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M.E. Boulton, S. Ali, J. Cai; Pigment Epithelial-Derived Factor (PEDF) Regulates Angiogenesis via Vascular Endothelial Growth Factor Receptor-1 . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2099.
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Purpose: To identify the mechanism by which PEDF can inhibit VEGF- induced angiogenesis. Methods: Retinal microvascular endothelial cells (MEC) were isolated from bovine eyes. Cultures were treated with 100ng/ml VEGF, 200ng/ml PEDF or a combination of VEGF and PEDF for 48hours. Control cultures were maintained in the absence of added growth factor. Proliferation was quantified using the crystal violet assay, migration was assessed following scrape wounding and the degree of tubule formation was measured in a Matrigel matrix. Western blotting was undertaken on Triton-soluble and Triton-insoluble fractions of cell proteins to examine the cleavage and translocation of the VEGF receptor-1 (VEGFR1). Results: VEGF increased MEC proliferation by 150% which was reduced by 50% after addition of PEDF. Similarly, VEGF stimulated MEC migration by 140% and this was reduced by 80% in the presence of PEDF. PEDF decreased VEGF-induced tubule formation by 90%. PEDF had no significant effect on either proliferation, migration or tubule formation in the absence of VEGF. Western blotting demonstrated that while PEDF did not affect the overall level of VEGFR1 it caused an increased translocation of VEGFR1 from the Triton-insoluble fraction to the Triton-soluble fraction. However, addition of γ-secretase which cleaves the transmembrane domain of VEGFR1 abolished the inhibitory effect of PEDF on VEGF induced angiogenesis and the translocation of VEGFR1. Conclusions: PEDF inhibits angiogenesis by enhancing the cleavage of VEGFR1 by γ-secretase leading to negative regulation of angiogenesis.
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