Induction Of MAPK Phosphatase-1 By Mild Hypothermia Inhibits TNF-α-induced Endothelial Barrier Dysfunction And Apoptosis

Aims:Hypothermia therapy has been shown to confer robust protection against numerous diseases inclusing stroke, brain injury, and cardiac arrest, and vascular endothelial cells are involved in stress-induced pathogenesis under insult conditions. However, the mechanisms underlying protective effect of mild hypothermia on endothelial cells have not yet been completely elucidated. Here, we investigated molecular effects of mild hypothermia on tumor necrosis factor-alpha (TNF-α)-induced endothelial barrier dysfunction and apoptosis.Methods:Human umbilical vein endothelial cells (HUVECs) treated with TNF-a were incubated under normothermia (37℃) or mild hypothermia (33℃). Endothelial permeability was detemined by fluorescein-isothiocyanate-labeled bovine serum albumin (FITC-BSA); actin cytoskeleton alterations was stained and tested with rhodamine-phalloidin; apoptosis was examined by terminal dUTP nick-end labeling (TUNEL) assay; the protein levels were determined by immunoblot. Then, the cells were transfected with MKP-1 siRNA, and were treated with TNF-α. The former detections were also performed.Results:(1) Treatment of HUVECs with TNF-αresulted in a significant increase of nomo-endothelial permeability, actin reorganization and apoptosis. Mild hypothermia markedly attenuated TNF-α-induced these effects.(2) Mild hypothermia inhibits TNF-α-induced activation of p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways.(3) The inhibitory action of mild hypothermia on stress fiber formation was mediated via inactivation of p38 MAPK/heat shock protein 27 (HSP27).(4) The decrease in TNF-α-induced apoptosis by mild hypothermia was associated with inhibition of p38 MAPK and JNK activity.(5) Mild hypothermia had no action on p38 MAPK and JNK upstream kinases MAPK kinase 3/6 (MKK3/6) and MAPK kinase 7 (MKK7), but markedly induced the expression of MAPK phosphatase-1 (MKP-1). (6) siRNA experiments showed that MKP-1 was an important mediator of mild hypothermia in attenuatting TNF-α-induced activation of p38 MAPK and JNK, endothelial barrier dysfunction, and apoptosis in HUVECs.Conclusion:These results for the first time demonstrate that mild hypothermia protects against TNF-α-induced endothelial barrier dysfunction and apoptosis through MKP-1-dependent mechanism.