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S. Gurunathan, E. Duh; PEDF Inhibits VEGF Activation of PI3 Kinase/Akt and Src in Human Retinal Endothelial Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5152.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Pigment epithelium–derived factor (PEDF) is a potent inhibitor of angiogenesis and VEGF action on endothelial cells. Our lab has previously described the suppression of retinal neovascularization in the oxygen–induced retinopathy model by PEDF. The aim of this study was to investigate whether PEDF modulates VEGF signaling in retinal endothelial cells. In particular, we examined the effect of VEGF on two important signaling pathways in endothelial cells: phosphatidylinositol 3–kinase (PI3K)/ Akt and Src . Methods: Human retinal endothelial cells (HRECs) and human umbilical vein endothelial cells (HUVECs) were cultured in EGM2–MV medium (Clonetics, San Diego, CA) and used between passages 5 and 10. HRECs were cultured on fibronectin–coated dishes. Cells were grown in 5% CO2 at 37° C and media changed every 2 days. The cells were starved for 5 hrs in OPTIMEM with 0.5% FBS. Cells were then incubated with VEGF and/or PEDF. After cytokine treatment, cells were harvested, washed, and lysed in buffer containing 10 mM TRIS, 50 mM NaCl, Na–pyrophosphate, 1% Triton, 1 mM Na3VO4, 1 mM PMSF and protein inhibitor cocktail (Roche). Cell lysates were subjected to 10% SDS–PAGE and transferred onto nitrocellulose membranes (Sigma,USA). The blots were probed with antiphospho–Akt (Ser 473) and anti–Src (Tyr 416). Results: Consistent with previous results in other endothelial cell types, VEGF treatment activated Akt phosphorylation at Ser 473 and Src phosphorylation at Tyr 416 in retinal endothelial cells. PEDF treatment significantly inhibited VEGF activation of both Akt and Src in a dose–dependent manner. PEDF treatment had a similar inhibitory effect on VEGF activation of these proteins in human umbilical vein endothelial cells. Conclusions: These results indicate that PEDF can block VEGF activation of important signaling pathways, specifically PI3–kinase/Akt and Src in both retinal and umbilical vein endothelial cells. Modulation of these pathways may contribute to PEDF’s ability to block VEGF–induced retinal neovascularization. Commercial relationship: None Grant Identification: NIH/NEI, Juvenile Diabetes Research Foundation, Research to Prevent Blindness
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