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Aim: 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity., Methods: Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A 3 , trioxilin A 3 and trioxilin C 3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK)., Results: All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A 3 , C 3 and hepoxilin A 3 (3 μm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A 3 , C 3 and hepoxilin A 3 (10 μm) inhibited U46619 (10 nM)-induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B 3 and hepoxilin B 3 were not synthesized in arteries and exhibited little biological activity., Conclusion: Trioxilin A 3 and C 3 and hepoxilin A 3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists., Competing Interests: The authors declared no conflicts of interest, financial or otherwise., (© 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

Epoxyeicosatrienoic acids (EETs) are endogenous ligands that undergo hydrolysis by soluble epoxide hydrolase (sEH). The responses of 11, 12-EET in comparison with other vasodilator agonists including carbachol and sodium nitroprusside (SNP) were investigated. The effect of 1-cyclohexyl-3-dodecyl urea (CDU), a sEH, was tested on the vasodilator effect induced by 11, 12-EET in the perfused mesenteric beds isolated from normo-glycaemic and type-1 STZ-diabetic rats. In the perfused mesenteric beds of control and diabetic animals, 11, 12-EET produced vasodilation in a dose-dependent manner. The vasodilator response induced by 11, 12-EET was significantly decreased in tissues obtained from diabetic animals, but this was significantly corrected through inhibition of sEH. The effects of nitric oxide synthase inhibitor, cyclo-oxygenase inhibitor, specific potassium channel inhibitors, soluble guanylyl cyclase inhibitor and transient receptor potential channel V4 inhibitor, on vasodilator response to 11, 12-EET were investigated. In tissues isolated from control animals, vasodilator responses to 11, 12-EET were not inhibited by acute incubation with l-NAME, l-NAME with indomethacin, glibenclamide, iberiotoxin, charybdotoxin, apamin or ODQ. Incubation with the transient receptor potential channel V4 inhibitor ruthenium red caused significantly reduced vasodilator responses induced by 11, 12-EET. In conclusion, results from this study indicate that 11, 12-EET has a vasodilator effect in the perfused mesenteric bed, partly through activation of vanilloid receptor. A strategy to elevate the levels of EETs may have a significant impact in correcting microvascular abnormality associated with diabetes., (© 2017 The Authors. Autonomic & Autacoid Pharmacology Published by John Wiley & Sons Ltd.)

The objective of the present study was to assess the effect of elevating epoxygenated fatty acids on retinal vascular inflammation. To stimulate inflammation we utilized TNFα, a potent pro-inflammatory mediator that is elevated in the serum and vitreous of diabetic patients. In TNFα-stimulated primary human retinal microvascular endothelial cells, total levels of epoxyeicosatrienoic acids (EETs), but not epoxydocosapentaenoic acids (EDPs), were significantly decreased. Exogenous addition of 11,12-EET or 19,20-EDP when combined with 12-(3-adamantane-1-yl-ureido)-dodecanoic acid (AUDA), an inhibitor of epoxide hydrolysis, inhibited VCAM-1 and ICAM-1 expression and protein levels; conversely the diol product of 19,20-EDP hydrolysis, 19,20-DHDP, induced VCAM1 and ICAM1 expression. 11,12-EET and 19,20-EDP also inhibited leukocyte adherence to human retinal microvascular endothelial cell monolayers and leukostasis in an acute mouse model of retinal inflammation. Our results indicate that this inhibition may be mediated through an indirect effect on NFκB activation. This is the first study demonstrating a direct comparison of EET and EDP on vascular inflammatory endpoints, and we have confirmed a comparable efficacy from each isomer, suggesting a similar mechanism of action. Taken together, these data establish that epoxygenated fatty acid elevation will inhibit early pathology related to TNFα-induced inflammation in retinal vascular diseases.

Cardiovascular disease remains the leading cause of death worldwide. Among many potential targets for pharmacological intervention, a promising strategy involves epoxyeicosatrienoic acid (EET) and soluble epoxide hydroxylase (sEH) inhibition. sEH is the enzyme that converts EET to its less potent metabolite; therefore, EET is upregulated by its inhibitor. EET has pleotropic effects that collectively reduce inflammation, while increasing vasodilation and insulin sensitivity. Recent reports indicate that EET agonists and sEH inhibitors are capable of not only reversing endothelial dysfunction and hypertension, but also of reversing cardiac remodeling, which is a hallmark of cardiomyopathy and the metabolic syndrome. EET agonists and sEH inhibitors are in development as potential therapies, and at least one drug is already in clinical trials. This review examines the activity of EET in biological systems, proposes a series of pathways to explain its mechanism of action, and discusses how these might be exploited for potential therapeutic use., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Epoxyeicosatrienoic acids (EETs) are metabolic products of free arachidonic acid, which are produced through cytochrome P-450 (CYP) epoxygenases. EETs have anti-inflammatory, antiapoptotic, and antioxidative activities. However, the effect of EETs on cigarette smoke-induced lung inflammation is not clear. Autophagy is believed to be involved in the pathogenesis of chronic obstructive pulmonary disease. In addition, nuclear erythroid-related factor 2 (Nrf2), a transcription factor that regulates many antioxidant genes, is thought to regulate antioxidant defenses in several lung diseases. In addition, interaction between EETs, autophagy, and Nrf2 has been reported. The aim of this study was to explore the effect of 14,15-EET on cigarette smoke condensate (CSC)-induced inflammation in a human bronchial epithelial cell line (Beas-2B), and to determine whether the underlying mechanisms involved in the regulation of Nrf2 through inhibition of autophagy. Autophagy and expression of autophagy signaling pathway proteins (LC3B, p62, PI3K, Akt, p-Akt, and p-mTOR) and anti-inflammatory proteins (Nrf2 and HO-1) were assessed via Western blot analysis. Autophagosomes and autolysosomes were detected by adenoviral mRFP-GFP-LC3 transfection. Inflammatory factors (IL-6, IL-8, and MCP-1) were detected by ELISA. Lentiviral vectors carrying p62 short hairpin RNA were used to interfere with p62 expression to evaluate the effect of p62 on Nrf2 expression. Nrf2 expression was determined through immunocytochemistry. 14,15-EET treatment resulted in a significant reduction in IL-6, IL-8, and MCP-1 secretion, and increased accumulation of Nrf2 and expression of HO-1. In addition, 14,15-EET inhibited CSC-induced autophagy in Beas-2B cells. The mechanism of the anti-inflammatory effect of 14,15-EET involved inhibition of autophagy and an increase in p62 levels, followed by translocation of Nrf2 into the nucleus, which then upregulated expression of the antioxidant enzyme HO-1. 14,15-EET protects against CSC-induced lung inflammation by promoting accumulation of Nrf2 via inhibition of autophagy., (Copyright © 2016 the American Physiological Society.)

Objective: Dietary supplementation with polyunsaturated fatty acids has been widely used for primary and secondary prevention of cardiovascular disease in individuals at risk; however, the cardioprotective benefits of polyunsaturated fatty acids remain controversial because of lack of mechanistic and in vivo evidence. We present direct evidence that an omega-6 polyunsaturated fatty acid, dihomo-γ-linolenic acid (DGLA), exhibits in vivo cardioprotection through 12-lipoxygenase (12-LOX) oxidation of DGLA to its reduced oxidized lipid form, 12(S)-hydroxy-8Z,10E,14Z-eicosatrienoic acid (12(S)-HETrE), inhibiting platelet activation and thrombosis., Approach and Results: DGLA inhibited ex vivo platelet aggregation and Rap1 activation in wild-type mice, but not in mice lacking 12-LOX expression (12-LOX(-/-)). Similarly, wild-type mice treated with DGLA were able to reduce thrombus growth (platelet and fibrin accumulation) after laser-induced injury of the arteriole of the cremaster muscle, but not 12-LOX(-/-) mice, supporting a 12-LOX requirement for mediating the inhibitory effects of DGLA on platelet-mediated thrombus formation. Platelet activation and thrombus formation were also suppressed when directly treated with 12(S)-HETrE. Importantly, 2 hemostatic models, tail bleeding and arteriole rupture of the cremaster muscle, showed no alteration in hemostasis after 12(S)-HETrE treatment. Finally, the mechanism for 12(S)-HETrE protection was shown to be mediated via a Gαs-linked G-protein-coupled receptor pathway in human platelets., Conclusions: This study provides the direct evidence that an omega-6 polyunsaturated fatty acid, DGLA, inhibits injury-induced thrombosis through its 12-LOX oxylipin, 12(S)-HETrE, which strongly supports the potential cardioprotective benefits of DGLA supplementation through its regulation of platelet function. Furthermore, this is the first evidence of a 12-LOX oxylipin regulating platelet function in a Gs α subunit-linked G-protein-coupled receptor-dependent manner., (© 2016 American Heart Association, Inc.)

