P133	Epoxyeicosatrienoic acids act as second messengers in VEGF-induced angiogenesis.
1R.Michaelis, 1B.Fisslthaler, 2J.R.Falck, 1I.Fleming, 1R.Busse
1Institut für Kardiovaskuläre Physiologie, J.W.Goethe Universität, Frankfurt/Main, DE; 2Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, US.

Cytochrome P450 (CYP) epoxygenases of the 2C family metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs), which are now recognized as intracellular signaling molecules involved in endothelial cell proliferation and angiogenesis. As the expression of CYP 2C enzymes, like that of the vascular endothelial growth factor (VEGF) can be enhanced by hypoxia we determined whether EETs may act as second messengers in the VEGF signaling pathway. Stimulation of human umbilical vein endothelial cells with VEGF induced CYP 2C (RNA and protein) expression by approximately 2-fold. This response was even more pronounced in bovine retina endothelial cells. Moreover, an increase in CYP activity was also able to affect specific components of the VEGF pathway and a gene array revealed an increase in VEGF receptor 2 mRNA levels in endothelial cells overexpressing CYP 2C9. RT-PCR confirmed these results and the CYP 2C9-induced increase in VEGF receptor 2 expression could be inhibited by the CYP 2C9 inhibitor sulfaphenazole as well as by the EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Functional evidence for a role of EETs in VEGF-induced angiogenesis was obtained in a fibrin gel assay in which 14,15-EEZE reduced VEGF-induced tube formation by approximately 30%, without affecting tube formation induced by basic fibroblast growth factor (bFGF) or epidermal growth factor. VEGF-induced angiogenesis in mice in vivo (Matrigel plug assay) was also abolished by 14,15-EEZE while the response to bFGF was not affected. Taken together, these data indicate, that CYP 2C-derived EETs act as second messengers in the VEGF signaling pathway that promotes angiogenesis but not in the angiogenic signaling cascade activated by other growth factors.

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