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The effect of a high glycemic diet (HGD) on brain microvasculature is a crucial, yet understudied research topic, especially in females. This study aimed to determine the transcriptomic changes in female brain hippocampal microvasculature induced by a HGD and characterize the response to a soluble epoxide hydrolase inhibitor (sEHI) as a mechanism for increased epoxyeicosatrienoic acids (EETs) levels shown to be protective in prior models of brain injury. We fed mice a HGD or a low glycemic diet (LGD), with/without the sEHI (t-AUCB), for 12 weeks. Using microarray, we assessed differentially expressed protein-coding and noncoding genes, functional pathways, and transcription factors from laser-captured hippocampal microvessels. We demonstrated for the first time in females that the HGD had an opposite gene expression profile compared to the LGD and differentially expressed 506 genes, primarily downregulated, with functions related to cell signaling, cell adhesion, cellular metabolism, and neurodegenerative diseases. The sEHI modified the transcriptome of female mice consuming the LGD more than the HGD by modulating genes involved in metabolic pathways that synthesize neuroprotective EETs and associated with a higher EETs/dihydroxyeicosatrienoic acids (DHETs) ratio. Our findings have implications for sEHIs as promising therapeutic targets for the microvascular dysfunction that accompanies vascular dementia. [ABSTRACT FROM AUTHOR]

Oxylipins are metabolites of polyunsaturated fatty acids that mediate cardiovascular health by attenuation of inflammation, vascular tone, hemostasis, and thrombosis. Very low‐density lipoproteins (VLDL) contain oxylipins, but it is unknown whether the liver regulates their concentrations. In this study, we used a perfused liver model to observe the effect of inflammatory lipopolysaccharide (LPS) challenge and soluble epoxide hydrolase inhibition (sEHi) on VLDL oxylipins. A compartmental model of deuterium‐labeled linoleic acid and palmitic acid incorporation into VLDL was also developed to assess the dependence of VLDL oxylipins on fatty acid incorporation rates. LPS decreased the total fatty acid VLDL content by 30% [6%,47%], and decreased final concentration of several oxylipins by a similar amount (13‐HOTrE, 35% [4%,55%], −1.3 nM; 9(10)‐EpODE, 29% [3%,49%], −2.0 nM; 15(16)‐EpODE, 29% [2%,49%], −1.6 nM; AA‐derived diols, 32% [5%,52%], −2.4 nM; 19(20)‐DiHDPA, 31% [7%,50%], −1.0 nM). However, the EPA‐derived epoxide, 17(18)‐EpETE, was decreased by 75% [49%,88%], (−0.52 nM) with LPS, double the suppression of other oxylipins. sEHi increased final concentration of DHA epoxide, 16(17)‐EpDPE, by 99% [35%,193%], (2.0 nM). Final VLDL‐oxylipin concentrations with LPS treatment were not correlated with linoleic acid kinetics, suggesting they were independently regulated under inflammatory conditions. We conclude that the liver regulates oxylipin incorporation into VLDL, and the oxylipin content is altered by LPS challenge and by inhibition of the epoxide hydrolase pathway. This provides evidence for delivery of systemic oxylipin signals by VLDL transport. [ABSTRACT FROM AUTHOR]

Acute kidney injury (AKI) frequently complicates major surgery and can be associated with hypertension and progress to chronic kidney disease, but reports on blood pressure normalization in AKI are conflicting. In the present study, we investigated the effects of an angiotensin-converting enzyme inhibitor, enalapril, and a soluble epoxide hydrolase inhibitor, 1-trifluoromethoxyphenyl- 3-(1-propionylpiperidin-4-yl)urea (TPPU), on renal inflammation, fibrosis, and glomerulosclerosis in a mouse model of ischemia-reperfusion injury (IRI)-induced AKI. Male CD1 mice underwent unilateral IRI for 35 min. Blood pressure was measured by tail cuff, and mesangial matrix expansion was quantified on methenamine silver-stained sections. Renal perfusion was assessed by functional MRI in vehicle- and TPPU-treated mice. Immunohistochemistry was performed to study the severity of AKI and inflammation. Leukocyte subsets were analyzed by flow cytometry, and proinflammatory cytokines were analyzed by quantitative PCR. Plasma and tissue levels of TPPU and lipid mediators were analyzed by liquid chromatography mass spectrometry. IRI resulted in a blood pressure increase of 20 mmHg in the vehicle-treated group. TPPU and enalapril normalized blood pressure and reduced mesangial matrix expansion. However, inflammation and progressive renal fibrosis were severe in all groups. TPPU further reduced renal perfusion on days 1 and 14. In conclusion, early antihypertensive treatment worsened renal outcome after AKI by further reducing renal perfusion despite reduced glomerulosclerosis. [ABSTRACT FROM AUTHOR]

