Arachidonic Acid Epoxides Research Articles

Cytochrome P‐450 epoxygenase 2J2 modulates adenosine receptor‐mediated vascular response in mouse mesenteric arteries

Cytochrome P‐450 epoxygenases including CYP2J2 are expressed in vascular endothelial cells that generate epoxyeicosatrienoic acids (EETs) through epoxidation of arachidonic acid. EETs are endothelium‐derived hyperpolarizing factors which are protective in nature and are degraded by soluble epoxide hydrolase (sEH). In aorta from sEH−/− mice, we reported upregulation of A2A adenosine receptor (AR), CYP2J2, and PPARg, and the downregulation of A1AR and PPARa with enhanced vascular relaxation. In this study, we explored whether over‐expression of CYP2J2 in endothelium (Tie2‐CYP2J2 Tr) regulates AR dependent vascular responses in mouse mesenteric arteries (MAs). We performed DMT myograph experiments in WT and Tie2‐CYP2J2 Tr mice. Our findings revealed that acetylcholine‐dependent response was unchanged. Phenylephrine and KCl induced contractions were significantly higher in Tie2‐CYP2J2 Tr vs. WT mice (p<0.05). Importantly, CCPA (selective A1AR agonist)‐induced contractions were significantly reduced in Tie2‐CYP2J2 Tr vs. WT mice (p<0.05). NECA (non‐selective AR agonist)‐induced relaxation was similar Tie2‐sEH Tr vs. WT mice. Especially, pinacidil (KATP channel opener) induced relaxation was also significantly enhanced in Tie2‐CYP2J2 Tr vs. WT mice. Our data suggest that over‐expression of CYP2J2 enhances vascular relaxation possibly due to the combination of reduction in A1AR‐dependent contraction and enhancement of relaxation through KATP channel, indicating a possible interaction among CYP2J2, ARs and KATP channels (HL‐114559, Z01‐ES025034, HL027339 & HL094447).

The Biological Actions of 11,12‐Epoxyeicosatrienoic Acid in Endothelial Cells are Specific to the R/S Enantiomer and Require the Gs Protein

Cytochrome P450 (CYP)‐derived epoxides of arachidonic acid, i.e., the epoxyeicosatrienoic acids (EETs), are important lipid signaling molecules. Since many actions of 11,12‐EET are dependent on the activation of protein kinase A (PKA), the existence of a cell surface Gs‐coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12‐EET are enantiomer specific and linked to a potential G protein‐coupled receptor, we assessed 11,12‐EET‐induced, PKA‐dependent translocation of transient receptor potential (TRP) C6 channels as well as angiogenesis. In primary cultures of human endothelial cells, (±)‐11,12‐EET led to the rapid (30 seconds) translocation a TRPC6V5 fusion protein, an effect reproduced by 11(R),12(S)‐EET, but not 11(S),12(R)‐EET or (±)‐14,15‐EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)‐11,12‐EET and 11(R),12(S)‐EET while 11(S),12(R)‐EET and 11,12‐dihydroxyeicosatrienoic acid were without effect. The effects of (±)‐11,12‐EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists and TRP channel trafficking was also prevented by a PKA inhibitor. The siRNA‐mediated downregulation of Gs in endothelial cells had no significant effect on responses stimulated by VEGF or a PKA activator but abolished responses to (±)‐11,12‐EET. The down regulation of Gq/11 failed to prevent 11,12‐EET‐induced TRPC6 channel translocation or the formation of capillary like structures. Taken together, our results suggest that a Gs‐coupled receptor in the endothelial cell membrane responds to 11(R),12(S)‐EET, mediates the PKA‐dependent translocation and activation of TRPC6 channels, as well as angiogenesis.

