Absolute protein assay for the simultaneous quantification of two epoxide hydrolases in rats by mass spectrometry-based targeted proteomics.

Epoxide hydrolases catalyze the hydrolysis of both exogenous and endogenous epoxides to the corresponding vicinal diols by adding water. Microsomal and soluble epoxide hydrolase are two main mammalian enzymes that have been intensely characterized. The purpose of this investigation was to develop and validate a proteomics assay allowing the simultaneous quantification of microsomal and soluble epoxide hydrolase in rats. Protein quantification was realized through targeted proteomics using liquid chromatography with tandem mass spectrometry for the determination of trypsin-specific surrogate peptides after digestion. Stable isotope-labeled peptides were used as the internal standards. The chromatography of the surrogate peptides was performed on an Agilent SB C18 column (100 mm × 4.6 mm, 1.8 µm) with gradient elution. Acetonitrile containing 0.1% formic acid and 0.1% formic acid aqueous solution were used as mobile phases. A multiple reaction monitoring method in a positive ionization mode was used for the simultaneous detection of the peptides. The method was validated concerning the specificity, linearity, within-day and between-day accuracy and precision, matrix effect, stability, and digestion efficiency. The developed assay was successfully used to quantify the protein levels of microsomal and soluble epoxide hydrolase in rat liver, kidney, and heart S9 samples.

Cortical Proteins Associated With Cognitive Resilience in Community-Dwelling Older Persons.

Importance: Identifying genes and proteins for cognitive resilience (ie, targets that may be associated with slowing or preventing cognitive decline regardless of the presence, number, or combination of common neuropathologic conditions) provides a complementary approach to developing novel therapeutics for the treatment and prevention of Alzheimer disease and related dementias. Objective: To identify proteins associated with cognitive resilience via a proteome-wide association study of the human dorsolateral prefrontal cortex. Design, Setting, and Participants: This study used data from 391 community-dwelling older persons who participated in the Religious Orders Study and the Rush Memory and Aging Project. The Religious Orders Study began enrollment January 1, 1994, and the Rush Memory and Aging Project began enrollment September 1, 1997, and data were collected and analyzed through October 23, 2019. Exposures: Participants had undergone annual detailed clinical examinations, postmortem evaluations, and tandem mass tag proteomics analyses. Main Outcomes and Measures: The outcome of cognitive resilience was defined as a longitudinal change in cognition over time after controlling for common age-related neuropathologic indices, including Alzheimer disease, Lewy bodies, transactive response DNA-binding protein 43, hippocampal sclerosis, infarcts, and vessel diseases. More than 8000 high abundance proteins were quantified from frozen dorsolateral prefrontal cortex tissue using tandem mass tag and liquid chromatography-mass spectrometry. Results: There were 391 participants (273 women); their mean (SD) age was 79.7 (6.7) years at baseline and 89.2 (6.5) years at death. Eight cortical proteins were identified in association with cognitive resilience: a higher level of NRN1 (estimate, 0.140; SE, 0.024; P = 7.35 × 10-9), ACTN4 (estimate, 0.321; SE, 0.065; P = 9.94 × 10-7), EPHX4 (estimate, 0.198; SE, 0.042; P = 2.13 × 10-6), RPH3A (estimate, 0.148; SE, 0.031; P = 2.58 × 10-6), SGTB (estimate, 0.211; SE, 0.045; P = 3.28 × 10-6), CPLX1 (estimate, 0.136; SE, 0.029; P = 4.06 × 10-6), and SH3GL1 (estimate, 0.179; SE, 0.039; P = 4.21 × 10-6) and a lower level of UBA1 (estimate, -0.366; SE, 0.076; P = 1.43 × 10-6) were associated with greater resilience. Conclusions and Relevance: These protein signals may represent novel targets for the maintenance of cognition in old age.

Development of Improved Double-Nanobody Sandwich ELISAs for Human Soluble Epoxide Hydrolase Detection in Peripheral Blood Mononuclear Cells of Diabetic Patients and the Prefrontal Cortex of Multiple Sclerosis Patients.

