EET Levels Research Articles

Paricalcitol ameliorates diabetic nephropathy by promoting EETs and M2 macrophage polarization and inhibiting inflammation by regulating VDR/CYP2J2 axis.

Previous studies have shown that paricalcitol (PA) has a protective effect on the kidneys. However, the exact molecular mechanism by which PA affects diabetic nephropathy (DN) progression remains uncertain. PBMCs of patients with DN were isolated, and CYP2J2 and VDR levels were detected by qPCR. Pearson correlation analysis was utilized to detect the relationship between uACR and CYP2J2 and VDR and between CYP2J2 and VDR. The protective effects of PA on DN have been examined by TUNEL, HE staining, ELISA, and Flow cytometry assays in STZ-induced mice. Moreover, THP-1 cells were stimulated with HG/LPS for invitro studies. ELISA, qPCR, western blot, and Flow cytometry assays were utilized to assess the effects of PA on DN progression by regulating CYP2J2. The interaction between CYP2J2 and VDR was analyzed by CHIP-qPCR and luciferase experiments. CYP2J2 and VDR levels were downregulated and uACR level was upregulated in DN patients. CYP2J2 and VDR were positively correlated in PBMCs. Both CYP2J2 and VDR are inversely correlated with uACR. Moreover, after PA treatment, 11, 12-EET levels increased, inflammatory factor levels decreased, and M2 macrophage polarization was promoted in STZ-induced mice and HG/LPS-triggered THP-1 cells. Depletion of CYP2J2 and VDR decreased 11, 12-EET level, enhanced inflammatory factor levels, and inhibited M2 macrophage polarization, which were reversed by CYP2J2 overexpression in HG/LPS-treated cells. Furthermore, VDR bound to the CYP2J2 promoter and promoted CYP2J2 transcriptional expression. The present work pointed out a new use for PA to inhibit DN progression by increasing EET level, inhibiting inflammatory response, and inducing M2 macrophage polarization via regulating the VDR/CYP2J2 axis.

Inhibiting the soluble epoxide hydrolase increases the EpFAs and ERK1/2 expression in the hippocampus of LiCl-pilocarpine post-status epilepticus rat model

PurposeThis study aimed to investigate the enzyme activity of soluble epoxide hydrolase (sEH) and quantify its metabolic substrates, namely epoxygenated fatty acids (EpFAs), and products of sEH in the hippocampus after administering TPPU [1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea], an inhibitor of sEH. Furthermore, it explored whether the extracellular signal-activated protein kinase 1/2 (ERK1/2) is involved in the anti-seizure effects of TPPU in the lithium chloride (LiCl)-pilocarpine induced post-status epilepticus (SE) rat model. MethodsThe rats were intraperitoneally (I.P.) injected with LiCl and pilocarpine to induce SE and then spontaneous recurrent seizures (SRS) were observed. Rats were randomly assigned into SRS + TPPU group (intragastrically administering 0.1 mg/kg/d TPPU), SRS + Vehicle group (administering the vehicle instead), and Control group. Enzyme-linked immunosorbent assay, Western-blot analysis, and ultra-high-performance liquid chromatography/mass spectrometry (LC/MS) were performed to measure the enzyme activity of sEH, the protein level of sEH and ERK1/2, and the concentration of TPPU and polyunsaturated fatty acids (PUFAs) metabolisms in the hippocampus. ResultsThe frequency of SRS events of Racine stage 3 or higher ranged from 0 to 19 per week in the SRS + Vehicle group, compared to 0–5 per week in the SRS + TPPU group. sEH enzyme activity and protein levels were significantly elevated in the SRS + Vehicle group compared to the Control group. After TPPU administration, the hippocampal TPPU concentration reached 10.94 ± 4.37 nmol/kg. sEH enzyme activity was significantly reduced in the LiCl-pilocarpine-induced post-SE rat model, although sEH protein levels did not decrease significantly. The regioisomers 8,9-, 11,12-, and 14,15-EETs, total EETs, the EETs/DHETs ratio, other EpFAs including 16(17)-EpDPA, and the 19(20)-EpDPA/19,20-DiHDPA ratio in the hippocampus were significantly increased. Additionally, the p-ERK1/2 to ERK1/2 ratio in the hippocampus was significantly elevated following TPPU administration. ConclusionThis study demonstrates that inhibiting sEH with TPPU increases the levels of EETs, other EpFAs, and ERK1/2 expression in the hippocampus of a LiCl-pilocarpine-induced post-SE rat model. These findings suggest that the anti-seizure effect of TPPU may be mediated through the EETs-ERK1/2 pathway.