The Change in Human Microvascular Function and its Relationship to Plasma Epoxide Content After Short‐Term Walnut Intake

Walnuts are a source of alpha‐linolenic acid (ALA) and linoleic acid (LA). We examined the effects of short‐term walnut intake on the reactive hyperemia index (RHI), a measure of microvascular function. We hypothesized that 4 weeks of 40g/day of walnuts would improve RHI compared to 5g/day of walnuts in hypercholesterolemic postmenopausal women. Microvascular function is influenced, in part, by locally produced vasodilators, such as nitric oxide and endothelial‐derived hyperpolarizing factor (EDHF). Putative EDHF include cytochrome P450 epoxides of arachidonic acid (AA) known as the EpETrEs. Epoxides are also derived from ALA (EpODE) and LA (EpODE); however, their vascular influence is currently undefined. Four weeks of 40g/day of walnut intake increased RHI compared to 5g/day (2.61 ± 0.10 versus 2.24 ± 0.12, respectively, p = 0.027). Plasma EpODE and EpOME were significantly increased with 40g/day compared to 5g/day of walnut intake. The total change in plasma epoxides was strongly associated with the change in RHI (r=0.65, p = 0.002). There was a strong association between the change in EpODE, EpOME, and EpETrE, as well as, these epoxides and the change in RHI (|r|=0.56, p < 0.01); with the strongest association observed for 14(15)‐EpETrE (r=0.72, p < 0.001). These data are consistent with reports of improved vascular function with walnut consumption, and provide new insights into potential relationships between changes in microvascular function and plasma epoxides after walnut intake.

Research/review: Insights into the mutation-induced dysfunction of arachidonic acid metabolism from modeling of human CYP2J2.

As a kind of monooxygenase with the function of catalyzing many reactions involved in drug metabolism and synthesis of cholesterol, steroids and other lipids, CYP2J2 is an important member of the cytochrome P450 superfamily. Located at the endoplasmic reticulum, CYP2J2 is responsible for epoxidation of endogenous arachidonic acid in cardiac tissue to produce cis-epoxyeicosatrienoic acids (EETs), which have anti-inflammatory and antifibrinolytic properties, and can protect endothelial cells from ischemic or hypoxic injuries. Some polymorphisms, e.g., CYP2J2 with mutation T143A, R158C, I192N or N404Y, could significantly reduce the metabolism of the arachidonic acid, causing or deteriorating the coronary artery disease. However, so far the detailed mechanism for the mutationinduced dysfunction of arachidonic metabolism is still unknown. To reveal its mechanism, a 3D (three-dimensional) structure for human CYP2J2 was developed, followed by docking the arachidonic acid ligand into the active site of the receptor. It was observed based on the binding mode thus found that Gly486 and Leu378 in the active site of the receptor played a key role in recognizing and positioning the carboxyl group of the ligand via hydrogen bonding interactions, and that any of the aforementioned five mutations might have, either directly or indirectly, impact to their role and hence causing the mutation-induced dysfunction of CYP2J2-mediated arachidonic acid metabolism. It is anticipated that the findings as reported in this review article may stimulate new strategy for finding novel therapeutic approaches to treat coronary artery disease.

An Omega-3 Epoxide of Docosahexaenoic Acid Lowers Blood Pressure in Angiotensin-II–Dependent Hypertension

Mediators of antihypertensive actions of docosahexaenoic acid (DHA) are largely unknown. The omega-3 epoxide of DHA, 19, 20-EDP (epoxy docosapentaenoic acid), is metabolized by soluble epoxide hydrolase (sEH), which also metabolizes the anti-inflammatory and antihypertensive arachidonic acid epoxides, epoxyeicosatrienoic acids (EETs). Based in part on plasma levels of EDPs after a DHA-rich diet, we hypothesized that 19, 20-EDP contributes to the antihypertensive actions of DHA in angiotensin-II (Ang-II)-dependent hypertension. Treatment individually with 19, 20-EDP and a potent sEH inhibitor TPPU (1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea) significantly lowered blood pressure (BP) as compared with Ang-II-infused animals. The largest reduction in BP was obtained with the combination of 19, 20-EDP and TPPU, which was more efficacious than the combination of 14, 15-EET and TPPU. Oxylipin profiling revealed that 19, 20-EDP and 14, 15-EET infusion affected not only most metabolites of the P450 pathway but also renal levels of prostaglandin-E2. Our findings suggest that 19, 20-EDP is a mediator of the antihypertensive effects of DHA in Ang-II-dependent hypertension. It seems that 19, 20-EDP requires metabolic stabilization with a sEH inhibitor to be most effective in lowering BP, although both TPPU and 19, 20-EDP are so effective on their own that demonstrating additive or synergistic interactions is difficult.

The biological actions of 11,12-epoxyeicosatrienoic acid in endothelial cells are specific to the R/S-enantiomer and require the G(s) protein.