Nanobodies have been progressively replacing traditional antibodies in various immunological methods. However, the use of nanobodies as capture antibodies is greatly hampered by their poor performance after passive adsorption to polystyrene microplates, and this restricts the full use of double nanobodies in sandwich enzyme-linked immunosorbent assays (ELISAs). Herein, using the human soluble epoxide hydrolase (sEH) as a model analyte, we found that both the immobilization format and the blocking agent have a significant influence on the performance of capture nanobodies immobilized on polystyrene and the subsequent development of double-nanobody sandwich ELISAs. We first conducted epitope mapping for pairing nanobodies and then prepared a horseradish-peroxidase-labeled nanobody using a mild conjugation procedure as a detection antibody throughout the work. The resulting sandwich ELISA using a capture nanobody (A9, 1.25 µg/mL) after passive adsorption and bovine serum albumin (BSA) as a blocking agent generated a moderate sensitivity of 0.0164 OD·mL/ng and a limit of detection (LOD) of 0.74 ng/mL. However, the introduction of streptavidin as a linker to the capture nanobody at the same working concentration demonstrated a dramatic 16-fold increase in sensitivity (0.262 OD·mL/ng) and a 25-fold decrease in the LOD for sEH (0.03 ng/mL). The streptavidin-bridged double-nanobody ELISA was then successfully applied to tests for recovery, cross-reactivity, and real samples. Meanwhile, we accidentally found that blocking with skim milk could severely damage the performance of the capture nanobody by an order of magnitude compared with BSA. This work provides guidelines to retain the high effectiveness of the capture nanobody and thus to further develop the double-nanobody ELISA for various analytes.

Soluble epoxide hydrolase derived lipid mediators are elevated in bronchoalveolar lavage fluid from patients with sarcoidosis: a cross-sectional study.

BACKGROUND: Sarcoidosis is a systemic inflammatory multi-organ disease almost always affecting the lungs. The etiology remains unknown, but the hallmark of sarcoidosis is formation of non-caseating epithelioid cells granulomas in involved organs. In Scandinavia, > 30% of sarcoidosis patients have Löfgren's syndrome (LS), an acute disease onset mostly indicating a favorable prognosis. The impact of dysregulation of lipid mediators, which has been investigated in other inflammatory disorders, is still unknown. METHODS: Using three different liquid chromatography coupled to tandem mass spectrometry targeted platforms (LC-MS/MS), we quantified a broad suite of lipid mediators including eicosanoids, sphingolipids and endocannabinoids in bronchoalveolar lavage (BAL) fluid from pulmonary sarcoidosis patients (n = 41) and healthy controls (n = 16). RESULTS: A total of 47 lipid mediators were consistently detected in BAL fluid of patients and controls. After false discovery rate adjustment, two products of the soluble epoxide hydrolase (sEH) enzyme, 11,12-dihydroxyeicosa-5,8,14-trienoic acid (11,12-DiHETrE, p = 4.4E-5, q = 1.2E-3, median fold change = 6.0) and its regioisomer 14,15-dihydroxyeicosa-5,8,11-trienoic acid (14,15-DiHETrE, p = 3.6E-3, q = 3.2E-2, median fold change = 1.8) increased in patients with sarcoidosis. Additional shifts were observed in sphingolipid metabolism, with a significant increase in palmitic acid-derived sphingomyelin (SM16:0, p = 1.3E-3, q = 1.7E-2, median fold change = 1.3). No associations were found between these 3 lipid mediators and LS, whereas levels of SM 16:0 and 11,12-DiHETrE associated with radiological stage (p < 0.05), and levels of 14,15-DiHETrE were associated with the BAL fluid CD4/CD8 ratio. CONCLUSIONS: These observed shifts in lipid mediators provide new insights into the pathobiology of sarcoidosis and in particular highlight the sEH pathway to be dysregulated in disease.

Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

Arabidopsis thaliana EPOXIDE HYDROLASE1 (AtEH1) is a cytosolic epoxide hydrolase involved in the synthesis of poly-hydroxylated cutin monomers.

Epoxide hydrolases (EHs) are present in all living organisms. They have been extensively characterized in mammals; however, their biological functions in plants have not been demonstrated. Based on in silico analysis, we identified AtEH1 (At3g05600), a putative Arabidopsis thaliana epoxide hydrolase possibly involved in cutin monomer synthesis. We expressed AtEH1 in yeast and studied its localization in vivo. We also analyzed the composition of cutin from A. thaliana lines in which this gene was knocked out. Incubation of recombinant AtEH1 with epoxy fatty acids confirmed its capacity to hydrolyze epoxides of C18 fatty acids into vicinal diols. Transfection of Nicotiana benthamiana leaves with constructs expressing AtEH1 fused to enhanced green fluorescent protein (EGFP) indicated that AtEH1 is localized in the cytosol. Analysis of cutin monomers in loss-of-function Ateh1-1 and Ateh1-2 mutants showed an accumulation of 18-hydroxy-9,10-epoxyoctadecenoic acid and a concomitant decrease in corresponding vicinal diols in leaf and seed cutin. Compared with wild-type seeds, Ateh1 seeds showed delayed germination under osmotic stress conditions and increased seed coat permeability to tetrazolium red. This work reports a physiological role for a plant EH and identifies AtEH1 as a new member of the complex machinery involved in cutin synthesis.

18F-FNDP for PET Imaging of Soluble Epoxide Hydrolase.

Soluble epoxide hydrolase (sEH) is a bifunctional enzyme located within cytosol and peroxisomes that converts epoxides to the corresponding diols and hydrolyzes phosphate monoesters. It serves to inactivate epoxyeicosatrienoic acids (EETs), which are generated in the brain to couple neuronal activity and cerebral blood flow in normal and pathologic states. Altered regulation of sEH was observed previously in various neuropathologic disorders including vascular dementia and stroke. Inhibitors of sEH are pursued as agents to mitigate neuronal damage after stroke. We developed N-(3,3-diphenylpropyl)-6-18F-fluoronicotinamide (18F-FNDP), which proved highly specific for imaging of sEH in the mouse and nonhuman primate brain with PET. METHODS: 18F-FNDP was synthesized from the corresponding bromo precursor. sEH inhibitory activity of 18F-FNDP was measured using an sEH inhibitor screening assay kit. Biodistribution was undertaken in CD-1 mice. Binding specificity was assayed in CD-1 and sEH knock-out mice and Papio anubis (baboon) through pretreatment with an sEH inhibitor to block sEH binding. Dynamic PET imaging with arterial blood sampling was performed in 3 baboons, with regional tracer binding quantified using distribution volume. The metabolism of 18F-FNDP in baboons was assessed using high-performance liquid chromatography. RESULTS: 18F-FNDP (inhibition binding affinity constant, 1.73 nM) was prepared in 1 step in a radiochemical yield of 14% ± 7%, specific radioactivity in the range of 888-3,774 GBq/µmol, and a radiochemical purity greater than 99% using an automatic radiosynthesis module. The time of preparation was about 75 min. In CD-1 mice, regional uptake followed the pattern of striatum > cortex > hippocampus > cerebellum, consistent with the known brain distribution of sEH, with 5.2% injected dose per gram of tissue at peak uptake. Blockade of 80%-90% was demonstrated in all brain regions. Minimal radiotracer uptake was present in sEH knock-out mice. PET baboon brain distribution paralleled that seen in mouse, with a marked blockade (95%) noted in all regions indicating sEH-mediated uptake of 18F-FNDP. Two hydrophilic metabolites were identified, with 20% parent compound present at 90 min after injection in baboon plasma. CONCLUSION: 18F-FNDP can be synthesized in suitable radiochemical yield and high specific radioactivity and purity. In vivo imaging experiments demonstrated that 18F-FNDP targeted sEH in murine and nonhuman primate brain specifically. 18F-FNDP is a promising PET radiotracer likely to be useful for understanding the role of sEH in a variety of conditions affecting the central nervous system.