There is growing evidence that soluble epoxide hydrolase (sEH) may play a role in cell differentiation. sEH metabolizes biologically highly active and generally cytoprotective epoxyeicosatrienoic acids (EETs), generated from arachidonic acid metabolism by CYP epoxygenases (CYP2C and CYP2J subfamilies), to less active corresponding diols. We investigated the effect of sEH inhibitor (TPPU) on the expression of villin, CYP2C8, CYP2C9, CYP2J2, and sEH in undifferentiated and in vitro differentiated HT-29 and Caco2 cell lines. The administration of 10 μM TPPU on differentiated HT-29 and Caco2 cells resulted in a significant decrease in expression of villin, a marker for intestinal cell differentiation. It was accompanied by a disruption of the brush border when microvilli appeared sparse and short in atomic force microscope scans of HT-29 cells. Although inhibition of sEH in differentiated HT-29 and Caco2 cells led to an increase in sEH expression in both cell lines, this treatment had an opposite effect on CYP2J2 expression in HT-29 and Caco2 cells. In addition, tissue samples of colorectal carcinoma and adjacent normal tissues from 45 patients were immunostained for sEH and villin. We detected a significant decrease in the expression of both proteins in colorectal carcinoma in comparison to adjacent normal tissue, and the decrease in both sEH and villin expression revealed a moderate positive association. Taken together, our results showed that sEH is an important player in intestinal cell differentiation. [ABSTRACT FROM AUTHOR]

Introduction: Previous studies in Ren-2 transgenic hypertensive rats (TGR) after 5/6 renal ablation (5/6 NX) have shown that besides pharmacological blockade of the renin-angiotensin system (RAS) also increasing kidney tissue epoxyeicosatrienoic acids (EET) levels by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for degradation of EETs, and endothelin type A (ETA) receptor blockade retards chronic kidney disease (CKD) progression. This prompted us to evaluate if this progression will be alleviated by the addition of sEH inhibitor and ETA receptor antagonist to the standard complex blockade of RAS (angiotensin-converting enzyme inhibitor plus angiotensin II type 1 receptor blocker) in rats with established CKD. Methods: The treatment regimens were initiated 6 weeks after 5/6 NX in TGR, and the follow-up period was 60 weeks. Results: The addition of sEH inhibition to RAS blockade improved survival rate, further reduced albuminuria and renal glomerular and kidney tubulointerstitial injury, and attenuated the decline in creatinine clearance – all this as compared with 5/6 NX TGR treated with RAS blockade alone. Addition of ETA receptor antagonist to the combined RAS and sEH blockade not only offered no additional renoprotection but, surprisingly, also abolished the beneficial effects of adding sEH inhibitor to the RAS blockade. Conclusion: These data indicate that pharmacological strategies that combine the blockade of RAS and sEH could be a novel tool to combat the progression of CKD. Any attempts to further extend this therapeutic regimen should be made with extreme caution. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the potential role and mechanism of TUPS, a soluble epoxide hydrolase inhibitor, in cardiac hypertrophy. Methods: Rat and H9C2 cell models of cardiac hypertrophy were induced by isoproterenol and angiotensin II, respectively, followed by TUPS treatment. The expression of hypertrophic markers, ANP and BNP, was determined by quantitative real‐time PCR. The abundance of Beclin‐1, LC3, p‐AMPK and phosphorylated‐mammalian target of rapamycin (p‐mTOR) proteins was analysed by Western blot and immunohistocytology. Cell morphology and viability were evaluated by F‐actin staining and MTS. H9C2 cells were transfected with GFP‐LC3 to evaluate autophagy flux. Key findings: TUPS significantly inhibited rat heart size, heart weight‐to‐body weight ratio, heart wall thickness, hypertrophic H9C2 cell swelling and viability suppression as well as the expression of ANP and BNP genes in hypertrophic models. In addition, autophagic markers Beclin‐1 and LC3 were elevated in both cellular and animal models, which were suppressed by TUPS, with corresponding changes of autophagy flux. The abundance of p‐AMPK was increased, while p‐mTOR was decreased in hypertrophic cells, which were abolished by TUPS. Rapamycin decreased p‐mTOR level, increased Beclin‐1 and LC3 expression and induced cell size enlargement and cell viability inhibition in hypertrophic H9C2 cells treated with TUPS. Conclusions: TUPS inhibits cardiac hypertrophy by regulating mTOR/autophagy axis. [ABSTRACT FROM AUTHOR]

