20-HETE increases superoxide production and activates NAPDH oxidase in pulmonary artery endothelial cells

Abstract

oxygen species (ROS) signal vital physiological processes including cell growth, angiogenesis, contraction, and relaxation of vascular smooth muscle. Because cytochrome P-450 family 4 (CYP4)/20-hydroxyeicosatetraenoic acid (20-HETE) has been reported to enhance angiogenesis, pulmonary vascular tone, and endothelial nitric oxide synthase function, we explored the potential of this system to stimulate bovine pulmonary artery endothelial cell (BPAEC) ROS production. Our data are the first to demonstrate that 20-HETE increases ROS in BPAECs in a time-and concentration-dependent manner as detected by enhanced fluorescence of oxidation products of dihydroethidium (DHE) and dichlorofluorescein diacetate. An analog of 20-HETE elicits no increase in ROS and blocks 20-HETE-evoked increments in DHE fluorescence, supporting its function as an antagonist. Endothelial cells derived from bovine aortas exhibit enhanced ROS production to 20-HETE quantitatively similar to that of BPAECs. 20-HETE-induced ROS production in BPAECs is blunted by pretreatment with polyethylene-glycolated SOD, apocynin, inhibition of Rac1, and a peptide-based inhibitor of NADPH oxidase subunit p47 phox association with gp91. These data support 20-HETE-stimulated, NADPH oxidase-derived, and Rac1/2-dependent ROS production in BPAECs. 20-HETE promotes translocation of p47 phox and tyrosine phosphorylation of p47 phox in a time-dependent manner as well as increased activated Rac1/2, providing at least three mechanisms through which 20-HETE activates NADPH oxidase. These observations suggest that 20-HETE stimulates ROS production in BPAECs at least in part through activation of NADPH oxidase within minutes of application of the lipid. superoxide; Rac1/2; hydrogen peroxide; CYP4A; reactive oxygen species PRODUCTS OF CYTOCHROME P-450 (CYP) -hydroxylases (including CYP4 isoforms) mediate key physiological functions including autoregulation of blood flow, tubuloglomerular feedback, Na ϩ reabsorption in the kidney, and relaxation of pulmonary arterioles (29,38). Our studies have focused on the role of CYP4 and its arachidonic acid (AA) metabolite, 20-hydroxyeicosatetraenoic acid (20-HETE), in pulmonary vascular function and biology. 20-HETE is the CYP -hydroxylation product of AA cleaved from membrane phospholipid sources. Enzymes of the CYP4A, -4B, and -4F families catalyze the -hydroxylation of fatty acids, and several isoforms in these families produce 20-HETE when incubated with AA. For example, rat CYP4A1, -4A2, and -4A3 catalyze AA -and -1-hydroxylations with the highest catalytic efficiency accruing to CYP4A1 (35). Although CYP4A2 and CYP4A3 exhibit an additional arachidonate 11,12-epoxidation activity, CYP4A1 operates solely as an -hydroxylase. Most investigators suggest that CYP4 isoforms constitute the major source of 20-HETE synthesis in extrahepatic tissues, including the lung (35, 38). Accordingly, we have investigated the effects of CYP4 product, 20-HETE, in our studies of this system in pulmonary vascular biology.We have identified a unique role f...

20-HETE induces remodeling of renal resistance arteries independent of blood pressure elevation in hypertension

“…It has also been shown to exert proinflammatory actions. In vascular endothelial cells, 20-HETE increases ROS, including superoxide (3,8,16), and stimulates NF-B activity (4,13), resulting in endothelial activation with increased expression of ICAM-1 and IL-8 levels (13). 20-HETE-dependent increases in NADPH oxidase, ROS, and NF-B activity are also seen in the renal microvasculature of androgen-treated rats (28,39).…”

Activation of JNK/c-Jun is required for the proliferation, survival, and angiogenesis induced by EET in pulmonary artery endothelial cells

“…Fresh bovine PAECs were isolated as previously described ( 11 ). The pulmonary tissues were obtained from a local slaughterhouse with all protocols reviewed and approved by the Ethical Committee of Laboratory Animals at Harbin Medical University.…”

Increase of 20-HETE Synthase after Brain Ischemia in Rats Revealed by PET Study with 11C-Labeled 20-HETE Synthase-Specific Inhibitor

“…It has been reported that 20-HETE acts as a potent vasoconstrictor of cerebral microvessels (Harder et al, 1994), and has an important role in the autoregulation of cerebral blood flow (Gebremedhin et al, 2000). 20-Hydroxyeicosatetraenoic acid has also been reported to induce the formation of oxygen radicals by interacting with nicotinamide adenine dinucleotide phosphate-oxidase and nitric oxide synthase (Wang et al, 2006;Cheng et al, 2008;Medhora et al, 2008). Previously, we reported that brain and plasma 20-HETE levels were significantly increased in the rat transient middle cerebral artery occlusion (tMCAO) model (Tanaka et al, 2007), and inhibition of 20-HETE synthesis by N-(3-chloro-4-morpholin-4-yl)phenyl-N'-hydroxyimido formamide (TS-011), a selective inhibitor of CYP4s, improved the autoregulatory dysfunction in the peri-infarct microcirculation and the neurologic outcomes in rat and monkey stroke models by reducing the infarct volume of the brain after ischemia (Miyata et al, 2005;Omura et al, 2006;Tanaka et al, 2007;Marumo et al, 2010).…”