19-Hydroxyeicosatetraenoic acid analogs: Antagonism of 20-hydroxyeicosatetraenoic acid-induced vascular sensitization and hypertension.

19-Hydroxyeicosatetraenoic acid (19-HETE, 1), a metabolically and chemically labile cytochrome P450 eicosanoid, has diverse biological activities including antagonism of the vasoconstrictor 20-hydroxyeicosatetraenoic acid (20-HETE, 2). A SAR study was conducted to develop robust analogs of 1 with improved in vitro and in vivo efficacy. Analogs were screened in vitro for inhibition of 20-HETE-induced sensitization of rat renal preglomerular microvessels toward phenylephrine and demonstrated to normalize the blood pressure of male Cyp4a14(-/-) mice that display androgen-driven, 20-HETE-dependent hypertension.

12(S)-hydroxyeicosatetraenoic acid impairs vascular endothelial permeability by altering adherens junction phosphorylation levels and affecting the binding and dissociation of its components in high glucose-induced vascular injury.

AIMS/INTRODUCTION: Diabetes is an important risk factor for atherosclerotic disease. The initiating factor of atherosclerosis is local endothelial cell injury. The arachidonic acid metabolite, 12(S)-hydroxyeicosatetraenoic acid (12[S]-HETE), might be involved in this process. In recent years, some studies have discussed the effect of 12(S)-HETE on vascular endothelial cell function. In the present study, we investigated the effect of 12(S)-HETE on vascular endothelial cell function in high-glucose conditions and the mechanisms involved. MATERIALS AND METHODS: Human umbilical vein endothelial cells were cultured in conventional M199 medium and high-glucose M199 medium. Human umbilical vein endothelial cells were stimulated with 12(S)-HETE and cinnamyl-3,4-dihydroxy-α-cyanocinnamate (a 12/15-lipoxygenases inhibitor). A type 1 diabetes mellitus model was established in C57BL/6 or 12/15-lipoxygenases knockout mice with streptozotocin. Aortic tissue was harvested for subsequent testing. The transmembrane transport of dextran and human acute monocytic leukaemia cell line (THP-1) cells was measured. The adherens junction protein, IkBα, nuclear factor kappa Bp65 (P65), intercellular adhesion molecule 1 and vascular cell adhesion protein 1 expression and phosphorylation, and the binding/dissociation of endothelial cell components were observed. RESULTS: Transendothelial migration of dextran and THP-1 cells was significantly increased by stimulation of human umbilical vein endothelial cell monolayers with high glucose and 12(S)-HETE (P < 0.05). High glucose and 12(S)-HETE altered the vascular endothelial cadherin and ß-catenin phosphorylation level, and promoted the dissociation of ß-catenin and vascular endothelial cadherin. Expression levels of P-Ikbα, P-P65, intercellular adhesion molecule 1 and vascular cell adhesion protein 1 were elevated in high glucose and 12(S)-HETE treated cells and diabetic mice compared with controls (P < 0.05). CONCLUSIONS: The lipoxygenases metabolite, 12(S)-HETE, can impair vascular endothelial permeability by altering adherens junction phosphorylation levels, and affecting the binding and dissociation of its components in high-glucose conditions.

20-HETE Signals Through G-Protein-Coupled Receptor GPR75 (Gq) to Affect Vascular Function and Trigger Hypertension.

