Abstract
Cytochrome P450 arachidonic acid (AA) epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), dilate arteries via hyperpolarization of smooth muscle cells and also have nonvasodilatory effects within the vasculature. The present study investigated the angiogenic effects of endogenous and exogenous EETs and the relevant signaling mechanisms involved. Bovine aortic endothelial cells (BAECs) were incubated with synthetic EETs or infected with recombinant adeno-associated viruses (rAAVs) containing CYP2C11-NADPH-cytochrome P450 oxidoreductase (CYPOR), CYP2J2, or CYP102 F87V mutant to increase endogenous levels of EETs. The following endpoints were measured: BAEC proliferation, migration, capillary formation, and in vivo angiogenesis. The potential involvement of various signaling pathways was explored using selective inhibitors. The results showed that transfection with either rAAV-CYP2C11-CYPOR, rAAV-CYP2J2, or rAAV-CYP102 F87V, or incubation with EETs promoted BAEC proliferation, increased migration of BAECs as assessed by Transwell analysis and wound healing assays, and enhanced capillary tubule formation as determined by chicken embryo chorioallantoic membrane assays and tube formation tests on matrigel. The effects of EETs on proliferation, migration, and capillary tubule formation were attenuated by inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 (PI3)-kinase/Akt pathways and partially attenuated by an endothelial nitricoxide synthase (eNOS) inhibitor but not by a protein kinase C inhibitor. In a rat ischemic hind limb model, rAAV-mediated AA epoxygenase transfection induced angiogenesis. We conclude that AA epoxygenase metabolites can promote angiogenesis, which may provide protection to ischemic tissues. The results also suggest that the angiogenic effects of EETs involve the MAPK and PI3-kinase/Akt signaling pathways, and to some extent, the eNOS pathway.
Footnotes
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This work was supported by Grants 30430320, 30270561, and 30340067 from the National Nature Science Foundation Committee of China, National 973 Project (G2000056901), and the National Institute of Environmental Health Sciences Division of Intramural Research.
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doi:10.1124/jpet.105.083477.
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ABBREVIATIONS: AA, arachidonic acid; P450, cytochrome P450; EET, epoxyeicosatrienoic acid; eNOS, endothelial nitric-oxide synthase; ERK, extracellular signal-regulated kinase; PKC, protein kinase C; PI3, phosphatidylinositol 3; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PD98059, 2-amino-3-methoxyflavone; H-7, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine; MAPK, mitogen-activated protein kinase; EGFR, epidermal growth factor receptor; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; 17-ODYA, 17-octadecynoic acid; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; rAAV, recombinant adeno-associated virus; GFP, green fluorescent protein; CYPOR, NADPH-cytochrome P450 oxidoreductase; BAEC, bovine aortic endothelial cell; PBS, phosphate-buffered saline; HPF, high-powered field; CAM, chicken embryo chorioallantoic membrane; MEK, mitogen-activated protein kinase kinase; l-NMMA, NG-monomethyl-l-arginine; VEGF, vascular endothelial growth factor.
- Received January 13, 2005.
- Accepted April 15, 2005.
- The American Society for Pharmacology and Experimental Therapeutics