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This Article Full Text Full Text (PDF) All Versions of this Article: 297/2/H602    most recent 00874.2008v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Google Scholar Articles by Guo, A. M. Articles by Scicli, A. G. PubMed PubMed Citation Articles by Guo, A. M. Articles by Scicli, A. G.

20-HETE can act as a nonhypoxic regulator of HIF-1 in human microvascular endothelial cells

¹Eye Care Services, Henry Ford Hospital, and ²Department of Anesthesiology, Wayne State University, Detroit, Michigan; and ³Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas

Submitted 6 August 2008 ; accepted in final form 27 May 2009

20-HETE increases the expression of VEGF in human dermal microvascular endothelial cells (ECs). Since VEGF is regulated by hypoxia inducible factor (HIF)-1, we studied whether 20-HETE also upregulates HIF-1 using the stable 20-HETE analog 20-hydroxyeicosa-5(Z),14(Z)dienoic acid (WIT003; 1–10 µM) and found that it induced a marked increase in HIF-1 protein levels. The increases in VEGF after the addition of WIT003 preceded the changes in HIF-1, and the increases in HIF-1 were prevented by a VEGF neutralizing antibody. This suggests that 20-HETE first causes increases in VEGF, which then, in turn, cause the upregulation of HIF-1. Stimulation with exogenously added VEGF also led to an upregulation of HIF-1. Incubation with the MEK1/ERK1/2 inhibitor U-0126 (10 µM) completely abolished the increases in VEGF and thus HIF-1, suggesting the involvement of ERK1/2 activation. The addition of WIT003 resulted in a rapid and sustained increase in superoxide formation. When WIT003 was added in the presence of the nitric oxide (NO) synthase (NOS) inhibitor N-nitro-L-arginine, no changes in superoxide, VEGF, or HIF-1 were observed. This suggests that NOS is responsible for the early changes in superoxide induced by WIT003. Furthermore, WIT003 induced the expression of the NADPH oxidase subunit p47^phox in ECs before the increases in HIF-1. Incubation with polyethylene glycol-superoxide dismutase (400 U/ml), apocynin (100 µM), diphenylene iodonium (10 µM), or p47^phox downregulation with small interfering (si)RNA all inhibited the increases in HIF-1 expression. This indicates that the early changes in superoxide lead to VEGF increases and thereby NADPH oxidase-dependent superoxide production, which is required for HIF-1 upregulation. We also found that the higher HIF-1 expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1 because their levels were markedly decreased by siRNA downregulation of HIF-1. 20-HETE may be a novel nonhypoxic regulator of HIF-1 and HIF-1-regulated genes in ECs.

cytochrome P-4504A; 20-hydroxyeicosatrienoic acid; hypoxia-inducible factor; nitric oxide synthase; superoxide; vascular endothelial growth factor