Endothelial cells are constantly exposed to high or low shear stress in arteries and veins by the flowing blood. Angiopoietin-2 (Ang-2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. In this study, flow-dependent regulation of Ang-2 was analyzed in vitro and in vivo. Ang-2 mRNA, protein expression and release was upregulated by 24 h of low (1 dyne/cm(2)), but downregulated by high flow (30 dyne/cm(2)) in human endothelial cells. Increased endothelial NO synthase expression and NO formation was not affecting regulation of Ang-2 by low or high flow. Low and high flow increased VEGF-A expression. Inhibition of VEGFR-2 prevented upregulation of Ang-2 by low flow, but not downregulation of Ang-2 by high flow. Furthermore, upregulation of Ang-2 by VEGF was reduced by application of high flow. Forkhead box O (FOXO) transcription factor FOXO1 has been shown to regulate Ang-2 expression in endothelial cells. FOXO1 binding activity was reduced by high flow. Nuclear localization of transcription factor FOXO1 was not changed by low flow, but reduced by high flow. In vivo, Ang-2 was higher expressed in veins compared to arteries. Arterial ligation augmented Ang-2 expression in distal arterial low flow areas. Our results support a VEGF-dependent induction of Ang-2 in low flow areas, and FOXO1-dependent downregulation of Ang-2 in high flow areas. These data suggest a new mechanism of flow-dependent regulation of vessel stability and differentiation.

Background: Endothelial cells are constantly exposed to high or low shear stress in arteries and veins by the flowing blood. Angiopoietin-2 (Ang-2) is acting as a critical regulator of vessel maturation and endothelial cell quiescence. In this study, we determined the effect of low and high laminar shear stress on the expression and release of angiopoietin (Ang-)2 in human endothelial cells, studied the role of nitric oxide and protein kinases in this context, determined the impact of VEGF on Ang-2, studied the expression, activity and translocation of the forkhead box O transcription factor FOXO1 by low and high shear stress, and analyzed the vessel- and flow-dependent expression of Ang-2 in vivo . Methods and Results: Primary cultures of human umbilical vein endothelial cells (HUVEC) were subjected to laminar shear stress at different physiological levels of laminar shear stress of 1, 5, 10, 15, and 30 dyne/cm 2 in a cone-and-plate viscometer. Ang-2 mRNA, protein expression and release was upregulated by 24 h of low (1 dyne/cm 2 ), but downregulated by high flow (30 dyne/cm 2 ) in human endothelial cells. Increased endothelial NO synthase expression and NO formation was not affecting regulation of Ang-2 by low or high flow. Tie2 protein expression, but not Tie2 phosphorylation was induced by high flow. Furthermore, low and high flow increased VEGF-A expression. Inhibition of VEGFR-2 prevented upregulation of Ang-2 by low flow, but not downregulation of Ang-2 by high flow. Upregulation of Ang-2 by VEGF was reduced by application of high flow. Forkhead box O (FOXO) transcription factor FOXO1 has been shown to regulate Ang-2 expression in endothelial cells. FOXO1 binding activity was reduced by high flow. Nuclear localization of transcription factor FOXO1 was not changed by low flow, but reduced by high flow. In vivo , Ang-2 was higher expressed in veins compared to arteries. Arterial ligation augmented Ang-2 expression in distal arterial low flow areas. Conclusion: Our results support a VEGF-dependent induction of Ang-2 in low flow areas, and FOXO1-dependent downregulation of Ang-2 in high flow areas. These data suggest a new mechanism of flow-dependent regulation of vessel stability and differentiation.