Deletion Of sEH Research Articles

Soluble epoxide hydrolase deletion rescues behavioral and synaptic deficits. By AMPK-mTOR pathway in autism animals

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by social defects often accompanied with emotional comorbidities. Aberrations in synaptic function and plasticity are the core feature in the pathophysiology of ASD. Targeting soluble epoxide hydrolase (sEH) has been found to exert protection in a wide-range of pathological conditions. However, the regulation of sEH deficiency on the synaptic deficits of ASD and the underlying mechanisms remain unclear. The valproate (VPA)-treated ASD animal model with genetic sEH knockout was applied in the present study. The results showed that the sEH expression was significantly increased in the prefrontal cortex of VPA-treated animals. Although no effect was found on tail malformation and body weight loss, genetic sEH deletion alleviated social deficits, and fear learning and memory extinction in the VPA-treated mice. After a series of electrophysiological assessments, we found that the beneficial effects of sEH deletion focused on the long-term synaptic plasticity, rather than presynaptic efficiency, in the VPA-treated mice. Furthermore, we observed that the dysregulated AMPK-mTOR pathway was restored under genetic sEH deletion in VPA-treated mice. Taken together, these findings uncovered an important role of sEH deficiency in the synaptic dysfunctions of ASD mediated by AMPK-mTOR pathway, providing a novel therapeutic target for ASD.

Role of the soluble epoxide hydrolase in keratinocyte proliferation and sensitivity of skin to inflammatory stimuli

The lipid content of skin plays a determinant role in its barrier function with a particularly important role attributed to linoleic acid and its derivatives. Here we explored the consequences of interfering with the soluble epoxide hydrolase (sEH) on skin homeostasis. sEH; which converts fatty acid epoxides generated by cytochrome P450 enzymes to their corresponding diols, was largely restricted to the epidermis which was enriched in sEH-generated diols. Global deletion of the sEH increased levels of epoxides, including the linoleic acid-derived epoxide; 12,13-epoxyoctadecenoic acid (12,13-EpOME), and increased basal keratinocyte proliferation. sEH deletion (sEH-/- mice) resulted in thicker differentiated spinous and corneocyte layers compared to wild-type mice, a hyperkeratosis phenotype that was reproduced in wild-type mice treated with a sEH inhibitor. sEH deletion made the skin sensitive to inflammation and sEH-/- mice developed thicker imiquimod-induced psoriasis plaques than the control group and were more prone to inflammation triggered by mechanical stress with pronounced infiltration and activation of neutrophils as well as vascular leak and increased 12,13-EpOME and leukotriene (LT) B4 levels. Topical treatment of LTB4 antagonist after stripping successfully inhibited inflammation and neutrophil infiltration both in wild type and sEH-/- skin. While 12,13-EpoME had no effect on the trans-endothelial migration of neutrophils, like LTB4, it effectively induced neutrophil adhesion and activation. These observations indicate that while the increased accumulation of neutrophils in sEH-deficient skin could be attributed to the increase in LTB4 levels, both 12,13-EpOME and LTB4 contribute to neutrophil activation. Our observations identify a protective role of the sEH in the skin and should be taken into account when designing future clinical trials with sEH inhibitors.

Role of the soluble epoxide hydrolase in the hair follicle stem cell homeostasis and hair growth

Polyunsaturated fatty acids (PUFAs) are used as traditional remedies to treat hair loss, but the mechanisms underlying their beneficial effects are not well understood. Here, we explored the role of PUFA metabolites generated by the cytochrome P450/soluble epoxide hydrolase (sEH) pathway in the regulation of the hair follicle cycle. Histological analysis of the skin from wild-type and sEH−/− mice revealed that sEH deletion delayed telogen to anagen transition, and the associated activation of hair follicle stem cells. Interestingly, EdU labeling during the late anagen stage revealed that hair matrix cells from sEH−/− mice proliferated at a greater rate which translated into increased hair growth. Similar effects were observed in in vitro studies using hair follicle explants, where a sEH inhibitor was also able to augment whisker growth in follicles from wild-type mice. sEH activity in the dorsal skin was not constant but altered with the cell cycle, having the most prominent effects on levels of the linoleic acid derivatives 12,13-epoxyoctadecenoic acid (12,13-EpOME), and 12,13-dihydroxyoctadecenoic acid (12,13-DiHOME). Fitting with this, the sEH substrate 12,13-EpOME significantly increased hair shaft growth in isolated anagen stage hair follicles, while its diol; 12,13-DiHOME, had no effect. RNA sequencing of isolated hair matrix cells implicated altered Wnt signaling in the changes associated with sEH deletion. Taken together, our data indicate that the activity of the sEH in hair follicle changes during the hair follicle cycle and impacts on two stem cell populations, i.e., hair follicle stem cells and matrix cells to affect telogen to anagen transition and hair growth.