Cytochrome P450-derived epoxides of arachidonic acid [i.e., the epoxyeicosatrienoic acids (EETs)] are important lipid signaling molecules involved in the regulation of vascular tone and angiogenesis. Because many actions of 11,12-cis-epoxyeicosatrienoic acid (EET) are dependent on the activation of protein kinase A (PKA), the existence of a cell-surface G(s)-coupled receptor has been postulated. To assess whether the responses of endothelial cells to 11,12-EET are enantiomer specific and linked to a potential G protein-coupled receptor, we assessed 11,12-EET-induced, PKA-dependent translocation of transient receptor potential (TRP) C6 channels, as well as angiogenesis. In primary cultures of human endothelial cells, (±)-11,12-EET led to the rapid (30 seconds) translocation a TRPC6-V5 fusion protein, an effect reproduced by 11(R),12(S)-EET, but not by 11(S),12(R)-EET or (±)-14,15-EET. Similarly, endothelial cell migration and tube formation were stimulated by (±)-11,12-EET and 11(R),12(S)-EET, whereas 11(S),12(R)-EET and 11,12-dihydroxyeicosatrienoic acid were without effect. The effects of (±)-11,12-EET on TRP channel translocation and angiogenesis were sensitive to EET antagonists, and TRP channel trafficking was also prevented by a PKA inhibitor. The small interfering RNA-mediated downregulation of G(s) in endothelial cells had no significant effect on responses stimulated by vascular endothelial growth or a PKA activator but abolished responses to (±)-11,12-EET. The downregulation of G(q)/11 failed to prevent 11,12-EET-induced TRPC6 channel translocation or the formation of capillary-like structures. Taken together, our results suggest that a G(s)-coupled receptor in the endothelial cell membrane responds to 11(R),12(S)-EET and mediates the PKA-dependent translocation and activation of TRPC6 channels, as well as angiogenesis.

An omega‐3 epoxide of docosahexaenoic acid lowers blood pressure in angiotensin II‐dependent hypertension (832.5)

Mediators of anti‐hypertensive actions of docosahexaenoic acid (DHA) are largely unknown. The omega‐3 epoxide of DHA, 19, 20‐EDP (epoxydocosapentaenoic acid) is metabolized by soluble epoxide hydrolase (sEH), which also metabolizes the anti‐inflammatory and anti‐hypertensive arachidonic acid (ARA) epoxides, EETs (epoxyeicosatrienoic acids). Based in part on plasma levels of EDPs following a DHA‐rich diet, we hypothesized that 19, 20‐EDP contributes to the anti‐hypertensive actions of DHA in angiotensin‐II dependent hypertension. Treatment individually with 19, 20‐EDP, and a potent sEH inhibitor (sEHI) TPPU (1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea) significantly lowered blood pressure (BP) as compared to angiotensin‐II infused animals. The largest reduction in BP was obtained with the combination of 19, 20‐EDP and TPPU, which was more efficacious than the combination of 14, 15‐EET and TPPU. Oxylipin profiling revealed that 19, 20‐EDP and 14, 15‐EET infusion affected mostly metabolites of the P450 pathway but also renal levels of prostaglandin‐E2. Our findings suggest that 19, 20‐ EDP is a mediator of the anti‐hypertensive and anti‐inflammatory effects of DHA in angiotensin‐II dependent hypertension. It appears that 19, 20‐ EDP requires metabolic stabilization with a sEHI to be most effective in lowering BP, although both TPPU and 19, 20‐ EDP are so effective on their own that demonstrating additive or synergistic interactions is difficult.Grant Funding Source: Supported by NIEHS R01 ES002710, NIEHS Superfund Research Program grant P42 ES004699

Activity, Inhibition, and Induction of Cytochrome P450 2J2 in Adult Human Primary Cardiomyocytes

