14,15-EET involved in the development of diabetic cardiac hypertrophy mediated by PPARs.

Cardiac hypertrophy is a key structural change in diabetic cardiomyopathy, which mechanism is unknown. 14,15-Epoxyeicosatrienoic acid (14,15-EET) generated from arachidonic acid by CYP2J2 has beneficial effects in metabolic syndrome, which also plays vital roles in inflammatory response. Peroxisome proliferator activated receptors (PPARs) are members of the nuclear receptor superfamily and have three subtypes of α, ß (or δ) and γ. Studies have found that 14,15-EET can perform various biological functions by activating PPARs, but its role in diabetic cardiac hypertrophy is unknown. This study aimed to investigate the role of 14,15-EET-PPARs signaling pathway in the development of diabetic cardiac hypertrophy. Diabetic cardiac hypertrophy was developed by high-fat diet feeding combined with streptozotocin (40 mg/kg/d for 5 days, i.p.) in mice and was induced by glucose at 25.5 mmol/L (high glucose, HG) in H9c2 cells. The decreased level of 14,15-EET and the down-regulated expression of PPARα, PPARß and PPARγ were found following diabetic cardiac hypertrophy in mice. Similarly, both the level of 14,15-EET and the PPARs expression were also reduced in HG-induced hypertrophic cardiomyocytes. Supplementation with 14,15-EET improved the cardiomyocyte hypertrophy and up-regulated PPARs expression, which were nullified by 14,15-EEZE, a 14,15-EET antagonist. Taken together, we conclude that the decreased 14,15-EET is involved in the development of diabetic cardiac hypertrophy through the down-regulation of PPARs.

Antinociception role of 14,15-epoxyeicosatrienoic acid in a central post-stroke pain model in rats mediated by anti-inflammation and anti-apoptosis effect.

Central post stroke pain (CPSP) is an intractable neuropathic pain syndrome that occurs after the acute focal lesion of the central nervous system (CNS) due to a cerebrovascular cause. Epoxyeicosatrienoic acids (EETs) exert many pharmacological effects in vivo and in vitro, such as anti-apoptosis, anti-inflammatory, and anti-oxidative stress. Neuroinflammation and apoptosis are the potential pathophysiological mechanisms of neuropathic pain. This study aimed to investigate whether 14,15-EET has an antinociception effect on CPSP rats through its anti-inflammation and anti-apoptosis mechanisms. Rats were treated with type IV collagenase (CPSP group) or saline (Sham group) via injection with a Hamilton syringe into the ventral posterior lateral nucleus (VPL) according to the stereotaxic coordinates. We first tested the mechanical withdrawal threshold, as well as neuroinflammation- and apoptosis-related protein expressions in the per-lesion site of CPSP and Sham rats. Sprague-Dawley rats were randomly divided into five groups, as follows: vehicle; EET at 0.025, 0.05, and 0.1 µg; and EET (0.1 µg) + EEZE (3.25 ng). EET or and vehicle were administered into VPL nuclei three consecutive days after hemorrhagic stroke. Immunostaining, ELISA, and Western blot were performed to evaluate neuroinflammation and apoptosis. Hemorrhagic stroke induced mechanical allodynia, glial activation, neuroinflammation, and apoptosis-related protein upregulation. However, early treatment with 14,15-EET inhibited glial cell activation, decreased proinflammatory cytokines and apoptosis-related protein, and alleviated the pain behavior of CPSP rats. Our results provided strong evidence that antinociception produced by 14,15-EET is partly mediated by the inhibition of neuroinflammation and apoptosis.

New Alkoxy- Analogues of Epoxyeicosatrienoic Acids Attenuate Cisplatin Nephrotoxicity In Vitro via Reduction of Mitochondrial Dysfunction, Oxidative Stress, Mitogen-Activated Protein Kinase Signaling, and Caspase Activation.

