PEDF Downregulates Tyrosine Phosphorylation Status of Vascular Endothelial Growth Factor Receptor1

Abstract

Abstract: : Purpose: To examine the mechanisms involved in the inhibitory effect of pigment epithelium–derived factor in angiogenesis in mammalian eyes. Methods: Bovine retinal microvascular endothelial cells were prepared and treated with VEGF, PEDF or VEGF and PEDF for 48 hours. In vitro angiogenesis analysis was performed. Immunoprecipiation and Western blotting was used to examine the effect of PEDF on the tyrosine phosphorylation status of VEGFR1. Results: Angiogenesis analysis indicated that PEDF only inhibited VEGF–induced in vitro vessel formation while PEDF alone had no effect. Western blotting results showed that PEDF does not regulate overall level of VEGFR1 in endothelial cells. VEGF induced an increase in autophosphorylation of VEGFR–1 compared to control with bands at 250 and 180 kDa in whole cell lysates. PEDF greatly reduced VEGFR1 phosphorylation in both the 250 and 180 kDa bands and this was greatest when PEDF was used in combination with VEGF. Analysis of the subcellular fractions demonstrated that VEGF induced an increase in autophosphorylation of VEGFR1 in membrane, cytoskeleton and nuclear fractions. Interestingly, PEDF almost completely blocked the VEGFR–1 tyrosine phosphorylation in all fractions regardless of whether the endothelial cells were cultured in the presence or absence of VEGF. Conclusions: The results from this study indicated that the inhibitory effect of PEDF on VEGF–induced angiogenesis is a consequence of enhancing γ–secretase–dependent cleavage of VEGFR1 and dephosphorylation of VEGFR1. This identifies a novel regulatory role for VEGFR1 in angiogenesis.

Keywords: growth factors/growth factor receptors