Regulation of soluble epoxide hydrolase in renal-associated diseases: insights from potential mechanisms to clinical researches.

In recent years, numerous experimental studies have underscored the pivotal role of soluble epoxide hydrolase (sEH) in renal diseases, demonstrating the reno-protective effects of sEH inhibitors. The nexus between sEH and renal-associated diseases has garnered escalating attention. This review endeavors to elucidate the potential molecular mechanisms of sEH in renal diseases and emphasize the critical role of sEH inhibitors as a prospective treatment modality. Initially, we expound upon the correlation between sEH and Epoxyeicosatrienoic acids (EETs) and also addressing the impact of sEH on other epoxy fatty acids, delineate prevalent EPHX2 single nucleotide polymorphisms (SNPs) associated with renal diseases, and delve into sEH-mediated potential mechanisms, encompassing oxidative stress, inflammation, ER stress, and autophagy. Subsequently, we delineate clinical research pertaining to sEH inhibition or co-inhibition of sEH with other inhibitors for the regulation of renal-associated diseases, covering conditions such as acute kidney injury, chronic kidney diseases, diabetic nephropathy, and hypertension-induced renal injury. Our objective is to validate the potential role of sEH inhibitors in the treatment of renal injuries. We contend that a comprehensive comprehension of the salient attributes of sEH, coupled with insights from clinical experiments, provides invaluable guidance for clinicians and presents promising therapeutic avenues for patients suffering from renal diseases.

Hepatic Transcriptome and Its Regulation Following Soluble Epoxide Hydrolase Inhibition in Alcohol-Associated Liver Disease.

Alcohol-associated liver disease (ALD) is a serious public health problem with limited pharmacologic options. The goal of the current study was to investigate the efficacy of pharmacologic inhibition of soluble epoxide hydrolase (sEH), an enzyme involved in lipid metabolism, in experimental ALD, and to examine the underlying mechanisms. C57BL/6J male mice were subjected to acute-on-chronic ethanol (EtOH) feeding with or without the sEH inhibitor 4-[[trans-4-[[[[4-trifluoromethoxy phenyl]amino]carbonyl]-amino]cyclohexyl]oxy]-benzoic acid (TUCB). Liver injury was assessed by multiple end points. Liver epoxy fatty acids and dihydroxy fatty acids were measured by targeted metabolomics. Whole-liver RNA sequencing was performed, and free modified RNA bases were measured by mass spectrometry. EtOH-induced liver injury was ameliorated by TUCB treatment as evidenced by reduced plasma alanine aminotransferase levels and was associated with attenuated alcohol-induced endoplasmic reticulum stress, reduced neutrophil infiltration, and increased numbers of hepatic M2 macrophages. TUCB altered liver epoxy and dihydroxy fatty acids and led to a unique hepatic transcriptional profile characterized by decreased expression of genes involved in apoptosis, inflammation, fibrosis, and carcinogenesis. Several modified RNA bases were robustly changed by TUCB, including N6-methyladenosine and 2-methylthio-N6-threonylcarbamoyladenosine. These findings show the beneficial effects of sEH inhibition by TUCB in experimental EtOH-induced liver injury, warranting further mechanistic studies to explore the underlying mechanisms, and highlighting the translational potential of sEH as a drug target for this disease.

Overexpression of Human Soluble Epoxide Hydrolase Exacerbates Coronary Reactive Hyperemia Reduction in Angiotensin-II-Treated Mouse Hearts.