RATIONALE: 20-Hydroxyeicosatetraenoic acid (20-HETE), one of the principle cytochrome P450 eicosanoids, is a potent vasoactive lipid whose vascular effects include stimulation of smooth muscle contractility, migration, and proliferation, as well as endothelial cell dysfunction and inflammation. Increased levels of 20-HETE in experimental animals and in humans are associated with hypertension, stroke, myocardial infarction, and vascular diseases. OBJECTIVE: To date, a receptor/binding site for 20-HETE has been implicated based on the use of specific agonists and antagonists. The present study was undertaken to identify a receptor to which 20-HETE binds and through which it activates a signaling cascade that culminates in many of the functional outcomes attributed to 20-HETE in vitro and in vivo. METHODS AND RESULTS: Using crosslinking analogs, click chemistry, binding assays, and functional assays, we identified G-protein receptor 75 (GPR75), currently an orphan G-protein-coupled receptor (GPCR), as a specific target of 20-HETE. In cultured human endothelial cells, 20-HETE binding to GPR75 stimulated Gαq/11 protein dissociation and increased inositol phosphate accumulation and GPCR-kinase interacting protein-1-GPR75 binding, which further facilitated the c-Src-mediated transactivation of epidermal growth factor receptor. This results in downstream signaling pathways that induce angiotensin-converting enzyme expression and endothelial dysfunction. Knockdown of GPR75 or GPCR-kinase interacting protein-1 prevented 20-HETE-mediated endothelial growth factor receptor phosphorylation and angiotensin-converting enzyme induction. In vascular smooth muscle cells, GPR75-20-HETE pairing is associated with Gαq/11- and GPCR-kinase interacting protein-1-mediated protein kinase C-stimulated phosphorylation of MaxiKß, linking GPR75 activation to 20-HETE-mediated vasoconstriction. GPR75 knockdown in a mouse model of 20-HETE-dependent hypertension prevented blood pressure elevation and 20-HETE-mediated increases in angiotensin-converting enzyme expression, endothelial dysfunction, smooth muscle contractility, and vascular remodeling. CONCLUSIONS: This is the first report to identify a GPCR target for an eicosanoid of this class. The discovery of 20-HETE-GPR75 pairing presented here provides the molecular basis for the signaling and pathophysiological functions mediated by 20-HETE in hypertension and cardiovascular diseases.

Development of cellular hypertrophy by 8-hydroxyeicosatetraenoic acid in the human ventricular cardiomyocyte, RL-14 cell line, is implicated by MAPK and NF-κB.

Recent studies have established the role of mid-chain hydroxyeicosatetraenoic acids (mid-chain HETEs) in the development of cardiovascular disease. Among these mid-chains, 8-HETE has been reported to have a proliferator and proinflammatory action. However, whether 8-HETE can induce cardiac hypertrophy has never been investigated before. Therefore, the overall objectives of the present study are to elucidate the potential hypertrophic effect of 8-HETE in the human ventricular cardiomyocytes, RL-14 cells, and to explore the mechanism(s) involved. Our results showed that 8-HETE induced cellular hypertrophy in RL-14 cells as evidenced by the induction of cardiac hypertrophy markers ANP, BNP, α-MHC, and ß-MHC in a concentration- and time-dependent manner as well as the increase in cell surface area. Mechanistically, 8-HETE was able to induce the NF-κB activity as well as it significantly induced the phosphorylation of ERK1/2. The induction of cellular hypertrophy was associated with a proportional increase in the formation of dihydroxyeicosatrienoic acids (DHETs) parallel to the increase of soluble epoxide hydrolase (sEH) enzyme activity. Blocking the induction of NF-κB, ERK1/2, and sEH signaling pathways significantly inhibited 8-HETE-induced cellular hypertrophy. Our study provides the first evidence that 8-HETE induces cellular hypertrophy in RL-14 cells through MAPK- and NF-κB-dependent mechanism

Cytochrome P450 epoxygenase metabolite, 14,15-EET, protects against isoproterenol-induced cellular hypertrophy in H9c2 rat cell line.

