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R-Ras interacts with filamin a to maintain endothelial barrier function.

G S Griffiths1, M Grundl, J S Allen

  • 1Department of Cell and Molecular Biology, Cardiovascular Research Center and the John A Burns School of Medicine, University of Hawaii at Manoa, Honolulu, Hawaii 96813, USA.

Journal of Cellular Physiology
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Active R-Ras and its association with Filamin A (FLNa) are crucial for maintaining endothelial barrier integrity. Loss of this interaction increases vascular permeability by affecting VE-Cadherin and Src signaling.

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Area of Science:

  • Endothelial biology
  • Molecular mechanisms of vascular permeability
  • Cellular signaling pathways

Background:

  • Vascular barrier integrity is critical for tissue homeostasis and is regulated by complex molecular mechanisms.
  • Previous research linked the GTPase R-Ras to Filamin A (FLNa), and FLNa deficiency is associated with increased vascular permeability.
  • The specific role of the R-Ras-FLNa interaction in endothelial barrier function remained unclear.

Purpose of the Study:

  • To investigate whether the association between R-Ras and FLNa influences endothelial barrier function.
  • To elucidate the molecular mechanisms by which R-Ras and FLNa regulate vascular permeability.

Main Methods:

  • Co-immunoprecipitation and pulldown assays to confirm R-Ras and FLNa interaction.
  • siRNA-mediated knockdown of R-Ras and FLNa in endothelial cells.
  • TransEndothelial Electrical Resistance (TEER) and FITC-dextran transwell assays to measure vascular permeability.
  • Re-expression studies with wild-type FLNa and a repeat 3 deletion mutant (FLNaΔ3).
  • Immunostaining for VE-Cadherin and Western blotting for phosphorylated proteins (VE-Cadherin, Src).
  • Pharmacological inhibition of R-Ras and Src signaling pathways.

Main Results:

  • Endogenous R-Ras interacts with FLNa in endothelial cells, specifically involving FLNa repeat 3.
  • Loss of R-Ras or FLNa, or disruption of their interaction (using FLNaΔ3), significantly increased vascular permeability.
  • Re-expression of FLNa restored barrier function, while FLNaΔ3 did not.
  • Knockdown of R-Ras/FLNa led to VE-Cadherin disorganization at adherens junctions.
  • Inhibition of R-Ras or FLNa loss increased phosphorylation of VE-Cadherin (Y731) and Src (Y416).
  • Dominant-negative R-Ras expression induced permeability, which was blocked by a Src inhibitor.

Conclusions:

  • The interaction between R-Ras and FLNa is essential for maintaining endothelial barrier function.
  • Disruption of the R-Ras-FLNa association promotes vascular permeability through VE-Cadherin and Src signaling.
  • Active R-Ras and its interaction with FLNa are critical for preventing endothelial leakiness.