Capillary leakage and edema formation is a complication of hypoxia and/or ischemia, which can compromise the outcome of reperfusion and the recovery of organs. AMP-activated protein kinase (AMPK) is an intracellular energy sensor, which regulates cellular metabolism to maintain the energy homeostasis. Recent studies, however, show that AMPK plays an important role in other physiological functions. Here the hypothesis was addressed whether AMPK is involved in the regulation of endothelial barrier function and a targeted activation of AMPK at the onset of reperfusion can protect against reperfusion-induced endothelial barrier failure. Therefore, the role of AMPK in endothelial barrier function was analyzed in human umbilical vein endothelial cells under physiological and pathophysiological (hypoxiareperfusion) conditions. It was found that the downregulation of AMPKalpha protein or its isoforms (alpha1 and alpha2) with siRNA resulted in a significant increase in basal permeability, disintegration of adherens junctions, and enhanced actin stress fiber formation in cultured endothelial monolayers. Exposure of endothelial cells to hypoxia (60 min, Po2<5 mmHg) led to an increase in interendothelial gap formation, activation of the contractile machinery (MLC and MYPT1 phosphorylation), F-actin stress fiber formation, and loss of VE-cadherin and beta- catenin from cell-cell junctions. These parameters were further aggravated during reperfusion.Moreover, reperfusion also elicited translocation of both AMPKalpha isoforms from cytoplasm to the nucleus. Hypoxia caused a marked increase in AMPK phosphorylation which is associated with activation of the enzyme, however during reperfusion AMPK phosphorylation declined rapidly. Targeted activation of AMPK by the adenosine analog AICAR at the onset of reperfusion reduced interendothelial gap formation. Simultaneously MLC and MYPT1 phosphorylation was abolished, cortical actin rearranged at cell borders, VE-cadherin/beta catenin re-established at cell junctions, and cytonuclear translocation of AMPKalpha isoforms was prevented. This protective effect of AICAR on all parameters was abolished by the AMPK inhibitor Ara-A or by AMPKalpha1/2 siRNA transfection. In isolated reperfused mouse hearts, pharmacological activation of AMPK led to a significant reduction in ischemia-reperfusion induced increase in water content denoting reduction of edema formation. The data of this study show that AMPK is involved in the regulation of endothelial barrier function. Targeted activation at the onset of reperfusion can protect against ischemia/hypoxia-reperfusion induced endothelial barrier failure. Therefore, activation of AMPK may be a promising new therapeutic option to prevent reperfusion-induced endothelial barrier dysfunction.
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