The usage of cisplatin, a highly potent chemotherapeutic, is limited by its severe nephrotoxicity. Arachidonic acid (ARA)-derived epoxyeicosatrienoic acids (EETs) and soluble epoxide hydrolase (sEH) inhibitors were shown to ameliorate this dose-limiting side effect, but both approaches have some pharmacological limitations. Analogues of EETs are an alternative avenue with unique benefits, but the current series of analogues face concerns regarding their structure and mimetic functionality. Hence, in this study, regioisomeric mixtures of four new ARA alkyl ethers were synthesized, characterized, and assessed as EET analogues against the concentration- and time-dependent toxicities of cisplatin in porcine proximal tubular epithelial cells. All four ether groups displayed bioisostere activity, ranging from marginal for methoxy- (1), good for n-propoxy- (4), and excellent for ethoxy- (2) and i-propoxy- (3). Compounds 2 and 3 displayed cytoprotective effects comparable to that of an EET regioisomeric mixture (5) against high, acute cisplatin exposures but were more potent against low to moderate, chronic exposures. Compounds 2 and 3 (and 5) acted through stabilization of the mitochondrial transmembrane potential and attenuation of reactive oxygen species, leading to reduced phosphorylation of mitogen-activated protein kinases p38 and JNK and decreased activation of caspase-9 and caspase-3. This study demonstrates that alkoxy- groups are potent and more metabolically stable bioisostere alternatives to the epoxide within EETs that enable sEH-independent activity. It also illustrates the potential of ether-based mimics of EETs and other epoxy fatty acids as promising nephroprotective agents to tackle the clinically relevant side effect of cisplatin without compromising its antineoplastic function.

11,12 Epoxyeicosatrienoic Acid Rescues Deteriorated Wound Healing in Diabetes.

Epoxyeicosatrienoic acids (EET) facilitate regeneration in different tissues, and their benefit in dermal wound healing has been proven under normal conditions. In this study, we investigated the effect of 11,12 EET on dermal wound healing in diabetes. We induced diabetes by i.p. injection of streptozotocin 2 weeks prior to wound creation on the dorsal side of the mouse ear. 11,12 EET was applied every second day on the wound, whereas the control groups received only solvent. Epithelialization was monitored every second day intravitally up to wound closure. Wounds were stained for VEGF, CD31, TGF-ß, TNF-α, SDF-1α, NF-κB, and Ki-67, and fibroblasts were counted after hematoxylin-eosin stain on days 3, 6, 9, and 16 after wounding. After induction of diabetes, wounds closed on day 13.00 ± 2.20 standard deviation (SD). Local 11,12 ETT application improved wound closure significantly to day 8.40 ± 1.39 SD. EET treatment enhanced VEGF and CD31 expression in wounds on day 3. It also seemed to raise TNF-α level on all days investigated as well as TGF-ß level on days 3 and 6. A decrease in NF-κB could be observed on days 9 and 16 after EET application. The latter findings were not significant. SDF-1α expression was not influenced by EET application, and Ki-67 was significantly less in the EET group on day 9 after EET application. The number of fibroblasts was significantly increased on day 9 after the 11,12 EET application. 11,12 EET improve deteriorated wound healing in diabetes by enhancing neoangiogenesis, especially in the early phase of wound healing. Furthermore, they contribute to the dissolution of the initial inflammatory reaction, allowing the crucial transition from the inflammatory to proliferative phase in wound healing.

14,15-EET Reduced Brain Injury from Cerebral Ischemia and Reperfusion via Suppressing Neuronal Parthanatos.

To investigate the effect of 14,15-EET on the parthanatos in neurons induced by cerebral ischemia and reperfusion, middle cerebral artery occlusion and reperfusion (MCAO/R) and oxygen glucose deprivation/reoxygenation (OGD/R) were used to simulate cerebral ischemia reperfusion in vivo and in vitro, respectively. TTC staining and the Tunel method were used to detect cerebral infarct volume and neuronal apoptosis. Western blot and immunofluorescence were used to detect poly (ADP-ribose) polymerase-1 (PARP-1) activation and AIF nuclear translocation. The production of reactive oxygen species (ROS) and the expression of antioxidant genes were detected by Mito SOX, DCFH-DA and qPCR methods. MCAO/R increased cerebral infarct volume and neuronal apoptosis in mice, while 14,15-EET pretreatment increased cerebral infarct volume and neuronal apoptosis. OGD/R induced reactive oxygen species generation, PARP-1 cleavage, and AIF nuclear translocation in cortical neurons. 14,15-EET pretreatment could enhance the antioxidant gene expression of glutathione peroxidase (GSH-Px), heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) in cortical neurons after ischemia and reperfusion. 14,15-EET inhibits the neuronal parthanatos induced by MCAO/R through upregulation of the expression of antioxidant genes and by reducing the generation of reactive oxygen species. This study advances the EET neuroprotection theory and provides a scientific basis for targeted clinical drugs that reduce neuronal parthanatos following cerebral ischemia and reperfusion.