Cytochrome P450 epoyxgenase 2J2 and epoxyeicosatrienoic acids (EETs) are known to protect against cardiac hypertrophy and heart failure, which involve the activation of 5'-AMP-activated protein kinase (AMPK) and Akt. Although the functional roles of AMPK and Akt are well established, the significance of cross talk between them in the development of cardiac hypertrophy and antihypertrophy of CYP2J2 and EETs remains unclear. We investigated whether CYP2J2 and its metabolites EETs protected against cardiac hypertrophy by activating AMPKα2 and Akt1. Moreover, we tested whether EETs enhanced cross talk between AMPKα2 and phosphorylated Akt1 (p-Akt1), and stimulated nuclear translocation of p-Akt1, to exert their antihypertrophic effects. AMPKα2(-/-) mice that overexpressed CYP2J2 in heart were treated with Ang II for 2 weeks. Interestingly, overexpression of CYP2J2 suppressed cardiac hypertrophy and increased levels of atrial natriuretic peptide (ANP) in the heart tissue and plasma of wild-type mice but not AMPKα2(-/-) mice. The CYP2J2 metabolites, 11,12-EET, activated AMPKα2 to induce nuclear translocation of p-Akt1 selectively, which increased the production of ANP and therefore inhibited the development of cardiac hypertrophy. Furthermore, by co-immunoprecipitation analysis, we found that AMPKα2β2γ1 and p-Akt1 interact through the direct binding of the AMPKγ1 subunit to the Akt1 protein kinase domain. This interaction was enhanced by 11,12-EET. Our studies reveal a novel mechanism in which CYP2J2 and EETs enhanced Akt1 nuclear translocation through interaction with AMPKα2β2γ1 and protect against cardiac hypertrophy and suggest that overexpression of CYP2J2 might have clinical potential to suppress cardiac hypertrophy and heart failure., (© 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Maintenance of a healthy pool of mitochondria is important for the function and survival of terminally differentiated cells such as cardiomyocytes. Epoxyeicosatrienoic acids (EETs) are epoxy lipids derived from metabolism of arachidonic acid by cytochrome P450 epoxygenases. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. The aim of this study was to investigate whether EET-mediated effects influence mitochondrial quality in HL-1 cardiac cells during starvation. HL-1 cells were subjected to serum- and amino acid free conditions for 24h. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EET-mediated events significantly improved mitochondrial function as assessed by preservation of the ADP/ATP ratio and oxidative respiratory capacity. Starvation induced mitochondrial hyperfusion observed in control cells was attenuated by UA-8. However, EET-mediated events did not affect the expression of mitochondrial dynamic proteins Fis1, DRP-1 or Mfn2. Rather we observed increased levels of OPA-1 oligomers and increased mitochondrial cristae density, which correlated with the preserved mitochondrial function. Increased DNA binding activity of pCREB and Nrf1/2 and increased SIRT1 activity together with elevated mitochondrial proteins suggest EET-mediated events led to preserved mitobiogenesis. Thus, we provide new evidence for EET-mediated events that preserve a healthier pool of mitochondria in cardiac cells following starvation-induced stress.

Recent research has demonstrated that colon cancer cell proliferation can be suppressed in the cells that overexpress COX-2 via generating 8-hydroxyoctanoic acid (a free radical byproduct) during dihomo-γ-linolenic acid (DGLA, an ω-6 fatty acid) peroxidation from knocking down cellular delta-5-desaturase (D5D, the key enzyme for converting DGLA to the downstream ω-6, arachidonic acid). Here, this novel research finding is extended to pancreatic cancer growth, as COX-2 is also commonly overexpressed in pancreatic cancer. The pancreatic cancer cell line, BxPC-3 (with high COX-2 expression and mutated p53), was used to assess not only the inhibitory effects of the enhanced formation of 8-hydroxyoctanoic acid from cellular COX-2-catalyzed DGLA peroxidation but also its potential synergistic and/or additive effect on current chemotherapy drugs. This work demonstrated that, by inducing DNA damage through inhibition of histone deacetylase, a threshold level of 8-hydroxyoctanoic acid achieved in DGLA-treated and D5D-knockdown BxPC-3 cells subsequently induce cancer cell apoptosis. Furthermore, it was shown that a combination of D5D knockdown along with DGLA treatment could also significantly sensitize BxPC-3 cells to various chemotherapy drugs, likely via a p53-independent pathway through downregulating of anti-apoptotic proteins (e.g., Bcl-2) and activating pro-apoptotic proteins (e.g., caspase 3, -9). This study reinforces the supposition that using commonly overexpressed COX-2 for molecular targeting, a strategy conceptually distinct from the prevailing COX-2 inhibition strategy used in cancer treatment, is an important as well as viable alternative to inhibit cancer cell growth. Based on the COX-2 metabolic cascade, the outcomes presented here could guide the development of a novel ω-6-based dietary care strategy in combination with chemotherapy for pancreatic cancer., (Copyright © 2016. Published by Elsevier Inc.)

Infiltrating neutrophils are known to promote in the development of tumor. However, it is unclear whether and how neutrophils are involved in triggering the growth of dormant metastases. Here we show that 14,15-epoxyeicosatrienoic acid (14,15-EET) can trigger the growth of dormant micrometastases by inducing neutrophilic infiltration and converting neutrophil function. 14,15-EET triggered neutrophil infiltration in metastatic lesions by activating STAT3 and JNK pathways to induce the expression of human IL-8 and murine CXCL15 in corresponding tumor cells. The continuous expression of hIL-8/mCXCL15 was maintained by the sustained and enhanced activation of JNK pathway. 14,15-EET up-regulated miR-155 expression by activating STAT3 and JNK pathways. miR-155 in turn down-regulated the expression of SHIP1 and DET1, thus augmenting the activation of JNK and c-Jun. Moreover, the function of neutrophils was converted from tumor-suppressing to tumor-promoting by 14,15-EET in vivo. By inducing the production of G-CSF/IL-6 in vivo, 14,15-EET induced the enhancement of STAT3 activation in neutrophils to increase MMP-9 expression and decrease TRAIL expression. Neutrophil-derived MMP-9 was required for 14,15-EET to induce angiogenesis during the growth of dormant micrometastases. Depleting neutrophils or inhibiting hIL-8/mCXCL15 up-regulation resulted in the failure of 14,15-EET to promote the development of micrometastases. These findings reveal a mechanism through which the infiltration and tumor-promoting function of neutrophils could be induced to trigger the growth of dormant metastases, which might be a driving force for the tumor recurrence based on dormant metastases., Competing Interests: The authors declare that there are no conflicts of interest to disclose.

Accumulating data suggest that epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid, both cytochrome P450 (P450) enzyme metabolites of arachidonic acid (AA), play important roles in cardiovascular diseases. For many years, the cardiotonic pill (CP), an herbal preparation derived from Salviae Miltiorrhizae Radix et Rhizoma, Notoginseng Radix et Rhizoma, and Borneolum Syntheticum, has been widely used in China for the treatment of coronary artery disease. However, its pharmacological mechanism has not been well elucidated. The purpose of this study was to investigate the chronic effects of the CP on myocardial ischemia-reperfusion injury (MIRI) and AA P450 enzyme metabolism in rats (in vivo) and H9c2 cells (in vitro). The results showed that CP dose dependently (10, 20, and 40 mg/kg/d; 7 days) mitigated MIRI in rats. The plasma concentrations of EETs in CP-treated ischemia-reperfusion (I/R) rats (40 mg/kg/d; 7 days) were significantly higher (P < 0.05) than those in controls. Cardiac Cyp1b1, Cyp2b1, Cyp2e1, Cyp2j3, and Cyp4f6 were significantly induced (P < 0.05); CYP2J and CYP2C11 proteins were upregulated (P < 0.05); and AA-epoxygenases activity was significantly increased (P < 0.05) after CP (40 mg/kg/d; 7 days) administration in rats. In H9c2 cells, the CP also increased (P < 0.05) the EET concentrations and showed protection in hypoxia-reoxygenation (H/R) cells. However, an antagonist of EETs, 14,15-epoxyeicosa-5(Z)-enoic acid, displayed a dose-dependent depression of the CP's protective effects in H/R cells. In conclusion, upregulation of cardiac epoxygenases after multiple doses of the CP-leading to elevated concentrations of cardioprotective EETs after myocardial I/R-may be the underlying mechanism, at least in part, for the CP's cardioprotective effect in rats., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid via CYP/epoxygenases, which are catabolized by soluble epoxide hydrolase (sEH) and known to possess cardioprotective properties. To date, the role of sEH in the modulation of pressure-induced myogenic response/constriction in coronary arteries, an important regulatory mechanism in the coronary circulation, and the issue as to whether the disruption of the sEH gene affects the myogenic response sex differentially have never been addressed. To this end, experiments were conducted on male (M) and female (F) wild-type (WT) and sEH-knockout (KO) mice. Pressure-diameter relationships were assessed in isolated and cannulated coronary arteries. All vessels constricted in response to increases in intraluminal pressure from 60 to 120 mmHg. Myogenic vasoconstriction was significantly attenuated, expressed as an upward shift in the pressure-diameter curve of vessels, associated with higher cardiac EETs in M-KO, F-WT, and F-KO mice compared with M-WT controls. Blockade of EETs via exposure of vessels to 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) prevented the attenuated myogenic constriction in sEH-KO mice. In the presence of 14,15-EEZE, pressure-diameter curves of females presented an upward shift from those of males, exhibiting a sex-different phenotype. Additional administration of N(ω)-nitro-l-arginine methyl ester eliminated the sex difference in myogenic responses, leading to four overlapped pressure-diameter curves. Cardiac sEH was downregulated in F-WT compared with M-WT mice, whereas expression of endothelial nitric oxide synthase and CYP4A (20-HETE synthase) was comparable among all groups. In summary, in combination with NO, the increased EET bioavailability as a function of genetic deletion and/or downregulation of sEH accounts for the female-favorable attenuation of pressure-induced vasoconstriction., (Copyright © 2016 the American Physiological Society.)

