20-Hydroxy-5,8,11,14-eicosatetraenoic Acid Mediates Endothelial Dysfunction via I B Kinase-Dependent Endothelial Nitric-Oxide Synthase Uncoupling

Endothelial dysfunction and activation occur in the vasculature and are believed to contribute to the pathogenesis of cardiovascular diseases. We have shown that 20-hydroxy5,8,11,14-eicosatetraenoic acid (20-HETE), a cytochrome P450 4A-derived eicosanoid that promotes vasoconstriction in the microcirculation, uncouples endothelial nitric-oxide synthase (eNOS) and reduces nitric oxide (NO) levels via the dissociation of the 90-kDa heat shock protein (HSP90) from eNOS. It also causes endothelial activation by stimulating nuclear factorB (NFB) and increasing levels of proinflammatory cytokines. In this study, we examined signaling mechanisms that may link 20-HETE-induced endothelial dysfunction and activation. Under conditions in which 20HETE inhibited NO production, it also stimulated inhibitor of NFB (I B) phosphorylation. Both effects were prevented by inhibition of tyrosine kinases and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK). It is noteworthy that inhibitor of I B kinase (IKK) activity negated the 20-HETE-mediated inhibition of NO production. Immunoprecipitation experiments revealed that treatment of ionophore-stimulated cells with 20-HETE brings about a decrease in HSP90-eNOS association and an increase in HSP90-IKK association, suggesting that the activation by 20-HETE of NFB is linked to its action on eNOS. Furthermore, addition of inhibitors of tyrosine kinase MAPK and IKK restored the 20-HETE-mediated impairment of acetylcholine-induced relaxation in rat renal interlobar arteries. The results indicate that 20-HETE mediates eNOS uncoupling and endothelial dysfunction via the activation of tyrosine kinase, MAPK, and IKK, and these effects are linked to 20-HETE-mediated endothelial activation. The integrity of the vascular endothelium is vital to the regulation of the cardiovascular system. The endothelium serves as a protective barrier between tissues and circulating blood and functions to maintain vascular homeostasis by releasing bioactive factors in response to hemodynamic changes and blood-borne signals. An uncontrolled endothelial cell response is involved in many disease processes, including hypertension, atherosclerosis, and diabetes. These diseases are related to endothelial injury, dysfunction and activation. Nitric oxide (NO), generated from L-arginine by endothelial nitric-oxide synthase (eNOS), is a key endothelial-derived factor, the bioavailability of which is essential to the integrity and function of the endothelium. Endothelial dysfunction occurs as a result of a loss of NO bioavailability that is due to either reduced formation or accelerated degradation of NO, and it is often associated with endothelial activation. NO actively mediates many functions of the endothelium. In addition to its This work was supported by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL34300] (to M.L.S.); the National Institutes of Health National Institute of General Medical Sciences [Grant GM31278] (to J.R.F.); the Robert A. Welch Foundation (to J.R.F.); and a American Heart Association Predoctoral Fellowship [Grant 0715781T] (to J.C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.109.159863. ABBREVIATIONS: NO, nitric oxide; eNOS, endothelial nitric-oxide synthase; SMC, smooth muscle cell; 20-HETE, 20-hydroxyeicosatetraenoic acid; HSP90, 90-kDa heat shock protein; MAPK, mitogen-associated protein kinase; ERK, extracellular signal-regulated kinase; BAEC, bovine aortic endothelial cell; FBS, fetal bovine serum; L-NMMA, N-monomethyl-L-arginine; AG82, cyano-(3,4,5-trihydroxy)cinnamonitrile; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)butadiene; 17-AAG, 17-(allylamino)-17-demethoxygeldanamycin; SC514, 4-amino-[2 ,3 -bithiophene]-5-carboxamide; NBD, NEMO-binding domain peptide; NEMO, NFB essential modulating protein; A23187, calcium ionophore; tiron, 4,5-dihydroxy-1,3-benzene disulfonic acid; IKK, inhibitor of I B kinase; EET, epoxyeicosatrienoic acid; EGFR, epidermal growth factor receptor; IKK, I B kinase; I B, inhibitor of NFB. 0022-3565/10/3321-57–65$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 332, No. 1 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 159863/3545838 JPET 332:57–65, 2010 Printed in U.S.A. 57 at A PE T Jornals on A ril 4, 2017 jpet.asjournals.org D ow nladed from potent vasorelaxing effect, NO counteracts leukocyte adhesion to the endothelium, vascular smooth muscle cell (SMC) proliferation, and platelet aggregation that typically characterize endothelial activation. 