We found recently that in Ren-2 transgenic hypertensive rats (TGR) addition of soluble epoxide hydrolase inhibitor (sEHi) to treatment with angiotensin-converting enzyme inhibitor (ACEi), surprisingly, increased the mortality due to heart failure (HF) induced by creation of the aorto-caval fistula (ACF). Since TGR exhibit sex-related differences in mortality, we examined here if such differentiation exists also in the response to the treatment with ACEi (trandolapril), alone or combined with sEHi [cis-4-[4-(3-adamantan-1-yl-ureido)cyclohexyloxy]benzoic acid, (c-AUCB)]. ACEi improved survival in males to 74 % (vs. 0 %) and in females to 65 % (vs. 32 %). ACEi and sEHi combined also improved the survival in male ACF TGR, however, it was significantly less (38 %) than after ACEi alone. In contrast, in females the combined treatment significantly improved the final survival rate (84 %). There were no significant sex-linked differences in survival rate in untreated or treated normotensive Hannover Sprague-Dawley rats. In conclusion, in HF patients with co-existing hypertension and RAS hyperactivity, the sex may co-determine the rate of HF progression, and can influence the effectiveness of the therapeutic measures applied. Therefore, in the relevant pre-clinical studies the sex-linked differences should be seriously considered. Our data indicate that TGR might be an optimal model for such studies. [ABSTRACT FROM AUTHOR]

1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) is a potent soluble epoxide hydrolase (sEH) inhibitor that is used extensively in research for modulating inflammation and protecting against hypertension, neuropathic pain, and neurodegeneration. Despite its wide use in various animal disease models, the metabolism of TPPU has not been well-studied. A broader understanding of its metabolism is critical for determining contributions of metabolites to the overall safety and effectiveness of TPPU. Herein, we describe the identification of TPPU metabolites using LC-MS/MS strategies. Four metabolites of TPPU (M1–M4) were identified from rat urine by a sensitive and specific LC-MS/MS method with double precursor ion scans. Their structures were further supported by LC-MS/MS comparison with synthesized standards. Metabolites M1 and M2 were formed from hydroxylation on a propionyl group of TPPU; M3 was formed by amide hydrolysis of the 1-propionylpiperdinyl group on TPPU; and M4 was formed by further oxidation of the hydroxylated metabolite M2. Interestingly, the predicted α-keto amide metabolite and 4-(trifluoromethoxy)aniline (metabolite from urea cleavage) were not detected by the LC-MRM-MS method. This indicates that if formed, the two potential metabolites represent <0.01% of TPPU metabolism. Species differences in the formation of these four identified metabolites was assessed using liver S9 fractions from dog, monkey, rat, mouse, and human. M1, M2, and M3 were generated in liver S9 fractions from all species, and higher amounts of M3 were generated in monkey S9 fractions compared to other species. In addition, rat and human S9 metabolism showed the highest species similarity based on the quantities of each metabolite. The presence of all four metabolites were confirmed in vivo in rats over 72-h post single oral dose of TPPU. Urine and feces were major routes for TPPU excretion. M1, M4 and parent drug were detected as major substances, and M2 and M3 were minor substances. In blood, M1 accounted for ~9.6% of the total TPPU-related exposure, while metabolites M2, M3, and M4 accounted for <0.4%. All four metabolites were potent inhibitors of human sEH but were less potent than the parent TPPU. In conclusion, TPPU is metabolized via oxidation and amide hydrolysis without apparent breakdown of the urea. The aniline metabolites were not observed either in vitro or in vivo. Our findings increase the confidence in the ability to translate preclinical PK of TPPU in rats to humans and facilitates the potential clinical development of TPPU and other sEH inhibitors. [ABSTRACT FROM AUTHOR]

An association between congestive heart failure (CHF) and chronic kidney disease (CKD) results in extremely poor patient survival rates. Previous studies have shown that increasing kidney epoxyeicosatrienoic acids (EETs) by blocking soluble epoxide hydrolase (sEH), an enzyme responsible for EETs degradation, improves the survival rate in CHF induced by aorto-caval fistula (ACF) and attenuates CKD progression. This prompted us to examine if sEH inhibitor treatment would improve the outcome if both experimental conditions are combined. Fawn-hooded hypertensive (FHH) rats, a genetic model showing early CKD development was employed, and CHF was induced by ACF. Treatment with an sEH inhibitor was initiated 4 weeks after ACF creation, in FHH and in fawn-hooded low-pressure (FHL) rats, a control strain without renal damage. The follow-up period was 20 weeks. We found that ACF FHH rats exhibited substantially lower survival rates (all the animals died by week 14) as compared with the 64% survival rate observed in ACF FHL rats. The former group showed pronounced albuminuria (almost 30-fold higher than in FHL) and reduced intrarenal EET concentrations. The sEH inhibitor treatment improved survival rate and distinctly reduced increases in albuminuria in ACF FHH and in ACF FHL rats, however, all the beneficial actions were more pronounced in the hypertensive strain. These data indicate that pharmacological blockade of sEH could be a novel therapeutic approach for the treatment of CHF, particularly under conditions when it is associated with CKD. [ABSTRACT FROM AUTHOR]