ABSTRACT: Coronary reactive hyperemia (CRH) is impaired in cardiovascular diseases, and angiotensin-II (Ang-II) exacerbates it. However, it is unknown how Ang-II affects CRH in Tie2-sEH Tr (human-sEH-overexpressed) versus wild-type (WT) mice. sEH-overexpression resulted in CRH reduction in Tie2-sEH Tr versus WT. We hypothesized that Ang-II exacerbates CRH reduction in Tie2-sEH Tr versus WT. The Langendorff system measured coronary flow in Tie2-sEH Tr and WT. The hearts were exposed to 15-second ischemia, and CRH was assessed in 10 mice each. Repayment volume was reduced by 40.50% in WT treated with Ang-II versus WT (7.42 ± 0.8 to 4.49 ± 0.8 mL/g) and 48% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (5.18 ± 0.4 to 2.68 ± 0.3 mL/g). Ang-II decreased repayment duration by 50% in WT-treated with Ang-II versus WT (2.46 ± 0.5 to 1.24 ± 0.4 minutes) and 54% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (1.66 ± 0.4 to 0.76 ± 0.2 minutes). Peak repayment flow was reduced by 11.2% in WT treated with Ang-II versus WT (35.98 ± 0.7 to 32.11 ± 1.4 mL/g) and 4% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (32.18 ± 0.6 to 30.89 ± 1.5 mL/g). Furthermore, coronary flow was reduced by 43% in WT treated with Ang-II versus WT (14.2 ± 0.5 to 8.15 ± 0.8 mL/min/g) and 32% in Tie2-sEH Tr treated with Ang-II versus Tie2-sEH Tr (12.1 ± 0.8 to 8.3 ± 1.2 mL/min/g). Moreover, the Ang-II-AT 1 -receptor and CYP4A were increased in Tie2-sEHTr. Our results demonstrate that Ang-II exacerbates CRH reduction in Tie2-sEH Tr mice.

Genetic polymorphisms of microsomal epoxide hydrolase and UDP-glucuronosyltransferase (UGT) and its effects on plasma carbamazepine levels and metabolic ratio in persons with epilepsy of South India: A cross-sectional genetic association study.

OBJECTIVES: Carbamazepine (CBZ), an anti-seizure drug, is widely prescribed for the management of focal seizures. At a given therapeutic dose, CBZ exhibits marked interindividual variation in the plasma CBZ levels. The aim wasto study the influence of EPHX1 c.337 T>C and UGT2B7*2 genetic polymorphisms on plasma carbamazepine (CBZ) levels in persons with epilepsy (PWE) from South India. METHODS: 115 PWE belong to South India origin who are on carbamazepine monotherapy were recruited. Genotyping of the two variants weredone using RT-PCR method. PWE who had seizure freedom for one year and their last dose which was not changed for one year duration were included and their plasma levels of CBZ and its active metabolite CBZ 10,11 epoxide were analysed by reverse phase HPLC. RESULTS: In EPHX1 c. 337 (T>C) polymorphism, the PWE carrying CC had lower plasma CBZ levels when compared to CT genotype (2.45 µg/ml vs 3.15 µg/ml. In UGT2B7*2, PWE carrying homozygous mutant TT had higher levels when compared with CT (3.09 µg/ml vs 2.74 µg/ml) genotype but found no statistical significance. Mutant genotype of EPHX1 (CC) had higher metabolic ratio compared to TT genotype (1.33 vs 1.17) but not found to be statistically significant. Mutant genotype of UGT2B7*2 (TT) was found to be having lower metabolic ratio when compared with CC genotype (1.18 vs 1.35; p value =0.08). CONCLUSION: PWE carrying EPHX1 c.337 T>C (rs1051740) and UGT2B7*2 (rs7439366) genetic polymorphisms did not affect the plasma CBZ levels and metabolic ratio of PWE of South Indian origin. However, this finding should be confirmed in a larger sample size which may help in optimization and personalized CBZ therapy in South Indians.

Hepatic soluble epoxide hydrolase activity regulates cerebral Aß metabolism and the pathogenesis of Alzheimer's disease in mice.

