Kindlin-2 Maintains Vascular Barrier Integrity By Stabilizing Endothelial Adherens Junctions

Kindlin-2, a widely distributed cytoskeletal protein, has been implicated in integrin activation, angiogenesis, thrombosis and hemostasis. Its absence is embryonically lethal in mice, emphasizing its biological importance. In the present study, we examined the role of Kindlin-2 in regulation of vascular integrity. In vivo, Kindlin-2^+/- mice showed enhanced (70%) permeability of tracheal vasculature to fluorescent beads as compared to wild-type (WT) littermates under both basal conditions or when stimulated with Platelet Activating Factor (PAF). Consistent with these in vivo observations, confluent monolayers of aortic endothelial cells (ECs) isolated from Kindlin-2^+/- mice had increased (2-3-fold) baseline, PAF- and thrombin-induced permeability for Alexa-488-labeled bovine serum albumin (BSA) or FITC-dextran-10000 as compared to WT cells. Also, reduction of Kindlin-2 expression by 60-70% in human umbilical vein endothelial cell (HUVEC) monolayers with Kindlin-2-specific siRNA resulted in 3-fold increase in their baseline or thrombin/(PAF)-induced permeability of these markers. Mechanistically, Kindlin-2 co-localized with VE-cadherin and actin within adherens junctions in resting, confluent HUVEC monolayers as well as co-immunoprecipitated with these proteins and other components of adherens junctions, including α-, β- and γ-catenin. In contrast, Kindlin-2 did not co-localize or co-immunoprecipitate with any components of GAP or tight junctions. VE-cadherin-based complexes had less associated Kindlin-2 and actin in Kindlin-2^+/- ECs and in HUVECs treated with Kindlin-2-specifc siRNA. Also, upon stimulation of HUVECs with PAF or thrombin, Kindlin-2 along with actin dissociated from VE-cadherin-based complexes, suggesting that Kindlin-2 stabilizes adherens junctions. In direct binding studies with recombinant proteins the SPR sensograms revealed direct interaction of Kindlin-2 with β- and γ-catenin, but not with α-catenin nor the VE-cadherin cytoplasmic tail. In addition, using actin spin-down assays we demonstrated that Kindlin-2 directly interacted with actin filaments and linked them to β- or γ-catenin. Using WT and deletion mutants of Kindlin-2 we mapped the β- and γ-catenin binding site to the F1 and F3 domains in Kindlin-2, while actin binding is very dependent on the F0 domain.

Taken together, these data identify a previously unappreciated function of Kindlin-2. It plays a crucial role in maintaining vascular integrity by linking adherens junctions to actin filaments.