Cytochrome P450 2J2 plays a significant role in the epoxidation of arachidonic acid to signaling molecules important in cardiovascular events. CYP2J2 also contributes to drug metabolism and is responsible for the intestinal clearance of ebastine. However, the interaction between arachidonic acid metabolism and drug metabolism in cardiac tissue, the main expression site of CYP2J2, has not been examined. Here we investigate an adult-derived human primary cardiac cell line as a suitable model to study metabolic drug interactions (inhibition and induction) of CYP2J2 in cardiac tissue. The primary human cardiomyocyte cell line demonstrated similar mRNA-expression profiles of P450 enzymes to adult human ventricular tissue. CYP2J2 was the dominant isozyme with minor contributions from CYP2D6 and CYP2E1. Both terfenadine and astemizole oxidation were observed in this cell line, whereas midazolam was not metabolized suggesting lack of CYP3A activity. Compared with recombinant CYP2J2, terfenadine was hydroxylated in cardiomyocytes at a similar K(m) value of 1.5 μM. The V(max) of terfenadine hydroxylation in recombinant enzyme was found to be 29.4 pmol/pmol P450 per minute and in the cells 6.0 pmol/pmol P450 per minute. CYP2J2 activity in the cell line was inhibited by danazol, astemizole, and ketoconazole in submicromolar range, but also by xenobiotics known to cause cardiac adverse effects. Of the 14 compounds tested for CYP2J2 induction, only rosiglitazone increased mRNA expression, by 1.8-fold. This cell model can be a useful in vitro model to investigate the role of CYP2J2-mediated drug metabolism, arachidonic acid metabolism, and their association to drug induced cardiotoxicity.

Cytochrome P4502S1: a novel monocyte/macrophage fatty acid epoxygenase in human atherosclerotic plaques

Cytochrome P450 (CYP) epoxygenases metabolize endogenous polyunsaturated fatty acids to their corresponding epoxides, generating bioactive lipid mediators. The latter play an important role in vascular homeostasis, angiogenesis, and inflammation. As little is known about the functional importance of extra-vascular sources of lipid epoxides, we focused on determining whether lipid epoxide-generating CYP isoforms are expressed in human monocytes/macrophages. Epoxides were generated by freshly isolated human monocytes and production increased markedly during differentiation to macrophages. Mass spectrometric analysis identified CYP2S1 as a novel macrophage CYP and CYP2S1-containing microsomes generated epoxides of arachidonic, linoleic and eicosapentaenoic acid. Macrophage CYP2S1 expression was increased by LPS and IFN-γ (classically activated), and oxidized LDL but not IL-4 and IL-13 (alternatively activated), and was colocalised with CD68 in inflamed human tonsils but not in breast cancer metastases. Prostaglandin (PG) E(2) is an immune modulator factor that promotes phagocytosis and CYP2S1 can metabolize its immediate precursors PGG(2) and PGH(2) to 12(S)-hydroxyheptadeca-5Z,8E,10E-trienoic acid (12-HHT). We found that CYP inhibition and siRNA-mediated downregulation of CYP2S1 increased macrophage phagocytosis and that the latter effect correlated with decreased 12-HHT formation. Although no Cyp2s1 protein was detected in aortae from wild-type mice it was expressed in aortae and macrophage foam cells from ApoE(-/-) mice. Consistent with these observations CYP2S1 was colocalised with the monocyte marker CD68 in human atherosclerotic lesions. Thus, CYP2S1 generates 12-HHT and is a novel regulator of macrophage function that is expressed in classical inflammatory macrophages, and can be found in murine and human atherosclerotic plaques.

Development of an HTS assay for EPHX2 phosphatase activity and screening of nontargeted libraries

The EPXH2 gene encodes soluble epoxide hydrolase (sEH), which has two distinct enzyme activities: epoxide hydrolase (Cterm-EH) and phosphatase (Nterm-phos). The Cterm-EH is involved in the metabolism of arachidonic acid epoxides that play important roles in blood pressure, cell growth, inflammation, and pain. While recent findings suggested complementary biological roles for Nterm-phos, research is limited by the lack of potent bioavailable inhibitors of this phosphatase activity. Also, a potent bioavailable inhibitor of this activity could be important in the development of therapy for cardiovascular diseases. We report herein the development of an HTS enzyme-based assay for Nterm-phos (Z′>0.9) using AttoPhos as the substrate. This assay was used to screen a wide variety of chemical entities, including a library of known drugs that have reached through clinical evaluation (Pharmakon 1600), as well as a library of pesticides and environmental toxins. We discovered that ebselen inhibits sEH phosphatase activity. Ebselen binds to the N-terminal domain of sEH (KI=550nM) and chemically reacts with the enzyme to quickly and irreversibly inhibit Nterm-phos, and subsequently Cterm-EH, and thus represents a new class of sEH inhibitor.