Highlights • Cerebral hypoperfusion impairs cognitive functions and inhibits synaptic plasticity. • Soluble epoxide hydrolase inhibition ameliorates cognitive dysfunction. • TPPU activates neuregulin-1/ErbB4 signaling and triggers PI3K-Akt pathways. Abstract Cerebral ischemic stroke is associated with a high rate of incidence, prevalence and mortality globally. Carotid artery stenosis, which is mainly caused by atherosclerosis plaque, results in chronic cerebral hypoperfusion and predominantly increases the risk of ischemic stroke. In the present study, we used bilateral common carotid artery stenosis (BCAS) model by placing microcoils of 0.18 mm diameter encompassing both common carotid arteries respectively, to mimic the pathogenesis of carotid artery atherosclerosis and intensively explore the pathology. We found that BCAS injury for 1 month impaired spatial cognitive functions significantly, and inhibited synaptic plasticity, including hippocampal long-term potentiation (LTP) inhibition, dendritic spine density reduction and synaptic associative proteins disorder. BCAS-induced cerebral hypoperfused mice treated with 1-(1-propanoylpiperidin-4-yl)-3-[4-(trifluoromethoxy)phenyl]urea (TPPU), a potent soluble epoxide hydrolase (sEH) inhibitor, exhibited amelioration of cognitive dysfunction and improved synaptic plasticity. The neural protective effects of TPPU on BCAS-induced cerebral hypoperfusion might due to activation of neuregulin-1 (NRG1)/ErbB4 signaling, and triggered PI3K-Akt pathways subsequently. Our results suggested that sEH inhibition could exert multi-target protective effects and alleviate spatial cognitive dysfunctions after chronic cerebral hypoperfusion in mice. [ABSTRACT FROM AUTHOR]

Abstract: Exercise training (ET) is a safe and efficacious therapeutic approach for myocardial infarction (MI). Given the numerous benefits of exercise, exercise‐induced mediators may be promising treatment targets for MI. C57BL/6 mice were fed 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidine‐4‐yl) urea (TPPU), a novel soluble epoxide hydrolase inhibitor (sEHI), to increase epoxyeicosatrienoic acid (EET) levels, for 1 week before undergoing MI surgery. After 1‐week recovery, the mice followed a prescribed exercise programme. Bone marrow‐derived endothelial progenitor cells (EPCs) were isolated from the mice after 4 weeks of exercise and cultured for 7 days. Angiogenesis around the ischaemic area, EPC functions, and the expression of microRNA‐126 (miR‐126) and its target gene Spred1 were measured. The results were confirmed in vitro by adding TPPU to EPC culture medium. ET significantly increased serum EET levels and promoted angiogenesis after MI. TPPU enhanced the effects of ET to reduce the infarct area and improve cardiac function after MI. ET increased EPC function and miR‐126 expression, which were further enhanced by TPPU, while Spred1 expression was significantly down‐regulated. Additionally, the protein kinase B/glycogen synthase kinase 3β (AKT/GSK3β) signalling pathway was activated after the administration of TPPU. EETs are a potential mediator of exercise‐induced cardioprotection in mice after MI. TPPU enhances exercise‐induced cardiac recovery in mice after MI by increasing EET levels and promoting angiogenesis around the ischaemic area. [ABSTRACT FROM AUTHOR]

Triggering receptors expressed on myeloid cell-1 (TREM-1) is a superimmunoglobulin receptor expressed on myeloid cells. TREM-1 amplifies the inflammatory response. Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 enzyme, have anti-inflammatory properties. However, the effects of EETs on TREM-1 expression under inflammatory stimulation remain unclear. Therefore, inhibition of soluble epoxide hydrolase (sEH) with a highly selective inhibitor [1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea, TPPU] was used to stabilize EETs. LPS was intratracheally injected into mice to induce pulmonary inflammation, after TPPU treatment for 3 h. Histological examination showed TPPU treatment-alleviated LPS-induced pulmonary inflammation. TPPU decreased TREM-1 expression, but not DAP12 or MyD88 expression. Murine peritoneal macrophages were challenged with LPS in vitro. We found that TPPU reduced LPS-induced TREM-1 expression in a dose-dependent manner, but not DAP12 or MyD88 expression. TPPU also decreased downstream signal from TREM-1, reducing pro-inflammatory cytokine TNF-α and IL-1β mRNA expression. Furthermore, TPPU treatment inhibited IkB degradation in vivo and in vitro. Our results indicate that the inhibition of sEH suppresses LPS-induced TREM-1 expression and inflammation via inhibiting NF-kB activation in murine macrophage. [ABSTRACT FROM AUTHOR]

Continuous efforts to identify sEH inhibitors using activity-guided fractionation have revealed 12 known compounds,2–13, fromRheum undulatum. Compounds2–13and1, which was obtained from the in-house library, were tested for inhibitory activity against sEH. Compounds1–9,11, and12were shown to have inhibitory activity, with IC50values ranging from 2.5 ± 0.5 to 53.2 ± 4.4 μM. They were subjected to enzyme kinetic studies to explore the binding mode between the ligand and receptor. Based on our results, compounds1,2,5–9,and11(mixed type) and compounds3and12(noncompetitive type) had a preferred allosteric site. Compound4was identified as a competitive-type interaction in the active site. Molecular docking studies revealed the interacting residues and binding energy between sEH and inhibitors. Additionally, molecular dynamics provided detailed information on the interaction between the ligand and receptor. [ABSTRACT FROM AUTHOR]

