Abstract
Abstract: :
Purpose:To elucidate the mechanism by which PEDF–induced cleavage of the intracellular domain of VEGFR1 can inhibit neovascularisation. Methods:Retinal microvascular endothelial cells were isolated from bovine eyes. Cultures were treated with 100ng/ml VEGF, 100ng/ml PEDF or a combination of VEGF and PEDF for 48hours. Control cultures were maintained in the absence of added growth factor. Subcellular protein extractions were performed and the localisation of the extracellular and intracellular domains of VEGFR1 determined by Western blotting. Since γ–secretase is responsible for the cleavage of the intracellular domain of VEGFR1 we also assessed the effect of γ –secretase inhibition on the intracellular translocation of VEGFR1. Results:Western blotting revealed the presence of both the extracellular and intracellular domains of VEGFR1 in the membrane, cytoskeletal and nuclear fractions in control cultures and those exposed to VEGF or PEDF alone. No detectable VEGFR1 was observed in the cytosolic fraction. By contrast, cells treated with both VEGF and PEDF demonstrated significant levels of the intracellular VEGFR1 domain in the cyotsol with a concommitant reduction in the membrane, cytoskeletal and nuclear fractions. Although the extracellular domain of VEGFR1 was reduced in the cytoskeletal and nuclear fraction this was not due to relocation to the cytosol since the extracellular domain could not be identified in the cytosol. The cytosolic translocation of the intracellular domain was blocked by addition of a γ–secretase inhibitor and was associated with increased levels of VEGFR1 in the cytoskeletal and the nuclear fractions in cells treated with both VEGF and PEDF. Conclusions:PEDF inhibitis VEGF–induced angiogenesis by shifting the translocation of the intracellular domain of VEGFR1 from the nucleus to the cytosol.
Keywords: growth factors/growth factor receptors • retinal neovascularization • vascular cells