EETs/sEHi alleviates nociception by blocking the crosslink between endoplasmic reticulum stress and neuroinflammation in a central poststroke pain model.

BACKGROUND: Central post-stroke pain (CPSP) is a chronic and intolerable neuropathic pain syndrome following a cerebral vascular insult, which negatively impacts the quality of life of stroke survivors but currently lacks efficacious treatments. Though its underlying mechanism remains unclear, clinical features of hyperalgesia and allodynia indicate central sensitization due to excessive neuroinflammation. Recently, the crosslink between neuroinflammation and endoplasmic reticulum (ER) stress has been identified in diverse types of diseases. Nevertheless, whether this interaction contributes to pain development remains unanswered. Epoxyeicosatrienoic acids (EETs)/soluble epoxy hydrolase inhibitors (sEHi) are emerging targets that play a significant role in pain and neuroinflammatory regulation. Moreover, recent studies have revealed that EETs are effective in attenuating ER stress. In this study, we hypothesized that ER stress around the stroke site may activate glial cells and lead to further inflammatory cascades, which constitute a positive feedback loop resulting in central sensitization and CPSP. Additionally, we tested whether EETs/sEHi could attenuate CPSP by suppressing ER stress and neuroinflammation, as well as their vicious cycle, in a rat model of CPSP. METHODS: Young male SD rats were used to induce CPSP using a model of thalamic hemorrhage and were then treated with TPPU (sEHi) alone or in combination with 14,15-EET or 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, the EET antagonist), tunicamycin (Tm, ER stress inducer), or 4-PBA (ER stress inhibitor). Nociceptive behaviors, ER stress markers, JNK and p38 (two well-recognized inflammatory kinases of mitogen-activated protein kinase (MAPK) signaling) expression, and glial cell activation were assessed. In addition, some healthy rats were intrathalamically microinjected with Tm or lipopolysaccharide (LPS) to test the interaction between ER stress and neuroinflammation in central pain. RESULTS: Analysis of the perithalamic lesion tissue from the brain of CPSP rats demonstrated decreased soluble epoxy hydrolase (sEH) expression, which was accompanied by increased expression of ER stress markers, including BIP, p-IRE, p-PERK, and ATF6. In addition, inflammatory kinases (p-p38 and p-JNK) were upregulated and glial cells were activated. Intrathalamic injection of sEHi (TPPU) increased the paw withdrawal mechanical threshold (PWMT), reduced hallmarks of ER stress and MAPK signaling, and restrained the activation of microglia and astrocytes around the lesion site. However, the analgesic effect of TPPU was completely abolished by 14,15-EEZE. Moreover, microinjection of Tm into the thalamic ventral posterior lateral (VPL) nucleus of healthy rats induced mechanical allodynia and activated MAPK-mediated neuroinflammatory signaling; lipopolysaccharide (LPS) administration led to activation of ER stress along the injected site in healthy rats. CONCLUSIONS: The present study provides evidence that the interaction between ER stress and neuroinflammation is involved in the mechanism of CPSP. Combined with the previously reported EET/sEHi effects on antinociception and neuroprotection, therapy with agents that target EET signaling may serve as a multi-functional approach in central neuropathic pain by attenuating ER stress, excessive neuroinflammation, and subsequent central sensitization. The use of these agents within a proper time window could not only curtail further nerve injury but also produce an analgesic effect.

TPPU treatment of burned mice dampens inflammation and generation of bioactive DHET which impairs neutrophil function.

