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

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-1alpha using the stable 20-HETE analog 20-hydroxyeicosa-5(Z),14(Z)dienoic acid (WIT003; 1-10 microM) and found that it induced a marked increase in HIF-1alpha protein levels. The increases in VEGF after the addition of WIT003 preceded the changes in HIF-1alpha, and the increases in HIF-1alpha 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-1alpha. Stimulation with exogenously added VEGF also led to an upregulation of HIF-1alpha. Incubation with the MEK1/ERK1/2 inhibitor U-0126 (10 microM) completely abolished the increases in VEGF and thus HIF-1alpha, 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-1alpha 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-1alpha. Incubation with polyethylene glycol-superoxide dismutase (400 U/ml), apocynin (100 microM), diphenylene iodonium (10 microM), or p47(phox) downregulation with small interfering (si)RNA all inhibited the increases in HIF-1alpha 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-1alpha upregulation. We also found that the higher HIF-1alpha expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1alpha because their levels were markedly decreased by siRNA downregulation of HIF-1alpha. 20-HETE may be a novel nonhypoxic regulator of HIF-1alpha and HIF-1alpha-regulated genes in ECs.