Alzheimer's disease (AD) is caused by a complex interaction between genetic and environmental factors. However, how the role of peripheral organ changes in response to environmental stimuli during aging in AD pathogenesis remains unknown. Hepatic soluble epoxide hydrolase (sEH) activity increases with age. Hepatic sEH manipulation bidirectionally attenuates brain amyloid-ß (Aß) burden, tauopathy, and cognitive deficits in AD mouse models. Moreover, hepatic sEH manipulation bidirectionally regulates the plasma level of 14,15-epoxyeicosatrienoic acid (-EET), which rapidly crosses the blood-brain barrier and modulates brain Aß metabolism through multiple pathways. A balance between the brain levels of 14,15-EET and Aß is essential for preventing Aß deposition. In AD models, 14,15-EET infusion mimicked the neuroprotective effects of hepatic sEH ablation at biological and behavioral levels. These results highlight the liver's key role in AD pathology, and targeting the liver-brain axis in response to environmental stimuli may constitute a promising therapeutic approach for AD prevention.

Genetic deletion or pharmacological inhibition of soluble epoxide hydrolase attenuated particulate matter 2.5 exposure mediated lung injury.

Air pollution represented by particulate matter 2.5 (PM2.5) is closely related to diseases of the respiratory system. Although the understanding of its mechanism is limited, pulmonary inflammation is closely correlated with PM2.5-mediated lung injury. Soluble epoxide hydrolase (sEH) and epoxy fatty acids play a vital role in the inflammation. Herein, we attempted to use the metabolomics of oxidized lipids for analyzing the relationship of oxylipins with lung injury in a PM2.5-mediated mouse model, and found that the cytochrome P450 oxidases/sEH mediated metabolic pathway was involved in lung injury. Furthermore, the sEH overexpression was revealed in lung injury mice. Interestingly, sEH genetic deletion or the selective sEH inhibitor TPPU increased levels of epoxyeicosatrienoic acids (EETs) in lung injury mice, and inactivated pulmonary macrophages based on the MAPK/NF-κB pathway, resulting in protection against PM2.5-mediated lung injury. Additionally, a natural sEH inhibitor luteolin from Inula japonica displayed a pulmonary protective effect towards lung injury mediated by PM2.5 as well. Our results are consistent with the sEH message and protein being both a marker and mechanism for PM2.5-induced inflammation, which suggest its potential as a pharmaceutical target for treating diseases of the respiratory system.

Soluble epoxide hydrolase deficiency promotes liver regeneration and ameliorates liver injury in mice by regulating angiocrine factors and angiogenesis.

BACKGROUND: Soluble epoxide hydrolase (sEH) is a key enzyme for the hydrolysis of epoxyeicosatrienoic acids (EETs) and has been implicated in the pathogenesis of hepatic inflammation, fibrosis, cancer, and nonalcoholic fatty liver disease. However, the role of sEH in liver regeneration and injury remains unclear. METHODS: This study used sEH-deficient (sEH-/-) mice and wild-type (WT) mice. Hepatocyte proliferation was assessed by immunohistochemical (IHC) staining for Ki67. Liver injury was evaluated by histological staining with hematoxylin and eosin (H&E), Masson's trichrome, and Sirius red, as well as IHC staining for α-SMA. Hepatic macrophage infiltration and angiogenesis were reflected by IHC staining for CD68 and CD31. Liver angiocrine levels were detected by ELISA. The mRNA levels of angiocrine or cell cycle-related genes were measured by quantitative real-time RT-PCR (qPCR). The protein levels of cell proliferation-related protein and phosphorylated signal transducer and activator of transcription 3 (STAT3) were detected by western blotting. RESULTS: sEH mRNA and protein levels were significantly upregulated in mice after 2/3 partial hepatectomy (PHx). Compared with WT mice, sEH-/- mice exhibited a higher liver/body weight ratio and more Ki67-positive cells on days 2 and 3 after PHx. The accelerated liver regeneration in sEH-/- mice was attributed to angiogenesis and endothelial-derived angiocrine (HGF) production. Subsequently, hepatic protein expression of cyclinD1 (CYCD1) and the downstream direct targets of the STAT3 pathway, such as c-fos, c-jun, and c-myc, were also suppressed post-PHx in sEH-/- compared to WT mice. Furthermore, sEH deficiency attenuated CCl4-induced acute liver injury and reduced fibrosis in both CCl4 and bile duct ligation (BDL)-induced liver fibrosis rodent models. Compared with WT mice, sEH-/- mice had slightly decreased hepatic macrophage infiltration and angiogenesis. Meanwhile, sEH-/- BDL mice had more Ki67-positive cells in the liver than WT BDL mice. CONCLUSIONS: sEH deficiency alters the angiocrine profile of liver endothelial to accelerate hepatocyte proliferation and liver regeneration, and blunts acute liver injury and fibrosis by inhibiting inflammation and angiogenesis. sEH inhibition is a promising target for liver diseases to improve liver regeneration and damage.