A Distinct Metabolically Defined Central Nucleus Circuit Bidirectionally Controls Anxiety-Related Behaviors.

Anxiety disorders are debilitating psychiatric diseases that affect ∼16% of the world's population. Although it has been proposed that the central nucleus of the amygdala (CeA) plays a role in anxiety, the molecular and circuit mechanisms through which CeA neurons modulate anxiety-related behaviors are largely uncharacterized. Soluble epoxide hydrolase (sEH) is a key enzyme in the metabolism of polyunsaturated fatty acids (PUFAs), and has been shown to play a role in psychiatric disorders. Here, we reported that sEH was enriched in neurons in the CeA and regulated anxiety-related behaviors in adult male mice. Deletion of sEH in CeA neurons but not astrocytes induced anxiety-like behaviors. Mechanistic studies indicated that sEH was required for maintaining the the excitability of sEH positive neurons (sEHCeA neurons) in the CeA. Using chemogenetic manipulations, we found that sEHCeA neurons bidirectionally regulated anxiety-related behaviors. Notably, we identified that sEHCeA neurons directly projected to the bed nucleus of the stria terminalis (BNST; sEHCeA-BNST). Optogenetic activation and inhibition of the sEHCeA-BNST pathway produced anxiolytic and anxiogenic effects, respectively. In summary, our studies reveal a set of molecular and circuit mechanisms of sEHCeA neurons underlying anxiety.SIGNIFICANCE STATEMENT Soluble epoxide hydrolase (sEH), a key enzyme that catalyzes the degradation of EETs, is shown to play a key role in mood disorders. It is well known that sEH is mostly localized in astrocytes in the prefrontal cortex and regulates depressive-like behaviors. Notably, sEH is also expressed in central nucleus of the amygdala (CeA) neurons. While the CeA has been studied for its role in the regulation of anxiety, the molecular and circuit mechanism is quite complex. In the present study, we explored a previously unknown cellular and circuitry mechanism that guides sEHCeA neurons response to anxiety. Our findings reveal a critical role of sEH in the CeA, sEHCeA neurons and CeA-bed nucleus of the stria terminalis (BNST) pathway in regulation of anxiety-related behaviors.

Deletion of soluble epoxide hydrolase suppressed chronic kidney disease-related vascular calcification by restoring Sirtuin 3 expression

Vascular calcification is common in chronic kidney disease (CKD) and contributes to cardiovascular disease (CVD) without any effective therapies available up to date. The expression of soluble epoxide hydrolase (sEH) is different in patients with and without vascular calcification. The present study investigates the role of sEH as a potential mediator of vascular calcification in CKD. Both Ephx2−/− and wild-type (WT) mice fed with high adenine and phosphate (AP) diet were used to explore the vascular calcification in CKD. Compared with WT, deletion of sEH inhibited vascular calcification induced by AP. sEH deletion also abolished high phosphorus (Pi)-induced phenotypic transition of vascular smooth muscle cells (VSMCs) independent of its epoxyeicosatrienoic acids (EETs) hydrolysis. Further gene expression analysis identified the potential role of Sirtuin 3 (Sirt3) in the sEH-regulated VSMC calcification. Under high Pi treatment, sEH interacted with Sirt3, which might destabilize Sirt3 and accelerate the degradation of Sirt3. Deletion of sEH may preserve the expression of Sirt3, and thus maintain the mitochondrial adenosine triphosphate (ATP) synthesis and morphology, significantly suppressing VSMC calcification. Our data supported that sEH deletion inhibited vascular calcification and indicated a promising target of sEH inhibition in vascular calcification prevention.

