Generation and characterization of epoxide hydrolase 3 (EPHX3)-deficient mice.

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play an important role in blood pressure regulation, protection against ischemia-reperfusion injury, angiogenesis, and inflammation. Epoxide hydrolases metabolize EETs to their corresponding diols (dihydroxyeicosatrienoic acids; DHETs) which are biologically less active. Microsomal epoxide hydrolase (EPHX1, mEH) and soluble epoxide hydrolase (EPHX2, sEH) were identified >30 years ago and are capable of hydrolyzing EETs to DHETs. A novel epoxide hydrolase, EPHX3, was recently identified by sequence homology and also exhibits epoxide hydrolase activity in vitro with a substrate preference for 9,10-epoxyoctadecamonoenoic acid (EpOME) and 11,12-EET. EPHX3 is highly expressed in the skin, lung, stomach, esophagus, and tongue; however, its endogenous function is unknown. Therefore, we investigated the impact of genetic disruption of Ephx3 on fatty acid epoxide hydrolysis and EET-related physiology in mice. Ephx3-/- mice were generated by excising the promoter and first four exons of the Ephx3 gene using Cre-LoxP methodology. LC-MS/MS analysis of Ephx3-/- heart, lung, and skin lysates revealed no differences in endogenous epoxide:diol ratios compared to wild type (WT). Ephx3-/- mice also exhibited no change in plasma levels of fatty acid epoxides and diols relative to WT. Incubations of cytosolic and microsomal fractions prepared from Ephx3-/- and WT stomach, lung, and skin with synthetic 8,9-EET, 11,12-EET, and 9,10-EpOME revealed no significant differences in rates of fatty acid diol formation between the genotypes. Ephx3-/- hearts had similar functional recovery compared to WT hearts following ischemia/reperfusion injury. Following intranasal lipopolysaccharide (LPS) exposure, Ephx3-/- mice were not different from WT in terms of lung histology, bronchoalveolar lavage fluid cell counts, or fatty acid epoxide and diol levels. We conclude that genetic disruption of Ephx3 does not result in an overt phenotype and has no significant effects on the metabolism of EETs or EpOMEs in vivo

Epoxide hydrolase 1 (EPHX1) hydrolyzes epoxyeicosanoids and impairs cardiac recovery after ischemia

Stimuli such as inflammation or hypoxia induce cytochrome P450 epoxygenase-mediated production of arachidonic acid-derived epoxyeicosatrienoic acids (EETs). EETs have cardioprotective, vasodilatory, angiogenic, anti-inflammatory, and analgesic effects, which are diminished by EET hydrolysis yielding biologically less active dihydroxyeicosatrienoic acids (DHETs). Previous in vitro assays have suggested that epoxide hydrolase 2 (EPHX2) is responsible for nearly all EET hydrolysis; EPHX1, which exhibits slow EET hydrolysis in vitro, is thought to contribute only marginally to EET hydrolysis. Using Ephx1-/-, Ephx2-/-, and Ephx1-/-/Ephx2-/- mice, we show herein that EPHX1 significantly contributes to EET hydrolysis in vivo. Disruption of Ephx1 and/or Ephx2 genes did not induce compensatory changes in expression of other Ephx genes or CYP2 family epoxygenases. Plasma levels of 8,9-, 11,12-, and 14,15-DHET were reduced by 38%, 44%, and 67% in Ephx2-/- mice compared with wild-type (WT) mice, respectively; however, plasma from Ephx1-/-/Ephx2-/- mice exhibited significantly greater reduction (100%, 99%, and 96%) of those respective DHETs. Kinetic assays and FRET experiments indicated that EPHX1 is a slow EET scavenger, but hydrolyzes EETs in a coupled reaction with P450s to limit basal EET levels. Moreover, we also found that EPHX1 activities are biologically relevant, as Ephx1-/-/Ephx2-/- hearts had significantly better postischemic functional recovery (71%) than both WT (31%) and Ephx2-/- (51%) hearts. These findings indicate that Ephx1-/-/Ephx2-/- mice are a valuable model for assessing EET-mediated effects, uncover a new paradigm for EET metabolism, and suggest that dual EPHX1 and EPHX2 inhibition may represent a therapeutic approach to manage human pathologies such as myocardial infarction

Generation of <i>Ephx3</i>^-/- mice.