Epoxyeicosatrienoic acids (EETs), the metabolites of arachidonic acid derived from the cytochrome P450 (CYP450) epoxygenases, are mainly metabolized by soluble epoxide hydrolase (sEH) to their corresponding diols. EETs but not their diols, have anti-inflammatory properties and inhibition of sEH might provide protective effects against inflammatory fibrosis. We test the effects of a selected sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), on bleomycin-induced pulmonary fibrosis (PF) in mice. A mouse model of PF was established by intratracheal injection of bleomycin and TPPU was administered for 21 days after bleomycin injection. We found TPPU treatment improved the body weight loss and survival rate of bleomycin-stimulated mice. Histological examination showed that TPPU treatment alleviated bleomycin-induced inflammation and maintained the alveolar structure of the pulmonary tissues. TPPU also decreased the bleomycin-induced deposition of collagen and the expression of procollagen I mRNA in lung tissues of mice. TPPU decreased the transforming growth factor-β1 (TGF-β1), interleukin-1β (IL-1β) and IL-6 levels in the serum of bleomycin-stimulated mice. Furthermore, TPPU inhibited the proliferation and collagen synthesis of mouse fibroblasts and partially reversed TGF-β1-induced α-smooth muscle actin expression. Our results indicate that the inhibition of sEH attenuates bleomycin-induced inflammation and collagen deposition and therefore prevents bleomycin-induced PF in a mouse model. [ABSTRACT FROM AUTHOR]

[Display omitted] • Benzamide derivatives containing urea moiety were designed and synthesized, and the structure–activity relationship was fully explored. • Compound A34 exhibited outstanding inhibitory activity against human sEH, with an IC 50 value of 0.04 ± 0.01 nM. • Compound A34 showed moderate systemic drug exposure and oral bioavailability in vivo metabolism studies. • In carrageenan-induced inflammatory pain rat model, compound A34 exhibited a better therapeutic effect compared to t -AUCB and Celecoxib. The elevation of epoxy-fatty acids through inhibition of soluble epoxide hydrolase (sEH) is efficient for the treatment of inflammatory and pain-related diseases. Herein, we reported the discovery of a series of benzamide derivatives containing urea moiety as sEH inhibitors. Intensive structural modifications led to the identification of compound A34 as a potent sEH inhibitor with good physicochemical properties. Molecular docking revealed an additional hydrogen-bonding interaction between the unique amide scaffold and Phe497, contributing to sEH inhibition potency enhancement. Compound A34 exhibited outstanding inhibitory activity against human sEH, with an IC 50 value of 0.04 ± 0.01 nM and a K i value of 0.2 ± 0.1 nM. It also showed moderate systemic drug exposure and oral bioavailability in vivo metabolism studies. In carrageenan-induced inflammatory pain rat model, compound A34 exhibited a better therapeutic effect compared to t -AUCB and Celecoxib. Metabolism studies in vivo together with an inflammatory pain evaluation suggest that A34 may be a viable lead compound for the development of highly potent sEH inhibitors. [ABSTRACT FROM AUTHOR]

Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by plateletderived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus- mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease. [ABSTRACT FROM AUTHOR]

Objectives To determine the protective role of a soluble epoxide hydrolase( sEH) inhibitor, trans-4-{4-[3-(4-trifluoromethoxyphenyl)-ureido] cyclohexyloxy} benzoic acid ( t-TUCB), in isoproterenol ( ISO)-induced myocardial ischaemic injury in vivo. Methods Cardioprotective activity of t-TUCB was studied against ISO-induced myocardial ischaemic injury in male Wistar rats. Cardioprotection was assessed by measuring elecrocardiographic ( EKG), serum lactate dehydrogenase ( LDH) and creatine kinase ( CK-MB) levels, cardiac calcium and antioxidant levels, and also by measuring infarct size in the cardiac tissue. Key findings Pretreatment with t-TUCB at 3, 10 and 30 mg/kg orally for a period of 14 days significantly prevented the changes in EKG parameters ( QTc interval prolongation, ST height depression, pathological Q waves formation and T-wave inversion), serum cardiac biomarkers ( CK-MB and LDH), relative heart weight, myocardial calcium levels, infarct size and the oxidative status in the cardiac tissue (lipid peroxidation, catalase and superoxide dismutase levels) when compared with the untreated control animals ( P < 0.05). Conclusion The sEH inhibitor t-TUCB significantly prevents ISO-induced myocardial ischaemic injury in rats. This study provides a preliminary confirmation of the efficacy of t-TUCB by oral administration in rats. [ABSTRACT FROM AUTHOR]

