EPHX 1 Regulates EET Hydrolysis and Postischemic Recovery 1 Epoxide hydrolase 1 ( EPHX 1 ) 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, antiinflammatory, 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 cytochromes P450 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. ________________________________________ Cytochrome P450 epoxygenases can oxidize arachidonic acid (AA) to form epoxyeicosatrienoic acids (EETs) which have potent cardiovascular effects. The biological effects of EETs are shortlived in that they are rapidly hydrolyzed to less active dihydroxyeicosatrienoic acids (DHETs) by EPHX2, also known as soluble epoxide hydrolase (sEH) (1). Indeed, Ephx2-/mice have increased EETs, decreased DHETs and improved outcomes in vascular disease models, which provide the basis for development of pharmacological EPHX2 inhibitors (1,2). EPHX2 inhibition increases EET levels in vivo, and leads to cardioprotective, vasodilatory, angiogenic, anti-inflammatory and analgesic effects (1-3). http://www.jbc.org/cgi/doi/10.1074/jbc.RA117.000298 The latest version is at JBC Papers in Press. Published on January 3, 2018 as Manuscript RA117.000298 Copyright 2018 by The American Society for Biochemistry and Molecular Biology, Inc. at FU B E R L IN /B IB L IO T H E K C H E M IE on Jauary 5, 2018 hp://w w w .jb.org/ D ow nladed from EPHX1 Regulates EET Hydrolysis and Postischemic Recovery 2 EPHX2 inhibitors, which have completed phase I clinical trials, are under investigation for treatment of neuropathic pain, and may hold promise for treatment of other ailments. However, genetic disruption of Ephx2 or EPHX2 pharmacological inhibition does not completely abolish EET hydrolysis in vivo. Among the EETs, EPHX2 has the biggest effect on levels of its preferred substrate, 14,15-EET, but has diminishing effects on 11,12and 8,9-EET which are equal or more potent in cardiovascular physiology (4-6). More complete or more broad inhibition of fatty acid epoxide hydrolysis could further potentiate the beneficial cardiovascular effects of endogenously produced EETs. Others have searched for additional enzymes capable of hydrolyzing EETs (7). EPHX1, also known as microsomal epoxide hydrolase (mEH), is known to be capable of EET hydrolysis; however, it is thought to play a minimal role. Compared to EPHX2, EPHX1 is 10sto 1000s-fold slower in in vitro EET hydrolysis assays, and is also less abundant in vivo (1,7,8). In some tissues, such as the brain, which has low EPHX2 expression, EPHX1 is believed to be responsible for hydrolysis of only a small fraction of its preferred substrate, 11,12-EET (1,8,9). Three other enzymes, EPHX3, EPHX4, and PEG1/MEST, were identified based on homology to the catalytic sites of EPHX1 and EPHX2 (7). EPHX3 was reported to have high catalytic efficiency towards fatty acid epoxides in vitro; however, Ephx3-/mice exhibit no alterations of fatty acid epoxide hydrolysis (10). EPHX4 and PEG1/MEST have not yet been examined for fatty acid epoxide hydrolase activity. In this study, we demonstrate that EPHX1 is responsible for a substantial proportion of EET hydrolysis in vivo. Ephx1-/-Ephx2-/have nearly complete loss of DHET in plasma or tissue lysates. Compared to Ephx1-/and Ephx2-/mice, Ephx1-/Ephx2-/mice have significantly increased plasma levels of nearly all fatty acid epoxide examined. Kinetic and FRET analyses suggest that EPHX1 interacts directly with P450 epoxygenases and likely participates in a coupled-reaction during basal EET formation. These surprising findings explain the large residual EET hydrolysis observed in Ephx2-/mice, reveal a mouse model with improved potentiation of EET effects, present a new paradigm for the role of EPHXs in EET metabolism, and suggest that combined inhibition of EPHX1 and EPHX2 represents a novel approach to treatment of disorders such as ischemic heart disease. RESULTS In this study, we examined fatty acid epoxide levels and metabolism in tissues and body fluids from WT, Ephx1-/-, Ephx2-/and Ephx1-/Ephx2-/mice. Ephx1-/and Ephx2-/mice were previously generated and maintained on a C57BL/6 background (11,12). In-crossing of Ephx1+/Ephx2+/mice yielded pups with no overt phenotypes; pups were born in normal Mendelian