Epoxygenase Cyp2c44 Regulates Hepatic Lipid Metabolism and Insulin Signaling by Controlling FATP2 Localization and Activation of the DAG/PKCδ Axis.

Cytochrome P450 epoxygenase Cyp2c44, a murine epoxyeicosatrienoic acid (EET) producing enzyme, promotes insulin sensitivity and Cyp2c44(-/-) mice show hepatic insulin resistance. Because insulin resistance leads to hepatic lipid accumulation and hyperlipidemia, we hypothesized that Cyp2c44 regulates hepatic lipid metabolism. Standard chow diet (SD) fed male Cyp2c44(-/-) mice had significantly decreased EET levels and increased hepatic and plasma lipid levels compared to wild-type mice. We showed increased hepatic plasma membrane localization of the FA transporter 2 (FATP2) and total unsaturated fatty acids and diacylglycerol levels. Cyp2c44(-/-) mice had impaired glucose tolerance and increased hepatic plasma membraneassociated PKCδ and phosphorylated IRS-1, two negative regulators of insulin signaling. Surprisingly, SD and high fat diet fed (HFD) Cyp2c44(-/-) mice had similar glucose tolerance and hepatic plasma membrane PKCδ levels, suggesting that SD-fed Cyp2c44(-/-) mice have reached their maximal glucose intolerance. Inhibition of PKCδ resulted in decreased IRS-1 serine phosphorylation and improved insulin-mediated signaling in Cyp2c44(-/-) hepatocytes. Finally, Cyp2c44(-/-) HFD-fed mice treated with the analog EET-A showed decreased hepatic plasma membrane FATP2 and PCKDδ levels with improved glucose tolerance and insulin signaling. In conclusion, loss of Cyp2c44 with concomitant decreased EET levels leads to increased hepatic FATP2 plasma membrane localization, diacylglycerol accumulation, and PKCδ-mediated attenuation of insulin signaling. Thus, Cyp2c44 acts as a regulator of lipid metabolism by linking it to insulin signaling.

Abstract LB334: Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis

Abstract Introduction: Eicosanoid mediated mechanisms of hormone therapy resistance in ER+HER2- breast cancer are poorly understood. Cytochrome P450 arachidonic acid (AA) epoxygenase-derived epoxyeicosatrienoic acids (EETs) contribute to breast cancer progression by promoting mitochondrial oxidative phosphorylation (OXPHOS). However, it remains unclear how EETs contribute to hormonal therapy resistance. In this study, the well-known letrozole resistant (LR) MCF-7 AC1 cell line overexpressing aromatase was selected for fulvestrant resistance (FR). A clonal cell line resistant to letrozole and fulvestrant (LR/FR) also exhibited resistance to palbociclib (PR) (MCF-7 AC1 LR/FR/PR). Xenograft tumors of this cell line were resistant to letrozole, fulvestrant, and palbociclib. Structure activity studies and modeling led to the development of CYP3A4 AA epoxygenase inhibiting biguanides hexyl cuban-1-yl biguanide (HCB) and fluorinated hexyl derivatives C5F2-HCB and C6F3-HCB. Fluorination on the hexyl moiety led to broader inhibition of EET regioisomer biosynthesis. These agents were tested for inhibition of proliferation, signaling, and OXPHOS/glycolysis balance in the MCF-7 AC1 LR/FR/PR cell line. Results: In the MCF-7 AC1 LR/FR/PR clonal cell line cyclin D1 and estrogen receptor (ER) expression were undetectable, while cyclin E1, CDK4, CDK6, CYP3A4, and c-MYC were upregulated 11, 40, 13, 10, and 12- fold (n=3, p<0.001) compared to the MCF-7 cell line. LC-MS analysis demonstrated 1.8, 1.4, and 1.2- fold higher total cellular levels of (±)8,9, (±)11,12, and (±)14,15-EET in the MCF-7 AC1 LR/FR/PR cell line compared to the MCF-7 cell line (n=3, p<0.05). C6F3-HCB suppressed cellular levels of EETs: (±)8,9-EET by 55% (n=3, p<0.031), (±)11,12-EET by 53% (n=3, p<0.029), and (±)14,15-EET by 60% (n=3, P<0.0001). The MCF-7 AC1 LR/FR/PR cell line was more potently inhibited by hexyl-cuban-1-yl biguanides compared to the MCF-7 cell line, exhibiting lower IC50 values (MCF-7 AC1 LR/FR/PR IC50 vs. MCF-7 IC50; HCB 4.1 vs. 7.0 uM; for C5F2-HCB 10 vs. 25 uM; C6F3-HCB: 5.4 vs. 12 uM; P values all <0.05). Measurement of spare respiratory capacity revealed that the MCF-7 AC1 LR/FR/PR cell line had 4.2% spare respiratory capacity compared to 34% for MCF-7, indicating a lower OXPHOS reserve for the resistant cells. Additionally, the MCF-7 AC1 LR/FR/PR cell line displayed a 2.4-fold higher extracellular acidification rate (ECAR) than the MCF-7 cell line, indicating a higher glycolysis rate (Warburg effect) in the resistant cells. Conclusion: Selection for fulvestrant resistance of MCF-7 AC1 cells resulted in upregulation of CYP3A4 and biosynthesis of hormone therapy resistance associated EETs while correlating with greater sensitivity to fluorinated hexyl-cuban-1-yl biguanides that inhibit EET biosynthesis. Citation Format: Zhijun Guo, Jianxun Lei, Allison Makovec, Swaathi Jayaraman, John R. Hawse, Carol Lange, Elizabeth Ambrose, Gunda I. Georg, Tony D'Assoro, Goetz P. Matthew, David A. Potter. Mechanisms of endocrine resistance to letrozole, fulvestrant, and palbociclib are associated with increased sensitivity to hexyl-cuban-1-yl biguanide inhibitors of CYP3A4 mediated epoxyeicosatrienoic acid biosynthesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB334.

