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

Abstract

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-1α 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-1α protein levels. The increases in VEGF after the addition of WIT003 preceded the changes in HIF-1α, and the increases in HIF-1α 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-1α. Stimulation with exogenously added VEGF also led to an upregulation of HIF-1α. Incubation with the MEK1/ERK1/2 inhibitor U-0126 (10 μM) completely abolished the increases in VEGF and thus HIF-1α, 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-1α 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-1α. 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-1α 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-1α upregulation. We also found that the higher HIF-1α expression induced by WIT003 was accompanied by higher expression of erythropoietin receptor and angiopoietin-2 proteins. These increases were caused by HIF-1α because their levels were markedly decreased by siRNA downregulation of HIF-1α. 20-HETE may be a novel nonhypoxic regulator of HIF-1α and HIF-1α-regulated genes in ECs.

20-HETE Regulates the Angiogenic Functions of Human Endothelial Progenitor Cells and Contributes to Angiogenesis In Vivo

“…These findings are consistent with other reports that 20-HETE is proangiogenic (Amaral et al, 2003;Jiang et al, 2004;Chen et al, 2005Chen et al, , 2012Miyata and Roman, 2005;Guo et al, 2007Guo et al, , 2009. In this study, we further demonstrated that 20-HETE contributes to the angiogenic functions in vivo.…”

“…Accumulating evidence has implicated the existence of interactions between the VEGF signaling pathway and the CYP4A-20-HETE system. 20-HETE induces the expression of HIF-1a and its downstream target VEGF in EPC and EC (Guo et al, 2007(Guo et al, , 2009(Guo et al, , 2011. Production of SDF1a and VEGF is regulated by upstream HIF-1a (Ceradini et al, 2004;De Falco et al, 2004;Hoenig et al, 2008), and 20-HETE increases the expression of both SDF-1 and VEGF (Guo et al, 2011), suggesting that 20-HETE may be upstream of VEGF.…”

“…It promotes EPC proliferation, migration, and secretion of proangiogenic molecules, such as hypoxia-inducible factor-1-a(HIF-1a), vascular endothelial growth factor (VEGF), and stroma-derived factor (SDF)-1, as well as EPC-mediated endothelial cell tube formation (Guo et al, 2011). Furthermore, 20-HETE increases EC proliferation and migration; both are essential steps in the angiogenic cascade (Guo et al, 2007;Jiang et al, 2004;Guo et al, 2009), However, the underlying cellular mechanisms by which 20-HETE regulates EPC angiogenic functions and its contribution to physiologic and pathologic neovascularization in vivo is largely unknown.…”

The Cytochrome P450 4A/F-20-Hydroxyeicosatetraenoic Acid System: A Regulator of Endothelial Precursor Cells Derived from Human Umbilical Cord Blood

“…We have shown that 20-HETE stimulates production of both VEGF and hypoxia-inducible factor-1␣ (HIF-1␣), essential regulators of angiogenic responses in ECs. In addition, 20-HETE increases EC proliferation and migration, both essential steps in the angiogenic cascade (Guo et al, 2007(Guo et al, , 2009. We also have shown that 20-HETE induces neovascularization in the rat cornea (Chen et al, 2005).…”

“…20-HETE is angiogenic both in vitro and in vivo (Jiang et al, 2004;Guo et al, 2007Guo et al, , 2009) and activates VEGF and HIF-1␣ in ECs (Guo et al, 2007(Guo et al, , 2009). In the vasculature, the main source of 20-HETE is the smooth muscle layer (Gebremedhin et al, 2000), although in the lung it also is produced by ECs (Zhu et al, 2002).…”