Soluble epoxide hydrolase expression in a porcine model of arteriovenous graft stenosis and anti-inflammatory effects of a soluble epoxide hydrolase inhibitor.

Synthetic arteriovenous (AV) grafts, placed between an artery and vein, are used for hemodialysis but often fail due to stenosis, typically at the vein-graft anastomosis. This study recorded T lymphocyte and macrophage accumulation at the vein-graft anastomosis, suggesting a role for inflammation in stenosis development. Epoxyeicosatrienoic acids (EETs), products of cytochrome P-450 epoxidation of arachidonic acid, have vasculoprotective and anti-inflammatory effects including inhibition of platelet activation, cell migration, and adhesion. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to less active diols. The effects of a specific inhibitor of sEH (sEHI) on cytokine release from human monocytes and mouse bone marrow-derived macrophages (BMMΦ) from wild-type (WT) and sEH knockout (KO) animals were investigated. Expression of sEH protein increased over time at the anastomosis as evaluated by immunohistochemistry. Pre-exposure of adherent human monocytes to sEHI (5 μM) significantly inhibited lipopolysaccharide-induced release of monocyte chemotactic protein-1 (MCP-1) and tumor necrosis factor-α and enhanced the EET-to-diol ratio. Release of MCP-1 from WT BMMΦ was significantly inhibited but release from sEH KO BMMΦ was not attenuated indicating the specificity of the sEHI. In contrast, sEHI did not inhibit the release of macrophage inflammatory protein-1 or interleukin-6. Nuclear translocation of NF-κB, as assessed by immunocytochemical staining, was not decreased with sEHI in monocytes, but the phosphorylation of JNK was completely abrogated, suggesting this pathway is the target of sEHI effects in monocytes. These results suggest that sEHI may be useful for inhibition of inflammation and subsequently stenosis in AV grafts.

Targeting Soluble Epoxide Hydrolase for Inflammation and Pain - An Overview of Pharmacology and the Inhibitors

Chronic inflammation is an important contributing factor to a variety of human diseases including rheumatoid arthritis, inflammatory bowel disease, psoriasis and atherosclerosis. Epoxidation of arachidonic acid by cytochrome P450 enzymes during inflammation yields epoxyeicosatrienoic acids (EETs). EETs have a variety of biological effects including modulation of inflammation, vascular smooth muscle migration and platelet aggregation. The EETs levels are regulated by soluble epoxide hydrolase (sEH), the major enzyme responsible for their degradation and conversion to inactive dihydroxyeicosatrienoic acids (DHETs); thereby limiting many of the biological actions of EETs. The molecular and pharmacological inhibition of sEH has been studied extensively for benefits on the cardiovascular system. More recent studies suggest the importance of EETs and sEH in pain and inflammation. This review will discuss the current status and emerging evidence on the role of sEH and sEH inhibitors in chronic inflammatory conditions such as atherosclerosis, colitis and arthritis. Although steroids and non-steroidal anti-inflammatory drugs are effective, their chronic use is limited by the metabolic and cardiovascular side effects. Currently there are no small molecule drugs for treatment of chronic inflammation and associated pain and sEH inhibitors with their intrinsic cardiovascular protective effects can potentially fill this void.