Sorafenib reduces the production of epoxyeicosatrienoic acids and leads to cardiac injury by inhibiting CYP2J in rats

Sorafenib, an important cancer drug in clinical practice, has caused heart problems such as hypertension, myocardial infarction, and thrombosis. Although some mechanisms of sorafenib-induced cardiotoxicity have been proposed, there is still more research needed to reach a well-established definition of the causes of cardiotoxicity of sorafenib. In this report, we demonstrate that sorafenib is a potent inhibitor of the CYP2J enzyme. Sorafenib significantly inhibited the production of epoxyeicosatrienoic acids (EETs) in rat cardiac microsomes. The in vivo experimental results also showed that after the administration of sorafenib, the levels of 11,12-EET and 14,15-EET in rat plasma were significantly reduced, which was similar to the results of CYP2J gene knockout. Sorafenib decreased the levels of EETs, leading to abnormal expression of mitochondrial fusion and fission factors in heart tissue. In addition, the expression of mitochondrial energy metabolism factors (Pgc-1α, Pgc-1β, Ampk, and Sirt1) and cardiac mechanism factors (Scn5a and Prkag2) was significantly reduced, increasing the risk of arrhythmia and heart failure. Meanwhile, the increase in injury markers Anp, CK, and CK-MB further confirmed the cardiotoxicity of sorafenib. This study is of great significance for understanding the cardiotoxicity of sorafenib, and is also a model for studying the cardiotoxicity of other drugs that inhibit CYP2J activity.

Quercetin inhibits the metabolism of arachidonic acid by inhibiting the activity of CYP3A4, thereby inhibiting the progression of breast cancer

BackgroundRecent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron’s anti-breast carcinoma (BC) function.MethodsBioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo.ResultsQuercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice.ConclusionQuercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development.

Abstract P418: Antigen-presenting Cell-specific MAP-kinase Signaling Contributes To Inflammation And Salt-sensitivity Of Blood Pressure In Humans