Objective: Endothelial dysfunction, including upregulation of inflammatory adhesion molecules and impaired vasodilatation, is a key element in cardiovascular disease. Aging and estrogen withdrawal in women are associated with endothelial inflammation, vascular stiffness and increased cardiovascular disease. Epoxyecosatrienoic acids (EETs), the products of arachidonic acid metabolism mediated by cytochrome P450 (CYP) 2J, 2C and other isoforms, are regulated by soluble epoxide hydrolase (sEH)-catalyzed conversion into less active diols. We hypothesized that 11,12-EETs would reduce the endothelial dysfunction associated with aging and estrogen loss., Approach/results: When stabilized by an sEH inhibitor (seHi), 11,12-EET at a physiologically low dose (0.1nM) reduced cytokine-stimulated upregulation of adhesion molecules on human aorta endothelial cells (HAEC) and monocyte adhesion under shear flow through marked depolarization of the HAEC when combined with TNFα. Mechanistically, neither 11,12-EETs nor 17β-estradiol (E2) at physiologic concentrations prevented activation of NFκB by TNFα. E2 at physiological concentrations reduced sEH expression in HAEC, but did not alter CYP expression, and when combined with TNFα depolarized the cell. We also examined vascular dysfunction in adult and aged ovariectomized Norway brown rats (with and without E2 replacement) using an ex-vivo model to analyze endothelial function in an intact segment of artery. sEHi and 11,12-EET with or without E2 attenuated phenylephrine induced constriction and increased endothelial-dependent dilation of aortic rings from ovariectomized rats., Conclusions: Increasing 11,12-EETs through sEH inhibition effectively attenuates inflammation and may provide an effective strategy to preserve endothelial function and prevent atherosclerotic heart disease in postmenopausal women., (Published by Elsevier Ltd.)

Background: Small renal arteries have a significant role in the regulation of renal hemodynamics and blood pressure (BP). To study potential changes in the regulation of vascular function in hypertension, we examined renal vasodilatory responses of small arteries from nonclipped kidneys of the 2-kidney, 1-clip Goldblatt hypertensive rats to native epoxyeicosatrienoic acids (EETs) that are believed to be involved in the regulation of renal vascular function and BP. A total of 2 newly synthesized EET analogues were also examined., Materials and Methods: Renal interlobular arteries isolated from the nonclipped kidneys on day 28 after clipping were preconstricted with phenylephrine, pressurized and the effects of a 14,15-EET analogue, native 14,15-EET and 11,12-ether-EET-8ZE, an analogue of 11,12-EET, on the vascular diameter were determined and compared to the responses of arteries from the kidneys of sham-operated rats., Results: In the arteries from nonclipped kidneys isolated in the maintenance phase of Goldblatt hypertension, the maximal vasodilatory response to 14,15-EET analogue was 30.1 ± 2.8% versus 49.8 ± 7.2% in sham-operated rats; the respective values for 11,12-ther-EET-8ZE were 31.4 ± 6.4% versus 80.4 ± 6%, and for native EETs they were 41.7 ± 6.6% versus 62.8 ± 4.4% (P ≤ 0.05 for each difference)., Conclusions: We propose that reduced vasodilatory action and decreased intrarenal bioavailability of EETs combined with intrarenal angiotensin II levels that are inappropriately high for hypertensive rats underlie functional derangements of the nonclipped kidneys of 2-kidney, 1-clip Goldblatt hypertensive rats. These derangements could play an important role in pathophysiology of sustained BP elevation observed in this animal model of human renovascular hypertension., Competing Interests: Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (Copyright © 2016 Southern Society for Clinical Investigation. Published by Elsevier Inc. All rights reserved.)

Multiple players are involved in the highly complex pathophysiologic responses after stroke. Therefore, therapeutic approaches that target multiple cellular elements of the neurovascular unit in the damage cascade hold considerable promise for the treatment of stroke. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active eicosanoids called epoxyeicosatrienoic acids (EETs), which are further converted by soluble epoxide hydrolase (sEH) to less bioactive diols. EETs have been shown to exert direct cytoprotective effects upon several individual components of the neurovascular unit under simulated ischemic conditions in vitro. However, the cellular mechanism underlying EET-mediated neuroprotective effects after ischemia remains to be clarified. In this study, we investigated the effects of 14,15-EET and 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), a selective inhibitor of sEH, on multiple elements of neurovascular unit of the rat brain after middle cerebral artery occlusion-induced focal ischemia. The results showed that exogenous administration of 14,15-EET or AUDA could suppress astrogliosis and glial scar formation, inhibit microglia activation and inflammatory response, promote angiogenesis, attenuate neuronal apoptosis and infarct volume, and further promote the behavioral function recovery after focal ischemia. The results suggest that epoxyeicosanoid signaling is a promising multi-mechanism therapeutic target for the treatment of stroke.

Background: Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acids (AA) to form epoxyeicosatrienoic acids (EETs), which exert beneficial roles in the treatment of cardiovascular diseases, but little is known about its role on adventitial remodeling., Methods: We used C57BL/6J mice in vivo and primary rat adventitial fibroblasts (AFs) in vitro treated with Angiotensin II to investigate the effects of CYP2J2 gene delivery and exogenous EETs administration on adventitial remodeling., Results: CYP/sEH system was found to exist in human adventitia, and involved in adventitial remodeling process. Exogenous EETs administration significantly inhibited Ang II-induced AFs activation, characterized by differentiation, proliferation, migration, and collagen synthesis. These protective effects were partially reversed by PPARx03B3; antagonist GW9662 pretreatment or SOCS3 siRNA transfection. EETs suppressed Ang II-induced Ix03BA;Bα phosphorylation, subsequent NF-x03BA;B nuclear translocation via PPARx03B3; dependent signaling pathway in AFs. Additionally, EETs reduced Ang II-induced JAK2, STAT3 phosphorylation and subsequent phosphor-STAT3 nuclear translocation, which were mediated by SOCS3 induction but independent of PPARx03B3; activation. Furthermore, rAAV-CYP2J2 gene delivery reduced vessel wall thickening, AFs differentiation, proliferation and collagen deposition in aortic adventitia induced by Ang II infusion, which were mediated by NF-x03BA;B and SOCS3/JAK/STAT signaling pathways in blood pressure dependent and independent manner, respectively., Conclusion: We concluded that CYP2J2 overexpression attenuated Ang II-induced adventitial remodeling via PPARx03B3;-dependent NF-x03BA;B and PPARx03B3;-independent SOCS3/JAK/STAT inflammatory signaling pathways., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

We tested the hypothesis that suppression of epoxyeicosatrienoic acid (EET) metabolism via genetic knockout of the gene for soluble epoxide hydrolase (sEH-KO), or female-specific downregulation of sEH expression, plays a role in the potentiation of pulmonary hypertension. We used male (M) and female (F) wild-type (WT) and sEH-KO mice; the latter have high pulmonary EETs. Right ventricular systolic pressure (RVSP) and mean arterial blood pressure (MABP) in control and in response to in vivo administration of U46619 (thromboxane analog), 14,15-EET, and 14,15-EEZE [14,15-epoxyeicosa-5(z)-enoic acid; antagonist of EETs] were recorded. Basal RVSP was comparable among all groups of mice, whereas MABP was significantly lower in F-WT than M-WT mice and further reduced predominantly in F-KO compared with M-KO mice. U46619 dose dependently increased RVSP and MABP in all groups of mice. The increase in RVSP was significantly greater and coincided with smaller increases in MABP in M-KO and F-WT mice compared with M-WT mice. In F-KO mice, the elevation of RVSP by U46619 was even higher than in M-KO and F-WT mice, associated with the least increase in MABP. 14,15-EEZE prevented the augmentation of U46619-induced elevation of RVSP in sEH-KO mice, whereas 14,15-EET-induced pulmonary vasoconstriction was comparable in all groups of mice. sEH expression in the lungs was reduced, paralleled with higher levels of EETs in F-WT compared with M-WT mice. In summary, EETs initiate pulmonary vasoconstriction but act as vasodilators systemically. High pulmonary EETs, as a function of downregulation or deletion of sEH, potentiate U46619-induced increases in RVSP in a female-susceptible manner., (Copyright © 2015 the American Physiological Society.)

