Characterization of 5,6- and 8,9-Epoxyeicosatrienoic Acids (5,6- and 8,9-EET) as Potent in Vivo Angiogenic Lipids

Ambra Pozzi,Ines Macias-Perez,Tristin Abair,Shouzuo Wei,Yan Su,Roy Zent,John R Falck,Jorge H Capdevila

doi:10.1074/jbc.m501730200

Abstract

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.

Highlights

Interest in the biochemical mechanism of angiogenesis, the de novo formation of blood vessels from pre-existing vessels, stems from the critical roles played by this process in the pathophysiology of inflammation, cancer, and cardiovascular
Since the original description of the mitogenic properties of 14,15-epoxyeicosatrienoic acids (EET) [12, 19], several studies have characterized 11,12and 14,15-EET as powerful mitogens using cultured cells derived from kidney, brain, and endothelium (4,8 –12,18 –21), and a role of 14,15-EET in mediating the mitogenic responses of EGF and HB-EGF has been thoroughly documented in cultured LLCPk cells [20, 21]
We report here (i) the characterization of 5,6-and 8,9-EET as powerful mitogens, and selective chemotactic lipids for primary cultures of mouse pulmonary endothelial cells; (ii) the identification of the intracellular signaling pathways associated with their proliferative and chemotactic activities; and (iii) the demonstration of a role for these two EETs in promoting de novo angiogenesis in vivo

Summary

Introduction

Interest in the biochemical mechanism of angiogenesis, the de novo formation of blood vessels from pre-existing vessels, stems from the critical roles played by this process in the pathophysiology of inflammation, cancer, and cardiovascular. To study cellular EET synthase activity, semiconfluent endothelial cells (passage 3) were cultured in serum-free medium in the presence or absence of arachidonic acid (10 ␮M).

Results

Conclusion