Essential fatty acids and lipid mediators. Endocannabinoids

In 1929 Burr and Burr discovered the essential fatty acids omega-6 and omega-3. Since then, researchers have shown a growing interest in polyunsaturated fatty acids (PUFA) as precursors of "lipid mediator" molecules, often with opposing effects, prostaglandins, prostacyclins, thromboxanes, leukotrienes, lipossines, resolvines, protectines, maresins that regulate immunity, platelet aggregation, inflammation, etc. They showed that the balance between omega-3 and omega-6 acids has a profound influence on all the body's inflammatory responses and a raised level of PUFA omega-3 in tissue correlate with a reduced incidence of degenerative cardiovascular disease, some mental illnesses such as depression, and neuro-degenerative diseases such as Alzheimer's. The CYP-catalyzed epoxidation and hydroxylation of arachidonic acid (AA) were established recently as the so-called third branch of AGE cascade. Cytochrome P450 (CYP) epoxygenases convert AA to four epoxyeicosatrienoic acid (EET) regioisomers, that produce vascular relaxation anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are accessible to CYP enzymes in the same way as AA. Metabolites derived from EPA include epoxye-icosatetraenoic acids (EETR) and hydroxyeicosapentaenoic acids (19- and 20-HEPE), whereas DHA include epoxydocosapentaenoic acids (EDPs) hydroxydocosahexaenoic acids (21- and 22-HDoHE). For many of the CYP isoforms, the n-3 PUFAs are the preferred substrates and the available data suggest that some of the vasculo- and cardioprotective effects attributed to dietary n-3 PUFAs may be mediated by CYP-dependent metabolites of EPA and DHA. From AA derives also endocannabinoids like anandamide (N-arachidonoylethanolamine) and 2-arachidonoylglycerol, capable of mimicking the pharmacological actions of the active principle of Cannabis sativa preparations such as hashish and marijuana (-)-Delta9-tetrahydrocannabinol. They act as true 'endogenous cannabinoids' by binding and functionally activating one or both cannabinoid receptor present on nervous and peripheral cell membranes. Enzymes that carry out anandamide oxidation are the same fatty acid oxygenases that are known to act on endogenous arachidonic acid namely, the members of the COX, LOX, and P450 families of enzymes. Recent advances in the biochemistry and pharmacology of the endocannabinoid system, also for its central and peripheral roles in regulating food intake, will offer the development of novel therapeutic agents.

Cytochrome P450 2C24: expression, tissue distribution, high-throughput assay, and pharmacological inhibition

Cytochrome P450 (CYP)-mediated epoxidation of arachidonic acid (AA) contributes to important biological functions, including the pain-relieving responses produced by analgesic drugs. However, the relevant epoxygenase(s) remain unidentified. Presently, we describe the tissue distribution, high-throughput assay, and pharmacological characteristics of the rat epoxygenase CYP2C24. Following cloning from male rat liver, recombinant baculovirus containing the C-terminal His-tagged cDNA was constructed and used to express the protein in Spodoptera frugiperda (Sf9) cells. Enzymatic activity was detected with membranes, NADPH regenerating system and CYP reductase, and optimized for high throughput screening by use of the Vivid Blue© BOMCC fluorescence substrate. Quantitative real-time PCR identified CYP2C24 m-RNA in liver, kidney, heart, lung, gonad and brain. Screening of CYP2C24 activity against a panel of inhibitors showed a very strong correlation with activity against the human homologue CYP2C19. In agreement with recent findings on CYP2C19, the epoxygenase blockers PPOH and MS-PPOH inhibited CYP2C24 only weakly, confirming that these drugs are not universal epoxygenase inhibitors. Finally, comparisons of the CYP2C24 inhibitor profile with anti-analgesic activity suggests that this isoform does not contribute to brain analgesic drug action. The present methods and pharmacological data will aid in study of the biological significance of this CYP isoform.

Discovery of inhibitors of soluble epoxide hydrolase: a target with multiple potential therapeutic indications.

Discovery of inhibitors of soluble epoxide hydrolase: a target with multiple potential therapeutic indications.

Effect of cytochrome P450-dependent epoxyeicosanoids on Ristocetin-induced thrombocyte aggregation

Epoxyeicosatrienoic acids (EETs) produced by cytochrome P450 (CYP)-dependent epoxidation of arachidonic acid (AA) inhibit thrombocyte adhesion to the vascular wall. Upon dietary omega-3 fatty acid supplementation, EETs are partially replaced by eicosapentaenoic acid (EPA)-derived epoxyeicosatetraenoic acids (EEQs) and docosahexaenoic acid (DHA)-derived epoxydocosapentaenoic acids (EDPs). We hypothesized that the omega-3 epoxy-metabolites may exhibit superior anti-thrombogenic properties compared to their AA-derived counterparts. To test this hypothesis, we analyzed the effects of 11,12-EET, 17,18-EEQ and 19,20-EDP on Ristocetin-induced thrombocyte aggregation (RITA), a process that mimics thrombocyte adhesion to the vascular wall. The eicosanoids were added for 5, 30, or 60 minutes to thrombocyte-rich plasma freshly prepared immediately after blood collection from stringently selected apparently healthy subjects. Thrombocyte aggregation was then induced by Ristocetin (0.75 mg/mL) and assessed by turbidimetric measurements. After 60 minutes of preincubation, all three epoxy-metabolites significantly decreased the rate of RITA. 17,18-EEQ and 19,20-EDP were effective already at 1 μM, whereas 5-fold higher concentrations were required with 11,12-EET. Addition of AUDA, an inhibitor of the soluble epoxide hydrolase, potentiated the effect of 17,18-EEQ resulting in a significant further decrease of the velocity as well as amplitude of the aggregation process. In contrast to their profound effects on RITA, none of the epoxy-metabolites was effective in reducing collagen- or ADP-induced thrombocyte aggregation. These results indicate a highly specific role of CYP-eicosanoids in preventing thromboembolic events and suggest that the formation of 17,18-EEQ and 19,20-EDP may contribute to the anti-thrombotic effects of omega-3 fatty acids.