Increased epoxyeicosatrienoic acids and reduced soluble epoxide hydrolase expression in the preeclamptic placenta.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) derived from cytochrome P450 (CYP)-dependent metabolism of arachidonic acid are increased in the plasma of women with preeclampsia as compared with normal pregnancy and are significantly higher in fetal than in maternal plasma and erythrocytes. We hypothesized that differences in EET synthesis or metabolism in the feto-placental unit contributed to the observed differences in circulating EETs. METHOD: To evaluate EETs, formation as well as the expression of relevant CYP isoforms and the metabolizing enzyme, soluble epoxide hydrolase (sEH), biopsies of placenta were collected from 19 normal pregnancy and 10 preeclampsia at the time of cesarean section delivery. EETs were extracted from tissue homogenates and analyzed by liquid chromatography coupled with tandem mass spectrometry. RESULTS: Both cis-EETs and trans-EETs were detected in the placenta. Concentration of total EETs was higher in the placenta from preeclampsia compared with normal pregnancy (2.37 ±â€Š1.42 ng/mg vs. 1.20 ±â€Š0.72 ng/mg, mean ±â€ŠSD, P < 0.01), especially the 5,6-, 8,9- and 11,12-EETs, measured in a subgroup of tissue samples (normal pregnancy = 10, preeclampsia = 5). By immunohistochemistry, sEH, CYP2J2, CYP4A11 were present in placental villi with different pattern distribution, whereas CYP2C8 was not detectable. Neither were CYP2J2, CYP4A11, and CYP2C8 detected in the umbilical cord. Western blot analysis of placenta homogenates showed reduced expression of sEH in preeclampsia as compared with normal pregnancy. CONCLUSION: Increased EETs in the placenta and umbilical cord are associated with the presence of CYP2J2, whereas reduced expression of sEH in preeclampsia may be the key factor of increased EETs in the placenta.

Heavy chain single-domain antibodies to detect native human soluble epoxide hydrolase.

The soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, pain, cancer, and other diseases. However, there is not a simple, inexpensive, and reliable method to estimate levels of active sEH in tissues. Toward developing such an assay, a polyclonal variable domain of heavy chain antibody (VHH) sandwich immunoassay was developed. Ten VHHs, which are highly selective for native human sEH, were isolated from a phage-displayed library. The ten VHHs have no significant cross-reactivity with human microsomal epoxide hydrolase, rat and mouse sEH, and denatured human sEH. There is a high correlation between protein levels of the sEH determined by the enzyme-linked immunosorbent assay (ELISA) and the catalytic activity of the enzyme in S9 fractions of human tissues (liver, kidney, and lung). The VHH-based ELISA appears to be a new reliable method for monitoring the sEH and may be useful as a diagnostic tool for diseases influenced by sEH. This study also demonstrates the broad utility of VHH in biochemical and pharmacological research.

Single-Nucleotide Polymorphisms in Pig EPHX1 Gene are Associated with Pork Quality Traits.

Epoxide hydrolase 1 (EPHX1) plays an important role in both the activation and detoxification of exogenous chemicals. Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the highest level of EPHX1 expression occurred in Berkshire liver, which is an organ that plays a key role in detoxification. We examined EPHX1 SNPs to analyze effect on increased expression of EPHX1 gene in Berkshire liver by total of 192 pigs of a pure Berkshire line (males = 97; females = 95). As a result, two nonsynonymous SNPs (nsSNPs) of EPHX1 were found from c.685T>G and c.776C > T, and located in 5th and 6th exons, respectively, which constitute the A/b hydrolase 1 domain of epoxide hydrolase. The nsSNP c.685T > G was significant differences in meat color, protein content, collagen content, and pH24 hr. Especially, T and G alleles of the nsSNP c.685T > G were significantly associated with CIE a*/CIE b* and protein content/pH24 hr, respectively. The nsSNP c.776C > T was significant differences in drip loss and protein content. Among meat quality traits to associate with SNPs, the protein content was only significantly associated with sex. Therefore, it is suggested that nsSNP c.685T > G in EPHX1 gene is a potential to apply as appropriate DNA markers for improvement of porcine economic traits.

Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes.

The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography-tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.

Soluble epoxide hydrolase inhibitory and anti-inflammatory components from the leaves of Eucommia ulmoides Oliver (duzhong).

Eucommia ulmoides leaves have been used as a functional food and drink in China. The purpose of this study was to identify the bioactive constituents with soluble epoxide hydrolase (sEH) inhibitory activity and anti-inflammatory properties. Twenty-seven known compounds (1-27) were isolated from the leaves of E. ulmoides Oliver, and their structures were identified by NMR and ESIMS analysis; three of these, 2,5-dimethoxy-3-glucopyranosyl cinnamic alcohol (11), foliasalacioside E2 (26), and icariside F2 (27), were obtained from this plant for the first time. Compounds 1-7 exhibited soluble epoxide hydrolase (sEH) inhibitory activity at 100 µM; among them, quercetin (1) and kaempferol (5) displayed potential activities with IC50 values of 22.5 ± 0.9 and 31.3 ± 2.6 µM, respectively, with noncompetitive inhibition mode. Nuclear factor kappa B (NF-κB) inhibitory activity of the isolated compounds was evaluated by the NF-κB liciferase assay in HepG2 cells. Compounds 1, 9, 20, and 27 displayed potent NF-κB inhibitory effects, with IC50 values of 15.14 ± 2.29, 15.23 ± 2.34, 16.88 ± 2.17, and 16.25 ± 2.19 µM, respectively, whereas other compounds showed weak inhibition of NF-κB transcriptional activity ranging from 17.54 to 92.6 µM. A structure-activity relationship of flavonoids 1-9 was also discussed. The results obtained in this work might contribute to the understanding of pharmacological activities of E. ulmoides leaves and further investigation on its potential application values for food and drug.

Synthesis of leukotrienes in porcine uteri with endometritis induced by infection with Escherichia coli.

Leukotrienes (LTs) are lipid mediators that play a significant role in the inflammatory process. Their production in inflamed uteri is not fully understood. The present experiment aimed to determine LTB4 and LTC4 amounts, 5-lipooxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA levels and protein expression in inflamed porcine uteri. On Day 3 of the oestrous cycle (Day 0 of the study), either Escherichia coli suspension or saline were infused into uterine horns. Collection of uterine tissues and washings took place eight or sixteen days later. In gilts suffering from endometritis increased LTB4 and LTC4 levels in the endometrium and washings and 5-LO mRNA levels in the myometrium on Days 8 and 16, 5-LO protein levels in the endometrium and myometrium on Day 8, LTAH mRNA and protein levels in the endometrium and myometrium on Days 8 and 16, respectively. Although LTCS mRNA and protein expression in the myometrium and LTCS protein expression in the endometrium were enhanced on Day 16 after Escherichia coli inoculation, LTCS mRNA levels decreased on Day 8 in both tissues. Our study shows the upregulation of LT production in inflamed porcine uteri, which suggests the importance of these factors to the process of uterine inflammation.

Effect of allergen sensitization on external root resorption.

In orthodontic tooth movement (OTM), we should be concerned about external root resorption (ERR) as an undesirable iatrogenic problem, but its mechanisms are not fully understood. Since our previous epidemiologic studies found that patients with allergic diseases showed higher rates of ERR during orthodontic treatment, we explored the possible effect of allergic sensitization on ERR. In ovalbumin (OVA)-sensitized Brown-Norway rats, the amounts of ERR and OTM were greater than those in animals subjected to orthodontic force alone. The expression levels of RANKL and pro-inflammatory cytokines were increased in the periodontal tissues of sensitized rats with OTM, compared with control rats. Furthermore, leukotriene B4 (LTB4), a potent lipid mediator of allergic inflammation, and enzymes of the 5-lipoxygenase pathway, the biosynthetic pathway of leukotrienes, were also up-regulated. We found that low doses of aspirin suppressed ERR in allergen-sensitized rats, as well as the expressions of RANKL, pro-inflammatory cytokines, and LTB4. The present findings indicate that allergen sensitization has adverse effects on ERR under OTM, and that aspirin is a potential therapeutic agent for combating ERR.

