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

Abstract

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). EPHX1 Regulates EET Hydrolysis and Postischemic Recovery2 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 11,12-and 8,9-...

Chiral lipidomics of monoepoxy and monohydroxy metabolites derived from long-chain polyunsaturated fatty acids

“…Also, the enantioselectivity of mEH may provide a novel clue for understanding the role of this enzyme in human diseases (41,42). Indeed, mEH significantly determines the endogenous levels of monoepoxides and only a sEH/mEH-double KO was sufficient for almost completely preventing their hydrolysis to the corresponding vicinal diols in mice (23). Thus, it is tempting to speculate that polymorphic variants and/or xenobiotics leading to alterations in mEH expression and activity could have an important impact on bioactive lipid mediator formation via the CYP epoxygenase pathway.…”

“…Further studies are needed to better understand the complex interactions of CYP enzymes, epoxide hydrolases, and other factors in determining the profile of regio-and stereoisomeric fatty acid epoxides under in vivo conditions. It has been suggested that mEH primarily acts as a "firstpass" hydrolase for the monoepoxides generated by the adjacent CYP enzymes in the ER, whereas the sEH comes into play after the remaining monoepoxides have been released into the cytosol (23). Accordingly, the mEH is expected to make a significant contribution to total intracellular monoepoxide hydrolysis, in particular, under basal conditions, i.e., when monoepoxides are formed at low rates due to the limited availability of free fatty acids for CYP enzymes.…”

“…The sEH may take over a predominant role after mEH capacities are overwhelmed by increased monoepoxide formation in response to physiological or pathophysiological stimuli that activate phospholipases A2 and, thereby, make free AA, EPA, and DHA accessible to the CYP enzymes. The actual impact of mEH and sEH certainly also depends on the relative expression of the two epoxide hydrolases in a given cell type (23). A general question concerns the mechanisms of how monoepoxides generated by CYP enzymes can escape, at least in part, the attack of both mEH and sEH as required to exert their biological functions and to become endogenous constituents of the plasma, liver, and other tissues.…”

“…Specific mEH activities are by orders of magnitude lower than those of sEH enzymes, if determined with exogenously added substrates. However, a close physical association of microsomal CYP and mEH enzymes has been recently discovered that may largely facilitate the access to endogenously generated monoepoxides (22,23). Accordingly, we hypothesized that CYP-mEH interactions might also modulate the stereospecificity of microsomal metabolite formation.…”

Lipid mediators in immune regulation and resolution

“…Once formed, epoxygenase products in particular are quickly metabolized by epoxide hydrolases (EH) or reincorporated into membranes (Zeldin, ). Soluble EH (sEH) and microsomal EH (encoded by the ephx2 and ephx1 , respectively) combine to metabolize virtually all epoxygenase products in vivo (Edin et al, ). For example, EETs get converted to dihydroxy‐eicosatrienoic acids.…”