Vascular Permeability Is Regulated by -Secretase-Mediated Translocation of VEGFR-1 to the Adherens Junctions

Abstract

Purpose: : There is increasing evidence that γ-secretase plays an important role in VEGF-induced angiogenesis through regulation and translocation of VEGFR-1 (Cai et al, J Biol Chem. 2006). In this study we have investigated the role of VEGFR-1 and γ-secretase in the regulation of vascular permeability and determined how this changes under hypoxia.

Methods: : Primary retinal microvascular endothelial cells were isolated from bovine retina. Transendothelial resistance (TER) was measured using an EVOM volt-ohmmeter in the presence of VEGFA, PEDF or a combination (100ng/ml). In some experiments VEGF receptors were neutralized or a γ-secretase inhibitor was included. Western blotting was performed on lysates using antibodies against VEGFR-1, β-catenin, VE-cadherin, occludin, ZO1, presenilin and nicastrin. Immunoprecipitation was used to detect the association between these proteins. Hypoxia was achieved using a hypoxia workstation with 0.02% oxygen (pO₂=5mm Hg) and 5% oxygen (pO₂=40 mm Hg) representing tissue hypoxia and normoxia respectively.

Results: : VEGFA increased vascular permeability by disrupting the adherens junctional proteins but had no effect on tight junction integrity. The VEGF-induced decrease in TER could be prevented by PEDF (which upregulates γ-secretase in endothelial cells) and blockade of VEGFR-1 but not VEGFR-2. The effect of PEDF was blocked by addition of a γ-secretase inhibitor. Immunoprecipitation and Western blotting confirmed that a) VEGFR1 associates with the β-catenin-VE-cadherin complex, b) that the γ-secretase components presenilin and nicastrin are associated with VEGFR-1 and c) there is no change in the expression or localization of ZO1 or occludin. Western blot analysis showed that hypoxic treatment of cells increased the overall level of VEGFR-1 and γ-secretase but had no effect on β-catenin or VE-cadherin expression. Immunoprecipitation demonstrated that that the association between VEGFR-1 and VE-cadherin was increased under hypoxia and that this could be reversed by inhibition of γ-secretase.

Conclusions: : These results indicate VEGFR-1 plays a key role in vascular permeability and that this is regulated by γ-secretase. Hypoxia alters the balance of proteins and causes dysregulation in endothelial barrier properties.

Keywords: cell adhesions/cell junctions • growth factors/growth factor receptors • hypoxia