The vascular endothelium forms a semi-permeable barrier between blood and interstitium controlling the exchange of water, small solutes, ions, macromolecules and blood cells across the vessel wall. Loss of EC barrier function results in leakage of blood components to interstitium and finally life threatening edema formation during pathophysiological conditions like inflammation and ischemia-reperfusion. Thus, restoration of EC barrier function is important to regain vascular barrier integrity and to prevent edema formation. However, little is known about the mediators and mechanisms involved in the recovery of compromised EC barrier function.The maintenance of EC barrier integrity is highly dependent on the VE-cadherin-based AJs and actomyosin-based EC contractile machinery. It is now well-established that the disruption of endothelial AJs and activation of EC contractile machinery are the key steps leading towards EC barrier failure under hyper-permeable conditions, a typical EC response to inflammatory mediators such as thrombin.Insulin is an essential hormone and a key regulator of metabolism. Additionally, it also confers protective effects on the cardiovascular system. Inflammatory mediators like thrombin disrupts EC barrier function, which is recovered slowly. Here, the hypothesis was addressed whether insulin can mediate a faster restoration of EC barrier function and the underlying signaling mechanism by which insulin recovers the EC barrier function was elucidated.Our data demonstrate that thrombin-induced hyperpermeability of HUVEC monolayers has accelerated recovery in the presence of insulin in a concentration-dependent manner with maximal effect at 1 IU/ml of insulin concentration. This barrier restoration effect of insulin in response to thrombin-induced hyperpermeability was blocked by a specific insulin receptor antagonist. Insulin also abolished reperfusion-induced vascular leakage in isolated-saline perfused rat hearts. Insulin-induced a rapid increase in Akt phosphorylation in HUVECs. Accordingly, inhibition of PI3K with wortmannin completely abolished the insulin-mediated EC barrier restoration, further supporting that the EC barrier recovery function of Insulin is via activation of PI3K/Akt pathway. However, MLCK and eNOS/NO signaling pathways are not involved ininsulin-mediated recovery of EC barrier function. Insulin attenuated thrombin-induced increase in MLC and MYPT1 phosphorylation and also fastens the re-establishment of VE-cadherin dependent endothelial AJs in response to thrombin challenge indicating a contractile inactivation and stabilization of cell-cell adhesion structures. Remarkably, inhibition of Rock has additive effect on insulin-mediated accelerated restoration of failed barrier. Insulin activated Rho GTPase Rac1 and pharmacological inhibition of Rac1 activity by using a specific inhibitor (NSC23766) abrogated the EC barrier recovery effect of insulin on cultured EC monolayers, as well as on reperfusion-induced vascular leakage in intact isolated-saline perfused rat heart, suggesting a Rac1-dependent phenomenon.In conclusion, insulin accelerates restoration of EC barrier function in response to thrombin-induced EC barrier disruption via enhancement of cell-cell adhesion structures and inactivation of the EC contractile machinery. Insulin also abolished reperfusion-induced vascular leakage in isolated saline perfused rat hearts. These EC barrier restoration effects of insulin are mediated via PI3K/Akt and Rho GTPase-Rac1 activation which plays a decisive role in insulin-mediated recovery of EC barrier function both in HUVEC monolayers and in isolated perfused rat hearts.
Verknüpfung zu Publikationen oder weiteren Datensätzen