Aflatoxin B1 Increases Soluble Epoxide Hydrolase in the Brain and Induces Neuroinflammation and Dopaminergic Neurotoxicity.

Parkinson's disease (PD) is an increasingly common neurodegenerative movement disorder with contributing factors that are still largely unexplored and currently no effective intervention strategy. Epidemiological and pre-clinical studies support the close association between environmental toxicant exposure and PD incidence. Aflatoxin B1 (AFB1), a hazardous mycotoxin commonly present in food and environment, is alarmingly high in many areas of the world. Previous evidence suggests that chronic exposure to AFB1 leads to neurological disorders as well as cancer. However, whether and how aflatoxin B1 contributes to the pathogenesis of PD is poorly understood. Here, oral exposure to AFB1 is shown to induce neuroinflammation, trigger the α-synuclein pathology, and cause dopaminergic neurotoxicity. This was accompanied by the increased expression and enzymatic activity of soluble epoxide hydrolase (sEH) in the mouse brain. Importantly, genetic deletion or pharmacological inhibition of sEH alleviated the AFB1-induced neuroinflammation by reducing microglia activation and suppressing pro-inflammatory factors in the brain. Furthermore, blocking the action of sEH attenuated dopaminergic neuron dysfunction caused by AFB1 in vivo and in vitro. Together, our findings suggest a contributing role of AFB1 to PD etiology and highlight sEH as a potential pharmacological target for alleviating PD-related neuronal disorders caused by AFB1 exposure.

Enhancement of the liver's neuroprotective role ameliorates traumatic brain injury pathology.

Traumatic brain injury (TBI) is a pervasive problem worldwide for which no effective treatment is currently available. Although most studies have focused on the pathology of the injured brain, we have noted that the liver plays an important role in TBI. Using two mouse models of TBI, we found that the enzymatic activity of hepatic soluble epoxide hydrolase (sEH) was rapidly decreased and then returned to normal levels following TBI, whereas such changes were not observed in the kidney, heart, spleen, or lung. Interestingly, genetic downregulation of hepatic Ephx2 (which encodes sEH) ameliorates TBI-induced neurological deficits and promotes neurological function recovery, whereas overexpression of hepatic sEH exacerbates TBI-associated neurological impairments. Furthermore, hepatic sEH ablation was found to promote the generation of A2 phenotype astrocytes and facilitate the production of various neuroprotective factors associated with astrocytes following TBI. We also observed an inverted V-shaped alteration in the plasma levels of four EET (epoxyeicosatrienoic acid) isoforms (5,6-, 8,9-,11,12-, and 14,15-EET) following TBI which were negatively correlated with hepatic sEH activity. However, hepatic sEH manipulation bidirectionally regulates the plasma levels of 14,15-EET, which rapidly crosses the blood-brain barrier. Additionally, we found that the application of 14,15-EET mimicked the neuroprotective effect of hepatic sEH ablation, while 14,15-epoxyeicosa-5(Z)-enoic acid blocked this effect, indicating that the increased plasma levels of 14,15-EET mediated the neuroprotective effect observed after hepatic sEH ablation. These results highlight the neuroprotective role of the liver in TBI and suggest that targeting hepatic EET signaling could represent a promising therapeutic strategy for treating TBI.

Role of cytochrome P450-epoxygenase and soluble epoxide hydrolase in the regulation of vascular response.