Oxylipins modulate the behavior of immune cells in inflammation. Soluble epoxide hydrolase (sEH) converts anti-inflammatory epoxyeicosatrienoic acid (EET) to dihydroxyeicosatrienoic acid (DHET). An sEH-inhibitor, TPPU, has been demonstrated to ameliorate lipopolysaccharide (LPS)- and sepsis-induced inflammation via EETs. The immunomodulatory role of DHET is not well characterized. We hypothesized that TPPU dampens inflammation and that sEH-derived DHET alters neutrophil functionality in burn induced inflammation. Outbred mice were treated with vehicle, TPPU or 14,15-DHET and immediately subjected to either sham or dorsal scald 28% total body surface area burn injury. After 6 and 24 h, interleukin 6 (IL-6) serum levels and neutrophil activation were analyzed. For in vitro analyses, bone marrow derived neutrophil functionality and mRNA expression were examined. In vivo, 14,15-DHET and IL-6 serum concentrations were decreased after burn injury with TPPU administration. In vitro, 14,15-DHET impaired neutrophil chemotaxis, acidification, CXCR1/CXCR2 expression and reactive oxygen species (ROS) production, the latter independent from p38MAPK and PI3K signaling. We conclude that TPPU administration decreases DHET post-burn. Furthermore, DHET downregulates key neutrophil immune functions and mRNA expression. Altogether, these data reveal that TPPU not only increases anti-inflammatory and inflammation resolving EET levels, but also prevents potential impairment of neutrophils by DHET in trauma.

Mapping the Molecular Architecture Required for Lipid-Binding Pockets Using a Subset of Established and Orphan G-Protein Coupled Receptors.

G-protein coupled receptors (GPCRs) sense a wide variety of stimuli, including lipids, and transduce signals to the intracellular environment to exert various physiological responses. However, the structural features of GPCRs responsible for detecting and triggering responses to distinct lipid ligands have only recently begun to be revealed. 14,15-epoxyeicosatrienoic acid (14,15-EET) is one such lipid mediator that plays an essential role in the vascular system, displaying both vasodilatory and anti-inflammatory properties. We recently reported multiple low-affinity 14,15-EET-binding GPCRs, but the mechanism by which these receptors sense 14,15-EET remains unclear. Here, we have taken a combined computational and experimental approach to identify and confirm critical residues and properties within the lipid-binding pocket. Furthermore, we generated mutants to engineer selected GPCR-predicted binding sites to either confer or abolish 14,15-EET-induced signaling. Our structure-function analyses indicate that hydrophobic and positively charged residues of the receptor-binding pocket are prerequisites for recognizing lipid ligands such as 14,15-EET and possibly other eicosanoids.

Soluble epoxide hydrolase deletion attenuated nicotine-induced arterial stiffness via limiting the loss of SIRT1.

Arterial stiffness, a consequence of smoking, is an underlying risk factor of cardiovascular diseases. Epoxyeicosatrienoic acids (EETs), hydrolyzed by soluble epoxide hydrolase (sEH), have beneficial effects against vascular dysfunction. However, the role of sEH knockout in nicotine-induced arterial stiffness was not characterized. We hypothesized that sEH knockout could prevent nicotine-induced arterial stiffness. In the present study, Ephx2 (the gene encodes sEH enzyme) null (Ephx2-/-) mice and wild-type (WT) littermate mice were infused with or without nicotine and administered with or without nicotinamide [NAM, sirtuin-1 (SIRT1) inhibitor] simultaneously for 4 wk. Nicotine treatment increased sEH expression and activity in the aortas of WT mice. Nicotine infusion significantly induced vascular remodeling, arterial stiffness, and SIRT1 deactivation in WT mice, which was attenuated in Ephx2 knockout mice (Ephx2-/- mice) without NAM treatment. However, the arterial protective effects were gone in Ephx2-/- mice with NAM treatment. In vitro, 11,12-EET treatment attenuated nicotine-induced matrix metalloproteinase 2 (MMP2) upregulation via SIRT1-mediated yes-associated protein (YAP) deacetylation. In conclusion, sEH knockout attenuated nicotine-induced arterial stiffness and vascular remodeling via SIRT1-induced YAP deacetylation.NEW & NOTEWORTHY We presently show that sEH knockout repressed nicotine-induced arterial stiffness and extracellular matrix remodeling via SIRT1-induced YAP deacetylation, which highlights that sEH is a potential therapeutic target in smoking-induced arterial stiffness and vascular remodeling.