Salt-sensitivity of blood pressure (SSBP) is defined by the changes in blood pressure in concert with salt intake/depletion. SSBP affects 50 percent of hypertensive and 25 percent of normotensive individuals and is an independent risk for stroke and cardiovascular death in both groups. We recently found that phosphorylation of STAT3 and SMAD3 in antigen-presenting cells plays an important role in developing SSBP. We hypothesized that mitogen-activated protein kinases (MAPKs) signaling plays an important role in activating/phosphorylating JAK-STAT-SMAD and contributes to salt-sensitivity of blood pressure. We isolated monocytes from eleven healthy individuals and treated them with either normal salt (NS) or high salt (HS) and performed bulk-RNA-sequencing. The bulk-RNA transcriptomic data analysis suggested that out of 21 members of the MAPK family, eighteen were upregulated (1.7-fold) in monocytes exposed to high salt. Among these, MAPK7 (NS:818.18±54.96; HS:1473.18±151.50) and MAPKAPK2 (NS:3977.81±286.67; HS:6222.63±440.32) were significantly upregulated. In additional experiments, we enrolled eleven hypertensive patients and phenotyped them for salt-sensitivity using a rapid salt-load and depletion inpatient protocol. Blood was collected at baseline, after salt loading, and depletion, and CITE-Seq was performed on isolated PBMCs. The data analysis showed that MAPK7 and MAPKAPK2 expressions mirror the blood pressure changes following salt loading and depletion in salt-sensitive but not in salt-resistant patients. Moreover, we found a positive correlation between MAPKAPK2 expression and the pulse pressure ((PP) r=0.52, p=0.033) after salt-load. Epoxyeicosatrienoic acid (EET) has been shown to activate MAPKs resulting in inhibiting ENAC activity. The data showed a strong positive correlation between MAPK7 expression and levels of EETs in plasma (r=0.70, p=0.036), and urine (r=0.68, p=0.045) after salt-load. These results from bulk-RNA and single-cell CITE-Seq data indicate that MAPKs play an important role in human SSBP.

Baroreflex Sensitivity, Cardiac and Kidney Remodeling and Deterioration in Vasoactive Substances Content in Blood in Experimental Model of Renovascular Hypertension. Action of Natural Flavone, Luteolin

This study aimed to analyze pharmacological actions of phenolic compound luteolin on the renal and cardiac hypertrophy, blood pressure (BP), baroreflex sensitivity (BRS), levels of epoxyeicosatrienoic acids (EETs), prostaglandin E-2 (PGE-2) and endothelin-1 (E1) in plasma in the 2 kidney - 1 clip (2K-1C) model of renovascular hypertension (RVH). All animals, were randomized into 2 groups: control (normal) I - sham-operated, II- RVH male Wistar rats, which after 4 weeks of surgical intervention secondly randomized to control II group, treated 0.1% dimethyl sulphoxide (DMSO) and main group - with luteolin in 15 DMSO, 3 mg/kg body weight, intraperitonially, during 2 weeks. ET-1, EETs and PGE2 levels investigated in carotid artery blood plasma and analyzed using ELISA kits. All data statistically analyzed using the SPSS-10.0 program. In RVH rats BP increased by 32%, cardiac and right kidney hypertrophy and reduction in parasympathetic component of BRS by 40% and sympathetic by 39%. The plasma level of total trans-EETs and PGE2 in RVH rats decreased by 44% and 50% respectively, while the level of ET-1 increased by 67%. Two weeks treatment with luteolin lowered BP, improved parasympathetic, without marked changes in sympathetic component of BRS. Deremodeling of cardiac and renal hypertrophy under prolonged treatment with luteolin accompanied with increasing in the level of EETs by 44%, PGE-2 by 50% and markedly reducing of plasma content of ET-1 (by 60%). Inhibition of EET hydrolase using low doses of luteolin provides beneficial cardio and renoprotective action in experimental model of RVH.

Sorafenib increases cytochrome P450 lipid metabolites in patient with hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death, and medical treatment options are limited. The multikinase inhibitor sorafenib was the first approved drug widely used for systemic therapy in advanced HCC. Sorafenib might affect polyunsaturated fatty acids (PUFA)-derived epoxygenated metabolite levels, as it is also a potent inhibitor of the soluble epoxide hydrolase (sEH), which catalyzes the conversion of cytochrome-P450 (CYP)-derived epoxide metabolites derived from PUFA, such as omega-6 arachidonic acid (AA) and omega-3 docosahexaenoic acid (DHA), into their corresponding dihydroxy metabolites. Experimental studies with AA-derived epoxyeicosatrienoic acids (EETs) have shown that they can promote tumor growth and metastasis, while DHA-derived 19,20-epoxydocosapentaenoic acid (19,20-EDP) was shown to have anti-tumor activity in mice. In this study, we found a significant increase in EET levels in 43 HCC patients treated with sorafenib and a trend towards increased levels of DHA-derived 19,20-EDP. We demonstrate that the effect of sorafenib on CYP- metabolites led to an increase of 19,20-EDP and its dihydroxy metabolite, whereas DHA plasma levels decreased under sorafenib treatment. These data indicate that specific supplementation with DHA could be used to increase levels of the epoxy compound 19,20-EDP with potential anti-tumor activity in HCC patients receiving sorafenib therapy.

Macrophage Inactivation by Small Molecule Wedelolactone via Targeting sEH for the Treatment of LPS-Induced Acute Lung Injury.