The role of cytochrome P450-epoxygenase has been seen in cardiovascular physiology and pathophysiology. The aberration in CYP450-epoxygenase genes occur due to genetic polymorphisms, aging, or environmental factors, that increase susceptibility to cardiovascular diseases (CVDs). The actual role played by the CYP450-epoxygenases is the metabolism of arachidonic acid (AA) and linoleic acid (LA) into epoxyeicosatrienoic acids (EETs) and epoxyoctadecaenoic acid (EpOMEs) metabolites (oxylipins) and others, which is involved in vasodilation and myocardial-protection. But the genetic polymorphisms in CYP450-epoxygenases lose their beneficial cardiovascular effects of oxylipins, and the soluble epoxide hydrolase (sEH) antagonizes beneficial oxylipins into diols. Like sEH converts EETs into dihydroxyeicosatrienoic acid (DHETs), EpOMEs into dihydroxyoctadecaenoic acid (DiHOMEs), and reverses its beneficial effects, and the sEH gene (Ephx2) polymorphisms cause the enzyme to become overactive and convert epoxy-fatty acids into diols, making them vulnerable to CVDs, including hypertension. Other, enzymes like ω-hydroxylases (CYP450-4A11 & CYP450-4F2)-derived oxylipins from AA, ω-terminal-hydroxyeicosatetraenoic acids (19-, 20-HETE), lipoxygenase-derived oxylipins, mid-chain hydroxyeicosatetraenoic acids (5-, 11-, 12-, 15-HETEs), and the cyclooxygenase-derived prostanoids (prostaglandins: PGD2, PGF2α; thromboxane: Txs, oxylipins) are involved in vasoconstriction, hypertension, inflammation, and cardiac toxicity. Also, there are significant interactions were seen between adenosine receptors [adenosine A2A receptor (A2AAR) and adenosine A1 receptor (A1AR)] with CYP450-epoxygenases, ω-hydroxylases, sEH, and their derived oxylipins in the regulation of the cardiovascular response. Moreover, polymorphisms exist in CYP450-epoxygenases, ω-hydroxylases, sEH, and the adenosine receptor genes in populations associated with CVDs. This chapter will discuss the role of oxylipins' interactions with adenosine receptors in cardiovascular function/dysfunction in mice and humans.

Epoxide Hydrolases: Multipotential Biocatalysts.

Epoxide hydrolases are attractive and industrially important biocatalysts. They can catalyze the enantioselective hydrolysis of epoxides to the corresponding diols as chiral building blocks for bioactive compounds and drugs. In this review article, we discuss the state of the art and development potential of epoxide hydrolases as biocatalysts based on the most recent approaches and techniques. The review covers new approaches to discover epoxide hydrolases using genome mining and enzyme metagenomics, as well as improving enzyme activity, enantioselectivity, enantioconvergence, and thermostability by directed evolution and a rational design. Further improvements in operational and storage stabilization, reusability, pH stabilization, and thermal stabilization by immobilization techniques are discussed in this study. New possibilities for expanding the synthetic capabilities of epoxide hydrolases by their involvement in non-natural enzyme cascade reactions are described.

Enantioconvergent hydrolysis of racemic 1,2-epoxypentane and 1,2-epoxyhexane by an engineered Escherichia coli strain overexpressing a novel Streptomyces fradiae epoxide hydrolase.

In order to excavate microbial epoxide hydrolases (EHs) with desired catalytic properties, a novel EH, SfEH1, was identified based on the genome annotation of Streptomyces fradiae and sequence alignment analysis with local protein library. The SfEH1-encoding gene, sfeh1, was then cloned and over-expressed in soluble form in Escherichia coli/BL21(DE3). The optimal temperature and pH of recombinant SfEH1 (reSfEH1) and reSfEH1-expressing E. coli (E. coli/sfeh1) were both determined as 30 â„ƒ and 7.0, also indicating that the influences of temperature and pH on reSfEH1's activities were more obvious than those of E. coli/sfeh1 whole cells. Subsequently, using E. coli/sfeh1 as catalyst, its catalytic properties towards thirteen common mono-substituted epoxides were tested, in which E. coli/sfeh1 had the highest activity of 28.5 U/g dry cells for rac-1,2-epoxyoctane (rac-6a), and (R)-1,2-pentanediol ((R)-3b) (or (R)-1,2-hexanediol ((R)-4b)) with up to 92.5% (or 94.1%) eep was obtained at almost 100% conversion ratio. Regioselectivity coefficients (αS and ßR) displayed in the enantioconvergent hydrolysis of rac-3a (or rac-4a) were calculated to be 98.7% and 93.8% (or 95.2% and 98.9%). Finally, the reason of the high and complementary regioselectivity was confirmed by both kinetic parameter analysis and molecular docking simulations.