Genetic deletion of soluble epoxide hydrolase preserves cardiac function and limits inflammation in acute lipopolysaccharide injury

Objective Acute systemic inflammatory syndromes, such as endotoxemia, elicit detrimental multi-organ responses. The heart is particularly susceptible to an acute inflammatory response resulting in myocardial dysfunction which often leads to death. A stress-induced metabolism of N-3 and N-6 polyunsaturated fatty acids (PUFAs) will generate numerous bioactive lipid mediators with beneficial or adverse effects. Cytochrome P450 (CYP) derived epoxylipid mediators may be further hydrolyzed to diol metabolites by soluble epoxide hydrolase (sEH). Emerging evidence suggests epoxylipids can exert cardioprotective effects by modulating the NLRP3 inflammasome pathway. This study investigated whether cardiomyocyte specific sEH knockdown can preserve cardiac function and attenuate inflammation in a model of acute lipopolysaccharide (LPS) injury. Hypothesis Conditional knockdown of cardiomyocyte sEH protects the heart from acute LPS injury via modulation of the NLRP3 inflammasome pathway. Methods Cardiomyocyte targeted sEH knockdown mice were produced by crossing Ephx2 floxed mice with mice expressing Cre recombinase. Male sEHFl/Fl (knockdown) and sEH+/+ (Cre control) mice were treated with tamoxifen (45 mg/kg, 6 i.p injections over 8 days) 5 weeks prior to LPS injection (10mg/kg, i.p.). A comparator group of male wild type (WT) and global sEH null mice were subjected to LPS treatment. Echocardiography was conducted pre-injection and 6 or 24hr post-LPS followed by euthanization. Plasma cytokine and chemokine levels were determined with a multi-plex assay. In vitro studies employed neonatal rat cardiomyocytes treated with LPS (1µg/mL), 19, 20-epoxydocosapentaenoic acid (EDP, 1µM) or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB, 10µM), for 6hr. NLRP3 and pro-IL-1β expression was assessed using Western immunoblotting. Results Genetic deletion of sEH preserved cardiac function following 24hr LPS exposure indicating an initial early decline at 6hr which was recovered in both sEHFl/Fl and sEH null but not WT mice. The increased plasma levels of pro-inflammatory cytokines and chemokines following LPS treatment was attenuated in mice lacking sEH expression. Cardiomyocytes treated with LPS had increased NLRP3 inflammasome complex formation and pro-IL-1β expression, which was not attenuated by co-treatment with EDP or tAUCB. However, plasma levels of IL-1β were reduced in both sEHFl/Fl and global sEH null mice following LPS exposure. Conclusion Deletion of sEH protects cardiac function and limits pro-inflammatory response following exposure to LPS. Preliminary data suggest epoxylipids do not prevent the formation of the NLRP3 inflammasome complex in cardiomyocytes but may impact downstream effectors. Thus, limiting the NLRP3 inflammasome cascade to reduce LPS-induced cardiac and inflammatory injury.

Soluble Epoxide Hydrolase in Aged Female Mice and Human Explanted Hearts Following Ischemic Injury.

Myocardial infarction (MI) accounts for a significant proportion of death and morbidity in aged individuals. The risk for MI in females increases as they enter the peri-menopausal period, generally occurring in middle-age. Cytochrome (CYP) 450 metabolizes N-3 and N-6 polyunsaturated fatty acids (PUFA) into numerous lipid mediators, oxylipids, which are further metabolised by soluble epoxide hydrolase (sEH), reducing their activity. The objective of this study was to characterize oxylipid metabolism in the left ventricle (LV) following ischemic injury in females. Human LV specimens were procured from female patients with ischemic cardiomyopathy (ICM) or non-failing controls (NFC). Female C57BL6 (WT) and sEH null mice averaging 13–16 months old underwent permanent occlusion of the left anterior descending coronary artery (LAD) to induce myocardial infarction. WT (wild type) mice received vehicle or sEH inhibitor, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB), in their drinking water ad libitum for 28 days. Cardiac function was assessed using echocardiography and electrocardiogram. Protein expression was determined using immunoblotting, mitochondrial activity by spectrophotometry, and cardiac fibre respiration was measured using a Clark-type electrode. A full metabolite profile was determined by LC–MS/MS. sEH was significantly elevated in ischemic LV specimens from patients, associated with fundamental changes in oxylipid metabolite formation and significant decreases in mitochondrial enzymatic function. In mice, pre-treatment with tAUCB or genetic deletion of sEH significantly improved survival, preserved cardiac function, and maintained mitochondrial quality following MI in female mice. These data indicate that sEH may be a relevant pharmacologic target for women with MI. Although future studies are needed to determine the mechanisms, in this pilot study we suggest targeting sEH may be an effective strategy for reducing ischemic injury and mortality in middle-aged females.