The arachidonic acid metabolite 11,12-epoxyeicosatrienoic acid alleviates pulmonary fibrosis.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid that are rapidly metabolized into diols by soluble epoxide hydrolase (sEH). sEH inhibition has been shown to increase the biological activity of EETs, which are known to have anti-inflammatory properties. However, the role of EETs in pulmonary fibrosis remains unexplored. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to analyze EETs in the lung tissues of patients with idiopathic pulmonary fibrosis (IPF, n = 29) and controls (n = 15), and the function of 11,12-EET was evaluated in in vitro and in vivo in pulmonary fibrosis models. EET levels in IPF lung tissues, including those of 8,9-EET, 11,12-EET, and 14,15-EET, were significantly lower than those in control tissues. The 11,12-EET/11,12-DHET ratio in human lung tissues also differentiated IPF from control tissues. 11,12-EET significantly decreased transforming growth factor (TGF)-ß1-induced expression of α-smooth muscle actin (SMA) and collagen type-I in MRC-5 cells and primary fibroblasts from IPF patients. sEH-specific siRNA and 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU; sEH inhibitor) also decreased TGF-ß1-induced expression of α-SMA and collagen type-I in fibroblasts. Moreover, 11,12-EET and TPPU decreased TGF-ß1-induced p-Smad2/3 and extracellular-signal-regulated kinase (ERK) expression in primary fibroblasts from patients with IPF and fibronectin expression in Beas-2B cells. TPPU decreased the levels of hydroxyproline in the lungs of bleomycin-induced mice. 11,12-EET or sEH inhibitors could inhibit pulmonary fibrosis by regulating TGF-ß1-induced profibrotic signaling, suggesting that 11,12-EET and the regulation of EETs could serve as potential therapeutic targets for IPF treatment.

Role of endothelium-pericyte signaling in capillary blood flow response to neuronal activity.

Local blood flow in the brain is tightly coupled to metabolic demands, a phenomenon termed functional hyperemia. Both capillaries and arterioles contribute to the hyperemic response to neuronal activity via different mechanisms and timescales. The nature and specific signaling involved in the hyperemic response of capillaries versus arterioles, and their temporal relationship are not fully defined. We determined the time-dependent changes in capillary flux and diameter versus arteriolar velocity and flow following whisker stimulation using optical microangiography (OMAG) and two-photon microscopy. We further characterized depth-resolved responses of individual capillaries versus capillary networks. We hypothesized that capillaries respond first to neuronal activation, and that they exhibit a coordinated response mediated via endothelial-derived epoxyeicosatrienoates (EETs) acting on pericytes. To visualize peri-capillary pericytes, we used Tie2-GFP/NG2-DsRed mice, and to determine the role of endothelial-derived EETs, we compared cerebrovascular responses to whisker stimulation between wild-type mice and mice with lower endothelial EETs (Tie2-hsEH). We found that capillaries respond immediately to neuronal activation in an orchestrated network-level manner, a response attenuated in Tie2-hsEH and inhibited by blocking EETs action on pericytes. These results demonstrate that capillaries are first responders during functional hyperemia, and that they exhibit a network-level response mediated via endothelial-derived EETs' action on peri-capillary pericytes.

Kidney-Targeted Epoxyeicosatrienoic Acid Analog, EET-F01, Reduces Inflammation, Oxidative Stress, and Cisplatin-Induced Nephrotoxicity.

Although epoxyeicosatrienoic acid (EET) analogs have performed well in several acute and chronic kidney disease models, targeted delivery of EET analogs to the kidney can be reasonably expected to reduce the level of drug needed to achieve a therapeutic effect and obviate possible side effects. For EET analog kidney-targeted delivery, we conjugated a stable EET analog to folic acid via a PEG-diamine linker. Next, we compared the kidney targeted EET analog, EET-F01, to a well-studied EET analog, EET-A. EET-A or EET-F01 was infused i.v. and plasma and kidney tissue collected. EET-A was detected in the plasma but was undetectable in the kidney. On the other hand, EET-F01 was detected in the plasma and kidney. Experiments were conducted to compare the efficacy of EET-F01 and EET-A for decreasing cisplatin nephrotoxicity. Cisplatin was administered to WKY rats treated with vehicle, EET-A (10 mg/kg i.p.) or EET-F01 (20 mg/kg or 2 mg/kg i.p.). Cisplatin increased kidney injury markers, viz., blood urea nitrogen (BUN), N-acetyl-ß-(D)-glucosaminidase (NAG), kidney injury molecule-1 (KIM-1), and thiobarbituric acid reactive substances (TBARS). EET-F01 was as effective as EET-A in decreasing BUN, NAG, KIM-1, TBARS, and renal histological injury caused by cisplatin. Despite its almost 2×-greater molecular weight compared with EET-A, EET-F01 was comparably effective in decreasing renal injury at a 10-fold w/w lower dose. EET-F01 decreased cisplatin nephrotoxicity by reducing oxidative stress and inflammation. These data demonstrate that EET-F01 targets the kidney, allows for a lower effective dose, and combats cisplatin nephrotoxicity. In conclusion, we have developed a kidney targeted EET analog, EET-F01, that demonstrates excellent potential as a therapeutic for kidney diseases.