Leukotriene A4 hydrolase (LTA4H rs17525495) gene polymorphisms and paradoxical reactions in extrapulmonary tuberculosis.

Paradoxical reactions (PRs) are poorly studied complex immunological phenomena, among patients with tuberculosis (TB). When PRs involves critical structures like the central nervous system (CNS), immunomodulatory therapy is often required. Predictors for PRs in TB to pre-empt appropriate treatment strategies in high-risk groups are lacking. TT genotype of Leukotriene A4 hydrolase (LTA4H) promoter region rs17525495 polymorphisms are associated with exaggerated immune responses in Tuberculous meningitis (TBM), the most severe form of extrapulmonary tuberculosis (EPTB). The association of these polymorphisms with PRs is not known. We evaluated this plausibility among 113 patients with EPTB, at high risk of PRs. Majority [81 (71.7%)] had disseminated tuberculosis with prominent CNS [54 (47.8%)] and lymph node involvement [47 (41.6%)]. Human immunodeficiency Virus (HIV) co-infection was seen among 23 (20.3%) patients. PRs were noted in 38.9% patients, at a median duration of 3 months (IQR 2-4). LTA4H rs17525495 single nucleotide polymorphism (SNP) analysis showed 52 (46%) patients had CC, 43 (38.1%) had CT and 18 (15.9%) had TT genotypes. There was no statistically significant difference in occurrence [CC 38.5% vs CT 39.5% vs TT 38.7%] and time of onset [median (IQR)] of PRs across the genotypes [CC 3 (1-4.7), CT 3 (2-5), TT 2 (2-3)]. PRs was shown to be significantly linked with HIV co-infection (RR 0.6, 95% CI 0.29-1.28), culture positivity (RR 0.5, 95% CI 0.28-1.14), TB Lymphadenitis (RR 0.7, 95% CI 0.44-1.19) and CNS involvement RR 2.1, 95% CI 1.27-3.49) in the univariate analysis (p < 0.2). On multivariate analysis, CNS involvement alone was associated with PRs (aRR 3.8 (1.38-10.92); p < 0.01). PRs were associated with CNS involvement but not with LTA4H rs17525495 polymorphisms.

The soluble epoxide hydrolase inhibitor TPPU improves comorbidity of chronic pain and depression via the AHR and TSPO signaling.

BACKGROUND: Patients suffering from chronic pain often also exhibit depression symptoms. Soluble epoxide hydrolase (sEH) inhibitors can decrease blood levels of inflammatory cytokines. However, whether inhibiting sEH signaling is beneficial for the comorbidity of pain and depression is unknown. METHODS: According to a sucrose preference test (SPT), spared nerve injury (SNI) mice were classified into pain with or without an anhedonia phenotype. Then, sEH protein expression and inflammatory cytokines were assessed in selected tissues. Furthermore, we used sEH inhibitor TPPU to determine the role of sEH in chronic pain and depression. Importantly, agonists and antagonists of aryl hydrocarbon receptor (AHR) and translocator protein (TSPO) were used to explore the pathogenesis of sEH signaling. RESULTS: In anhedonia-susceptible mice, the tissue levels of sEH were significantly increased in the medial prefrontal cortex (mPFC), hippocampus, spinal cord, liver, kidney, and gut. Importantly, serum CYP1A1 and inflammatory cytokines, such as interleukin 1ß (IL-1ß) and the tumor necrosis factor α (TNF-α), were increased simultaneously. TPPU improved the scores of mechanical withdrawal threshold (MWT) and SPT, and decreased the levels of serum CYP1A1 and inflammatory cytokines. AHR antagonist relieved the anhedonia behaviors but not the algesia behaviors in anhedonia-susceptible mice, whereas an AHR agonist abolished the antidepressant-like effect of TPPU. In addition, a TSPO agonist exerted a similar therapeutic effect to that of TPPU, whereas pretreatment with a TSPO antagonist abolished the antidepressant-like and analgesic effects of TPPU. CONCLUSIONS: sEH underlies the mechanisms of the comorbidity of chronic pain and depression and that TPPU exerts a beneficial effect on anhedonia behaviors in a pain model via AHR and TSPO signaling.

