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Aims: Phosphorylation of forkhead box O (FoxO) transcription factors induces their nuclear exclusion and proteosomal degradation. Here, we investigated the effect of fluid shear stress on FoxO1a in primary cultures of human endothelial cells and the kinases that regulate its phosphorylation., Methods and Results: Shear stress (12 dynes/cm2) elicited the phosphorylation, nuclear exclusion, and degradation of FoxO1a. Inhibition of Akt signalling using either a dominant negative (DN) mutant of Akt or downregulation of Gab1 largely failed to affect the shear stress-induced changes in FoxO1a, while a DN-AMP-activated protein kinase (AMPK) abrogated its shear stress-induced phosphorylation and degradation. Similar effects were observed using the AMPK inhibitor compound C. Moreover, in an in vitro assay, the AMPK directly phosphorylated FoxO1a. As FoxO1a regulates the expression of angiopoietin-2 (Ang-2), we determined the role of shear stress and the AMPK in this phenomenon. Not only did the DN-AMPK increase the expression of Ang-2 in cells maintained under static conditions, it also abrogated the shear stress-induced decrease in FoxO1a and Ang-2 protein levels. Functionally, Ang-2 sensitizes endothelial cells to the effects of tumour necrosis factor (TNF)-alpha, and DN-AMPK increased basal endothelial cell E-selectin expression and permeability as well as the increase induced by TNF-alpha., Conclusion: These data indicate that the AMPK activated by fluid shear stress is a novel regulator of FoxO1a phosphorylation and protein levels. Moreover, as the AMPK-dependent phosphorylation and degradation of FoxO1a attenuates Ang-2 expression and protects against the pro-inflammatory actions of TNF-alpha, this kinase may be a useful target to prevent the progression of vascular diseases.