Cytochrome P-450 (CYP) epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acid (EET) regioisomers, which activate several signaling pathways to promote endothelial cell proliferation, migration, and angiogenesis. Since vascular endothelial growth factor (VEGF) plays a key role in angiogenesis, we assessed a possible role of EETs in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C promoter activity in endothelial cells and enhanced CYP2C8 mRNA and protein expression resulting in increased intracellular EET levels. VEGF-induced endothelial cell tube formation was inhibited by the EET antagonist 14,15-epoxyeicosa-5( Z)-enoicacid (14,15-EEZE), which did not affect the VEGF-induced phosphorylation of its receptor or basic fibroblast growth factor (bFGF)-stimulated tube formation. Moreover, VEGF-stimulated endothelial cell sprouting in a modified spheroid assay was reduced by CYP2C antisense oligonucleotides. Mechanistically, VEGF stimulated the phosphorylation of the AMP-activated protein kinase (AMPK), which has also been linked to CYP induction, and the overexpression of a constitutively active AMPK mutant increased CYP2C expression. On the other hand, a dominant-negative AMPK mutant prevented the VEGF-induced increase in CYP2C RNA and protein expression in human endothelial cells. In vivo (Matrigel plug assay) in mice, endothelial cells were recruited into VEGF-impregnated plugs; an effect that was sensitive to 14,15-EEZE and the inclusion of small interfering RNA directed against the AMPK. The EET antagonist did not affect responses observed in plugs containing bFGF. Taken together, our data indicate that CYP2C-derived EETs participate as second messengers in the angiogenic response initiated by VEGF and that preventing the increase in CYP expression curtails the angiogenic response to VEGF.

Objective— Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) are known to stimulate angiogenesis, but the mechanisms involved are incompletely understood. Because EphB4 is involved in vascular development, the aim of this study was to investigate whether, and to what extent, EphB4 is part of the signaling cascade that results in CYP2C9-mediated angiogenesis. Methods and Results— CYP2C9 overexpression as well as stimulation with 11,12-EET (up to 48 hours) time-dependently increased EphB4 expression in endothelial cells. This effect and the activation of the EphB4 promoter were mediated by the phosphatidylinositol-3-kinase (P13-K)/Akt pathway and sensitive to the P13-K inhibitor LY 294002 as well as to simultaneous transfection with dominant-negative Akt. 11,12-EET treatment also increased EphB4 expression in isolated mouse mesenteric arteries as well as in the vessels that developed in 11,12-EET-impregnated Matrigel plugs. Moreover, the CYP2C9-stimulated formation of capillary-like structures in a modified spheroid assay was markedly attenuated by EphB4 downregulation (antisense oligonucleotides). Using a parallel approach in vivo, the inclusion of siRNA directed against EphB4 in EET-impregnated Matrigel plugs prevented endothelial cell invasion and vascularization. Conclusions— Our data indicate that EphB4 is a critical component of the CYP2C9- and 11,12-EET-activated signaling cascade that promotes angiogenesis in vitro as well as in vivo.

Cytochrome P450 (CYP) epoxygenases of the 2C family metabolize arachidonic acid to various epoxyeicosatrienoic acid (EET) regioisomers, which play an important role in the regulation of vascular tone and homeostasis. EETs also promote angiogenesis, but the mechanisms involved are not completely understood. EphB4 and its ligand ephrinB2 are known to play a major role as neuronal guidance molecules, but are also involved in angiogenesis and vasculogenesis. Given this pro-angiogenic role we set out to investigate whether EETs modulate the expression of EphB4 and/or ephrinB2 . The overexpression of CYP2C9 in endothelial cells (EC) increased EphB4 expression over 72 hours to a level approximately 4-fold that detected in cells infected with a control (CYP2C9 antisense) adenovirus. This effect was sensitive to the epoxygenase inhibitor MSPPOH. CYP2C9 overexpression also resulted in an increase in expression of the EphB4-ligand ephrinB2 as determined Western blot analysis. When cells were stimulated with 11,12-EET (1μM, up to 48 hours) EphB4 expression increased in a time-dependent manner. Moreover endogenous EphB4 expression was down-regulated in cells transfected with CYP2C9 antisense oligonucleotides. In order to demonstrate that CYP2C9 metabolites activate transcription of EphB4 we assessed the consequences of CYP overexpression on the activity of a luciferase-coupled EphB4 promoter construct. Simultaneous transfection of EC with CYP2C9 and the EphB4 promoter resulted in a significant increase in luciferase activity compared to that observed in cells transfected with a control virus and the EphB4 promoter. To clarify whether EphB4 is a critical determinant of CYP2C9-induced angiogenesis, EC sprouting was assessed using a collagen gel-based in vitro angiogenesis assay. Following transfection with EphB4 antisense or scrambled oligonucleotides, capillary-like structures were clearly present after 24 hours in cells overexpressing CYP2C9, while EphB4 downregulation abolished CYP2C9-induced sprouting. Taken together, our data indicate that EphB4 is a component of the CYP2C9- and EET-activated signaling cascade that promotes angiogenesis.