Activation of vascular endothelial growth factor through reactive oxygen species mediates 20-hydroxyeicosatetraenoic acid-induced endothelial cell proliferation.
20-Hydroxyeicosatetraenoic acid (20-HETE) is formed by the omega-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 approximately 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.
Guo
Austin M
AM
Henry Ford Hospital, One Ford Place, 4D, Detroit, MI, USA. mguo1@hfhs.org
Arbab
Ali S
AS
Falck
John R
JR
Chen
Ping
P
Edwards
Paul A
PA
Roman
Richard J
RJ
Scicli
A Guillermo
AG
D000098:Acetophenones / Q000494:pharmacology; D000804:Angiotensin II / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D042783:Endothelial Cells / Q000187:drug effects*; D004789:Enzyme Activation / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism
C494665:20-hydroxyeicosa-5(Z),14(Z)-dienoic acid; D000098:Acetophenones; C497442:HIF1A protein, human; D006893:Hydroxyeicosatetraenoic Acids; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017382:Reactive Oxygen Species; D042461:Vascular Endothelial Growth Factor A; D013481:Superoxides; D000804:Angiotensin II; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; C056165:acetovanillone; D040301:Vascular Endothelial Growth Factor Receptor-2