<p>A) Wild type <i>Ephx3</i> locus on the reverse strand of chromosome 17:b1. B) Targeting vector to introduce <i>LoxP</i> sites flanking the promoter region and first four exons of <i>Ephx3</i>. Targeting vector constructed with <i>FRT</i>-site flanked neomycin resistance positive selection and MC1-DTA negative selection. C) Targeted <i>Ephx3</i> locus following homologous recombination. D) <i>Ephx3</i> “flox” locus following excision of neomycin resistance cassette via <i>in vivo</i> FLP recombinase exposure. E) <i>Ephx3</i> null allele generated by breeding to germline expressing Cre recombinase mouse line (Prm-Cre). F) Representative Southern blots using both the 5’ and 3’ probes to confirm proper recombination event. Only relevant restriction enzymes sites shown with expected fragment sizes.</p

Eicosanoids and Prostaglandins (pg/ml) in Plasma from <i>Ephx3</i>^<i>-/-</i> and WT mice treated with or without LPS.

<p>Eicosanoids and Prostaglandins (pg/ml) in Plasma from <i>Ephx3</i>^<i>-/-</i> and WT mice treated with or without LPS.</p

LC-MS/MS analysis shows no differences in endogenous epoxide:diol ratios in the <i>Ephx3</i>^<i>-/-</i> mice.

<p>Endogenous epoxide:diol ratios for (A) 8,9-EET/8,9-DHET, (B) 11,12-EET/11,12-DHET, (C) 14,15-EET/14,15-DHET, (D) 9,10-EpOME/9,10-DiHOME, (E) 12,13-EpOME/12,13-DiHOME, (F) 17,18-EpETE/17,18-DHET, and (G) 19,20-EpDPE/19,20-DiHDPA were determined in the heart, lung, skin, and plasma by LC-MS/MS in <i>Ephx3</i>^<i>-/-</i> and WT mice (n = 3–10, p = NS).</p

Incubation with synthetic EETs/EpOMEs showes no changes in diol formation rate in <i>Ephx3</i>^<i>-/-</i> mice.

<p>LC-MS/MS analysis of diol formation after a 5 minute incubation with synthetic EETs or EpOMEs in (A) lung, (B) skin, and (C) stomach lysates from <i>Ephx3</i>^<i>-/-</i> and WT mice. (n = 5–10, p = NS).</p

11,12-DHET formation rates are largely unchanged in microsomal and cytosolic fractions from various tissues of <i>Ephx3</i>^-/- mice.

<p>LC-MS/MS analysis of 11,12-DHET formation in microsomal and cytosolic fractions after a 5 minute incubation with synthetic 11,12-EET with/without the EPHX2 inhibitor t-AUCB in (A) lung, (B) skin, and (C) stomach samples (n = 3–6, *p<0.05).</p

Body weight and organ weight:body weight ratios are not altered in <i>Ephx3</i>^<i>-/-</i> <i>mice</i>.

<p>(A) Body weight of both male and female WT and <i>Ephx3</i>^<i>-/-</i> mice. (B) Heart weight:body weight ratio and (C) kidney weight:body weight ratio in <i>Ephx3</i>^<i>-/-</i> and WT mice (n = 5–10, p = NS).</p

Bronchoalveolar lavage fluid cells, lung histology, and plasma epoxide:diol ratios are unchanged in <i>Ephx3</i>^<i>-/-</i> mice after LPS treatment.

<p>(A) Bronchoalveolar lavage fluid cell counts and differentials, (B) lung histology, and (C) LC-MS/MS epoxide/diol ratios in plasma from <i>Ephx3</i>^<i>-/-</i> and WT mice four hours after intranasal LPS exposure (n = 4–6; p = NS, original magnification = 10x).</p