Role of CPI-17 in ischemia-reperfusion induced barrier failure
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Failure of endothelial barrier leading to capillary leakage and edema formation during reperfusion represents major impediment for the recovery of an organ. The endothelial barrier function is maintained by equilibrium of competing contractile and adhesive forces, generated by the actomyosin cytoskeleton and adherens junction proteins, respectively. The activity of the endothelial contractile machinery is regulated by the phosphorylation state of the myosin light chain (MLC), the activity of which is regulated by activities of MLC-kinase (MLCK) and MLC-phosphatase (MLCP). In the present study, the molecular mechanism of ischemia-reperfusion-induced activation of the contractile machinery was analyzed, focusing on the involvement of protein kinase C (PKC) and Rho-associated kinase (ROCK)-mediated inhibition of MLCP. Furthermore, rearrangement of the actin cytoskeleton, loss of VE-cadherin, and ß-catenin from cell-cell adhesions were analyzed during ischemia-reperfusion. Exposure of endothelial cells to severe ischemia (Po2 < 1 mmHg, pH 6.4) for 45 min, followed by 45 min of reperfusion, caused an increase in macromolecule permeability and phosphorylation of MLC, myosin-targeting subunit of MLCP (MYPT1), and C-kinase potentiated protein phosphatase-1 inhibitor (CPI-17). ML-7 (10 µM), a pharmacological inhibitor of MLCK, partially reduced ischemia-reperfusion-induced MLC phosphorylation and hyperpermeability. But also PKC inhibitor bisindolylmaleimide (8 µM) and Rho kinase inhibitor Y-27632 (20 µM) reduced ischemia-reperfusion-induced barrier dysfunction and phosphorylation of MLC, MYPT1, and CPI-17. Downregulation of CPI-17 by siRNA significantly abolished the ischemia-reperfusion-induced increase in permeability and led to actin cytoskeleton rearrangement and restoration of VE-cadherin at cell-cell adhesions.The data of the present study show for the first time that under ischemia-reperfusion CPI-17 is activated, it stimulates the contractile machinery and induces distortion of adherens junction proteins. These effects are prevented by downregulation of CPI-17. The development of therapeutic strategies targeting CPI-17 provides a new option to protect against ischemia-reperfusion-induced barrier failure.Verknüpfung zu Publikationen oder weiteren Datensätzen
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Giessen : VVB Laufersweiler