Inflammatory mediators like thrombin causes endothelial barrier failure via activation of contractile machinery due to inhibition of the myosin light chain phosphatase (MLCP) and disturbance of cell-cell contacts. Here the hypothesis was tested that CPI-17, a endogenous inhibitor of the PP1 catalytic subunit of the MLCP, mediates thrombin-induced activation of the contractile apparatus and loss of cell-cell contacts. In cultured human umbilical vein endothelial cell (EC) thrombin (0.2U/ml) increased permeability (albumin flux) by (210±12%), isometric tension (IT; force production of EC cultured on collagen gels) by 130±8%, and phosphorylation of regulatory myosin light chains (MLC~P) by 90±8%. Thrombin increased phosphorylation of CPI-17 by 50±6% and recruitment of CPI-17 to PP1 catalytic subunit (immunoprecipitation) (n = 5, P<0.05, for all further parameters). Depletion of CPI-17 by siRNA lead to a 33±7% reduction in thrombin-induced hyperpermeability and a reduction of MLC~P to the same extent. Moreover, thrombin-induced increase in stress- fibre formation, loss of VE-cadherin, and ß-catenin from cell-cell junctions were also attenuated (confocal microscopy).

Conclusion: CPI-17 is an important mediator of thrombin-induced activation of endothelial contractile machinery and loss of cell- cell contacts leading to barrier failure. CPI-17 may be an important therapeutic target for protection of endothelial barrier function against inflammatory mediators.