Novel Insights into the Role of Soluble Epoxide Hydrolase in Adipogenesis

Obesity is a growing epidemic that presents a major health problem worldwide. The past decade has seen advances in the identification of specific factors that contribute to this condition. However, despite these strides, there is still much to be learned about the underlying mechanisms. A better understanding of these biochemical mechanisms will enhance our ability to prevent and treat obesity. This study demonstrates that modulation of epoxy fatty acids (EpFA) metabolism in mouse white adipocytes alters cell cycle progression and clonal expansion. In addition, CRISPR/Cas9‐mediated deletion of soluble epoxide hydrolase (sEH), a cytosolic enzyme which hydrolyzes epoxy fatty acids (EpFA) into less active diols, resulted in alterations in adipogenesis. Similar findings were obtained using a selective sEH inhibitor 1‐trifluoromethoxyphenyl‐3‐(1‐propionylpiperidin‐4‐yl) urea (TPPU). Importantly, mice with specific deletion of sEH in the adipose tissue were resistant to high fat feeding and exhibited increased energy expenditure. Taken together, the present study demonstrates that modulation of sEH activity in white adipocytes alters adipocyte differentiation and improves glycemic control. Additionally, our findings suggest that sEH is a physiological modulator of adipogenesis and a potential target for the development of novel anti‐obesity therapeutic strategies.Support or Funding InformationThis work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases (R00DK100736) to A.B.

Soluble epoxide hydrolase promotes astrocyte survival in retinopathy of prematurity.

Polyunsaturated fatty acids such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability, we investigated the role of sEH in a mouse model of ROP. In WT mice, hyperoxia elicited tyrosine nitration and inhibition of sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (19,20-DHDP). Correspondingly, in a murine model of ROP, sEH-/- mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than did their WT littermates. Astrocytes were the cells most affected by sEH deletion, and hyperoxia increased astrocyte apoptosis. In rescue experiments, 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 and presenilin-1-associated protein to attenuate poly ADP-ribose polymerase activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss, but also reduced pathological vascular tuft formation in sEH-/- mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement for preventing retinopathy in preterm infants.

GENETIC DELETION OF SOLUBLE EPOXIDE HYDROLASE PRESERVES CARDIAC FUNCTION IN AGED FEMALE MICE

Biological aging is an inevitable part of life that has intrigued individuals for millennia. The progressive decline in biological systems impacts cardiac function and increases vulnerability to stress contributing to morbidity and mortality in elderly individuals. Yet, our understanding of the molecular, biochemical and physiological mechanisms as well as sex differences is limited. There is growing evidence indicating CYP450 epoxygenase-mediated metabolites of n-3 and n-6 polyunsaturated fatty acids are active lipid mediators regulating cardiac homeostasis. These epoxy metabolites are rapidly hydrolyzed and inactivated by the soluble epoxide hydrolase (sEH). The current study aims to characterize cardiac function in young and aged sEH null mice compared to the corresponding wild-type (WT) mice. Cardiac function was assessed in both young (3 month) and aged (16 month) WT and sEH null male and female mice by echocardiography. There were no significant differences in cardiac function observed between young WT and sEH null mice. However, there was a marked decline in cardiac function in aged WT mice, notably significant decreases in ejection fraction and fractional shortening in both female and males. Interestingly, female aged sEH null mice demonstrated preserved cardiac function compared to aged WT and sEH null males. As well, the development of left ventricular (LV) mass was lower in aged female sEH null mice compared to other mice. While the etiology of age-related cardiovascular pathogenesis is poorly understood, it has been shown that deterioration of mitochondria over time plays a critical role in age-related cardiac dysfunction. Increased oxidative stress associated with dysfunctional mitochondria correlated with increased expression of both cytosolic and mitochondrial phosphorylated Akt in all aged mice. Increased expression of acetyl Mn-SOD was observed in aged WT and aged male sEH null mice but not aged female sEH null mice. Together these data demonstrated deletion of sEH in aged female mice correlates with preserved cardiac function in mice.

The phosphatase activity of soluble epoxide hydrolase regulates ATP-binding cassette transporter-A1-dependent cholesterol efflux.

The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E‐deficient mice, primarily in macrophage foam cells. Oxidized low‐density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH (sEH −/−) in macrophages markedly exacerbated oxLDL‐induced lipid accumulation and decreased the expression of ATP‐binding cassette transporters‐A1 (ABCA1) and apolipoprotein AI‐dependent cholesterol efflux following oxLDL treatment. The down‐regulation of ABCA1 in sEH −/− macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH‐ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL‐induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL‐induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.