Soluble epoxide hydrolase (sEH) plays a critical role in inflammation by modulating levels of epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs). Here, we investigate the possible role of sEH in lipopolysaccharide (LPS)-mediated macrophage activation and acute lung injury (ALI). In this study, we found that a small molecule, wedelolactone (WED), targeted sEH and led to macrophage inactivation. Through the molecular interaction with amino acids Phe362 and Gln384, WED suppressed sEH activity to enhance levels of EETs, thus attenuating inflammation and oxidative stress by regulating glycogen synthase kinase 3beta (GSK3β)-mediated nuclear factor-kappa B (NF-κB) and nuclear factor E2-related factor 2 (Nrf2) pathways in vitro. In an LPS-stimulated ALI animal model, pharmacological sEH inhibition by WED or sEH knockout (KO) alleviated pulmonary damage, such as the increase in the alveolar wall thickness and collapse. Additionally, WED or sEH genetic KO both suppressed macrophage activation and attenuated inflammation and oxidative stress in vivo. These findings provided the broader prospects for ALI treatment by targeting sEH to alleviate inflammation and oxidative stress and suggested WED as a natural lead candidate for the development of novel synthetic sEH inhibitors.

TPPU inhibits inflammation-induced excessive autophagy to restore the osteogenic differentiation potential of stem cells and improves alveolar ridge preservation

Inflammation-induced autophagy is a double-edged sword. Dysfunction of autophagy impairs the differentiation capacity of mesenchymal stem cells and enhances inflammation-induced bone loss. Tooth extraction with periodontal and/or endodontic lesions exacerbates horizontal and vertical resorption of alveolar bone during the healing period. Alveolar socket preservation (ASP) procedure following tooth extraction has important clinical implications for future prosthodontic treatments. Studies have shown that epoxyeicosatrienoic acids (EETs) have significant anti-inflammatory effects and participate in autophagy. However, whether EETs can minimize alveolar bone resorption and contribute to ASP by regulating autophagy levels under inflammatory conditions remain elusive. Here, we figured out that LPS-induced inflammatory conditions increased the inflammatory cytokine and inhibited osteogenic differentiation of human dental pulp stem cells (hDPSCs), and led to excessive autophagy of hDPSCs. Moreover, we identified that increased EETs levels using TPPU, a soluble epoxide hydrolase inhibitor, reversed these negative outcomes. We further demonstrated the potential of TPPU to promote early healing of extraction sockets and ASP, and speculated that it was related to autophagy. Taken together, these results suggest that targeting inhibition of soluble epoxide hydrolase using TPPU plays a protective role in the differentiation and autophagy of mesenchymal stem cells and provides potential feasibility for applying TPPU for ASP, especially under inflammatory conditions.

Soluble epoxide hydrolase and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy: Interrelation and regulation by C/EBPβ

ObjectivesStudies in certain cardiac hypertrophy models suggested the individual role of soluble epoxide hydrolase (sEH) and canonical transient receptor potential 3 (TRPC3) channels, however, whether they jointly mediate hypertrophic process remains unexplored. Hyperhomocysteinemia promotes cardiac hypertrophy while the involvement of sEH and TRPC3 channels remains unknown. This study aimed to explore the role of, and interrelation between sEH and TRPC3 channels in homocysteine-induced cardiac hypertrophy. MethodsRats were fed methionine-enriched diet to induce hyperhomocysteinemia. H9c2 cells and neonatal rat cardiomyocytes were incubated with homocysteine. Cardiac hypertrophy was evaluated by echocardiography, histological examination, immunofluorescence imaging, and expressions of hypertrophic markers. Epoxyeicosatrienoic acids (EETs) were determined by ELISA. TRPC3 current was recorded by patch-clamp. Gene promotor activity was measured using dual-luciferase reporter assay. ResultsInhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU) reduced ventricular mass, lowered the expression of hypertrophic markers, decreased interstitial collagen deposition, and improved cardiac function in hyperhomocysteinemic rats, associated with restoration of EETs levels in myocardium. TPPU or knockdown of sEH suppressed TRPC3 transcription and translation as well as TRPC3 current that were enhanced by homocysteine. Exogenous 11,12-EET inhibited homocysteine-induced TRPC3 expression and cellular hypertrophy. Silencing C/EBPβ attenuated, while overexpressing C/EBPβ promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters. ConclusionssEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPβ. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner.

Cytochrome P450-derived eicosanoids in brain: From basic discovery to clinical translation.