20-Hydroxyeicosatetraenoic acid (20-HETE), the -hydroxylation product of arachidonic acid, is a primary eicosanoid in the microcirculation. Its synthesis is catalyzed by enzymes of the cytochrome P450 (P450) 4 gene family, which encodes multiple structurally and functionally similar P450 proteins (CYP4A, -4B, and -4F) that are controlled by factors such as age and sex hormones (Hardwick, 2008). The importance of this metabolic pathway emerged with the seminal observation that reduction in its activity lowers blood pressure in spontaneously hypertensive rats (Sacerdoti et al., 1989). Consequently, the hypothesis that this pathway participates in the regulation of blood pressure was substantiated in experimental animal models and in humans (Miyata and Roman, 2005; Ward et al., 2008). Its role in promoting pro-hypertensive mechanisms and participating in the regulation of vascular tone and homeostasis (Miyata and Roman, 2005) stems from findings that 20-HETE is a potent vasoactive eicosanoid. 20-HETE is a key constrictor eicosanoid in microcirculatory districts, most notably the renal and cerebral microcirculations (Miyata and Roman, 2005); in the pulmonary circulation, it is vasodilatory (Zhu et al., 2002). Its synthesis within the vascular wall is primarily localized to the SMCs, increases with decreased vessel diameter, is stimulated by vasoactive hormones such as angiotensin II and endothelin-1, and is inhibited by nitric oxide (Miyata and Roman, 2005). It elicits vasoconstriction largely via inhibition of the SMC large conductance Ca -activated K channel, leading to depolarization and elevation in cytosolic [Ca ], and in some blood vessels via the Rho-kinase phosphorylation of MLC20 and the sensitization of the contractile apparatus to Ca (Kroetz and Xu, 2005; Miyata and Roman, 2005). Besides its vasoactivity, 20-HETE has been shown to stimulate SMC migration and proliferation (Muthalif et al., 1998; Stec et al., 2007). Although endothelial synthesis of 20-HETE is questionable in most vascular beds, except for the pulmonary circulation (Zhu et al., 2002), its actions on endothelial function have been recently documented. 20-HETE is a potent angiogenic factor in vitro and in vivo (Medhora et al., 2007) and a mitogen to endothelial cells (Guo et al., 2007) as well as a modulator of eNOS-NO activation and function (Chen et al., 2006; Wang et al., 2006; Singh et al., 2007; Cheng et al., 2008). It is noteworthy that changes in the production of 20-HETE have been observed in numerous pathological conditions, including ischemic cerebrovascular diseases, kidney diseases, hypertension, diabetes, and toxemia of pregnancy (Miyata and Roman, 2005). The vascular phenotype in many of these conditions is that of injury typified by endothelial dysfunction and activation. We have provided substantial evidence that 20-HETE contributes to both endothelial dysfunction and endothelial activation. In two experimental models of increased vascular synthesis of 20-HETE, the resulting endothelial dysfunction (i.e., reduced acetylcholine-induced relaxation, oxidative stress, and hypertension) (Wang et al., 2006; Singh et al., 2007) has been linked to vascular 20-HETE; administration of a 20-HETE synthesis inhibitor in vivo reversed endothelial dysfunction and normalized blood pressure. We further demonstrated that 20-HETE impairs NO production in vitro and its function in vivo by uncoupling the eNOS activation process via inhibition of HSP90 association (Cheng et al., 2008). In addition to its actions on eNOS, 20-HETE potently stimulates endothelial activation by activating the NFB and MAPK-ERK1/2 pathways, leading to increased levels of proinflammatory cytokines such as interleukin-8 and adhesion molecules (Ishizuka et al., 2008). In the current study, we explore whether 20-HETE-induced endothelial dysfunction and activation are linked. The results suggest that 20-HETEmediated eNOS uncoupling and impairment of acetylcholineinduced relaxations are dependent on tyrosine kinase, MAPK, and I B kinase (IKK); these steps link 20-HETEinduced endothelial dysfunction to activation. Materials and Methods Cell Culture. Bovine aortic endothelial cells (BAEC; passages 3–5), cultured in microvascular endothelial cell growth medium (Lonza, Walkersville, MD), were placed in FBS-free medium for 24 h after achieving 70% confluence. On the day of the experiment, they were preincubated with or without 20-HETE (5 nM) and N-monomethyl-L-arginine (L-NMMA; 100 M) in the presence and absence of inhibitors for 30 min. The following inhibitors were used: genistein (30 M), AG82 (10 M), U0126 (10 M), wedelolactone (50 M), SC514 (10 M), geldanamycin (10 M; Calbiochem, Gibbstown, NJ), radicicol (10 M), 17-(allylamino)-17-demethoxygeldanamycin (17AAG; 10 M), NEMO-binding domain peptide (NBD; 100 M), and NBD control peptide (100 M; BIOMOL Research Laboratories, Plymouth Meeting, PA). Cells were then incubated in the presence of L-arginine (25 M) with and without calcium ionophore (A23187; 5 M) for 10 to 30 min. Cells and media were processed for the measurements described below. Measurement of NO. NO levels were evaluated by measuring total nitrite and nitrate (NOx) content in culture medium using the NO quantitation kit and following the manufacturer’s instructions (Active Motif Inc., Carlsbad, CA) as described previously (Cheng et