Abstract 625: Cytochrome P450 2C9-induced Angiogenesis Involves the Expression of EphB4 and ephrinB2 in Endothelial Cells
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.