Abstract: Background: Epoxyeicosatrienoic acids (EETs) are natural angiogenic mediators regulated by soluble epoxide hydrolase (sEH). Inhibitors of sEH can stabilize EETs levels and were reported to reduce atherosclerosis and inhibit myocardial infarction in animal models. In this work, we investigated whether increasing EETs with the sEH inhibitor t-AUCB would increase angiogenesis related function in endothelial progenitor cells (EPCs) from patients with acute myocardial infarction (AMI). Methods and results: EPCs were isolated from 50 AMI patients and 50 healthy subjects (control). EPCs were treated with different concentrations of t-AUCB for 24h with or without peroxisome proliferator activated receptor γ (PPARγ) inhibitor GW9662. Migration of EPCs was assayed in trans-well chambers. Angiogenesis assays were performed using a Matrigel-Matrix in vitro model. The expression of vascular endothelial growth factor (VEGF), hypoxia-inducible factor 1α (HIF-1α) mRNA and protein in EPCs was measured by real-time PCR or Western blot, respectively. Also, the concentration of EETs in the culture supernatant was detected by ELISA. The activity of EPCs in the AMI patient group was reduced compared to healthy controls. Whereas increasing EET levels with t-AUCB promoted a dose dependent angiogenesis and migration in EPCs from AMI patients. Additionally, the t-AUCB dose dependently increased the expression of the angiogenic factors VEGF and HIF-α. Lastly, we provide evidence that these effects were PPARγ dependent. Conclusion: The results demonstrate that the sEH inhibitor positively modulated the functions of EPCs in patients with AMI through the EETs–PPARγ pathway. The present study suggests the potential utility of sEHi in the therapy of ischemic heart disease. [Copyright &y& Elsevier]

Abstract: Soluble epoxide hydrolase (sEH, EPHX2) metabolizes eicosanoid epoxides, including epoxyeicosatrienoic acids (EETs) to the corresponding dihydroxyeicosatrienoic acids (DHETs), and leukotoxin (LTX) to leukotoxin diol (LTX diol). EETs, endothelium-derived hyperpolarizing factors, exhibit potentially beneficial properties, including anti-inflammatory effects and vasodilation. A novel, potent, selective inhibitor of recombinant human, rat and mouse sEH, GSK2256294A, exhibited potent cell-based activity, a concentration-dependent inhibition of the conversion of 14,15-EET to 14,15-DHET in human, rat and mouse whole blood in vitro, and a dose-dependent increase in the LTX/LTX diol ratio in rat plasma following oral administration. Mice receiving 10 days of cigarette smoke exposure concomitant with oral administration of GSK2256294A exhibited significant, dose-dependent reductions in pulmonary leukocytes and keratinocyte chemoattractant (KC, CXCL1) levels. Mice receiving oral administration of GSK2256294A following 10 days of cigarette smoke exposure exhibited significant reductions in pulmonary leukocytes compared to vehicle-treated mice. These data indicate that GSK2256294A attenuates cigarette smoke-induced inflammation by both inhibiting its initiation and/or maintenance and promoting its resolution. Collectively, these data indicate that GSK2256294A would be an appropriate agent to evaluate the role of sEH in clinical studies, for example in diseases where cigarette smoke is a risk factor, such as chronic obstructive pulmonary disease (COPD) and cardiovascular disease. [Copyright &y& Elsevier]

Abstract: Cardioprotective effects of epoxyeicosatrienoic acids (EETs) have been demonstrated in models of young mice with either the cardiomyocyte specific over-expression of cytochrome P450 2J2 (CYP2J2 Tr) or deletion of soluble epoxide hydrolase (sEH null). In this study we examined differences in EET-induced cardioprotection in young (2 months) and aged (12 months) CYP2J2 Tr and sEHnull mice using Langendorff isolated perfused heart model. Improved postischemic functional recovery was observed in both young and aged sEH null mice compared to age matched WT. Conversely, the cardioprotective effect observed in young CYP2J2 Tr was lost in aged CYP2J2 Tr mice. The loss of cardioprotection in aged CYP2J2 Tr was regained following perfusion with the sEH inhibitor t-AUCB. Data demonstrated increased levels of leukotoxin diol (DiHOME) and oxidative stress as well decreased protein phosphatase 2A (PP2A) activation in aged CYP2J2 Tr. In conclusion, inhibition of sEH and EET-induced cardioprotection is maintained in aged mice. However, the loss of protective effects observed in aged CYP2J2 Tr might be attributed to increased levels of DiHOME, oxidative stress and/or decreased PP2A activity. [Copyright &y& Elsevier]