Soluble Epoxide Hydrolase Inhibition Attenuates Excitotoxicity Involving 14,15-Epoxyeicosatrienoic Acid-Mediated Astrocytic Survival and Plasticity to Preserve Glutamate Homeostasis.

Astrocytes play pivotal roles in regulating glutamate homeostasis at tripartite synapses. Inhibition of soluble epoxide hydrolase (sEHi) provides neuroprotection by blocking the degradation of 14,15-epoxyeicosatrienoic acid (14,15-EET), a lipid mediator whose synthesis can be activated downstream from group 1 metabotropic glutamate receptor (mGluR) signaling in astrocytes. However, it is unclear how sEHi regulates glutamate excitotoxicity. Here, we used three primary rat cortical culture systems, neuron-enriched (NE), astrocyte-enriched glia-neuron mix (GN), and purified astrocytes, to delineate the underlying mechanism by which sEHi and 14,15-EET attenuate excitotoxicity. We found that sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA) and 14,15-EET both attenuated N-methyl-D-aspartate (NMDA)-induced neurite damage and cell death in GN, not NE, cortical cultures. The anti-excitotoxic effects of 14,15-EET and AUDA were both blocked by the group 1 mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), as were their protective effects against NMDA-disrupted perineuronal astrocyte processes expressing glutamate transporter-1 (GLT-1) and subsequent glutamate uptake. Knockdown of sEH expression also attenuated NMDA neurotoxicity in mGluR5- and GLT-1-dependent manners. The 14,15-EET/AUDA-preserved astroglial integrity was confirmed in glutamate-stimulated primary astrocytes along with the reduction of the c-Jun N-terminal kinase 1 phosphorylation, in which the 14,15-EET effect is mGluR5-dependent. In vivo studies validated that sEHi and genetic deletion of sEH (Ephx2-KO) ameliorated excitotoxic kainic acid-induced seizure, memory impairment, and neuronal loss while preserving GLT-1-expressing perineuronal astrocytes in hippocampal CA3 subregions. These results suggest that 14,15-EET mediates mGluR5-dependent anti-excitotoxicity by protecting astrocytes to maintain glutamate homeostasis, which may account for the beneficial effect of sEH inhibition in excitotoxic brain injury and diseases.

Soluble epoxide hydrolase modulates immune responses in activated astrocytes involving regulation of STAT3 activity

BackgroundAstrocyte activation is a common pathological feature in many brain diseases with neuroinflammation, and revealing the underlying mechanisms might shed light on the regulatory processes of the diseases. Recently, soluble epoxide hydrolase (sEH) has been proposed to affect neuroinflammation in brain injuries. However, the roles of astrocytic sEH in brains with neurodegeneration remain unclear.MethodsThe expression of astrocytic sEH in the brains of APPswe/PSEN1dE9 (APP/PS1) mice developing Alzheimer’s disease (AD)-like pathology was evaluated by confocal imaging. LPS-activated primary astrocytes with mRNA silencing or overexpression of sEH were used to investigate its regulatory roles in astrocyte activation and the induction of pro-inflammatory markers. Primary astrocytes isolated from a sEH knockout (sEH−/−) background were also applied.ResultsThe immunoreactivity of sEH was increased in activated astrocytes in parallel with the progression of AD in APP/PS1 mice. Our data from primary astrocyte cultures further demonstrate that the overexpression of sEH ameliorated, while the silencing of sEH mRNA enhanced, the lipopolysaccharides (LPS)-induced expression of pro-inflammatory markers, such as inducible nitric oxide, cyclooxygenase 2 (COX-2), and pro-inflammatory cytokines. These findings suggest that sEH negatively regulates astrocyte immune responses. Enhanced immune responses found in LPS-activated sEH−/− astrocytes also support the notion that the expression of sEH could suppress the immune responses during astrocyte activation. Similarly, sEH−/− mice that received intraperitoneal injection of LPS showed exacerbated astrocyte activation in the brain, as observed by the elevated expression of glial fibrillary acidic protein (GFAP) and pro-inflammatory markers. Moreover, our data show that the phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was upregulated in activated astrocytes from sEH mouse brains, and the pharmacological blockade of STAT3 activity alleviated the pro-inflammatory effects of sEH deletion in LPS-activated primary astrocytes.ConclusionsOur results provide evidence, for the first time, showing that sEH negatively regulates astrocytic immune responses and GFAP expression, while the underlying mechanism at least partly involves the downregulation of STAT3 phosphorylation. The discovery of a novel function for sEH in the negative control of astrocytic immune responses involving STAT3 activation confers further insights into the regulatory machinery of astrocyte activation during the development of neurodegeneration.

