RhoA regulation of endothelial monolayer integrity.

摘要

The endothelium forms a semi-permeable barrier between the blood (and its constituents) and the outlying tissue. RhoA regulation of the endothelium was investigated in order to establish the critical nature of this monomeric GTPase in mediating the maintenance of a normal endothelial barrier. Thrombin-induced activation of Rho as well as FAK-mediated regulation of Rho GTPase cycling was investigated in several endothelial cell-types.; Thrombin-induced phosphorylation of p115RhoGEF and subsequent activation of Rho was observed in HUVEC within 1 minute following stimulation. Further, inhibition of PKCalpha blocked phosphorylation of p115RhoGEF as well as inhibiting serum response factor, formation of actin stress and thrombin-induced barrier dysfunction.; Additionally, FAK regulation of Rho GTPase cycling was investigated in HPAE cells by competitive inhibition with FRNK (FAK related non-kinase). With this approach, it was shown that inhibition of FAK results in a breakdown of the endothelial barrier, constitutive activation of Rho and its downstream effects, and implicates a possible negative regulatory pathway mediated by FAK signaling through p190RhoGAP to switch Rho to its inactive form (Rho-GDP).; Overall, the findings in this thesis establish the critical nature of regulating RhoA activation in order to maintain a normal endothelial barrier between the blood and outlying tissue. Failure to properly regulate this barrier results in its breakdown which in turn may result in pathological conditions such as pulmonary edema and thrombosis.