N‐cadherin adhesion modulates RhoGTPase signaling to promote endothelial barrier integrity

Abstract

The vascular endothelium is comprised of a monolayer of endothelial cells (ECs) lining the blood vessels, which functions as a semi‐permeable barrier between blood and the surrounding tissue. Vascular Endothelial (VE)‐cadherin, which forms homotypic adhesion between ECs at cell‐cell contacts called adherens junctions (AJs) is the main adhesive molecule that restricts passage of protein rich fluids through the endothelial monolayer. Along with VE‐cadherin, ECs express Neural (N)‐cadherin, which is excluded from EC‐EC junctions and instead forms heterotypic adhesions between ECs and mural cells (such as pericytes or smooth muscle cells) at the abluminal surface of the endothelium. While the role of VE‐cadherin adhesion in regulating endothelial barrier function has been well established, the role of N‐cadherin in ECs remains mainly unknown. Recent evidence suggests that pericytes are critical to maintaining proper endothelial barrier function, however it is still unclear if this is solely due to pericyte recruitment to the endothelium or if N‐cadherin adhesion mediated signaling in ECs plays a role in strengthening the endothelial barrier. Using an EC specific, inducible knockout mouse model developed in our lab, we have shown that deletion of Cdh2 gene (N‐cadherin) results in increased permeability of pulmonary endothelial microvessels, suggesting that N‐cadherin adhesion restricts permeability in the endothelial barrier. To study N‐cadherin adhesion‐mediated signaling in endothelial monolayers, we have utilized surface chemistry techniques to mimic N‐cadherin homophilic interaction in vitro. Covalent attachment of N‐cadherin extracellular domain to an Ni‐NTA surface in an oriented manner induced formation of N‐cadherin adhesions proximal to tricellular junctions and promoted recruitment of VE‐cadherin to AJs. Depletion of N‐cadherin in ECs or denaturation of N‐cadherin extracellular domain abolished this effect, suggesting that N‐cadherin “outside‐in” signaling promotes assembly of VE‐cadherin adhesion. Using microscale biomimetic platforms combined with mass spectroscopy, we isolated and analyzed N‐cadherin complexes. We found that a dual Rac1 and RhoA activator, Trio, is recruited to N‐cadherin adhesion complexes to activate both Rac1 and RhoA signaling at AJs. This was consistent with increased acto‐myosin tension across VE‐cadherin analyzed with a FRET‐based biosensor as well as formation of lamellipodia protrusions above AJs. Furthermore, inhibition of ROCK signaling or depletion of Neuronal navigator 1 (Nav1) abolished recruitment of VE‐cadherin to AJs specifically in cells plated onto N‐cadherin but not gelatin‐coated surfaces. These data cumulatively demonstrate that N‐cadherin promotes assembly of VE‐cadherin adhesion by providing spatial control of RhoA and Rac1 activities through activation of Trio.Support or Funding InformationNIH R01 HL103922