VE‐cadherin restoration does not rescue barrier function in endothelial monolayers depleted of p120

We previously demonstrated that the association of p120‐catenin (p120) with the juxtamembrane domain (JMD) of vascular endothelial (VE)‐cadherin is required to maintain barrier function of endothelial cell monolayers. Specifically, overexpression of the JMD resulted in increased monolayer permeability, however levels of both VE‐cadherin and p120 were decreased. In order to distinguish if decreased barrier function was due to a loss of p120 and not VE‐cadherin, we established a system in which p120 was depleted by shRNA delivered by lentivirus and VE‐cadherin was restored via adenoviral delivery of VE‐cadherin fused to red fluorescent protein (RFP). Loss of p120 resulted in decreased barrier function as assessed by changed in trans‐endothelial resistance (TEER), which was associated with decreased expression of VE‐cadherin, β‐catenin, and α‐catenin. Decreased TEER was rescued by restoration of p120 but not by expression of VE‐cadherin‐RFP, despite localization of VE‐cadherin‐RFP at cell‐cell borders. Expression of VE‐cadherin‐RFP restored levels of β‐catenin and α‐catenin, implicating that a complex of VE‐cadherin, β‐catenin, and α‐catenin requires p120 to form a restrictive barrier. These results demonstrate that p120 is required for maintaining endothelial barrier function, and experiments are underway to determine the mechanism in which p120 maintains endothelial monolayer integrity. RO1‐HL77870