Inhibition of Soluble Epoxide Hydrolase for Renal Health.

A soluble epoxide hydrolase (sEH) mediates the metabolism of epoxy fatty acids to form the corresponding vicinal diols, which are usually inactive or less active than the epoxide substrates. The sEH enzyme presents in many organs, including but not limited to the liver, heart, spleen, lung, and kidney. Here we summarized the changes in the expression and activity of sEH in multiple renal diseases, such as acute kidney injury (AKI), diabetic nephrology (DN), chronic kidney diseases (CKD), hypertension-mediated renal damage, and other renal dysfunctions. We also discussed the pharmacologic effects and the underlying mechanisms of sEH inhibition by using an inhibitor of sEH and/or the generic deletion of sEH on multiple renal diseases. We believe that sEH is a potential therapeutic target for renal dysfunction although the target disease needs further investigation.

Genetic Deletion of Soluble Epoxide Hydroxylase Causes Anxiety-Like Behaviors in Mice.

Soluble epoxide hydrolase (sEH), an enzyme with COOH-terminal hydrolase and NH2-terminal lipid phosphatase activities, is expressed in regions of the brain such as the cortex, white matter, hippocampus, substantia nigra, and striatum. sEH is involved in the regulation of cerebrovascular and neuronal function upon pathological insults. However, the physiological significance of sEH and its underlying mechanism in modulating brain function are not fully understood. In this study, we investigated the role of sEH in anxiety and potential underlying mechanisms in mice. Western blot for protein phosphorylation and expression was performed. Immunohistochemical analyses and Nissl and Golgi staining were performed for histological examination. Mouse behaviors were evaluated by open field activity, elevated plus maze, classical fear conditioning, social preference test, and Morris water maze. Our results demonstrated that the expression of sEH was upregulated during postnatal development in wild-type (WT) mice. Genetic deletion of sEH (sEH-/-) in mice resulted in anxiety-like behavior and disrupted social preference. Increased olfactory bulb (OB) size and altered integrity of neurites were observed in sEH-/- mice. In addition, ablation of sEH in mice decreased protein expression of tyrosine hydroxylase and reduced dopamine production in the brain. Moreover, the level of phosphorylated calmodulin kinase II (CaMKII) and glycogen synthase kinase 3 α/β (GSK3α/β) was higher in sEH-/- mice than in WT mice. Collectively, these findings suggest that sEH is a key player in neurite outgrowth of neurons, OB development in the brain, and the development of anxiety-like behavior, by regulating the CaMKII-GSK3α/β signaling pathway.

Blockade of soluble epoxide hydrolase attenuates post-ischemic neuronal hyperexcitation and confers resilience against stroke with TrkB activation

Inhibition and deletion of soluble epoxide hydrolase (sEH) has been suggested to ameliorate infarction in experimental ischemic stroke possibly via vasoactive epoxyeicosatrienoic acids. However, it is unknown whether the neuroprotective mechanisms involve alteration of post-ischemic neuronal transmission and neurotrophic signaling. We used a permanent middle cerebral artery occlusion (MCAO) model in adult wild-type mice with the sEH inhibitor 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA) post-treatment and in sEH knockout (sEH KO) mice. We found that sensorimotor recovery was significantly enhanced after MCAO in both AUDA-treated and sEH KO mice, with decreased sEH activity and brain infarction. Decreased post-ischemic long-term potentiation (iLTP) was observed in an ex vivo hippocampal oxygen-glucose deprivation model. Tropomyosin receptor kinase B (TrkB) activation, rather than glutamate receptor alteration, was consistently found after the different manipulations. Immunohistochemistry further revealed peri-infarct neuronal TrkB activation and microvasculature augmentation in AUDA-treated and sEH KO mice, suggesting parallel neurovascular enhancement. Mechanistically, pretreatment with a selective TrkB antagonist ANA12 countered the effect of iLTP attenuation induced by sEH deletion ex vivo and abolished the infarct reduction in vivo. Together, the neuroprotective effects of sEH inhibition and gene deletion can both be mediated partially via enhancement of TrkB signaling which attenuated post-ischemic neuroexcitation and neurological deficits.