Plasma epoxyeicosatrienoic acids and dihydroxyeicosatrieonic acids, insulin, glucose and risk of diabetes: The strong heart study.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid with multiple biological functions. Rodent experiments suggest EETs play a role in insulin sensitivity and diabetes, but evidence in humans is limited. To address this knowledge gap, we conducted a case-cohort study in the Strong Heart Family Study, a prospective cohort among American Indians. METHODS: We measured 4 EET species and 4 species of corresponding downstream metabolites, dihydroxyeicosatrieonic acids (DHETs), in plasma samples from 1161 participants, including 310 with type 2 diabetes. We estimated the associations of total (esterified and free) EETs and DHETs with incident diabetes risk, adjusting for known risk factors. We also examined cross-sectional associations with plasma fasting insulin and glucose in the case-cohort and in 271 participants without diabetes from the older Strong Heart Study cohort, and meta-analyzed the results from the 2 cohorts. FINDINGS: We observed no significant association of total EET or DHET levels with incident diabetes. In addition, plasma EETs were not associated with plasma insulin or plasma glucose. However, higher plasma 14,15-DHET was associated with lower plasma insulin and lower plasma glucose. INTERPRETATION: In this first prospective study of EETs and diabetes, we found no evidence for a role of total plasma EETs in diabetes. The novel associations of 14,15-DHET with insulin and glucose warrant replication and exploration of possible mechanisms. FUNDING: US National Institutes of Health.

Pharmacological regulation of cytochrome P450 metabolites of arachidonic acid attenuates cardiac injury in diabetic rats.

Diabetic cardiomyopathy (DCM) is a well-established complication of type 1 and type 2 diabetes associated with a high rate of morbidity and mortality. DCM is diagnosed at advanced and irreversible stages. Therefore, it is of utmost need to identify novel mechanistic pathways involved at early stages to prevent or reverse the development of DCM. In vivo experiments were performed on type 1 diabetic rats (T1DM). Functional and structural studies of the heart were executed and correlated with mechanistic assessments exploring the role of cytochromes P450 metabolites, the 20-hydroxyeicosatetraenoic acids (20-HETEs) and epoxyeicosatrienoic acids (EETs), and their crosstalk with other homeostatic signaling molecules. Our data displays that hyperglycemia results in CYP4A upregulation and CYP2C11 downregulation in the left ventricles (LV) of T1DM rats, paralleled by a differential alteration in their metabolites 20-HETEs (increased) and EETs (decreased). These changes are concomitant with reductions in cardiac outputs, LV hypertrophy, fibrosis, and increased activation of cardiac fetal and hypertrophic genes. Besides, pro-fibrotic cytokine TGF-ß overexpression and NADPH (Nox4) dependent-ROS overproduction are also correlated with the observed cardiac functional and structural modifications. Of interest, these observations are attenuated when T1DM rats are treated with 12-(3-adamantan-1-yl-ureido) dodecanoic acid (AUDA), which blocks EETs metabolism, or N-hydroxy-N'-(4-butyl-2-methylphenol)Formamidine (HET0016), which inhibits 20-HETEs formation. Taken together, our findings confer pioneering evidence about a potential interplay between CYP450-derived metabolites and Nox4/TGF-ß axis leading to DCM. Pharmacologic interventions targeting the inhibition of 20-HETEs synthesis or the activation of EETs synthesis may offer novel therapeutic approaches to treat DCM.

Epoxyeicosatrienoic acids improve glucose homeostasis by preventing NF-κB-mediated transcription of SGLT2 in renal tubular epithelial cells.