We have previously shown that isoproterenol-induced cardiac hypertrophy causes significant changes to cytochromes P450 (CYPs) and soluble epoxide hydrolase (sEH) gene expression. Therefore, in this study, we examined the effect of isoproterenol in H9c2 cells, and the protective effects of 14,15-EET against isoproterenol-induced cellular hypertrophy. Isoproterenol was incubated with H9c2 cells for 24 and 48 h. To determine the protective effects of 14,15-EET, H9c2 cells were incubated with isoproterenol in the absence and presence of 14,15-EET. Thereafter, the expression of hypertrophic markers and different CYP genes were determined by real time-PCR. Our results demonstrated that isoproterenol significantly increased the expression of hypertrophic marker, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), parallel to a significant increase in cell surface area. Also, isoproterenol increased the mRNA expression of CYP1A1, CYP1B1, CYP2J3, CYP4F4 and CYP4F5, as well as the gene encoding sEH, EPHX2. On other hand, 14,15-EET significantly attenuated the isoproterenol-mediated induction of ANP, BNP, CYP1A1, CYP2J3, CYP4F4, CYP4F5 and EPHX2. Moreover 14,15-EET prevented the isoproterenol-mediated increase in cell surface area. Interestingly, 20-hydroxyeicosatetraenoic acid (20-HETE) treatment caused similar effects to that of isoproterenol treatment and induced cellular hypertrophy in H9c2 cells. In conclusion, isoproterenol induces cellular hypertrophy and modulates the expression of CYPs and EPHX2 in H9c2 cells. Furthermore, 14,15-EET exerts a protective effect against isoproterenol-induced cellular hypertrophy whereas, 20-HETE induced cellular hypertrophy in H9c2 cells.

Inhibition of cytochrome P450omega-hydroxylase: a novel endogenous cardioprotective pathway.

Cytochrome P450s (CYP) and their arachidonic acid (AA) metabolites have important roles in regulating vascular tone, but their function and specific pathways involved in modulating myocardial ischemia-reperfusion injury have not been clearly established. Thus, we characterized the effects of several selective CYPomega-hydroxylase inhibitors and a CYPomega-hydroxylase metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), on the extent of ischemia-reperfusion injury in canine hearts. During 60 minutes of ischemia and particularly after 3 hours of reperfusion, 20-HETE was produced at high concentrations. A nonspecific CYP inhibitor, miconazole, and 2 specific CYPomega-hydroxylase inhibitors, 17-octadecanoic acid (17-ODYA) and N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS), markedly inhibited 20-HETE production during ischemia-reperfusion and produced a profound reduction in myocardial infarct size (expressed as a percent of the area at risk) (19.6+/-1.7% [control], 8.4+/-2.5% [0.96 mg/kg miconazole], 5.9+/-2.2% [0.28 mg/kg 17-ODYA], and 10.8+/-1.8% [0.40 mg/kg DDMS], P<0.05, respectively). Conversely, exogenous 20-HETE administration significantly increased infarct size (26.9+/-1.9%, P<0.05). Several CYPomega-hydroxylase isoforms, which are known to produce 20-HETE such as CYP4A1, CYP4A2, and CYP4F, were demonstrated to be present in canine heart tissue and their activity was markedly inhibited by incubation with 17-ODYA. These results indicate an important endogenous role for CYPomega-hydroxylases and in particular their product, 20-HETE, in exacerbating myocardial injury in canine myocardium. The full text of this article is available online at http://circres.ahajournals.org.

Arachidonate has protumor-promoting action that is inhibited by linoleate in mouse skin carcinogenesis.

Previous studies demonstrated a requirement for arachidonic acid metabolites in tumor development in mouse skin. The goal of this study was to determine whether the arachidonate content of epidermal phospholipids could be altered by increasing dietary levels of linoleate and whether specific metabolites of linoleate and arachidonate have dissimilar biological effects. In a series of tumor studies in which the quantity of dietary linoleate was incrementally increased, a slight reduction in phospholipid levels of arachidonate was observed that correlated with an increased phospholipid level of linoleate and a suppression in tumor yield. A comparison of the arachidonate lipoxygenase metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) with the 13-hydroxyoctadecadienoic acid (13-HODE) lipoxygenase metabolite of linoleate revealed that 12-HETE has biological activities that mimic the phorbol ester tumor promoters, whereas 13-HODE has antithetical effects. Specifically, 12(S)-HETE enhanced the activation of protein kinase C by phorbol esters, mimicked phorbol ester-induced adhesion of keratinocytes to fibronectin and mimicked phorbol ester repression of expression of a differentiation-related gene, keratin-1. 13-HODE blocked 12-HETE-induced cell adhesion and prevented 12-HETE-induced suppression of keratin-1 expression. Overall, these studies suggest that arachidonate and linoleate have opposing functions in the epidermis, particularly with regard to events involved in tumor development.