Influence of EPHX1 c.337 T>C and UGT2B7*2 genetic polymorphisms on the requirement of carbamazepine maintenance dose in persons with epilepsy (PWE) of Southern part of India: a cross-sectional genetic association study.

OBJECTIVES: Carbamazepine (CBZ) is a first-line antiseizure drug used for focal onset seizures. It exhibits inter-individual variability in plasma carbamazepine levels and there are both genetic and non-genetic factors having a role in the requirement of CBZ maintenance dose. The aim was to study the influence of EPHX1 c.337 T>C and UGT2B7*2 genetic polymorphisms on CBZ maintenance dose requirement in persons with epilepsy. METHODS: Persons with epilepsy (PWE) of both gender of age 15-65 years on carbamazepine monotherapy who had been taking same maintenance dose for one year were eligible. Five milliliter of venous blood was collected in 10% EDTA under aseptic precautions. After centrifugation, the cellular component was used for DNA extraction and genotyping. For three genotypes of EPHX1 c.337 T>C and UGT2B7*2, the differences in mean carbamazepine dose were analyzed using Analysis of Variance (ANOVA). An unpaired t-test was used to draw a comparison between the genotypes and CBZ maintenance dose requirement for dominant and recessive models of EPHX1 c.337 T>C and UGT2B7*2. A value of p<0.05 was considered to be statistically significant. RESULTS: For UGT2B7*2 (rs 7439366), CT required a higher dose (CT 626 mg/day and TT 523 mg/day) but not found to be significant (p-value 0.167). PWE carrying CT genotype of EPHX1 c.337 T>C had 62 mg higher dose when compared to homozygous mutant CC (590 mg/day for CT and 528 mg/day for CC) but p-value was not found to be significant (p-value 0.835). CONCLUSIONS: The results of our study done in 115 PWE showed there was a lack of association between SNPs of EPHX1 c.337 T>C, UGT2B7*2 and CBZ maintenance dose requirement in Southern part of India and this finding has to be confirmed in a larger sample size.

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

OBJECTIVES: Studies 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. METHODS: Rats 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. RESULTS: Inhibition 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/EBPß attenuated, while overexpressing C/EBPß promoted homocysteine-induced hypertrophy and expressions of sEH and TRPC3, resulting respectively from inhibition or activation of sEH and TRPC3 gene promoters. CONCLUSIONS: sEH and TRPC3 channels jointly contribute to homocysteine-induced cardiac hypertrophy. Homocysteine transcriptionally activates sEH and TRPC3 genes through a common regulatory element C/EBPß. sEH activation leads to an upregulation of TRPC3 channels via a 11,12-EET-dependent manner.

Cloning and characterization of Aedes aegypti juvenile hormone epoxide hydrolases (JHEHs).

