Kindlin-2 interacts with endothelial adherens junctions to support vascular barrier integrity

Abstract

Endothelial cells (EC) establish a physical barrier between the blood and surrounding tissue. Impairment of this barrier can occur during inflammation, ischemia or sepsis and cause severe organ dysfunction. Kindlin-2, primarily recognized as a focal adhesion protein in EC, was not anticipated to have a role in vascular barrier. We tested the role of Kindlin-2 in regulating vascular integrity using several different approaches to decrease Kindlin-2 levels in EC. Reduced levels of Kindlin-2 in Kindlin-2^+/- mice, aortic endothelial cells (MAECs) from these⁻mice, and human umbilical ECs (HUVEC) treated with Kindlin-2 siRNA showed enhanced basal and Platelet Activating Factor (PAF) or lipopolysaccharide-stimulated vascular leakage compared to wild-type (WT) counterparts. PAF preferentially disrupted Kindlin-2^+/−MAECs barrier to bovine serum albumin (BSA) and dextran and reduced transendothelial resistance (TEER) compared to WT cells. Kindlin-2 co-localized and co-immunoprecipitated with VE-cadherin-based complexes, including β- and γ-catenin and actin, components of Adherens Junctions (AJ). Direct interaction of Kindlin-2 with β- and γ-catenin and actin was demonstrated in co-immunoprecipitation and surface plasmon resonance (SPR) experiments. In thrombin-stimulated HUVECs, Kindlin-2 and cortical actin dissociated from stable AJs and redistributed to radial actin stress fibres of remodelling focal AJs. The β- and γ-catenin binding site resides within the F1 and F3 subdomains of Kindlin-2, but not the integrin binding site in F3.

These results establish a previously unrecognized and vital role of Kindlin-2 in maintaining the vascular barrier by linking VE-cadherin-based complexes to cortical actin and thereby stabilizing AJ.

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