Institute of Physiology, Aulweg 129, 35392 Giessen Germany.

Background: Ischemia-induced capillary leakage leading to myocardial oedema is a severe complication and may jeopardize the outcome of reperfusion. The AMP-activated protein kinase (AMPK) is well known to be a fuel sensor which activated under ischemia. Now it is apparent that AMPK may also play an important role in stabilizing endothelial (EC) barrier function. Here it was tested, whether targeted activation of AMPK at the onset of reperfusion protects against reperfusion-induced endothelial barrier failure. Methods and Results:Downregulation of AMPK by siRNA (~80%) leads to disintegration of VE- cadherin/b-catenin –mediated cell adhesion structures, increased actin stress fiber formation (immunocytochemistry) and gap formation (GF:video-imaging technique) in cultured human umbilical vein endothelial cells. Exposure of EC to ischemia (40 min, Po2<5 mmHg; pH 6.4) caused a 3-fold increase in AMPK activity (AMPK~P: AMPK phosphorylation; Western blot) and GF after 40 min. Reperfusion (40 min,Po2=140 mm Hg; pH 7.4) leads to a further increase in GF by 307±9% (P<0.05, n=5), 3 fold myosin light chain phosphorylation (MLC~P), 2.5 fold phosphorylation of MYPT1 (Mypt1~P), actin stress fiber formation, loss of VE-cadherin/ b-catenin from cell-cell adhesions in 40 min. In contrast, AMPK~P declined to basal level within the first 10 min of reperfusion. Addition of AICAR at the onset of reperfusion enhanced AMPK~P above the end-ischemic level, led to rearrangement of cortical actin, suppressed MLC~P and Mypt1~P, re- establishment of VE-cadherin/b-catenin -mediated cell-cell adhesions, and abolished reperfusion- induced GF. This protective effect of AICAR on all parameters was completely abolished in AMPK- downregulated cells. Conclusions: The results of this study show that activation of AMPK pathway protects against reperfusion-induced endothelial barrier failure via stabilizing adhesion junctions and inactivation of the contractile machinery. Hence, targeting AMPK pathway may be a new therapeutic intervention to prevent ischemia-reperfusion- vascular hyperpermeabilty.