Genetic deletion or pharmacological inhibition of soluble epoxide hydrolase reduces brain damage and attenuates neuroinflammation after intracerebral hemorrhage

BackgroundInflammatory responses significantly contribute to neuronal damage and poor functional outcomes following intracerebral hemorrhage (ICH). Soluble epoxide hydrolase (sEH) is known to induce neuroinflammatory responses via degradation of anti-inflammatory epoxyeicosatrienoic acids (EET), and sEH is upregulated in response to brain injury. The present study investigated the involvement of sEH in ICH-induced neuroinflammation, brain damage, and functional deficits using a mouse ICH model and microglial cultures.MethodsICH was induced by injecting collagenase in both wild-type (WT) C57BL/6 mice and sEH knockout (KO) mice. WT mice were injected intracerebroventricularly with 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), a selective sEH inhibitor, 30 min before ICH. Expression of sEH in the hemorrhagic hemisphere was examined by immunofluorescence and Western blot analysis. The effects of genetic deletion or pharmacological inhibition of sEH by AUDA on neuroinflammatory responses, EET degradation, blood-brain barrier (BBB) permeability, histological damage, and functional deficits were evaluated. The anti-inflammatory mechanism of sEH inactivation was investigated in thrombin- or hemin-stimulated cultured microglia.ResultsICH induced an increase in sEH protein levels in the hemorrhagic hemisphere from 3 h to 4 days. sEH was expressed in microglia/macrophages, astrocytes, neurons, and endothelial cells in the perihematomal region. Genetic deletion of sEH significantly attenuated microglia/macrophage activation and expression of inflammatory mediators and reduced EET degradation at 1 and 4 days post-ICH. Deletion of sEH also reduced BBB permeability, matrix metalloproteinase (MMP)-9 activity, neutrophil infiltration, and neuronal damage at 1 and 4 days. Likewise, administration of AUDA attenuated proinflammatory microglia/macrophage activation and EET degradation at 1 day post-ICH. These findings were associated with a reduction in functional deficits and brain damage for up to 28 days. AUDA also ameliorated neuronal death, BBB disruption, MMP-9 activity, and neutrophil infiltration at 1 day. However, neither gene deletion nor pharmacological inhibition of sEH altered the hemorrhage volume following ICH. In primary microglial cultures, genetic deletion or pharmacological inhibition of sEH by AUDA reduced thrombin- and hemin-induced microglial activation. Furthermore, AUDA reduced thrombin- and hemin-induced P38 MAPK and NF-κB activation in BV2 microglia cultures. Ultimately, AUDA attenuated N2A neuronal death that was induced by BV2 microglial conditioned media.ConclusionsOur results suggest that inhibition of sEH may provide a potential therapy for ICH by suppressing microglia/macrophage-mediated neuroinflammation.

Deletion or inhibition of soluble epoxide hydrolase protects against brain damage and reduces microglia-mediated neuroinflammation in traumatic brain injury.

Traumatic brain injury (TBI) induces a series of inflammatory processes that contribute to neuronal damage. The present study investigated the involvement of soluble epoxide hydrolase (sEH) in neuroinflammation and brain damage in mouse TBI and in microglial cultures. The effects of genetic deletion of sEH and treatment with an sEH inhibitor, 12-(3-adamantan-1-yl-ureido)-dodecanoic acid (AUDA), on brain damage and inflammatory responses were evaluated in mice subjected to controlled cortical impact. The anti-inflammatory mechanism of sEH inhibition/deletion was investigated in vitro. TBI-induced an increase in sEH protein level in the injured cortex from 1 h to 4 days and sEH was expressed in microglia. Genetic deletion of sEH significantly attenuated functional deficits and brain damage up to 28 days post-TBI. Deletion of sEH also reduced neuronal death, apoptosis, brain edema, and BBB permeability at 1 and 4 day(s). These changes were associated with markedly reduced microglial/macrophage activation, neutrophil infiltration, matrix metalloproteinase-9 activity, inflammatory mediator expression at 1 and 4 day(s), and epoxyeicosatrienoic acid (EET) degradation at 1 and 4 day(s). Administration of AUDA attenuated brain edema, apoptosis, inflammatory mediator upregulation and EET degradation at 4 days. In primary microglial cultures, AUDA attenuated both LPS- or IFN-γ-stimulated nitric oxide (NO) production and reduced LPS- or IFN-γ-induced p38 MAPK and NF-κB signaling. Deletion of sEH also reduced IFN-γ-induced NO production. Moreover, AUDA attenuated N2A neuronal death induced by BV2 microglial-conditioned media. Our results suggest that inhibition of sEH may be a potential therapy for TBI by modulating the cytotoxic functions of microglia.

