Abstract
To determine the efficacy of cytochrome P450 2C9 metabolites of arachidonic acid, viz. 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs), in inducing angiogenesis, we have studied their effects on human dermal microvascular endothelial cell (HDMVEC) tube formation and migration. All four EETs stimulated HDMVEC tube formation and migration in a dose-dependent manner. Because 14,15-EET was found to be slightly more efficacious than 5,6-, 8,9-, and 11,12-EETs in stimulating HDMVEC tube formation and migration, we next focused on elucidation of the signaling mechanisms underlying its angiogenic activity. 14,15-EET stimulated Akt and S6K1 phosphorylation in Src- and phosphatidylinositol 3-kinase (PI3K)-dependent manner in HDMVECs. Inhibition of Src and PI3K-Akt-mTOR signaling by both pharmacological and dominant-negative mutant approaches suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. In addition, 14,15-EET induced the expression of fibroblast growth factor-2 (FGF-2) in Src- and PI3K-Akt-dependent and mTOR-independent manner in HDMVECs. Neutralizing anti-FGF-2 antibodies completely suppressed 14,15-EET-induced HDMVEC tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. Together, these results show for the first time that Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 activation plays an indispensable role in 14,15-EET-induced angiogenesis.
Highlights
Ulation of important biological activities such as the maintenance of vascular tone [5, 6]
Quiescent human dermal microvascular endothelial cell (HDMVEC) were added to the upper chamber of the culture inserts that were placed in a 24-well plate, and vehicle or various concentrations of the indicated epoxyeicosatrienoic acids (EETs) were added to the lower chamber
Because 14,15-EET appears to be slightly more efficacious than 5,6, 8,9, and 11,12-EETs in stimulating both HDMVEC tube formation and migration, we focused on investigating the mechanisms of its angiogenic effects
Summary
Ulation of important biological activities such as the maintenance of vascular tone [5, 6] These lipid molecules have been reported to mediate various intracellular signaling events in response to various external cues [7,8,9,10,11]. We report that 14,15-EET, the CYP2C9 metabolite of arachidonic acid, stimulates human dermal microvascular endothelial cell (HDMVEC) tube formation and migration in vitro and Matrigel plug angiogenesis in vivo. We present evidence that 14,15-EET-induced HDMVEC tube formation and migration and Matrigel plug angiogenesis require activation of Src and PI3K-Akt signaling via targeting in parallel with FGF-2 expression and mTOR-S6K1 stimulation
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