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.

Soluble epoxide hydrolase (sEH) inhibition has been demonstrated to have beneficial effects on various diseases, such as hypertension, diabetes, and brain ischemia. However, whether sEH inhibition has therapeutic potential in Parkinson's disease is still unknown. In this paper, we found that sEH expression is increased in 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP)-treated mice, and sEH deficiency and inhibition significantly attenuated tyrosine hydroxylase (TH)-positive cell loss and improved rotarod performance. The substrate of sEH, 14,15-epoxyeicosatrienoic acid (14,15-EET), protected TH-positive cells and alleviated the rotarod performance deficits of wild-type mice but not sEH-knockout mice. Moreover, the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) abolished the neuronal protective effects of sEH deficiency. In primary cultured cortical neurons, MPP(+) induced significant Akt inactivation in neurons from sEH wild-type mice, and this effect was not observed in neurons from knockout mice. Our data indicate that sEH deficiency and inhibition increased 14,15-EET in MPTP-treated mice, which activated the Akt-mediated protection of TH-positive neurons and behavioral functioning. We also found that sEH deficiency attenuated TH-positive cell loss in a paraquat-induced mouse model of Parkinson's. Our data suggest that sEH inhibition might be a powerful tool to protect dopaminergic neurons in Parkinson's disease.

Arterial smooth muscle (SM) cells respond autonomously to changes in intravascular pressure, adjusting tension to maintain vessel diameter. The values of membrane potential (Vm) and sarcoplasmic Ca(2+) concentration (Ca(in)) within minutes of a change in pressure are the results of two opposing pathways, both of which use Ca(2+) as a signal. This works because the two Ca(2+)-signaling pathways are confined to distinct microdomains in which the Ca(2+) concentrations needed to activate key channels are transiently higher than Ca(in). A mathematical model of an isolated arterial SM cell is presented that incorporates the two types of microdomains. The first type consists of junctions between cisternae of the peripheral sarcoplasmic reticulum (SR), containing ryanodine receptors (RyRs), and the sarcolemma, containing voltage- and Ca(2+)-activated K(+) (BK) channels. These junctional microdomains promote hyperpolarization, reduced Ca(in), and relaxation. The second type is postulated to form around stretch-activated nonspecific cation channels and neighboring Ca(2+)-activated Cl(-) channels, and promotes the opposite (depolarization, increased Ca(in), and contraction). The model includes three additional compartments: the sarcoplasm, the central SR lumen, and the peripheral SR lumen. It incorporates 37 protein components. In addition to pressure, the model accommodates inputs of α- and β-adrenergic agonists, ATP, 11,12-epoxyeicosatrienoic acid, and nitric oxide (NO). The parameters of the equations were adjusted to obtain a close fit to reported Vm and Ca(in) as functions of pressure, which have been determined in cerebral arteries. The simulations were insensitive to ± 10% changes in most of the parameters. The model also simulated the effects of inhibiting RyR, BK, or voltage-activated Ca(2+) channels on Vm and Ca(in). Deletion of BK β1 subunits is known to increase arterial-SM tension. In the model, deletion of β1 raised Ca(in) at all pressures, and these increases were reversed by NO., (© 2015 Karlin.)

Background: Atopic dermatitis (AD) is a chronic and relapsing skin disorder with pruritic skin symptoms. We previously reported that dihomo-γ-linolenic acid (DGLA) prevented the development of AD in NC/Tnd mice, though the mechanism remained unclear., Objective: We attempted to investigate the mechanism of preventive effect of DGLA on AD development in NC/Tnd mice., Methods: The clinical outcomes of NC/Tnd mice that were given diets containing DGLA, arachidonic acid, or eicosapentaenoic acid were compared. Lipid mediator contents in the skin in each group were also quantified. In addition, release of lipid mediators from RBL-2H3 mast cells treated with either DGLA or prostaglandin D1 (PGD1) was measured. Furthermore, effect of PGD1 on gene expression of thymic stromal lymphopoietin (TSLP) in PAM212 keratinocyte cells was determined., Results: Only DGLA containing diet suppressed the development of dermatitis in vivo. By quantifying the 20-carbon fatty acid-derived eicosanoids in the skin, the application of DGLA was found to upregulate PGD1, which correlated with a better outcome in NC/Tnd mice. Moreover, we confirmed that mast cells produced PGD1 after DGLA exposure, thereby exerting a suppressive effect on immunoglobulin E-mediated degranulation. PGD1 also suppressed gene expression of TSLP in keratinocytes., Conclusion: These results suggest that oral administration of DGLA causes preventive effects on AD development in NC/Tnd mice by regulating the PGD1 supply., (Copyright © 2015 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.)

Cytochrome P-450 epoxygenase (EPOX)-derived epoxyeicosatrienoic acids (EETs), 5-lipoxygenase (5-LO), and leukotriene B4 (LTB4), the product of 5-LO, all play a pivotal role in the vascular inflammatory process. We have previously shown that EETs can alleviate oxidized low-density lipoprotein (ox-LDL)-induced endothelial inflammation in primary rat pulmonary artery endothelial cells (RPAECs). Here, we investigated whether ox-LDL can promote LTB4 production through the 5-LO pathway. We further explored how exogenous EETs influence ox-LDL-induced LTB4 production and activity. We found that treatment with ox-LDL increased the production of LTB4 and further led to the expression and release of both monocyte chemoattractant protein-1 (MCP-1/CCL2) and intercellular adhesion molecule-1 (ICAM-1). All of the above ox-LDL-induced changes were attenuated by the presence of 11,12-EET and 14,15-EET, as these molecules inhibited the 5-LO pathway. Furthermore, the LTB4 receptor 1 (BLT1 receptor) antagonist U75302 attenuated ox-LDL-induced ICAM-1 and MCP-1/CCL2 expression and production, whereas LY255283, a LTB4 receptor 2 (BLT2 receptor) antagonist, produced no such effects. Moreover, in RPAECs, we demonstrated that the increased expression of 5-LO and BLT1 following ox-LDL treatment resulted from the activation of nuclear factor-κB (NF-κB) via the p38 mitogen-activated protein kinase (MAPK) pathway. Our results indicated that EETs suppress ox-LDL-induced LTB4 production and subsequent inflammatory responses by downregulating the 5-LO/BLT1 receptor pathway, in which p38 MAPK phosphorylation activates NF-κB. These results suggest that the metabolism of arachidonic acid via the 5-LO and EPOX pathways may present a mutual constraint on the physiological regulation of vascular endothelial cells.

There is increasing evidence from various scientific groups that hepoxilins represent novel inflammatory mediators. In vitro studies have shown that the hepoxilins cause mobilization of intracellular calcium in human neutrophils, cause plasma leakage, and potently stimulate chemotaxis of human neutrophils. In vivo, the hepoxilin pathway is activated in conditions of inflammation, e.g. after pathogen infection, in inflamed conditions (psoriasis, arthritis), and hepoxilins promote inflammatory hyperalgesia and allodynia. Although much work has demonstrated an effect of hepoxilins on neutrophils, the hepoxilin pathway has been demonstrated in a variety of tissues, including the lung, brain, pituitary, pancreatic islets, skin, etc. A genetic defect linked to a deficiency in hepoxilin formation has been described and believed to be responsible for the scaly skin observed in ichthyosis. Despite their biological and chemical instability, the involvement of the hepoxilin pathway in pathology has been demonstrated in vitro and in vivo through either isolation of the hepoxilins themselves (or their metabolites) or implied through the use of stable hepoxilin analogs. These analogs have additionally shown efficacy in animal models of lung fibrosis, cancer, thrombosis and diabetes. Research on these compounds has merely scratched the surface, but results published to date have suggested that the hepoxilin pathway is a distinct and novel pathway leading to inflammation and hepoxilin antagonists may provide the means of controlling early aspects of the acute inflammatory phase. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance"., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Human left internal mammary arteries (LIMAs) are commonly used as donor grafts for coronary bypass surgery. Previous reports suggested that 11,12-epoxyeicosatrienoic acid (11,12-EET) is an important endothelial-derived hyperpolarizing factor (EDHF) in human LIMAs and that EETs act through large conductance Ca²⁺-activated K⁺ channels (KCa1.1) to induce smooth muscle cell hyperpolarization and relaxation in these tissues. In this study, we aimed to explore the role of vanilloid transient receptor potential channel 4 (TRPV4) and canonical transient receptor potential channel 1 (TRPC1) channels in the EET-induced smooth muscle hyperpolarization and vascular relaxation in human LIMAs. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1, and KCa1.1 physically interacted with each other to form a complex. Sharp microelectrode and vascular tension studies demonstrated that 11,12-EET (300 nmol/L) and 4α-phorbol 12,13-didecanoate (5 μmol/L) were able to induce smooth muscle membrane hyperpolarization and vascular relaxation in isolated human LIMA segments. The hyperpolarizing and relaxant effects were markedly reduced by treatments that could suppress the expression/activity of TRPV4, TRPC1, or KCa1.1. With the use of human embryonic kidney 293 cells that over-expressed with TRPV4, TRPC1 and KCa1.1, we found that TRPC1 is the linker through which TRPV4 and KCa1.1(α) can interact. The present study revealed that 11,12-EET targets the TRPV4-TRPC1-KCa1.1 complex to induce smooth muscle cell hyperpolarization and vascular relaxation in human LIMAs. This finding provides novel mechanistic insights for the EET action in human LIMAs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Epoxyeicosatrienoic acids (EETs) are products of arachidonic acid metabolism catalyzed by cytochrome P450 epoxygenases. These small molecules are autocrine and paracrine lipid mediators with important roles in inflammation, cardiovascular function, and angiogenesis. Recent evidence has highlighted EETs as potent promoters of organ regeneration and malignant metastasis. We speculated that EETs might impact osteoclastogenesis and bone loss. Using both in vitro and in vivo studies, we observed that EETs significantly attenuated bone loss and inhibited osteoclast formation and activity, which were associated with a decreased receptor activator of NF-κB ligand (RANKL):osteoprotegerin ratio and serum levels of TNF-α and IL-1β. At the molecular level, EETs abrogated RANKL-induced activation of NF-κB, activator protein-1 (AP-1), and MAPKs, including ERK and JNK, but not p38, during osteoclast formation. EETs also prevented the production of reactive oxygen species (ROS) following RANKL stimulation. As a result, EETs suppressed osteoclast-specific gene expression, including tartrate resistant acid phosphatase (TRAP), cathepsin K (CK), matrix metalloproteinase (MMP)-9, and receptor activator of NF-κB (RANK). In conclusion, our findings demonstrate that EETs inhibit osteoclastogenesis through modulation of multiple pathways both upstream and downstream of RANKL signaling. The administration or stabilized endogenous levels of EETs could represent a novel therapeutic strategy for osteoclast-related disorders, such as rheumatoid arthritis and postmenopausal osteoporosis., (© FASEB.)