Deletion of soluble epoxide hydrolase enhances coronary reactive hyperemia in isolated mouse heart: role of oxylipins and PPARγ.

The relationship between soluble epoxide hydrolase (sEH) and coronary reactive hyperemia (CRH) response to a brief ischemic insult is not known. Epoxyeicosatrienoic acids (EETs) exert cardioprotective effects in ischemia/reperfusion injury. sEH converts EETs into dihydroxyeicosatrienoic-acids (DHETs). Therefore, we hypothesized that knocking out sEH enhances CRH through modulation of oxylipin profiles, including an increase in EET/DHET ratio. Compared with sEH+/+, sEH-/- mice showed enhanced CRH, including greater repayment volume (RV; 28% higher, P < 0.001) and repayment/debt ratio (32% higher, P < 0.001). Oxylipins from the heart perfusates were analyzed by LC-MS/MS. The 14,15-EET/14,15-DHET ratio was 3.7-fold higher at baseline (P < 0.001) and 5.6-fold higher post-ischemia (P < 0.001) in sEH-/- compared with sEH+/+ mice. Likewise, the baseline 9,10- and 12,13-EpOME/DiHOME ratios were 3.2-fold (P < 0.01) and 3.7-fold (P < 0.001) higher, respectively in sEH-/- compared with sEH+/+ mice. 13-HODE was also significantly increased at baseline by 71% (P < 0.01) in sEH-/- vs. sEH+/+ mice. Levels of 5-, 11-, 12-, and 15-hydroxyeicosatetraenoic acids were not significantly different between the two strains (P > 0.05), but were decreased postischemia in both groups (P = 0.02, P = 0.04, P = 0.05, P = 0.03, respectively). Modulation of CRH by peroxisome proliferator-activated receptor gamma (PPARγ) was demonstrated using a PPARγ-antagonist (T0070907), which reduced repayment volume by 25% in sEH+/+ (P < 0.001) and 33% in sEH-/- mice (P < 0.01), and a PPARγ-agonist (rosiglitazone), which increased repayment volume by 37% in both sEH+/+ (P = 0.04) and sEH-/- mice (P = 0.04). l-NAME attenuated CRH in both sEH-/- and sEH+/+ These data demonstrate that genetic deletion of sEH resulted in an altered oxylipin profile, which may have led to an enhanced CRH response.

Inhibition of Soluble Epoxide Hydrolase Limits Mitochondrial Damage and Preserves Function Following Ischemic Injury

Aims: Myocardial ischemia can result in marked mitochondrial damage leading to cardiac dysfunction, as such identifying novel mechanisms to limit mitochondrial injury is important. This study investigated the hypothesis that inhibiting soluble epoxide hydrolase (sEH), responsible for converting epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids protects mitochondrial from injury caused by myocardial infarction.Methods: sEH null and WT littermate mice were subjected to surgical occlusion of the left anterior descending (LAD) artery or sham operation. A parallel group of WT mice received an sEH inhibitor, trans-4-[4-(3-adamantan-1-y1-ureido)-cyclohexyloxy]-benzoic acid (tAUCB; 10 mg/L) or vehicle in the drinking water 4 days prior and 7 days post-MI. Cardiac function was assessed by echocardiography prior- and 7-days post-surgery. Heart tissues were dissected into infarct, peri-, and non-infarct regions to assess ultrastructure by electron microscopy. Complexes I, II, IV, citrate synthase, PI3K activities, and mitochondrial respiration were assessed in non-infarct regions. Isolated working hearts were used to measure the rates of glucose and palmitate oxidation.Results: Echocardiography revealed that tAUCB treatment or sEH deficiency significantly improved systolic and diastolic function post-MI compared to controls. Reduced infarct expansion and less adverse cardiac remodeling were observed in tAUCB-treated and sEH null groups. EM data demonstrated mitochondrial ultrastructure damage occurred in infarct and peri-infarct regions but not in non-infarct regions. Inhibition of sEH resulted in significant improvements in mitochondrial respiration, ATP content, mitochondrial enzymatic activities and restored insulin sensitivity and PI3K activity.Conclusion: Inhibition or genetic deletion of sEH protects against long-term ischemia by preserving cardiac function and maintaining mitochondrial efficiency.