Background and Purpose We have previously shown that isoprenaline-induced cardiac hypertrophy causes significant changes in the expression of cytochromes P450 ( CYP) and soluble epoxide hydrolase ( sEH) genes. Therefore, it is important to examine whether the inhibition of sEH by 1-(1-methanesulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea ( TUPS) will protect against isoprenaline-induced cardiac hypertrophy. Experimental Approach Male Sprague- Dawley rats were treated with TUPS (0.65 mg kg−1 day−1, p.o.), isoprenaline (5 mg kg−1 day−1, i.p.) or the combination of both. In vitro H9c2 cells were treated with isoprenaline (100 μM) in the presence and absence of either TUPS (1 μM) or 11,12 EET (1 μM). The expression of hypertrophic, fibrotic markers and different CYP genes were determined by real-time PCR. Key Results Isoprenaline significantly induced the hypertrophic, fibrotic markers as well as the heart to body weight ratio, which was significantly reversed by TUPS. Isoprenaline also caused an induction of CYP1A1, CYP1B1, CYP2B1, CYP2B2, CYP4A3 and CYP4F4 gene expression and TUPS significantly inhibited this isoprenaline-mediated effect. Moreover, isoprenaline significantly reduced 5,6-, 8,9-, 11,12- and 14,15- EET and increased their corresponding 8,9-, 11,12- and 14,15-dihydroxyeicosatrienoic acid ( DHET) and the 20- HETE metabolites. TUPS abolished these isoprenaline-mediated changes in arachidonic acid ( AA) metabolites. In H9c2 cells, isoprenaline caused a significant induction of ANP, BNP and EPHX2 mRNA levels. Both TUPS and 11,12- EET significantly decreased this isoprenaline-mediated induction of ANP, BNP and EPHX2. Conclusions and Implications TUPS partially protects against isoprenaline-induced cardiac hypertrophy, which confirms the role of sEH and CYP enzymes in the development of cardiac hypertrophy. [ABSTRACT FROM AUTHOR]

Background: Researches have shown that soluble epoxide hydrolase inhibitors (sEHi) can protect against the development of atherosclerosis. Simultaneously, emerging evidences have implicated the association between fatty acid synthase (FAS) and acute coronary syndrome (ACS). We tested the hypothesis that sEHi could reduce the occurrence of ACS by regulating FAS. Methods: Hospitalized ACS patients were selected as the ACS group (n = 65) while healthy normal subjects as the control group (n = 65). The blood levels of lipoproteins, fasting glucose, myocardial enzyme and high-sensitivity C-reactive protein (hs-CRP) were measured within 24 hours after admission. The peripheral blood mononuclear cells (PBMCs) were isolated and cultured. Trans-4-[4-(3-Adamantan-1-ylureido)cyclohexyloxy] benzoic acid (t-AUCB), a kind of sEHi, was then added to cells in various concentrations (0, 10, 50, 100 μmol/L). The expression of FAS, interleukin-6 (IL-6) mRNA and protein was detected by real-time PCR or Western blot, respectively. Results: (1) Compared with the control group, the serum concentration of hs-CRP in the ACS group was increased (P<0.05). The expression of FAS, IL-6 mRNA and protein were significantly increased in PBMCs from the ACS group (all P<0.05). Moreover, the levels of FAS and IL-6 mRNA were positively correlated with the serum concentration of hs-CRP (r = 0.685, P<0.01; r = 0.715, P<0.01) respectively. (2) The expression of FAS, IL-6 mRNA and protein in PBMCs from the ACS group were dose-dependently inhibited by sEHi (all P<0.05). Conclusions: sEH inhibition regulated FAS and inhibited inflammation in cultured PBMCs from ACS patients, a mechanism that might prevent rupture of atherosclerotic lesions and protect against development of ACS. [ABSTRACT FROM AUTHOR]

17,18-Epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP) are bioactive epoxides produced from n-3 polyunsaturated fatty acid eicosapentaenoic acid and docosahexaenoic acid, respectively. However, these epoxides are quickly metabolized into less active diols by soluble epoxide hydrolase (sEH). We have previously demonstrated that an sEH inhibitor, t-TUCB, decreased serum triglycerides (TG) and increased lipid metabolic protein expression in the brown adipose tissue (BAT) of diet-induced obese mice. This study investigates the preventive effects of t-TUCB (T) alone or combined with 19,20-EDP (T + EDP) or 17,18-EEQ (T + EEQ) on BAT activation in the development of diet-induced obesity and metabolic disorders via osmotic minipump delivery in mice. Both T + EDP and T + EEQ groups showed significant improvement in fasting glucose, serum triglycerides, and higher core body temperature, whereas heat production was only significantly increased in the T + EEQ group. Moreover, both the T + EDP and T + EEQ groups showed less lipid accumulation in the BAT. Although UCP1 expression was not changed, PGC1α expression was increased in all three treated groups. In contrast, the expression of CPT1A and CPT1B, which are responsible for the rate-limiting step for fatty acid oxidation, was only increased in the T + EDP and T + EEQ groups. Interestingly, as a fatty acid transporter, CD36 expression was only increased in the T + EEQ group. Furthermore, both the T + EDP and T + EEQ groups showed decreased inflammatory NFκB signaling in the BAT. Our results suggest that 17,18-EEQ or 19,20-EDP combined with t-TUCB may prevent high-fat diet-induced metabolic disorders, in part through increased thermogenesis, upregulating lipid metabolic protein expression, and decreasing inflammation in the BAT. [ABSTRACT FROM AUTHOR]

