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L. Chen, S.S. Zhang, C.J. Barnstable, J. Tombran–Tink; PEDF Binds to VEGF–r2 and Inhibits VEGF–Induced Phosphorylation of This Receptor . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5334.
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© ARVO (1962-2015); The Authors (2016-present)
Pigment epithelium derived factor (PEDF) is a powerful endogenous inhibitor of angiogenesis and has potential application for the treatment of neovascular diseases. We previously showed that PEDF inhibited VEGF– promoted survival of human umbilical vein endothelial cells (HUVECs), and blocked VEGF–induced phosphorylation of several pathways. The purpose of the present study was to determine the mechanism by which PEDF abrogates VEGF function.
Proteins were extracted from HUVECs grown in the presence of various concentrations of PEDF, VEGF, or both for times up to 3 hr. Western blotting was used to measure the phosphorylation of the receptor at Tyr 951, 996, and 1175 and downstream signal molecules. A specific kinase assay was used to measure the activity of VEGF–R2. ELISA assays were carried out using recombinant extracellular regions of VEGF–R2 immobilized onto plastic to determine binding of PEDF to VEGF–R2. Immunoprecipitations with anti–VEGF–R2 were carried out on solubilised HUVEC extracts and co–precipitated molecules detected on Western blots.
PEDF inhibited VEGF–induced phosphorylation of VEGF–R2 in a time and dosage dependent pattern at all three tyrosine sites analysed. This PEDF effect could not be reversed with the general phosphatase inhibitor, orthavanadate. Phosphorylation of the major down stream signaling molecules of activated VEGF–R2 including PI3 kinase/Akt, PLCγ, FAK and Src were significantly reduced by PEDF. ELISA assays showed that PEDF specifically bind to the extracelluar domain of VEGF–R2 with an apparent affinity of 2 nM. PEDF did not block VEGF binding to this receptor suggesting that binding of the two molecules to the receptor occurs at different sites. Immunoprecipitation of VEGF–R2 from HUVEC lysates co–precipitated PEDF, and vice versa.
PEDF binds to the extracellular domain of VEGF–R2 and inhibits its VEGF–induced phosphorylation as well as subsequent downstream signaling pathways. We propose that PEDF inhibits angiogenesis, in part, by specifically binding to VEGFR2 and interfering with VEGF mediated mitogenic signals on endothelial cells.
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