Swelling in the isolated perfused cornea induced by 12(R)hydroxyeicosatetraenoic acid.

PURPOSE: To evaluate the effect of 12(R)hydroxyeicosatetraenoic acid (12(R)HETE) on corneal swelling when directly perfused to human and rabbit corneal endothelium. METHOD: Excised rabbit and human corneas were mounted in the in vitro specular microscope and the endothelium was perfused with 12(R)HETE at 10(-5), 10(-6), and 10(-7) mol/l. Both 12(R)HETE and 12(S)HETE were compared at equal molar (10(-6) mol/l) concentrations. The reversal of 12(R)HETE and ouabain corneal swelling was also compared. Endothelial permeability to carboxyfluorescein was measured after 12(R)HETE perfusion. High-performance liquid chromatographic analysis confirmed that 12(R)HETE remained in the perfusion media. RESULTS: 12(R)HETE caused a dose-dependent corneal swelling of 25 +/- 2, 24 +/- 1, and 14 +/- 0.5 microns/hr at 10(-5), 10(-6), and 10(-7) mol/l, respectively. Equal molar concentrations (10(-6) mol/l) of 12(S)HETE did not cause corneal swelling. Removal of the 12(R)HETE from the perfusion media resulted in reversal of corneal swelling whereas corneal swelling induced by ouabain did not reverse after ouabain removal. 12(R)HETE (10(-6) mol/l) perfused to the human corneal endothelium inhibited temperature reversal corneal thinning when compared to the paired corneal endothelium perfused with BSS Plus (Alcon Laboratories, Inc., Fort Worth, TX). Na/K adenosine triphosphatase activity was inhibited by 10(-6) mol/l ouabain by 35%, 10(-6) mol/l 12(R)HETE by 54%, and 10(-6) mol/l 12(S)HETE by 0.5%. Endothelial permeability to carboxyfluorescein was unaffected by 12(R)HETE. CONCLUSION: 12(R)HETE causes corneal swelling by inhibiting endothelial pump function. This inhibition of transport appears to be at least partly mediated by inhibition of endothelial Na/K adenosine triphosphatase.

Effects of chronic intracutaneous administration of arachidonic acid and its metabolites. Induction of leukocytoclastic vasculitis by leukotriene B4 and 12-hydroxyeicosatetraenoic acid and its prevention by prostaglandin E2.

Despite the postulated role of arachidonic acid-derived metabolites in the pathophysiology of chronic inflammatory dermatoses such as psoriasis and atopic or contact dermatitis, the cutaneous effects of their chronic application have not yet been investigated. We therefore studied systematically the effects of chronic intracutaneous administration of arachidonic acid, prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and 12-hydroxyeicosatetraenoic acid (12-HETE) in guinea pigs, and describe previously unrecognized findings partly different from those reported in the past for short-term or topical application of these inflammatory mediators. Leukotriene B4 and 12-HETE led to massive histologic changes characteristic of leukocytoclastic vasculitis. These changes could be prevented by concomitant PGE2 administration. In epidermis, LTB4 and 12-HETE caused some spongiosis as well as hyperplasia and increased tritiated thymidine autoradiographic labeling index. Arachidonic acid and PGE2 alone had little effect. These data suggest that in addition to other inflammatory or hyperproliferative dermatoses, arachidonic acid metabolites formed via lipoxygenase pathways could play a major role in leukocytoclastic vasculitis, whereas PGs could exert a tissue-protective effect.