In response to endothelial cell activation, arachidonic acid can be converted by cytochrome P450 (CYP) epoxygenases to epoxyeicosatrienoic acids (EETs), which have potent vasodilator and anti-inflammatory properties. In this study, we investigated the effects of exogenous EETs on cigarette smoke extract (CSE)-induced inflammation in human bronchial epithelial cells (NCI-H292). We found that CSE inhibited the expression of CYP2C8 and mildly stimulated the expression of epoxide hydrolase 2 (EPHX2) but did not change the expression of CYP2J2. Treatment with 11,12-EET or 14,15-EET attenuated the CSE-induced release of interleukin (IL)-8 by inhibiting the phosphorylation of p38 mitogen-activated protein kinases (MAPKs). Our results demonstrated that CSE may reduce the anti-inflammatory ability of epithelial cells themselves by lowering the EET level. EETs from pulmonary epithelial cells may play a critical protective role on epithelial cell injury., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: Cardiac remodelling is one of the key pathological changes that occur with cardiovascular disease. Previous studies have demonstrated the beneficial effects of CYP2J2 expression on cardiac injury. In the present study, we investigated the effects of cardiomyocyte-specific CYP2J2 expression and EET treatment on angiotensin II-induced cardiac remodelling and sought to determine the underlying molecular mechanisms involved in this process., Methods and Results: Eight-week-old mice with cardiomyocyte-specific CYP2J2 expression (αMHC-CYP2J2-Tr) and wild-type (WT) control mice were treated with Ang-II. Ang-II treatment of WT mice induced changes in heart morphology, cardiac hypertrophy and dysfunction, as well as collagen accumulation; however, cardiomyocyte-specific expression of CYP2J2 attenuated these effects. The cardioprotective effects observed in α-MHC-CYP2J2-Tr mice were associated with peroxisome proliferator-activated receptor (PPAR)-γ activation, reduced oxidative stress, reduced NF-κB p65 nuclear translocation, and inhibition of TGF-β1/smad pathway. The effects seen with cardiomyocyte-specific expression of CYP2J2 were partially blocked by treatment with PPAR-γ antagonist GW9662. In in vitro studies, 11,12-EET(1 μmol/L) treatment attenuated cardiomyocyte hypertrophy and remodelling-related protein (collagen I, TGF-β1, TIMP1) expression by inhibiting the oxidative stress-mediated NF-κB pathway via PPAR-γ activation. Furthermore, conditioned media from neonatal cardiomyocytes treated with 11,12-EET inhibited activation of cardiac fibroblasts and TGF-β1/smad pathway., Conclusion: Cardiomyocyte-specific expression of CYP2J2 or treatment with EETs protects against cardiac remodelling by attenuating oxidative stress-mediated NF-κBp65 nuclear translocation via PPAR-γ activation., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22-24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infection. This α-Toc-induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae-challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Maternal intake of omega-3 polyunsaturated fatty acids (n-3 PUFA) is critical during perinatal development of the brain. Docosahexaenoic acid (DHA) is the most abundant n-3 PUFA in the brain and influences neuronal membrane function and neuroprotection. The present study aims to assess the effect of dietary n-3 PUFA availability during the gestational and postnatal period on cognition, brain metabolism and neurohistology in C57BL/6J mice. Female wild-type C57BL/6J mice at day 0 of gestation were randomly assigned to either an n-3 PUFA deficient diet (0.05% of total fatty acids) or an n-3 PUFA adequate diet (3.83% of total fatty acids) containing preformed DHA and its precursor α-linolenic acid. Male offspring remained on diet and performed cognitive tests during puberty and adulthood. In adulthood, animals underwent (31)P magnetic resonance spectroscopy to assess brain energy metabolites. Thereafter, biochemical and immunohistochemical analyses were performed assessing inflammation, neurogenesis and synaptic plasticity. Compared to the n-3 PUFA deficient group, pubertal n-3 PUFA adequate fed mice demonstrated increased motor coordination. Adult n-3 PUFA adequate fed mice exhibited increased exploratory behavior, sensorimotor integration and spatial memory, while neurogenesis in the hippocampus was decreased. Selected brain regions of n-3 PUFA adequate fed mice contained significantly lower levels of arachidonic acid and higher levels of DHA and dihomo-γ-linolenic acid. Our data suggest that dietary n-3 PUFA can modify neural maturation and enhance brain functioning in healthy C57BL/6J mice. This indicates that availability of n-3 PUFA in infant diet during early development may have a significant impact on brain development., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Injurious inflammatory response is critical to the development of lung ischemia/reperfusion injury (LIRI). The cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acid to epoxyeicosatrienoic acids (EETs), which exert an anti-inflammatory effect on the cardiovascular system. We therefore cytochrome hypothesized that CYP2J2 overexpression and pretreatment with exogenous EETs may have the potential to reduce LIRI., Methods: A rat model was used to mimic the condition of LIRI by clamping the left pulmonary hilum for 60 minutes, followed by reperfusion for 2 hours. Moreover, we developed a cell model using human pulmonary artery endothelial cells by anoxia for 8 hours, followed by reoxygenation for 16 hours to determine the anti-inflammatory effect and mechanism of CYP2J2 overexpression and exogenous 11,12-EET., Results: Lung ischemia/reperfusion increased lung wet/dry and lung weight/body weight ratios, protein concentration in bronchoalveolar lavage fluid and concentrations of pro-inflammatory, including mediators in serum IL-1β, IL-8, TNF-α, sP- and sE-selectin, and decreased concentration of anti-inflammatory mediator IL-10. Ischemia/reperfusion also leaded to pulmonary edema and inflammation under light microscopy. Furthermore, activation of NF-κB p65 and degradation of IκBα were remarkably increased in ischemia/reperfusion lung tissues. While CYP2J2 overexpression significantly inhibited the above effects (p<0.05). In vitro data further confirmed the anti-inflammatory effect of CYP2J2 overexpression and 11,12-EET, an effect that may probably be mediated by PPARγ activation., Conclusion: CYP2J2 overexpression and administration of exogenous EETs can protect against LIRI via anti-inflammatory effects. This can be a novel potential strategy for prevention and treatment of LIRI., (© 2015 S. Karger AG, Basel.)