Studies have shown that epoxyeicosatrienoic acids (EETs) can regulate glucose homeostasis, but the specific mechanisms need further exploration. The sodium-glucose co-transporter 2 (SGLT2) is highly expressed in diabetic kidneys, which further promotes renal reabsorption of glucose to respond to the hyperglycemic state of diabetes. Herein, whether EETs can be a latent inhibitor of SGLT2 to regulate glucose homeostasis in diabetic state needs to be elucidated. Our study demonstrated that EETs attenuated the glucose reabsorption via renal tubular epithelial cells in diabetic mice, which partly accounted for the beneficial effects of EETs on glucose homeostasis. Moreover, 14,15-EET suppressed SGLT2 expression in both diabetic kidney and renal tubular epithelial cells. Further, inhibition of NF-κB with BAY 11-7082 decreased insulin-induced SGLT2 expression while NF-κB overexpression reversed the above effects. In addition, 14,15-EET attenuated SGLT2 expression via inactivating NF-κB. Mechanistically, 14,15-EET attenuated NF-κB mediated SGLT2 transcription at the -1821/-1812 P65-binding site. These results showed that EETs ameliorated glucose homeostasis via preventing NF-κB-mediated transcription of SGLT2 in renal tubular epithelial cells, providing a unique therapeutic strategy for insulin resistance and diabetes.

14,15-Epoxyeicosatrienoic Acid Alleviates Pathology in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of late-onset dementia, and there exists an unmet medical need for effective treatments for AD. The accumulation of neurotoxic amyloid-ß (Aß) plaques contributes to the pathophysiology of AD. EPHX2 encoding soluble epoxide hydrolase (sEH)-a key enzyme for epoxyeicosatrienoic acid (EET) signaling that is mainly expressed in lysosomes of astrocytes in the adult brain-is cosited at a locus associated with AD, but it is unclear whether and how it contributes to the pathophysiology of AD. In this report, we show that the pharmacologic inhibition of sEH with 1-trifluoromethoxyphenyl- 3-(1-propionylpiperidin-4-yl) urea (TPPU) or the genetic deletion of Ephx2 reduces Aß deposition in the brains of both male and female familial Alzheimer's disease (5×FAD) model mice. The inhibition of sEH with TPPU or the genetic deletion of Ephx2 alleviated cognitive deficits and prevented astrocyte reactivation in the brains of 6-month-old male 5×FAD mice. 14,15-EET levels in the brains of these mice were also increased by sEH inhibition. In cultured adult astrocytes treated with TPPU or 14,15-EET, astrocyte Aß clearance was increased through enhanced lysosomal biogenesis. Infusion of 14,15-EET into the hippocampus of 5×FAD mice prevented the aggregation of Aß. Notably, a higher concentration of 14,15-EET (200 ng/ml) infusion into the hippocampus reversed Aß deposition in the brains of 6-month-old male 5×FAD mice. These results indicate that EET signaling, especially 14,15-EET, plays a key role in the pathophysiology of AD, and that targeting this pathway is a potential therapeutic strategy for the treatment of AD.SIGNIFICANCE STATEMENT There are limited treatment options for Alzheimer's disease (AD). EPHX2 encoding soluble epoxide hydrolase (sEH) is located at a locus that is linked to late-onset AD, but its contribution to the pathophysiology of AD is unclear. Here, we demonstrate that sEH inhibition or Ephx2 deletion alleviates pathology in familial Alzheimer's disease (5×FAD) mice. Inhibiting sEH or increasing 14,15-epoxyeicosatrienoic acid (EET) enhanced lysosomal biogenesis and amyloid-ß (Aß) clearance in cultured adult astrocytes. Moreover, the infusion of 14,15-EET into the hippocampus of 5×FAD mice not only prevented the aggregation of Aß, but also reversed the deposition of Aß. Thus, 14,15-EET plays a key role in the pathophysiology of AD and therapeutic strategies that target this pathway may be an effective treatment.

Bioconversion of arachidonic acid into human 14,15-hepoxilin B3 and 13,14,15-trioxilin B3 by recombinant cells expressing microbial 15-lipoxygenase without and with epoxide hydrolase.