Juvenile hormone epoxide hydrolase (JHEH) plays an important role in the metabolism of juvenile hormone III (JH III) in insects. To study the role that JHEH plays in female Aedes aegypti JHEH 1, 2, and 3 complementary DNA (cDNAs) were cloned and sequenced. Northern blot analyses show that the three transcripts are expressed in the head thorax, the gut, the ovaries, and the fat body of females. Molecular modeling shows that the enzyme is a homodimer that binds JH III acid (JH IIIA) at the catalytic groove better than JH III. The cDNA of JHEH 1 and 2 are very similar indicating close relationship. Knocking down of jheh 1, 2, and 3 in adult female and larval Ae. aegypti using double-stranded RNA (dsRNA) did not affect egg development or caused adult mortality. Larvae that were fed bacterial cells expressing dsRNA against jheh 1, 2, and 3 grew normally. Treating blood-fed female Ae. aegypti with [12-3 H](10R) JH III and analyzing the metabolites by C18 reversed phase chromatography showed that JHEH preferred substrate is not JH III but JH IIIA. Genomic analysis of jheh 1, 2, and 3 indicate that jheh 1 and 2 are transcribed from a 1.53 kb DNA whereas jheh 3 is transcribed from a 10.9 kb DNA. All three genes are found on chromosome two at distinct locations. JHEH 2 was expressed in bacterial cells and purified by Ni affinity chromatography. Sequencing of the recombinant protein by MS/MS identified JHEH 2 as the expressed recombinant protein.

CRISPR/Cas9-mediated inactivation of the phosphatase activity of soluble epoxide hydrolase prevents obesity and cardiac ischemic injury.

INTRODUCTION: Although the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown. OBJECTIVES: This study aimed to assess in vivo the physiological role of sEH-P. METHODS: CRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity. RESULTS: The sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury. CONCLUSION: Our study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.

Wide-spread enhancer effect of SNP rs2279590 on regulating epoxide hydrolase-2 and protein tyrosine kinase 2-beta gene expression.

Polymorphisms in the PTK2B-CLU locus have been associated with various neurodegenerative disorders including pseudoexfoliation glaucoma, Alzheimer's and Parkinson's. Many of these genomic variants are within enhancer elements and modulate genes associated with the disease pathogenesis. However, mechanisms by which they control the gene expression is unknown. Previously, we have shown that clusterin enhancer element surrounding rs2279590 intronic variant, a risk factor in the pathogenesis of pseudoexfoliation glaucoma modulates gene expression of clusterin (CLU), protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase 2 (EPHX2). Here, we explored the mechanism by which rs2279590 enhancer regulates their gene expression through chromosome conformation capture assays. 3C assays revealed a strong enhancer-promoter chromatin interaction between rs2279590 enhancer and promoters of genes CLU, PTK2B and EPHX2 in the HEK293 wild type cells. Moreover, genomic knockout of rs2279590 element significantly decreases the chromatin-chromatin cross-linking frequency suggesting gene regulation at transcriptional level through formation of chromatin loop. In addition, molecular assays showed a significantly decreased expression of EPHX2 but not PTK2B at both mRNA and protein level in the lens capsule of pseudoexfoliation affected patients in comparison to control subjects implying a role of EPHX2 in the pathogenesis of pseudoexfoliation.

Decreased Expression of Soluble Epoxide Hydrolase Suppresses Murine Choroidal Neovascularization.

Neovascular or "wet" age-related macular degeneration (nAMD) is a leading cause of blindness among older adults. Choroidal neovascularization (CNV) is a major pathological feature of nAMD, in which abnormal new blood vessel growth from the choroid leads to irreversible vision loss. There is a critical need to develop novel therapeutic strategies to address limitations of the current anti-vascular endothelial growth factor biologics. Previously, we identified soluble epoxide hydrolase (sEH) as a possible therapeutic target for CNV through a forward chemical genetic approach. The purpose of this study was to validate sEH as a target by examining retinal expression of sEH protein and mRNA by immunohistochemistry and RNAscope in situ hybridization, respectively, and to assess the efficacy of an adeno-associated virus (AAV) vector designed to knock down the sEH gene, Ephx2, in the murine laser-induced (L-) CNV model. nAMD patient postmortem eye tissue and murine L-CNV showed overexpression of sEH in photoreceptors and retinal pigment epithelial cells. Ephx2 knockdown significantly reduced CNV and normalized mRNA expression levels of CNV-related inflammatory markers. Thus, this study further establishes sEH as a promising therapeutic target against CNV associated with nAMD.