Characterization of 5,6- and 8,9-epoxyeicosatrienoic acids (5,6- and 8,9-EET) as potent in vivo angiogenic lipids.

The cytochrome P450 arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs) are powerful, nonregioselective, stimulators of cell proliferation. In this study we compared the ability of the four EETs (5,6-, 8,9-, 11,12-, and 14,15-EETs) to regulate endothelial cell proliferation in vitro and angiogenesis in vivo and determined the molecular mechanism by which EETs control these events. Inhibition of the epoxygenase blocked serum-induced endothelial cell proliferation, and exogenously added EETs rescued cell proliferation from epoxygenase inhibition. Studies with selective ERK, p38 MAPK, or PI3K inhibitors revealed that whereas activation of p38 MAPK is required for the proliferative responses to 8,9- and 11,12-EET, activation of PI3K is necessary for the cell proliferation induced by 5,6- and 14,15-EET. Among the four EETs, only 5,6- and 8,9-EET are capable of promoting endothelial cell migration and the formation of capillary-like structures, events that are dependent on EET-mediated activation of ERK and PI3K. Using subcutaneous sponge models, we showed that 5,6- and 8,9-EET are pro-angiogenic in mice and that their neo-vascularization effects are enhanced by the co-administration of an inhibitor of EET enzymatic hydration, presumably because of reduced EET metabolism and inactivation. These studies identify 5,6- and 8,9-EET as powerful and selective angiogenic lipids, provide a functional link between the EET proliferative chemotactic properties and their angiogenic activity, and suggest a physiological role for them in angiogenesis and de novo vascularization.