OBJECTIVE: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B3, 14,15-HXB3) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B3, 13,14,15-TrXB3) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively. RESULTS: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB3 and 13,14,15-TrXB3, respectively. Under the optimal conditions of 30 g cells L-1, 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB3 and 100 mM 13,14,15-TrXB3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h-1, respectively. CONCLUSION: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

Cytochrome P450 epoxygenase-derived 5,6-epoxyeicosatrienoic acid relaxes pulmonary arteries in normoxia but promotes sustained pulmonary vasoconstriction in hypoxia.

AIMS: The aim of the study was to investigate the role of cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in sustained hypoxic pulmonary vasoconstriction (HPV). METHODS: Vasomotor responses of isolated mouse intrapulmonary arteries (IPAs) were assessed using wire myography. Key findings were verified by haemodynamic measurements in isolated perfused and ventilated mouse lungs. RESULTS: Pharmacological inhibition of EET synthesis with MS-PPOH, application of the EET antagonist 14,15-EEZE or deficiency of CYP2J isoforms suppressed sustained HPV. In contrast, knockdown of EET-degrading soluble epoxide hydrolase or its inhibition with TPPU augmented sustained HPV almost twofold. All EET regioisomers elicited relaxation in IPAs pre-contracted with thromboxane mimetic U46619. However, in the presence of KCl-induced depolarization, 5,6-EET caused biphasic contraction in IPAs and elevation of pulmonary vascular tone in isolated lungs, whereas other regioisomers had no effect. In patch-clamp experiments, hypoxia elicited depolarization in pulmonary artery smooth muscle cells (PASMCs), and 5,6-EET evoked inward whole cell currents in PASMCs depolarized to the hypoxic level, but not at their resting membrane potential. CONCLUSIONS: The EET pathway substantially contributes to sustained HPV in mouse pulmonary arteries. 5,6-EET specifically appears to be involved in HPV, as it is the only EET regioisomer able to elicit not only relaxation, but also sustained contraction in these vessels. 5,6-EET-induced pulmonary vasoconstriction is enabled by PASMC depolarization, which occurs in hypoxia. The discovery of the dual role of 5,6-EET in the regulation of pulmonary vascular tone may provide a basis for the development of novel therapeutic strategies for treatment of HPV-related diseases.

NLRX1 knockout aggravates lipopolysaccharide (LPS)-induced heart injury and attenuates the anti-LPS cardioprotective effect of CYP2J2/11,12-EET by enhancing activation of NF-κB and NLRP3 inflammasome.

NLRX1 weakens lipopolysaccharide (LPS)-induced NF-κB activation on immune cells. Cytochrome P450 epoxygenase 2J2 (CYP2J2) attenuates LPS-induced cardiac injury by inhibiting NF-κB activation. However, it is still unclear whether NLRX1 could reduce LPS-induced heart damage and whether it is involved in the anti-LPS cardioprotective effect of CYP2J2. In this study, we found that NLRX1 knockout further exacerbated LPS-induced heart injury and up-regulated the proinflammatory cytokines in serum and heart tissue, and weakened the inhibitory effect of CYP2J2 on the harmful effects caused by LPS. We also found that LPS treatment induced ubiquitination of NLRX1 and promoted its binding to IKKα/ß in myocardial tissue, which should theoretically inhibit NF-κB activation. However, LPS eventually leads to activation of NF-κB and NLRP3 inflammasome. Under the action of LPS, CYP2J2 further promoted the ubiquitination of NLRX1 and its binding to IKKα/ß, impaired NF-κB activation and NLRP3 inflammasome activation. NLRX1 knockout notably aggravated LPS-induced NF-κB activation and NLRP3 inflammasome activation, and attenuated the inhibitory effects of CYP2J2 on NF-κB signal and NLRP3 inflammasome. More, CYP2J2 reduced LPS-induced reactive oxygen species (ROS) production and mitochondrial depolarization in heart cells, thereby inhibiting NLRP3 inflammasome activation. NLRX1 knockdown aggravated mitochondrial depolarization induced by LPS and weakened the protective effect of CYP2J2 on mitochondrial potential, although it had no significant effect on reactive oxygen species production. Together, these findings demonstrated that NLRX1 knockout aggravated LPS-induced heart injury and weakened the anti-LPS cardioprotective effect of CYP2J2 by enhancing activation of NF-κB and NLRP3 inflammasome.