ratios (Table S1), had similar body weights and organ/body weight ratios (Table S2), and exhibited no gross anatomical or histological abnormalities. Mice showed expected alterations of gene expression (Figure 1A-B). Ephx1-/mice have been reported to express low levels of a truncated, noncoding, Ephx1 mRNA transcript (11). Ephx1 mRNA levels were reduced in Ephx1-/and Ephx1/Ephx2-/mice relative to WT. Ephx2mRNA levels were nearly absent in Ephx2-/and Ephx1-/Ephx2-/mice. Likewise, EPHX1 protein was absent from Ephx1-/and Ephx1-/Ephx2-/mice, and EPHX2 protein was absent from Ephx2-/and Ephx1-/Ephx2-/mice. EPHX1 was detected in both the S-9 fraction, which contains both cytosol and microsomes, and in microsomes. EPHX2 was detected in both S-9 and microsomal fractions. This is consistent with previous findings of nearly equal distribution of EPHX2 between cytosolic and microsomal fractions (13-15). We observed no compensatory regulation of hepatic Ephx1, Ephx2, Ephx3 or Ephx4 mRNAs (Figure 1A, Figure S1). In addition, hepatic expression of Cyp2c and Cyp2j family members was also comparable across genotypes. Similar Ephx1, Ephx2, Ephx3 and Ephx4 expression profiles were observed in mouse heart (Figure S2). We quantified oxylipin levels in plasma from WT, Ephx1-/-, Ephx2-/and Ephx1-/-Ephx2-/mice using LC-MS/MS (Figure 2A-B, Table S3). Disruption of Ephx1 alone did not alter plasma 14,15-EET, 11,12-EET, 8,9-EET or 5,6-EET levels compared to WT, nor did it alter levels of epoxides derived from linoleic acid (12,13-EpOME and 9,10-EpOME), docasahexaenoic acid (19,20-EpDPE) or eicosapentaenoic acid (17,18EpETE). In contrast, Ephx1-/plasma had significantly lower levels of 8,9-DHET, 5,6-DHET, 19,20-DiHDPA and 17,18-DHET compared to at FU B E R L IN /B IB L IO T H E K C H E M IE on Jauary 5, 2018 hp://w w w .jb.org/ D ow nladed from EPHX1 Regulates EET Hydrolysis and Postischemic Recovery 3 WT. Plasma from Ephx2-/mice had significantly higher levels of 14,15-EET, 11,12-EET, 8,9-EET, 17,18-EpETE, 12,13-EpOME and 9,10-EpOME, and significantly lower levels of 14,15-DHET, 11,12-DHET, 8,9-DHET and 12,13-DiHOME compared to WT. Disruption of both Ephx1 and Ephx2 more dramatically altered plasma oxylipin levels. Compared to WT, Ephx1-/Ephx2-/-mice had significantly higher levels of every CYP-derived epoxide measured except 5,6-EET. Importantly, in Ephx1-/Ephx2-/mouse plasma, levels of all the fatty acid diols were low or undetectable. Moreover, compared to Ephx2-/mice, plasma from Ephx1-/Ephx2-/mice had significantly higher levels of 11,12-EET, 17,18-EpETE, and 19,20EpDPE, and significantly lower levels of all diols measured except 12,13-DiHOME. This data suggest functional compensation of EPHX1 and EPHX2 with regard to fatty acid epoxide hydrolysis in vivo. Together, EPHX1 and EPHX2 account for hydrolysis of the nearly all circulating epoxides derived from arachidonic, linoleic, eicosapentaenoic and docosahexaenoic acids. Our in vivo data suggest a substantial role of EPHX1 in hydrolysis of endogenous fatty acid epoxides that was not predicted by prior in vitro assays. In vitro kinetic assays, typically performed at supraphysiologic (μM) substrate concentrations (7,8), may underestimate the capacity for EPHX1 to hydrolyze fatty acid epoxides in vivo. Indeed, intracellular EET levels are estimated to be in the low nM range (16,17). To determine the relative contribution of EPHX1 and EPHX2 to EET hydrolysis at physiologically relevant concentrations, liver lysates from all four genotypes were incubated with exogenous 11,12-EET at 11000 nM concentration (Figure 3). In this assay, EPHX2 was the dominant hydrolase. Ephx1-/lysates showed no decrease in enzymatic activity compared to WT, while Ephx2-/lysates show nearly complete reduction of hydrolysis (94-99%) at all 11,12-EET concentrations. Ephx1-/Ephx2-/lysates had a small additional decrease in 11,12EET hydrolysis compared to Ephx2-/lysates. This data suggest that EPHX1 is responsible for only a small fraction (1-4%) of 11,12-EET hydrolysis in the liver under these experimental conditions. Our in vitro 11,12-EET hydrolysis assays do not reflect the pattern observed in vivo where EPHX1 contributes significantly to EET hydrolysis