Abstract
20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the ω-hydroxylation of arachidonic acid by cytochrome P450 4A and 4F enzymes, and it induces angiogenic responses in vivo. To test the hypothesis that 20-HETE increases endothelial cell (EC) proliferation via vascular endothelial growth factor (VEGF), we studied the effects of WIT003 [20-hydroxyeicosa-5(Z),14(Z)-dienoic acid], a 20-HETE analog on human macrovascular or microvascular EC. WIT003, as well as pure 20-HETE, stimulated EC proliferation by ∼40%. These proliferative effects were accompanied by increased VEGF expression and release that were observed as early as 4 h after 20-HETE agonist addition. This was accompanied by increased phosphorylation of the VEGF receptor 2. The proliferative effects of 20-HETE were markedly inhibited by a VEGF-neutralizing antibody. Polyethylene glycol-superoxide dismutase (PEG-SOD) markedly inhibited both the increases in VEGF expression and the proliferative effects of 20-HETE. In contrast, administration of the NAD(P)H oxidase inhibitor apocynin had no effect to the proliferative response to 20-HETE. The 20-HETE agonist markedly increased superoxide formation as reflected by an increase in dihydroethidium staining of EC, and this increase was inhibited by PEG-SOD but not by apocynin. 20-HETE also increased the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in EC, whereas an inhibitor of MAPK [U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene] suppressed the proliferative and the VEGF changes but not the pro-oxidant effects of 20-HETE. These data suggest that 20-HETE stimulates superoxide formation by pathways other than apocynin-sensitive NAD(P)H oxidase, thereby activating MAPK and then enhancing VEGF synthesis that drives EC proliferation. Thus, 20-HETE may be involved in the regulation of EC functions, such as angiogenesis.
Footnotes
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This work was supported by National Institutes of Health Grants EY014385 (to A.G.S.), GM31278 (to J.R.F.), and HL 036279 (to R.J.R.) and by a grant from the Robert A. Welch Foundation (to J.R.F.). This work was presented in part: Guo M, Roman R, Falck J, Chen P, and Scicli AG (2006) 20-HETE-induced proliferation of endothelial cells is mediated by activation of VEGF receptors, in The 8th Annual Winter Eicosanoid Conference; 2006 March 12–15; Baltimore, MD. New York Medical College, Valhalla, NY.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.106.115360.
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ABBREVIATIONS: 20-HETE, 20-hydroxyeicosatetraenoic acid; WIT003, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid; HUVEC, human umbilical vascular endothelial cells; HDMVEC, human dermal microvascular endothelial cells; PEG-SOD, polyethylene glycol-superoxide dismutase; DPI, diphenylene iodonium; DHE, dihydroethidium; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor; ANG II, angiotensin II; HIF, hypoxia-inducible factor; ROS, reactive oxygen species; MAPK, mitogen-activated protein kinases; ERK1/2, extracellular signal-regulated kinase; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; TRITC, tetramethylrhodamine B isothiocyanate; SU5416, 3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-iodolin-2-one.
- Received October 16, 2006.
- Accepted January 5, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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