Cytochrome P450 metabolism of arachidonic acid produces epoxyeicosatrienoates (EETs) and hydroxyeicosatetraenoates (HETEs). Both classes of eicosanoids play important and opposing roles in brain function and disease. EETs promote vasodilation and exhibit antiinflammatory and cytoprotective properties; their biological action is blunted by metabolism to less active diols by the enzyme soluble epoxide hydrolase (sEH). EETs levels are dysregulated in disease states, primarily due to increased activity of sEH. Inhibition of sEH is a promising therapeutic approach for multiple brain disorders including stroke, dementia, subarachnoid hemorrhage and epilepsy. In this chapter, we summarize evidence implicating P450 eicosanoids and their synthetic and metabolizing enzymes in brain health and disease, and experimental and clinical studies targeting these pathways for brain disorders. We also discuss the diagnostic utility of quantifying P450 eicosanoids and their enzymes as disease biomarkers. Remarkable progress has been achieved in translating basic science discoveries in this field clinically.

Glimepiride Use is Associated with Reduced Cardiovascular Mortality in Patients with Type 2 Diabetes and Chronic Heart Failure: A Prospective Cohort Study.

Glimepiride has good cardiovascular safety. However, whether glimepiride benefits clinical cardiovascular outcomes is unclear. A total of 21,451 inpatients with type 2 diabetes (T2D) and chronic heart failure (CHF) were analyzed, including 638 who received glimepiride treatment and 20,813 who did not. Propensity score matching yielded 509 pairs (glimepiride and non-glimepiride groups), and both groups were followed up. Kaplan-Meier and Cox regression analyses were used to compare all-cause mortality, cardiovascular mortality, hospitalizations and emergency visits for heart failure, and hospitalizations for acute myocardial infarction or stroke. During follow-up, the all-cause mortality (adjusted hazard ratio [HR], 0.47; 95% confidence interval [CI], 0.35-0.63; P < 0.001), cardiovascular mortality (adjusted HR, 0.34; 95% CI, 0.24-0.48; P < 0.001), and number of hospitalizations and emergency visits for heart failure (adjusted HR, 0.42; 95% CI, 0.36-0.50; P < 0.001) and hospitalizations for acute myocardial infarction or stroke (adjusted HR, 0.53; 95% CI, 0.38-0.73; P < 0.001) were significantly lower in the glimepiride group; the conclusion remained similar in all subgroups. Furthermore, high-dose glimepiride use (2-4 mg/day) was associated with lower cardiovascular mortality than low-dose (1 mg/day) (adjusted HR, 0.55; 95% CI, 0.31-0.99; P = 0.047). Glimepiride exhibited good molecular docking with soluble epoxide hydrolase (sEH) and increased the level epoxyeicosatrienoic acid (EET). Long-term continuous glimepiride use is associated with better survival, fewer hospitalizations and emergency visits for heart failure, and fewer hospitalizations for acute myocardial infarction or stroke in patients with T2D and CHF. High-dose glimepiride has greater cardiovascular protective advantages than low-dose glimepiride. The cardiovascular protective effect of glimepiride may be related to the EET level increase through sEH inhibition.

Cardiac Disease Alters Myocardial Tissue Levels of Epoxyeicosatrienoic Acids and Key Proteins Involved in Their Biosynthesis and Degradation.

CYP2J2 is the main epoxygenase in the heart that is responsible for oxidizing arachidonic acid to cis-epoxyeicosatrienoic acids (EETs). Once formed, EETs can then be hydrolyzed by soluble epoxide hydrolase (sEH, encoded by EPHX2) or re-esterified back to the membrane. EETs have several cardioprotective properties and higher levels are usually associated with better cardiac outcomes/prognosis. This study investigates how cardiovascular disease (CVD) can influence total EET levels by altering protein expression and activity of enzymes involved in their biosynthesis and degradation. Diseased ventricular cardiac tissues were collected from patients receiving Left Ventricular Assist Device (LVAD) or heart transplants and compared to ventricular tissue from controls free of CVD. EETs, and enzymes involved in EETs biosynthesis and degradation, were measured using mass spectrometric assays. Terfenadine hydroxylation was used to probe CYP2J2 activity. Significantly higher cis- and trans-EET levels were observed in control cardiac tissue (n = 17) relative to diseased tissue (n = 24). Control cardiac tissue had higher CYP2J2 protein levels, which resulted in higher rate of terfenadine hydroxylation, compared to diseased cardiac tissues. In addition, levels of both NADPH-Cytochrome P450 oxidoreductase (POR) and sEH proteins were significantly higher in control versus diseased cardiac tissue. Overall, alterations in protein and activity of enzymes involved in the biosynthesis and degradation of EETs provide a mechanistic understanding for decreased EET levels in diseased tissues.

