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M. E. Boulton, J. Cai, S. Caballero, M. B. Grant; -Secretase Regulates Vascular Permeability Through the Association of VEGFR-1 With VE-Cadherin and ß-Catenin in Response to PEDF. Invest. Ophthalmol. Vis. Sci. 2007;48(13):1734.
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We have previously shown that γ-secretase mediates the intramembranous proteolysis of VEGFR-1 (Cai et al. J Biol Chem. 2006). Since VEGFR-1 is associated with adherens junctions, we hypothesized that translocated VEGFR-1 can associate with the VE-cadherin complex to regulate vascular permeability.
Primary bovine retinal microvascular endothelial cells were prepared and treated with VEGFA (100ng/ml), PEDF (100ng/ml) and/or γ-secretase inhibitor (12.5 nM) for up to 24 hours. Total cell lysates were immunoprecipitated with an antibody against the C-terminus of VEGFR-1, and subsequent Western blots were probed with antibodies against adherens junction proteins (α-, ß-catenin, VE-cadherin) and two major components of the γ-secretase complex (presenilin-1 and nicastrin). Transendothelial resistance was measured in cells exposed to growth factors in the presence and absence of γ-secretase inhibitor. In addition, in vivo vascular permeability in the oxygen-induced retinopathy (OIR) model was assessed following modulation of γ-secretase activity.
Western blotting demonstrated constitutive association between VEGFR-1, α-catenin, ß-catenin and VE-cadherin in confluent microvascular endothelial cells. The addition of PEDF upregulated γ-secretase activity, blocked VEGF-induced permeability and decreased VEGF-induced-association of VEGFR-1 with ß-catenin and VE-cadherin. Addition of γ-secretase inhibitor abolished the response to PEDF. In the OIR model PEDF-modulated upregulation of γ-secretase reduced vascular permeability and this was partially reduced following intravitreal injection of γ-secretase inhibitor. The translocation of VEGFR-1 to the adherens junction was dependent on the association of presenilin and nicastrin with membrane bound VEGFR-1. Translocation of VEGFR-1 and changes in vascular permeability was blocked when presenilin-1 and nicastrin were prevented from binding to VEGFR-1.
γ-secretase regulates vascular endothelial permeability by modulating VEGFR1 association with the VE-cadherin, ß-catenin complex.
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