Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6 dyn/cm 2 , 24 h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.

ADAMTS1 inhibits capillary sprouting, and since capillary sprouts do not experience the shear stress caused by blood flow, this study undertook to clarify the relationship between shear stress and ADAMTS1. It was found that endothelial cells exposed to shear stress displayed a strong upregulation of ADAMTS1, dependent upon both the magnitude and duration of their exposure. Investigation of the underlying pathways demonstrated involvement of phospholipase C, phosphoinositide 3-kinase, and nitric oxide. Forkhead box protein O1 was identified as a likely inhibitor of the system, as its knockdown was followed by a slight increase in ADAMTS1 expression. In silico prediction displayed a transcriptional binding site for Forkhead box protein O1 in the promotor region of the ADAMTS1 gene, as well as sites for nuclear factor 1, SP1, and AP-1. The anti-angiogenic effects of ADAMTS1 were attributed to its cleavage of thrombospondin 1 into a 70-kDa fragment, and a significant enhancement of this fragment was indeed demonstrated by immunoblotting shear stress-treated cells. Accordingly, scratch wound closure displayed a slowdown in conditioned medium from shear stress-treated endothelial cells, an effect that could be completely blocked by a knockdown of thrombospondin 1 and partially blocked by a knockdown of ADAMTS1. Non-perfused capillary sprouts in rat mesenteries stained negative for ADAMTS1, while vessels in the microcirculation that had already experienced blood flow yielded the opposite results. The shear stress-dependent expression of ADAMTS1 in vitro was therefore also demonstrated in vivo and thereby confirmed as a mechanism connecting blood flow with the regulation of angiogenesis.

No abstract

While vascular endothelial cells are normally exposed to shear stress, there is no flow and consequently reduced shear stress in capillary sprouts. To explore, if this lack in mechano‐stimulation alters the endothelial phenotype, we analysed the influence of shear stress on the activity of transcription factor FOXO1 and the expression of its target gene angiopoietin‐2 (ang2), an antagonist of the endothelial tyrosine kinase receptor tie2.Endothelial cells were exposed to shear stress using a cone‐and‐plate system with or without inhibition of PI3K (using LY294,002) or FOXO1 (using siRNA) and analysed by real time RT‐PCR, Western blots, immunoprecipitation, and fluorescence microscopy.Shear stress (6°dyn/cm2) increased phosphorylation of Akt and FOXO1 in a PI3K‐dependent manner and FOXO1 was translocated out of the nucleus. In addition, expression of FOXO1 on the mRNA‐ and protein level was strongly reduced by shear stress. Suppression of FOXO1 by shear stress was accompanied by a decrease of ang2‐mRNA and ‐protein, which was similarly be achieved by siRNA against FOXO1. Following flow stop, ang2‐mRNA expression increased within two hours to steady state values under static conditions.So, in capillary sprouts, lacking shear stress activates endothelial FOXO1 by which ang2 is rapidly induced. Thus, the tie2‐system will be inactivated during capillary sprouting, which may turn the vessel wall more susceptible to additional angiogenic factors. By contrast, the onset of flow will activate the tie2‐system and thereby contribute to stabilize the vessel wall.