Quinazoline-4(3H)-one-7-carboxamide Derivatives as Human Soluble Epoxide Hydrolase Inhibitors with Developable 5-Lipoxygenase Activating Protein Inhibition.

Soluble epoxide hydrolase (sEH) metabolizes epoxyeicosatrienoic acids (EETs), which are endowed with beneficial biological activities as they reduce inflammation, regulate endothelial tone, improve mitochondrial function, and decrease oxidative stress. Therefore, inhibition of sEH for maintaining high EET levels is implicated as a new therapeutic modality with broad clinical applications for metabolic, renal, and cardiovascular disorders. In our search for new sEH inhibitors, we designed and synthesized novel amide analogues of the quinazolinone-7-carboxylic acid derivative 5, a previously discovered 5-lipoxygenase-activating protein (FLAP) inhibitor, to evaluate their potential for inhibiting sEH. As a result, we identified new quinazolinone-7-carboxamides that demonstrated selective sEH inhibition with decreased FLAP inhibitor properties. The tractable SAR results indicated that the amide and thiobenzyl fragments flanking the quinazolinone nucleus are critical features governing the potent sEH inhibition, and compounds 34, 35, 37, and 43 inhibited the sEH activity with IC50 values of 0.30–0.66 μM. Compound 34 also inhibited the FLAP-mediated leukotriene biosynthesis (IC50 = 2.91 μM). In conclusion, quinazolinone-7-carboxamides can be regarded as novel lead structures, and newer analogues with improved efficiency against sEH along with or without FLAP inhibition can be generated.

Total flavonoids of Inula japonica alleviated the inflammatory response and oxidative stress in LPS-induced acute lung injury via inhibiting the sEH activity: Insights from lipid metabolomics

BackgroundAcute lung injury (ALI) is a severe respiratory disease characterized by diffuse lung interstitial and respiratory distress and pulmonary edema with a mortality rate of 35%-40%. Inula japonica Thunb., known as “Xuan Fu Hua” in Chinese, is a traditional Chinese medicine Inulae Flos to use for relieving cough, eliminating expectorant, and preventing bacterial infections in the clinic, and possesses an anti-pulmonary fibrosis effect. However, the effect and action mechanism of I. japonica on ALI is still unclear. PurposeThis study aimed to investigate the protective effect and underlying mechanism of total flavonoids of I. japonica (TFIJ) in the treatment of ALI. Study design and methodsA mouse ALI model was established through administration of LPS by the intratracheal instillation. Protective effects of TFIJ in the inflammation and oxidative stress were studied in LPS-induced ALI mice based on inflammatory and oxidative stress factors, including MDA, MPO, SOD, and TNF-α. Lipid metabolomics, bioinformatics, Western blot, quantitative real-time PCR, and immunohistochemistry were performed to reveal the potential mechanism of TFIJ in the treatment of ALI. ResultsTFIJ significantly alleviated the interstitial infiltration of inflammatory cells and the collapse of the alveoli in LPS-induced ALI mice. Lipid metabolomics demonstrated that TFIJ could significantly affect the CYP2J/sEH-mediated arachidonic acid metabolism, such as 11,12-EET, 14,15-EET, 8,9-DHET, 11,12-DHET, and 14,15-DHET, revealing that sEH was the potential target of TFIJ, which was further supported by the recombinant sEH-mediated the substrate hydrolysis in vitro (IC50 = 1.18 μg/ml). Inhibition of sEH by TFIJ alleviated the inflammatory response and oxidative stress via the MAPK, NF-κB, and Nrf2 signaling pathways. ConclusionThese results demonstrated that TFIJ could suppress the sEH activity to stabilize the level of EETs, allowing the alleviation of the pathological course of lung injury in LPS-treated mice, which suggested that TFIJ could serve as the potential agents in the treatment of ALI.

Plasma epoxyeicosatrienoic acids and diabetes-related cardiovascular disease: The cardiovascular health study.

SummaryBackgroundEpoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid that may impact atherosclerosis, and animal experimental studies suggest EETs protect cardiac function. Plasma EETs are mostly esterified to phospholipids and part of an active pool. To address the limited information about EETs and CVD in humans, we conducted a prospective study of total plasma EETs (free + esterified) and diabetes-related CVD in the Cardiovascular Health Study (CHS).MethodsWe measured 4 EET species and their metabolites, dihydroxyepoxyeicosatrienoic acids (DHETs), in plasma samples from 892 CHS participants with type 2 diabetes. We determined the association of EETs and DHETs with incident myocardial infarction (MI) and ischemic stroke using Cox regression.FindingsDuring follow-up (median 7.5 years), we identified 150 MI and 134 ischemic strokes. In primary, multivariable analyses, elevated levels of each EET species were associated with non-significant lower risk of incident MI (for example, hazard ratio for 1 SD higher 14,15-EET: 0.86, 95% CI: 0.72–1.02; p=0.08). The EETs-MI associations became significant in analyses further adjusted for DHETs (hazard ratio for 1 SD higher 14,15-EET adjusted for 14,15-DHET: 0.76, 95% CI: 0.63–0.91; p=0.004). Elevated EET levels were associated with higher risk of ischemic stroke in primary but not secondary analyses. Three DHET species were associated with higher risk of ischemic stroke in all analyses.InterpretationFindings from this prospective study complement the extensive studies in animal models showing EETs protect cardiac function and provide new information in humans. Replication is needed to confirm the associations.FundingUS National Institutes of Health.

Soluble Epoxide Hydrolase Inhibitor t-AUCB Ameliorates Vascular Endothelial Dysfunction by Influencing the NF-κB/miR-155-5p/eNOS/NO/IκB Cycle in Hypertensive Rats.

Epoxyeicosatrienoic acids (EETs), angiogenic mediators degraded by soluble epoxide hydrolase (sEH), have been shown to exert beneficial effects on the cardiovascular system. The current study assessed the impact of increased EETs with an sEH inhibitor, t-AUCB, on two-kidney-one-clip (2K1C)-induced renovascular endothelial dysfunction, associated with hypertension, in rats. The hypertensive rats exhibited increased systolic blood pressure, reduced renal blood flow, impaired endothelium-dependent relaxation and eNOS phosphorylation in the renal arteries, elevated ROS production in the endothelium of the renal arteries, and decreased EET levels in plasma, the renal arteries, and endothelial cells; however, t-AUCB reversed all the deleterious effects. Moreover, we found that the stimulation of AMPK/UCP2 scavenged ROS and restored endothelial function in the renal arteries of hypertensive rats undergoing therapy with t-AUCB. In addition, we were the first to reveal the potential role of miR-155-5p in the occurrence and development of vascular endothelial dysfunction in hypertension. Importantly, t-AUCB recovered NO bioavailability by regulating the NF-κB/miR-155-5p/eNOS/NO/IκB cycle after the activation of AMPK/UCP2 and the subsequent inhibition of ROS in hypertensive rat renal artery endothelial cells. This study will provide evidence for this additional new mechanism, underlying the benefits of EETs and the related agents against hypertensive vasculopathy.

Abstract 2379: Gender-specific metabolome in bladder cancer: Role of EPHX2 in bladder cancer

Abstract Bladder cancer (BLCA) is the fourth most common cancer diagnosed in American men. In the U.S., men are four times more likely to be diagnosed with bladder cancer than women. Our research group has previously identified alterations in xenobiotic metabolism in BLCA. In this study, using a metabolomics approach, we identified alterations in the arachidonic acid pathway in bladder tumors from male versus female patients. Included within this altered arachidonic acid pathway was downregulation of EPHX2, an enzyme that converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DiHETs). Consistent with this, levels of EETs were significantly elevated in bladder tumors derived from male versus female patients. Cox proportional hazard regression analysis further revealed that reduced expression of EPHX2 was significantly associated with poor clinical outcome across multiple publicly available datasets only in male BLCA patients but not in females. Mechanistic studies revealed that EPHX2 over-expression in male-derived BLCA cell line reduced cell growth in vitro and tumor growth in vivo. Overall, these studies nominate EPHX2 as a potential tumor suppressor in male patients with BLCA. Citation Format: Mohammed Khurshidul Hassan, Roshan Borkar, Karthik Reddy Kami Reddy, Danthasinghe Waduge Badrajee Piyarathna, Chandra Shekar Amara, Allison Bellman, ChandraShekar R. Ambati, Vasanta Putluri, Abu Hena Mostafa Kamal, Roni J. Bollag, Martha K. Terris, Leomar Y. Ballester, Yair Lotan, Cristian Coarfa, Arun Sreekumar, Nagireddy Putluri. Gender-specific metabolome in bladder cancer: Role of EPHX2 in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2379.