Abstract 1305: Epoxyeicosatrienoic Acids Are Part Of The Vascular Endothelial Growth Factor-activated Signaling Cascade In Endothelial Cells Resulting In The Formation Of Fully Functional Vessels
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 act as intracellular signaling molecules activating a number of signaling pathways and promote endothelial cell proliferation, migration and angiogenesis. Since vascular endothelial growth factor (VEGF) is known to play a major role in endothelial cell (EC) proliferation and angiogenesis, we assessed whether or not EETs are implicated in the VEGF-activated signal transduction cascade. Stimulation with VEGF increased CYP2C9 promoter activity in endothelial cells and enhanced CYP2C mRNA and protein expression resulting in increased intracellular EET levels. To clarify whether EETs play a functional role in VEGF-induced angiogenesis, EC cell sprouting was assessed in fibrin as well as in collagen-based spheroid assays. VEGF-induced tube formation in these models was reduced by the EET-antagonist 14,15-epoxyeicosa-5(Z)-enoicacid (14,15-EEZE). To verify the role of EETs in VEGF-induced angiogenesis in vivo and to demonstrate that EET-induced vessels are fully functional, a Matrigel plug assay was performed. CD31-immunostaining revealed increased infiltration of EC in VEGF-impregnated plugs compared to control conditions and this response was inhibited by 14, 15-EEZE. However, while VEGF elicited the formation of EC tubes, these lacked smooth muscle cell/pericyte coating and the EC were not labeled by intravenously injected (tail vein) isolectin. In contrast, vessels that formed in EET-impregnated plugs were perfused and therefore fully functional as demonstrated by an uptake of isolectin. This was further confirmed by the significant accumulation of ultrasound contrast agent compared to control plugs. Moreover, only EC in those plugs containing EETs were also stabilized by smooth muscle cells/pericytes. Taken together, our data indicate that CYP2C-derived EETs not only participate as second messengers in the angiogenic response initiated by VEGF, but that EETs have the potential to influence much more than angiogenesis by enhancing pericyte recruitment to EC tubes to promote vascular maturation.