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

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

Protection of salvianolic acid A on rat brain from ischemic damage via soluble epoxide hydrolase inhibition.

Epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH) have been associated with ischemic stroke. Salvianolic acid A (SAA) is proved to display potent cerebroprotection. However, little information is available about the link between them. This study aimed to investigate whether SAA exhibits its protective effects in rats subjected to middle cerebral artery occlusion (MCAO) through sEH and EETs. The results showed that SAA treatment ameliorated neurological deficits and reduced infarct volume. Notably, the beneficial effects of SAA were attenuated by co-administration of (14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE)), a putative selective EETs antagonist. Furthermore, SAA increased the 14,15-EET levels in the blood and brain of sham and MCAO rats. Assay for hydrolase activity showed that 1 and 3 mg/kg of SAA significantly diminished brain sEH activity of MCAO rats. A fluorescent assay in vitro indicated that SAA could inhibit recombinant human sEH activity in a concentration-dependent manner (IC(50) = 1.62 µmol/l). Immunohistochemical analysis showed that SAA at the doses of 1 and 3 mg/kg significantly decreased sEH protein expression in hippocampus CA1 region of MCAO rats. In conclusion, cerebral protection of SAA is mediated, at least in part, via inhibiting sEH to increase EETs levels.

Soluble epoxide hydrolase: sex differences and role in endothelial cell survival.

OBJECTIVE: Sex differences in cerebral ischemic injury are, in part, attributable to the differences in cerebrovascular perfusion. We determined whether the brain microvascular endothelial cells (ECs) isolated from the female brain are more resistant to ischemic injury compared with male ECs, and whether the difference is attributable to lower expression of soluble epoxide hydrolase and higher levels of vasoprotective epoxyeicosatrienoic acids (EETs). We also determined whether protection by EETs is linked to the inhibition of rho-kinase (ROCK). METHODS AND RESULTS: EC ischemic damage was measured after oxygen-glucose deprivation (OGD) using propidium iodide (PI) and cleaved caspase-3 labeling. Expression of soluble epoxide hydrolase was determined by quantitative polymerase chain reaction and immunocytochemistry, EETs levels by liquid chromatography-tandem mass spectrometry, and ROCK activity by ELISA. EC damage was higher in males compared with females, which correlated with higher soluble epoxide hydrolase mRNA, stronger immunoreactivity, and lower EETs compared with female ECs. Inhibition of soluble epoxide hydrolase abolished the sex difference in EC damage. ROCK activity was higher in male versus female ECs after OGD, and sex differences in EC damage and ROCK activity were abolished by 14,15-EET and ROCK inhibition. CONCLUSIONS: Sex differences in ischemic brain injury are, in part, attributable to differences in EET-mediated inhibition of EC ROCK activation after ischemia.

Redox regulation of soluble epoxide hydrolase by 15-deoxy-delta-prostaglandin J2 controls coronary hypoxic vasodilation.

