P173	Induction of sprouting inhibitor METH-1 in endothelial cells by shear stress and oxygen tension.
A.Zakrzewicz, M.Bongrazio, J.Knöchel, A.Alter, W.Wunderlich, J.Abraham, A.R.Pries
Charité, Institute of Physiology, Berlin, DE.

Blood vessels grow by two main mechanisms, sprouting and splitting of capillaries. These types of angiogenesis occur during different hemodynamic situations. Sprouting is triggered by tissue hypoxia and creates a no-flow situation inside the capillary sprout while splitting is triggered by an increase in shear stress. Since both types of vessel growth can be induced by the same angiogenic factor VEGF, we teseted the hypothesis, that sprouting inhibitor METH-1 is differentially expressed in endothelial cells by shear stress and oxygen tension. Human umbilical vein endothelial cells (HUVEC) were freshly isolated, cultivated to confluency and exposed to varying shear stress (0,5 dyn/cm2 – 10 dyn/cm2) and oxygen tension (20 mmHg – 150 mmHg). Cells were then analysed for mRNA expression by RT-PCR and real time-PCR. METH-1 mRNA is greately induced by shear stress in a time- and force-dependent manner. Following flow stop, its expression goes back to basic levels again. Shear stress-induced up-regulation of METH-1 can be completely suppressed by inhibition of phospholipase C or NO-production and in part by inhibition of p53. In HUVEC, cultured without flow, METH-1 expression is reduced by lowering oxygen tension. By combinations of shear stress and oxygen tension, the highest expression of METH-1 mRNA was achieved with 10 dyn/cm2 and 100 mmHg oxygen tension, while the lowest expression was observed during flow stop and 20 mmHg oxygen tension. This expression pattern of METH-1 mRNA is in aggreement with the fact, that arteries and collaterals do not sprout and that shear stress-induced angiogenesis occurs by splitting rather than sprouting of capillaries. By the same trigger mechanisms, we would predict a suppression of METH-1 in capillary sprouts from our data. Thus, VEGF effects might be significantly modulated by acopmpanying factors like METH-1.

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