May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Vascular Permeability Is Regulated by -Secretase-Mediated Translocation of VEGFR-1 to the Adherens Junctions
Author Affiliations & Notes
  • M. E. Boulton
    Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas
  • M. B. Grant
    Pharmacology and Therapeutics, University of Florida, Gainesville, Florida
  • S. L. Rudrabhatla
    Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas
  • D. Nguyen
    Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas
  • J. Cai
    Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas
  • Footnotes
    Commercial Relationships  M.E. Boulton, None; M.B. Grant, None; S.L. Rudrabhatla, None; D. Nguyen, None; J. Cai, None.
  • Footnotes
    Support  NIH Grant EY018358-01
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 2653. doi:https://doi.org/
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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. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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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 
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