TPA-induced secretion of VEGF by RPE cells in a dose-dependent manner.
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