Background/aims: Epoxyeicosatrienoic acids (EETs), a type of lipid mediators produced by cytochrome P450 epoxygenases, exert anti-inflammatory, angiogenic, anti-oxidative and anti-apoptotic effects. However, the role of EETs in cigarette smoke-induced lung injury and the underlying mechanisms are not fully known. The aim of this study was to explore the effects of CYP2J2-EETs on cigarette smoke extracts (CSE)-induced apoptosis in human bronchial epithelial cell line (Beas-2B) and the possible mechanisms involved., Methods: Cytochrome P450 epoxygenase 2J2 (CYP2J2) and its metabolites EETs were assessed by western blotting or LC-MS-MS. Cell viability and apoptosis were determined by MTT assay and AnnexinV-PI staining. Reactive oxygen species (ROS) were assessed by measuring H2DCFDA. Caspase-3, HO-1, MAPK and endoplasmic reticulum (ER) stress-related markers GRP78, p-elF2a, and CHOP were evaluated by western blotting., Results: CSE suppressed expression of both CYP2J2 and EET by Beas-2B cells. CSE also induced apoptosis, the generation of ROS and the ER stress in Beas-2B cells. These changes were abolished by pretreatment with exogenous 14,15-EET while pretreatment with 14,15-EEZE, a selective EET antagonist, abolished the protective effects of 14,15-EET. In addition, EETs increased the expression of antioxidant enzyme HO-1. Furthermore, 14,15-EET reduced CSE-induced activation of p38 and JNK., Conclusion: The data suggest that CYP2J2-derived EETs protect against CSE-induced lung injury possibly through attenuating ER stress., (© 2015 S. Karger AG, Basel.)

Pulmonary exacerbations in cystic fibrosis airways are accompanied by inflammation, neutrophilia, and mucous thickening. Cystic fibrosis sputum contains a large amount of uncleared DNA contributed by neutrophil extracellular trap (NET) formation from neutrophils. The exact mechanisms of the induction of NETosis in cystic fibrosis airways remain unclear, especially in uninfected lungs of patients with early cystic fibrosis lung disease. Here we show that Hepoxilin A3, a proinflammatory eicosanoid, and the synthetic analog of Hepoxilin B3, PBT-3, directly induce NETosis in human neutrophils. Furthermore, we show that Hepoxilin A3-mediated NETosis is NADPH-oxidase-dependent at lower doses of Hepoxilin A3, while it is NADPH-oxidase-independent at higher doses. Together, these results demonstrate that Hepoxilin A3 is a previously unrecognized inducer of NETosis in cystic fibrosis lungs and may represent a new therapeutic target for treating cystic fibrosis and other inflammatory lung diseases.

Epoxyeicosatrienoic acids (EETs) contribute to haemodynamics, electrolyte homoeostasis and blood pressure regulation, leading to the concept that EETs can be therapeutically targeted for hypertension. In the present study, multiple structural EET analogues were synthesized based on the EET pharmacophore and vasodilator structure-activity studies. Four EET analogues with 91-119% vasodilatory activity in the isolated bovine coronary artery (EC50: 0.18-1.6 μM) were identified and studied for blood-pressure-lowering in hypertension. Two EET analogues in which the COOH group at carbon 1 of the EET pharmacophore was replaced with either an aspartic acid (EET-A) or a heterocyclic surrogate (EET-X) were administered for 14 days [10 mg/kg per day intraperitoneally (i.p.)]. Both EET-A and EET-X lowered blood pressure in spontaneously hypertensive rats (SHRs) and in angiotensin II (AngII) hypertension. On day 14, the mean arterial pressures in EET analogue-treated AngII-hypertensive and SHRs were 30-50 mmHg (EET-A) and 15-20 mmHg (EET-X) lower than those in vehicle-treated controls. These EET analogues (10 mg/kg per day) were further tested in AngII hypertension by administering orally in drinking water for 14 days and EET-A lowered blood pressure. Additional experiments demonstrated that EET-A inhibits epithelial sodium channel (ENaC) activity in cultured cortical collecting duct cells and reduced renal expression of ENaC subunits in AngII hypertension. In conclusion, we have characterized EET-A as an orally active antihypertensive EET analogue that protects vascular endothelial function and has ENaC inhibitory activity in AngII hypertension.

The effects of mead acid (MA; 5,8,11-eicosatrienoic acid) on the suppression of breast cancer cell growth and metastasis were examined in vitro and in vivo by using the KPL-1 human breast cancer cell line. MA suppressed KPL-1 cell growth in culture with an IC50 value of 214.2 µM (65.7 µg/ml) for 72 h, and MA significantly suppressed transplanted KPL-1 tumor growth (tumor volume and tumor weight: 872±103 mm3 and 1,000±116 mg vs. 376±66 mm3 and 517±84 mg) and regional (axillary) lymph node metastasis (67%, 10/15 vs. 10%, 1/10) in female athymic mice fed an MA-rich diet for 8 weeks. Tumor suppression was due to the suppression of cell proliferation. In ELISA, although vascular endothelial growth factor (VEGF) levels were unchanged, VEGF receptor (VEGFR)1 and VEGFR2 levels were significantly decreased after treatment with a 214.2-µM dose of MA for 72 h; E-cadherin levels were unchanged. As VEGF, VEGFR1 and VEGFR2 expression was co-localized in KPL-1 cells, the mechanism leading to cell growth suppression was VEGF signaling directly to KPL-1 cells by an autocrine process. In contrast, MA did not influence angiogenesis. The mechanisms of action were through VEGF signaling directly to cancer cells.

Aims: Vascular endothelial cells synthesize and release vasodilators such as nitric oxide (NO) and epoxyeicosatrienoic acids (EETs). NO is known to inhibit EET-induced smooth muscle hyperpolarization and relaxation. This study investigates the underlying mechanism of this inhibition., Methods and Results: Through measurements of membrane potential and arterial tension, we show that 11,12-EET induced membrane hyperpolarization and vascular relaxation in endothelium-denuded porcine coronary arteries. These responses were suppressed by S-nitroso-N-acetylpenicillamine (SNAP) and 8-Br-cGMP, an NO donor and a membrane-permeant analogue of cGMP, respectively. The inhibitory actions of SNAP and 8-Br-cGMP on 11,12-EET-induced membrane hyperpolarization and vascular relaxation were reversed by hydroxocobalamin, an NO scavenger; ODQ, a guanylyl cyclase inhibitor; and KT5823, a protein kinase G (PKG) inhibitor. The inhibitory actions of SNAP and 8-bromo cyclic GMP (8-Br-cGMP) on the EET responses were also abrogated by shielding TRPC1-PKG phosphorylation sites with an excessive supply of exogenous PKG substrates, TAT-TRPC1(S172) and TAT-TRPC1(T313). Furthermore, a phosphorylation assay demonstrated that PKG could directly phosphorylate TRPC1 at Ser(172) and Thr(313). In addition, 11,12-EET failed to induce membrane hyperpolarization and vascular relaxation when TRPV4, TRPC1, or KCa1.1 was selectively inhibited. Co-immunoprecipitation studies demonstrated that TRPV4, TRPC1, and KCa1.1 physically associated with each other in smooth muscle cells., Conclusion: Our findings demonstrate a novel role of the NO-cGMP-PKG pathway in the inhibition of 11,12-EET-induced smooth muscle hyperpolarization and relaxation via PKG-mediated phosphorylation of TRPC1., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

14,15-epoxyeicosatrienoic acid (14,15-EET) is implicated in regulating physiological functions of endothelial cells (ECs), yet the potential molecular mechanisms underlying the beneficial effects in ECs are not fully understood. In this study, we investigated whether transient receptor potential vanilloid receptor type 1 (TRPV1) is involved in 14,15-EET-mediated Ca(2+) influx, nitric oxide (NO) production and angiogenesis. In human microvascular endothelial cells (HMECs), 14,15-EET time-dependently increased the intracellular level of Ca(2+). Removal of extracellular Ca(2+), pharmacological inhibition or genetic disruption of TRPV1 abrogated 14,15-EET-mediated increase of intracellular Ca(2+) level in HMECs or TRPV1-transfected HEK293 cells. Furthermore, removal of extracellular Ca(2+) or pharmacological inhibition of TRPV1 decreased 14,15-EET-induced NO production. 14,15-EET-mediated tube formation was abolished by TRPV1 pharmacological inhibition. In an animal experiment, 14,15-EET-induced angiogenesis was diminished by inhibition of TRPV1 and in TRPV1-deficient mice. TRPV1 may play a crucial role in 14,15-EET-induced Ca(2+) influx, NO production and angiogenesis.

Epoxyeicosatrienoic acids (EETs) are generated from arachidonic acid catalysed by cytochrome P450 (CYP) epoxygenases. In addition to regulating vascular tone EETs may alleviate inflammation and ROS. The present study was conducted to determine whether CYP2C8 gene overexpression was able to increase the level of EETs, and subsequently prevent TNF-α induced inflammation and reactive oxygen species (ROS) in human umbilical vein endothelial cells (HUVECs) and macrophages. Peroxisome proliferator‑activated receptor γ (PPARγ) activation, nuclear factor-κB (NF-κB) activation, endothelial nitric oxide synthase (eNOS) activation, gp-91 activation, and inflammatory cytokine expression were detected by western blot analysis or enzyme-linked immuno-sorbent assay. Intracellular reactive oxygen species (ROS) was measured by flow cytometry, while the migration of vascular smooth muscle cells (VSMCs) was detected by transwell assay. pCMV-mediated CYP2C8 overexpression and its metabolites, EETs, markedly suppressed TNF-α induced inflammatory cytokines IL-6 and MCP-1 expression via the activation of NF-κB and degradation of IκBα. Moreover, pretreatment with 11,12-EET significantly blocked TNF-α-induced ROS production. CYP2C8‑derived EETs also effectively alleviated the migration of VSMCs and improved the function of endothelial cells through the upregulation of eNOS, which was significantly decreased under the stimulation of TNF-α. Furthermore, these protective effects observed were mediated by PPARγ activation. To the best of our knowledge, the results of the present study demonstrated for the first time that CYP2C8-derived EETs exerted antivascular inflammatory and anti-oxidative effects, at least in part, through the activation of PPARγ. Thus, the CYP2C8 gene may be useful in the prevention and treatment of vascular inflammatory diseases.

