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

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.