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M. E. Boulton, M. B. Grant, S. L. Rudrabhatla, D. Nguyen, J. Cai; Vascular Permeability Is Regulated by -Secretase-Mediated Translocation of VEGFR-1 to the Adherens Junctions. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2653.
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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.
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.
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.
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.
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