T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation via its metabolism leading to prostanoids, leukotrienes, and epoxyeicosatrienoic acids (EETs). However, the effects of these latter molecules on T-currents have not been investigated. Here, we describe the effects of the major cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products on the three human recombinant T-channels (Cav3.1, Cav3.2, and Cav3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Cav3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC50 = 0.54 μM), occurred in the minute range, and were reversible. The 5,6-EET inhibited the Cav3 currents at physiological resting membrane potentials mostly by inducing a large negative shift in their steady-state inactivation properties. Using knockout mice for Cav3.1 and Cav3.2, we demonstrated that the vasodilation of preconstricted mesenteric arteries induced by 5,6-EET was specifically impaired in Cav3.2 knockout mice. Overall, our results indicate that inhibition of Cav3 currents by 5,6-EET is an important mechanism controlling the vascular tone.

Cytochrome P-450, family 2, subfamily c, polypeptide 44 (Cyp2c44) epoxygenase metabolizes arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) in kidney and vascular tissues. In the present study, we used real-time quantitative PCR techniques to examine the effect of high salt or high K(+) (HK) intake on the expression of Cyp2c44, a major Cyp2c epoxygenase in the mouse kidney. We detected Cyp2c44 in the proximal convoluted tubule, thick ascending limb, distal convoluted tubule (DCT)/connecting tubule (CNT), and collecting duct (CD). A high-salt diet increased the expression of Cyp2c44 in the thick ascending limb and DCT/CNT but not in the proximal convoluted tubule and CD. In contrast, an increase in dietary K(+) intake augmented Cyp2c44 expression only in the DCT/CNT and CD. Neither high salt nor HK intake had a significant effect on the blood pressure (BP) of wild-type mice. However, HK but not high salt intake increased BP in CD-specific, Cyp2c44 conditional knockout (KO) mice. Amiloride, an epithelial Na(+) channel (ENaC) inhibitor, normalized the BP of KO mice fed HK diets, suggesting that lack of Cyp2c44 in the CD enhances ENaC activity and increases Na(+) absorption in KO mice fed HK diets. This notion was supported by metabolic cage experiments demonstrating that renal Na(+) excretion was compromised in KO mice fed HK diets. Also, patch-clamp experiments demonstrated that 11,12-EET, a major Cyp2c44 product, but not AA inhibited ENaC activity in the cortical CD of KO mice. We conclude that Cyp2c44 in the CD is required for preventing the excessive Na(+) absorption induced by HK intake by inhibition of ENaC and facilitating renal Na(+) excretion., (Copyright © 2014 the American Physiological Society.)

The objective of this study was to investigate the 14,15-epoxyeicosatrienoic acid (14,15-EET)-induced vasodilatations as well as the underlying signaling pathways in rat mesenteric arteries from young, adult and old normotensive (WKY) and hypertensive rats. Protein expressions for prostaglandin EP(1-4) receptors, large conductance Ca(2+)-activated K(+) (BK(Ca)) channels, and adenylate cyclase (AC) were determined together with 14,15-EET-induced vasodilatations in primary- versus secondary-branches of the mesenteric artery. Responses to 14,15-EET were greater in the smaller secondary- versus primary-branches (and also more sensitive with lower EC50) and were reduced in vessels from old (80 weeks) rats as well as from vessels from the spontaneous hypertensive rats (SHR). Regardless of age or hypertension responses to 14,15-EET were inhibited by the EP2 antagonist AH6809, BK(Ca) channel inhibitor iberiotoxin, or 3',5'-cyclic monophosphate (cAMP)-protein kinase A (PKA) pathway antagonists. These data indicate 14,15-EET-induced vasodilatation is mediated via the activation of EP2 receptors and opening of BK(Ca) channels. The expressions of the EP2 receptor and AC were markedly reduced in vessels from SHR as well as old rats, whereas BK(Ca) expression was reduced in old WKY and SHR, but not adult SHR. Furthermore, expression of the p53 protein, an indicator of cell senescence and apoptosis, was elevated in adult and old SHR as well as in old WKY. In summary, attenuated 14,15-EET-induced vasodilatation in mesenteric arteries from old normotensive WKY as well as adult and old SHR is associated with reduced expression of EP2 receptors and AC., (Copyright © 2014 Elsevier Inc. All rights reserved.)

14,15-Epoxyeicosatrienoic acid (14,15-EET), a metabolite of arachidonic acid, is enriched in the brain cortex and exerts protective effect against neuronal apoptosis induced by ischemia/reperfusion. Although apoptosis has been well recognized to be closely associated with mitochondrial biogenesis and function, it is still unclear whether the neuroprotective effect of 14,15-EET is mediated by promotion of mitochondrial biogenesis and function in cortical neurons under the condition of oxygen-glucose deprivation (OGD). In this study, we found that 14,15-EET improved cell viability and inhibited apoptosis of cortical neurons. 14,15-EET significantly increased the mitochondrial mass and the ratio of mitochondrial DNA to nuclear DNA. Key makers of mitochondrial biogenesis, peroxisome proliferator activator receptor gamma-coactivator 1 alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (TFAM), were elevated at both mRNA and protein levels in the cortical neurons treated with 14,15-EET. Moreover, 14,15-EET markedly attenuated the decline of mitochondrial membrane potential, reduced ROS, while increased ATP synthesis. Knockdown of cAMP-response element binding protein (CREB) by siRNA blunted the up-regulation of PGC-1α and NRF-1 stimulated by 14,15-EET, and consequently abolished the neuroprotective effect of 14,15-EET. Our results indicate that 14,15-EET protects neurons from OGD-induced apoptosis by promoting mitochondrial biogenesis and function through CREB mediated activation of PGC-1α and NRF-1., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Therapies to reverse the vascular endothelial aging process may play as a novel strategy for the treatment of cardiovascular diseases. 14,15-epoxyeicosatrienoic acid (14,15-EET) is a predominant cytochrome P450 epoxygenases-derived arachidonic acid metabolite and possesses multiple biological effects on the vascular system. The present study sought to investigate the roles of mammalian target of rapamycin complex 2 (mTORC2)/Akt signaling pathways in mediating the effect of 14,15-EET on endothelial senescence. By measuring the isometric tension in rat mesenteric arteries, we demonstrated that 14,15-EET improved the impaired endothelium-dependent vasodilatation in aged rats through activating mTORC2/Akt signaling pathway. Meanwhile, by promoting the formation of mTORC2 and the phosphorylation of Akt (Ser473), 14,15-EET inhibited the senescence of rat mesenteric arterial endothelial cells, which was not influenced by rapamycin but was significantly attenuated by Akt1/2 kinase inhibitor. The knockdown of Rictor gene by RNA interference abolished the inhibitory effect of 14,15-EET on endothelial senescence. Furthermore, 14,15-EET down-regulated the expression of p53 protein in aged endothelial cells. Meanwhile, the nuclear translocation of telomerase reverse transcriptase and the nuclear telomerase activity were also enhanced by treatment with 14,15-EET. Therefore, our present study suggests the crucial role of mTORC2/Akt signaling pathways in the inhibitory effects of 14,15-EET on the endothelial senescence. Our findings reveal important mechanistic clues to understanding of the effects of 14,15-EET on the endothelial functions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Oxidized low-density lipoprotein (Ox-LDL) is associated with atherosclerotic events through the modulation of arachidonic acid (AA) metabolism and activation of inflammatory signaling. Cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) mitigate inflammation through nuclear factor-κB (NF-κB). In this study, we explored the effects and mechanisms of exogenous EETs on the ox-LDL-induced inflammation of pulmonary artery endothelial cells (PAECs), which were cultured from rat pulmonary arteries. We determined that pre-treatment with 11,12-EET or 14,15-EET attenuated the ox-LDL-induced expression and release of intercellular adhesion molecule-1 (ICAM-1), E-selectin, and monocyte chemoattractant protein-1 (MCP-1) in a concentration-dependent manner. In addition, the ox-LDL-induced expression of CYP2J4 was upregulated by 11,12-EET and 14,15-EET (1μM). Furthermore, the endothelial receptor of lectin-like oxidized low-density lipoprotein (LOX-1) was downregulated in PAECs treated with EETs. The inflammatory responses evoked by ox-LDL (100μg/mL) were blocked by pharmacological inhibitors of Erk1/2 mitogen-activated protein kinase (MAPK) (U0126), p38 MAPK (SB203580), and NF-κB (PDTC). In addition, we confirmed that 11,12-EET suppresses phosphorylation of p38, degradation of IκBα, and activation of NF-κB (p65), whereas 14,15-EET can significantly suppress the phosphorylation of p38 and Erk1/2. Our results indicate that EETs exert beneficial effects on ox-LDL-induced inflammation primarily through the inhibition of LOX-1 receptor upregulation, MAPK phosphorylation, and NF-κB activation and through the upregulation of CYP2J4 expression. This study helps focus the current understanding of the contribution of EETs to the regulation of the inflammation of pulmonary vascular endothelial cells. Furthermore, the therapeutic potential of targeting the EET pathway in pulmonary vascular disease will be highlighted., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Natural products are the major sources of currently available anticancer drugs. We recently reported that phenanthrene-based tylophorine derivative-1 (PBT-1) may be a potential antitumor agent for lung adenocarcinoma. We therefore examined the direct targets of PBT-1 and their effects in inhibiting lung adenocarcinoma. We found that PBT-1 reduced the level of Slug and inhibits the migration, invasion, and filopodia formation of lung adenocarcinoma CL1-5 cells in vitro. In addition, PBT-1 displayed in vivo antitumor and antimetastasis activities against subcutaneous and orthotopic xenografts of CL1-5 cells in nude mice. Chemical proteomics showed that heat shock protein 90 (HSP90) and heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) bound PBT-1 in CL1-5 cells. Inhibition of HSP90 and hnRNP A2/B1 reduced the activation of AKT and Slug expression. Taken together, these findings suggest that PBT-1 binds to HSP90 and/or hnRNP A2/B1 and initiates antitumor activities by affecting Slug- and AKT-mediated metastasis and tumorigenesis.