Effects of Dynamic Exercise on Plasma Arachidonic Acid Epoxides and Diols in Human Volunteers

Metabolites of the cytochrome P450 (CYP) pathway may contribute to vasodilation of the vasculature. However, it is not known whether exercise affects their circulating concentrations. The authors determined effects of exercise intensity and duration on plasma concentrations of epoxy and dihydroxy metabolites of arachidonic acid. Their goal was to delineate the threshold workload, optimal workload, and duration required to produce increases in plasma concentrations of these vasoactive substances. Healthy volunteers (N = 14) performed maximal exercise testing on a bicycle ergometer during Visit 1. On separate days, subjects cycled for 20 min at 30%, 60%, and 80% of their maximal exercise intensity. The last day consisted of 40 min of exercise at 60% of maximal exercise intensity. Venous blood was obtained before, during, and after exercise for analysis. Compared with rest, increases were observed during the 80% workload at 20 min postexercise -14,15-DHET (0.77 ± 0.21 vs. 0.93 ± 0.27 nM) - and at 2 min postexercise: 11,12-DHET (0.64 ± 0.22 vs. 0.71 ± 0.24 nM; p < .05). Also compared with rest, 40-min values during the 60% workload were 14,15-DHET 0.79 ± 0.22 vs. 0.91 ± 0.31 nM and at 2 min post 14,15 EET 0.12 ± 0.06 vs. 0.21 ± 0.16 nM (p < .05). Results suggest the CYP metabolites (i.e., DHETs) are released during short-term high-intensity and long-term moderate-intensity exercise.

The soluble epoxide hydrolase as a pharmaceutical target for pain management.

ISSN 1758-1869 10.2217/PMT.11.47 © 2011 Future Medicine Ltd Pain Manage. (2011) 1(5), 383–386 A newly characterized drug target, the soluble epoxide hydrolase (sEH), offers hope for developing new agents for the control of both inflammatory and neuropathic pain. Although over 70% of the pharmaceuticals used worldwide are based on targets in the arachidonate cascade (including, among others, aspirin, cyclooxygenase [COX-2 inhibitors [COXIBs], NSAIDs, and blockers of the lipoxygenase 5 pathway), the cytochrome P450 branch of the cascade has been little studied and is currently the target of no pharmaceutical agents (Figure 1). Several P450 enzymes are involved in the production of both proand anti-inflammatory derivatives of arachidonic acid and of particular relevance to this editorial are the epoxides of arachidonic acid, also known as epoxyeicosatrienoic acids (EETs). EETs and other fatty acid epoxides are increasingly recognized as anti-inflammatory mediators acting directly to reduce NF-kB nuclear translocation but also to reduce a variety of inflammatory enzymes such as COX-2 which are induced in inflammation. It has proven difficult to modulate the P450s selectively in vivo with pharmaceuticals. However, the anti-inf lammatory EETs and other fatty acid epoxides are rapidly metabolized by a single enzyme known as the sEH (Figure 1). The resulting diols are far more polar and quickly move out of tissues into circulation. In most cases they are less biologically active than the epoxides, but in some cases the diols are actually proinf lammatory. Thus inhibitors of the sEH will stabilize the antiinflammatory EETs, and they have been evaluated in rodent models for a variety of inflammation-driven processes including cardiac hypertrophy, atherosclerosis, renal inflammation and fibrosis in addition to other indications [1]. There are a variety of small molecule transition state mimics available capable of inhibiting the human sEH in the low nanoto pico-molar concentration. Some of these inhibitors have good topical and oral availability and good pharmacokinetics including the sEH inhibitor shown in Figure 2. These compounds show good target engagement in vivo as demonstrated by their ability to stabilize and thus increase blood levels of epoxidized fatty acids while decreasing the concentrations of the corresponding diols [2]. The sEH inhibitors have been shown to reduce inf lammation with greater potency than NSAIDs in a number of