RATIONALE: 15-Deoxy-Δ-prostaglandin (15d-PG)J(2) is an electrophilic oxidant that dilates the coronary vasculature. This lipid can adduct to redox active protein thiols to induce oxidative posttranslational modifications that modulate protein and tissue function. OBJECTIVE: To investigate the role of oxidative protein modifications in 15d-PGJ(2)-mediated coronary vasodilation and define the distal signaling pathways leading to enhanced perfusion. METHODS AND RESULTS: Proteomic screening with biotinylated 15d-PGJ(2) identified novel vascular targets to which it adducts, most notably soluble epoxide hydrolase (sEH). 15d-PGJ(2) inhibited sEH by specifically adducting to a highly conserved thiol (Cys521) adjacent to the catalytic center of the hydrolase. Indeed a Cys521Ser sEH "redox-dead" mutant was resistant to 15d-PGJ(2)-induced hydrolase inhibition. 15d-PGJ(2) dilated coronary vessels and a role for hydrolase inhibition was supported by 2 structurally different sEH antagonists each independently inducing vasorelaxation. Furthermore, 15d-PGJ(2) and sEH antagonists also increased coronary effluent epoxyeicosatrienoic acids consistent with their vasodilatory actions. Indeed 14,15-EET alone induced relaxation and 15d-PGJ(2)-mediated vasodilation was blocked by the EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE). Additionally, the coronary vasculature of sEH-null mice was basally dilated compared to wild-type controls and failed to vasodilate in response to 15d-PGJ(2). Coronary vasodilation to hypoxia in wild-types was accompanied by 15d-PGJ(2) adduction to and inhibition of sEH. Consistent with the importance of hydrolase inhibition, sEH-null mice failed to vasodilate during hypoxia. CONCLUSION: This represents a new paradigm for the regulation of sEH by an endogenous lipid, which is integral to the fundamental physiological response of coronary hypoxic vasodilation.

A long-wavelength, fluorogenic probe for epoxide hydrolase: 7-(2-(oxiran-2-yl)ethoxy) resorufin.

Epoxide hydrolase plays an important role in the detoxification of genotoxic compounds and in the control of physiological signaling molecules. Altered levels of epoxide hydrolase activity are associated with many diseases, such as emphysema, lung cancer, ovarian cancer, and laryngeal carcinoma. We designed and synthesized a resorufin-based fluorogenic probe, 7-(2-(oxiran-2-yl)ethoxy) resorufin, which was hydrolyzed by microsomal epoxide hydrolase to form the corresponding diol, which upon further treatment with sodium periodate released the strongly fluorescent resorufin. The probe exhibits good biological compatibility and photophysical properties, such as long wavelength excitation (571 nm) and emission (585 nm) and a wide working pH range (from 6.0 to 10.0), and thus facilitates the determination of the activity of microsomal epoxide hydrolase.

Soluble epoxide hydrolase plays an essential role in angiotensin II-induced cardiac hypertrophy.

Pathophysiological cardiac hypertrophy is one of the most common causes of heart failure. Epoxyeicosatrienoic acids, hydrolyzed and degraded by soluble epoxide hydrolase (sEH), can function as endothelium-derived hyperpolarizing factors to induce dilation of coronary arteries and thus are cardioprotective. In this study, we investigated the role of sEH in two rodent models of angiotensin II (Ang II)-induced cardiac hypertrophy. The protein level of sEH was elevated in the heart of both spontaneously hypertensive rats and Ang II-infused Wistar rats. Blocking the Ang II type 1 receptor with losartan could abolish this induction. Administration of a potent sEH inhibitor (sEHI) prevented the pathogenesis of the Ang II-induced hypertrophy, as demonstrated by decreased left-ventricular hypertrophy assessed by echocardiography, reduced cardiomyocyte size, and attenuated expression of hypertrophy markers, including atrial natriuretic factor and beta-myosin heavy chain. Because sEH elevation was not observed in exercise- or norepinephrine-induced hypertrophy, the sEH induction was closely associated with Ang II-induced hypertrophy. In vitro, Ang II upregulated sEH and hypertrophy markers in neonatal cardiomyocytes isolated from rat and mouse. Expression of these marker genes was elevated with adenovirus-mediated sEH overexpression but decreased with sEHI treatment. These results were supported by studies in neonatal cardiomyocytes from sEH(-/-) mice. Our results suggest that sEH is specifically upregulated by Ang II, which directly mediates Ang II-induced cardiac hypertrophy. Thus, pharmacological inhibition of sEH would be a useful approach to prevent and treat Ang II-induced cardiac hypertrophy.