Prolonged endoplasmic reticulum (ER) stress causes apoptosis and is associated with heart failure. Whether CYP2J2 and its arachidonic acid metabolites [epoxyeicosatrienoic acids (EETs)] have a protective influence on ER stress and heart failure has not been studied. Assays of myocardial samples from patients with end-stage heart failure showed evidence of ER stress. Chronic infusion of isoproterenol (ISO) or angiotensin II (AngII) by osmotic mini-pump induced cardiac hypertrophy and heart failure in mice as evaluated by hemodynamic measurements and echocardiography. Interestingly, transgenic (Tr) mice with cardiomyocyte-specific CYP2J2 expression were protected against heart failure compared with wild-type mice. ISO or AngII administration induced ER stress and apoptosis, and increased levels of intracellular Ca(2+). These phenotypes were abolished by CYP2J2 overexpression in vivo or exogenous EETs treatment of cardiomyocytes in vitro. ISO or AngII reduced sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a) expression in hearts or isolated cardiomyocytes; however, loss of SERCA2a expression was prevented in CYP2J2 Tr hearts in vivo or in cardiomyocytes treated with EETs in vitro. The reduction of SERCA2a activity was concomitant with increased oxidation of SERCA2a. EETs reversed SERCA2a oxidation through increased expression of antioxidant enzymes and reduced reactive oxygen species levels. Tempol, a membrane-permeable radical scavenger, similarly decreased oxidized SERCA2a levels, restored SERCA2a activity, and markedly reduced ER stress response in the mice treated with ISO. In conclusion, CYP2J2-derived EETs suppress ER stress response in the heart and protect against cardiac failure by maintaining intracellular Ca(2+) homeostasis and SERCA2a expression and activity.

Background: Cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acids to epoxyeicosatrienoic acids (EETs). EETs exert various biological effects, including anti-inflammatory, anti-apoptotic, pro-proliferation, pro-angiogenesis, anti-oxidation, and anti-fibrosis effects. However, little is known about the role of CYP2J2 and EETs in lung ischemia/reperfusion injury. In this study, we examined the effects of exogenous EETs or CYP2J2 overexpression on lung ischemia/reperfusion injury in vivo and in vitro., Methods and Results: CYP2J2 gene was stably transfected into rat lungs via pcDNA3.1-CYP2J2 plasmid delivery, resulting in increased EETs levels in the serum and lung. A rat model of lung ischemia/reperfusion injury was developed by clamping the left lung hilum for 1 hour, followed by reperfusion for 2 hours. We found that CYP2J2 overexpression markedly decreased the levels of oxidative stress and cell apoptosis in lung tissues induced by ischemia/reperfusion. Moreover, we observed that exogenous EETs, or CYP2J2 overexpression, enhanced cell viability, decreased intracellular reactive oxygen species (ROS) generation, inhibited mitochondrial dysfunction, and attenuated several apoptotic signaling events in a human pulmonary artery endothelial cells (HPAECs)-based anoxia/reoxygenation model. These apoptotic events included activation of NADPH oxidase, collapse of mitochondrial transmembrane potential, and activation of pro-apoptotic proteins and caspase-3. These effects were mediated, at least partially, by the PI3K/Akt signaling pathway., Conclusion: These results reveal that CYP2J2 overexpression and exogenous EETs can protect against oxidative stress and apoptosis following lung ischemia/reperfusion in vivo and in vitro, suggesting that increasing the level of EETs may be a novel promising strategy to prevent and treat lung ischemia/reperfusion injury., (© 2014 S. Karger AG, Basel.)

We report the effects of two anti-cancer drugs, PBT-4, an experimental antagonist to the pro-inflammatory hepoxilins, and Gleevec (STI-571), an anti-leukaemic drug, on eicosanoid tumour levels in immunodeficient mice (NU/NU) xenografted with the leukaemic cell line, U937 bcl-xL. After the tumours had grown to 80-100mm(3) volume, an 8-day treatment with the drugs was initiated and the animals were monitored for 28 days. On various days, tumours were removed for measurement of 24 omega-6 eicosanoids. The data show remarkable direct correlation between inhibition of tumour AA release and 12-LOX products (including 12-HETE and hepoxilins) during PBT or STI treatment with tumour growth suppression. These findings suggest that inhibition of AA release may represent a novel underlying mechanism of action of PBT-4 (and STI) in vivo in suppressing tumour growth. As the PBT wears off, AA and 12-LOX products rise rapidly (Day 18) leading to the observed tumour growth spurt., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Soluble epoxide hydrolase (sEH) catalyzes the conversion of epoxyeicosatrienoic acids into less active eicosanoids, and inhibitors of sEH have anti-inflammatory and antiapoptotic properties. Based on previous observations that sEH inhibition attenuates cisplatin-induced nephrotoxicity by modulating nuclear factor-κB signaling, we hypothesized that this strategy would also attenuate cisplatin-induced renal apoptosis. Inhibition of sEH with AR9273 [1-adamantan-1-yl-3-(1-methylsulfonyl-piperidin-4-yl-urea)] reduced cisplatin-induced apoptosis through mechanisms involving mitochondrial apoptotic pathways and by reducing reactive oxygen species. Renal mitochondrial Bax induction following cisplatin treatment was significantly decreased by treatment of mice with AR9273 and these antiapoptotic effects involved p38 mitogen-activated protein kinase signaling. Similar mechanisms contributed to reduced apoptosis in Ephx2(-/-) mice treated with cisplatin. Moreover, in pig kidney proximal tubule cells, cisplatin-induced mitochondrial trafficking of Bax and cytochrome c, caspase-3 activation, and oxidative stress are significantly attenuated in the presence of epoxyeicosatrienoic acids (EETs). Collectively, these in vivo and in vitro studies demonstrate a role for EETs in limiting cisplatin-induced renal apoptosis. Inhibition of sEH represents a novel therapeutic strategy for protection against cisplatin-induced renal damage.

Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid involved in regulating pathways promoting cellular protection. We have previously shown that EETs trigger a protective response limiting mitochondrial dysfunction and reducing cellular death. Considering it is unknown how EETs regulate cell death processes, the major focus of the current study was to investigate their role in the autophagic response of HL-1 cells and neonatal cardiomyocytes (NCMs) during starvation. We employed a dual-acting synthetic analog UA-8 (13-(3-propylureido)tridec-8-enoic acid), possessing both EET-mimetic and soluble epoxide hydrolase (sEH) inhibitory properties, or 14,15-EET as model EET molecules. We demonstrated that EETs significantly improved viability and recovery of starved cardiac cells, whereas they lowered cellular stress responses such as caspase-3 and proteasome activities. Furthermore, treatment with EETs resulted in preservation of mitochondrial functional activity in starved cells. The protective effects of EETs were abolished by autophagy-related gene 7 (Atg7) short hairpin RNA (shRNA) or pharmacological inhibition of autophagy. Mechanistic evidence demonstrated that sarcolemmal ATP-sensitive potassium channels (pmKATP) and enhanced activation of AMP-activated protein kinase (AMPK) played a crucial role in the EET-mediated effect. Our data suggest that the protective effects of EETs involve regulating the autophagic response, which results in a healthier pool of mitochondria in the starved cardiac cells, thereby representing a novel mechanism of promoting survival of cardiac cells. Thus, we provide new evidence highlighting a central role of the autophagic response in linking EETs with promoting cell survival during deep metabolic stress such as starvation.