It is well established that exercise could protect against myocardial infarction (MI). Previously, we found that epoxyeicosatrienoic acids (EETs) could be induced by exercise and has been found to protect against MI via promoting angiogenic function of endothelial progenitor cells (EPCs). However, the underling mechanism of EETs in promoting EPC functions is unclear. C57BL/6 mice were fed with a novel soluble epoxide hydrolase inhibitor (sEHi), TPPU, to increase EET levels, for 1 week before undergoing MI surgery. Mice were then subjected to exercise training for 4 weeks. Bone marrow-derived EPCs were isolated and cultured in vitro. Exercise upregulated miR-126 expression but downregulated the protein levels of its target gene, Spred1 , in EPCs from MI mice. TPPU further enhanced the effects of exercise on EPCs. Spred1 overexpression abolished the protective effects of TPPU on EPC functions. Downregulation of miR-126 by antagomiR-126 impaired the inhibitor effects of TPPU on Spred1 mRNA and protein expression. Additionally, TPPU upregulated miR-126 is partially mediated through ERK/p38 MAPK pathway. This study showed that sEHi promoted miR-126 expression, which might be related to the beneficial effect of sEHi on EPC functions in MI mice under exercise conditions, by increasing ERK and p38 MAPK phosphorylation and inhibiting Spred1. [ABSTRACT FROM AUTHOR]

• Renal fibrosis is the most common pathophysiological event in chronic kidney disease (CKD) and is an independent predictor of long-term CKD prognosis. • A novel bifunctional molecule, RB394 concurrently activates PPAR-γ and inhibits soluble epoxide hydrolase, an enzyme that increases kidney protective epoxyeicosatrienoic acids (EETs) levels. • Interventional RB394 treatment attenuated renal fibrosis progression by acting on multiple pathophysiological renal fibrotic events. Renal fibrosis is a contributor to chronic kidney disease and an important predictor of long-term prognosis. We developed a dual soluble epoxide hydrolase inhibitor-PPAR-γ agonist (sEHi/PPAR-γ), RB394, and investigated its ability to attenuate renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. RB394 efficacy was compared to an sEH inhibitor (sEHi), a PPAR-γ agonist rosiglitazone (Rosi), or their combination (sEHi + Rosi). All interventional treatments were administrated in drinking water 3 days after UUO induction surgery and continued for 7 days. UUO mice developed renal fibrosis with higher collagen formation and RB394 significantly attenuated fibrosis (P < 0.05). Renal expression of α-smooth muscle actin (α-SMA) was elevated in UUO mice and all treatments except sEHi significantly attenuated renal α-SMA expression. Renal mRNA expression fibrotic and fibrosis regulators were higher in UUO mice and RB394 and sEHi + Rosi treatments attenuated their expression. Renal inflammation was evident in UUO mice with increased infiltration of CD45 and F4/80 positive cells. RB394 and sEHi + Rosi treatments attenuated renal inflammation in UUO mice. UUO mice had renal tubular and vascular injury. Renal tubular and vascular injuries were attenuated to a greater extent by RB394 and sEHi + Rosi than sEHi or Rosi treatment alone. Renal mRNA expression of oxidative stress markers were significantly higher in UUO mice (P < 0.05). RB394 and sEHi + Rosi attenuated expression of oxidative stress markers to a greater extent than other interventional treatments (P < 0.05). These findings demonstrate that RB394 can attenuate renal fibrosis by reducing renal inflammation, oxidative stress, tubular injury, and vascular injury. In conclusion, RB394 demonstrates exciting potential as a therapeutic for renal fibrosis and chronic kidney disease. [ABSTRACT FROM AUTHOR]

Brown adipose tissue (BAT) is an important target for obesity treatment and prevention. Soluble epoxide hydrolase (sEH) converts bioactive epoxy fatty acids (EpFAs) into less active diols. sEH inhibitors (sEHI) are beneficial in many chronic diseases by stabilizing EpFAs. However, roles of sEH and sEHI in brown adipogenesis and BAT activity in treating diet-induced obesity (DIO) have not been reported. sEH expression was studied in in vitro models of brown adipogenesis and the fat tissues of DIO mice. The effects of the sEHI, trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy-benzoic acid (t-TUCB), were studied in vitro and in the obese mice via mini osmotic pump delivery. sEH expression was increased in brown adipogenesis and the BAT of the DIO mice. t-TUCB promoted brown adipogenesis in vitro. Although t-TCUB did not change body weight, fat pad weight, or glucose and insulin tolerance in the obese mice, it decreased serum triglycerides and increased protein expression of genes important for lipid metabolism in the BAT. Our results suggest that sEH may play a critical role in brown adipogenesis, and sEHI may be beneficial in improving BAT protein expression involved in lipid metabolism. Further studies using the sEHI combined with EpFA generating diets for obesity treatment and prevention are warranted. [ABSTRACT FROM AUTHOR]