Soluble epoxide hydrolase in the generation and maintenance of high blood pressure in spontaneously hypertensive rats

We hypothesized that perinatal inhibition of soluble epoxide hydrolase (SEH), which metabolizes epoxyeicosatrienoic acids in the arachidonic acid (AA) cascade, with an orally active SEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), would persistently reduce blood pressure (BP) in adult SHR despite discontinuation of AUDA at 4 wk of age. Renal cytoplasmic epoxide hydrolase-2 (Ephx2) gene expression was enhanced in SHR vs. WKY from 2 days to 24 wk. Effects of perinatal treatment with AUDA, supplied to SHR dams until 4 wk after birth, on BP in female and male offspring and renal oxylipin metabolome in female offspring were observed and contrasted to female SHR for direct effects of AUDA (8-12 wk). Briefly, inhibition of SEH was effective in persistently reducing BP in female SHR when applied during the perinatal phase. This was accompanied by marked increases in major renal AA epoxides and decreases in renal lipoxygenase products of AA. Early inhibition of SEH induced a delayed increase in renal 5-HETE at 24 wk, in contrast to a decrease at 2 wk. Inhibition of SEH in female SHR from 8 to 12 wk did not reduce BP but caused profound decreases in renal 15(S)-HETrE, LTB4, TBX2, 5-HETE, and 20-HETE and increases in TriHOMEs. In male SHR, BP reduction after perinatal AUDA was transient. Thus, Ephx2 transcription and SEH activity in early life may initiate mechanisms that eventually contribute to high BP in adult female SHR. However, programmed BP-lowering effects of perinatal SEH inhibition in female SHR cannot be simply explained by persistent reduction in renal SEH activity but rather by more complex and temporally dynamic interactions between the renal SEH, lipoxygenase, and cyclooxygenase pathways.

The ω‐3 epoxide of eicosapentaenoic acid inhibits endothelial cell proliferation by p38 MAP kinase activation and cyclin D1/CDK4 down‐regulation

Dietary intake of ω-3 polyunsaturated fatty acids (ω-3 PUFAs) like eicosapentaenoic acid (EPA) decreases cancer risk, while arachidonic acid and other ω-6 PUFAs increase risk, but the underlying mechanisms are unclear. Cytochrome P450 (CYP)-derived epoxides contribute to enhanced tumourigenesis due to ω-6 PUFA intake. Thus, ω-6 arachidonic acid epoxides (EETs) inhibit apoptosis and stimulate proliferation by up-regulating cyclin D1 expression in cells. The present study evaluated the corresponding ω-3 PUFA epoxides and assessed their role in the regulation of cell proliferation. Four chemically stable EPA epoxides (formed at the 8,9-, 11,12-, 14,15- and 17,18-olefinic bonds) were synthesized and tested against growth-related signalling pathways in brain microvascular endothelial bEND.3 cells. Cell cycle distribution was determined by flow cytometry and cyclin gene expression by immunoblotting and real-time PCR. The role of the p38 mitogen-activated protein (MAP) kinase in cyclin D1 dysregulation was assessed using specific inhibitors and dominant-negative expression plasmids. The ω-3 17,18-epoxide of EPA decreased cell proliferation, interrupted the cell cycle in S-phase and down-regulated the cyclin D1/cyclin-dependent kinase (CDK)-4 complex, whereas the 8,9-, 11,12- and 14,15-epoxides were either inactive or enhanced proliferation. Cyclin D1 down-regulation by 17,18-epoxy-EPA was mediated by activation of the growth-suppressing p38 MAP kinase, but the alternate EPA-epoxides were inactive. The present findings suggest that the epoxide formed by CYP enzymes at the ω-3 olefinic bond may contribute to the beneficial effects of ω-3 PUFA by down-regulating cyclin D1 and suppressing cell proliferation.