Ding Ai

Opposite effects of gene deficiency and pharmacological inhibition of soluble epoxide hydrolase on cardiac fibrosis

Arachidonic acid-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; manipulation of their levels is a potentially useful pharmacological strategy. EETs are hydrolyzed by soluble epoxide hydrolase (sEH) to form the corresponding diols, thus altering and reducing the activity of these oxylipins. To better understand the phenotypic impact of sEH disruption, we compared the effect of EPHX2 gene knockout (EPHX2 −/−) and sEH inhibition in mouse models. Measurement of plasma oxylipin profiles confirmed that the ratio of EETs/DHETs was increased in EPHX2 −/− and sEH-inhibited mice. However, plasma concentrations of 9, 11, 15, 19-HETE were elevated in EPHX2 −/− but not sEH-inhibited mice. Next, we investigated the role of this difference in cardiac dysfunction induced by Angiotensin II (AngII). Both EPHX2 gene deletion and inhibition protected against AngII-induced cardiac hypertrophy. Interestingly, cardiac dysfunction was attenuated by sEH inhibition rather than gene deletion. Histochemical staining revealed that compared with pharmacological inhibition, EPHX2 deletion aggravated AngII-induced myocardial fibrosis; the mRNA levels of fibrotic-related genes were increased. Furthermore, cardiac inflammatory response was greater in EPHX2 −/− than sEH-inhibited mice with AngII treatment, as evidenced by increased macrophage infiltration and expression of MCP-1 and IL-6. In vitro, AngII-upregulated MCP-1 and IL-6 expression was significantly attenuated by sEH inhibition but promoted by EPHX2 deletion in cardiofibroblasts. Thus, compared with pharmacological inhibition of sEH, EPHX2 deletion caused the shift in arachidonic acid metabolism, which may led to pathological cardiac remodeling, especially cardiac fibrosis.

Systematic Metabolomic Analysis of Eicosanoids after Omega-3 Polyunsaturated Fatty Acid Supplementation by a Highly Specific Liquid Chromatography–Tandem Mass Spectrometry-Based Method

Omega-3 (ω-3) polyunsaturated fatty acids (PUFAs) have beneficial effects in many pathological processes, especially cardiovascular disease, and their protective eicosanoid metabolites are thought to play important roles. However, how ω-3 PUFAs affect the eicosanoid profile has not been elucidated comprehensively. Here, we systematically analyzed the eicosanoid metabolites induced by ω-3 PUFA supplementation. We developed an LC-MS/MS-based method covering 32 arachidonic acid (ARA) metabolites and 37 ω-3 PUFA-derived products. The limits of detection for eicosanoids were between 0.0625 and 1 pg and the detection specificity was optimized. We then quantified eicosanoids in mouse and human plasma and mouse aorta samples after ω-3 PUFA supplementation. Levels of EPA hydroxyl products, 4-HDoHE, 17,18-EEQ, 17,18-DiHETE, TXB2, and LXA4 were significantly changed in both mouse samples, and those of 2-series PGs, EDPs and DHA hydroxyl products were changed in aorta samples. Correlation network analysis of mouse plasma data revealed that some eicosanoids had higher connection degree or betweenness centrality score than others after ω-3 PUFA supplementation. Eicosanoids in human plasma were profiled across five time points after ω-3 PUFA supplementation. Fuzzy c-mean clustering algorithm suggested that the time curves of eicosanoid activity could be described with three kinetic patterns: sustained upregulation, short-term upregulation, and downregulation. This is the first systematic profiling of eicosanoids with ω-3 PUFA supplementation. The highly specific eicosanoid metabolomic and related data analysis methods would be powerful tools for comprehensive eicosanoid study.

Soluble epoxide hydrolase: A potential target for metabolic diseases.

Epoxyeicosatrienoic acids (EETs), important lipid mediators derived from arachidonic acid, have many beneficial effects in metabolic diseases, including atherosclerosis, hypertension, cardiac hypertrophy, diabetes, non‐alcoholic fatty liver disease, and kidney disease. Epoxyeicosatrienoic acids can be further hydrolyzed to less active diols by the enzyme soluble epoxide hydrolase (sEH). Increasing evidence suggests that inhibition of sEH increases levels of EETs, which have anti‐inflammatory effects and can prevent the development of hypertension, atherosclerosis, heart failure, fatty liver, and multiple organ fibrosis. Arachidonic acid is the most abundant omega‐6 polyunsaturated fatty acid (PUFA) and shares the same set of enzymes with omega‐3 PUFAs, such as docosahexaenoic acid and eicosapentaenoic acid. The omega‐3 PUFAs and metabolites, such as regioisomeric epoxyeicosatetraenoic acids and epoxydocosapentaenoic acids, have been reported to have strong vasodilatory and anti‐inflammatory effects. Therefore, sEH may be a potential therapeutic target for metabolic disorders. In this review, we focus on our and other recent studies of the functions of sEH, including the effects of its eicosanoid products from both omega‐3 and omega‐6 PUFAs, in various metabolic diseases. We also discuss the possible cellular and molecular mechanisms underlying the regulation of sEH.

Soluble epoxide hydrolase is involved in the development of atherosclerosis and arterial neointima formation by regulating smooth muscle cell migration

Epoxyeicosatrienoic acids (EETs) have beneficial effects on cardiovascular disease. Soluble epoxide hydrolase (sEH) metabolizes EETs to less active diols, thus diminishing their biological activity. sEH inhibitors can suppress the progression of atherosclerotic lesions in animal models. However, the regulation of sEH in vascular smooth muscle cells (VSMCs) and role of sEH in patients with atherosclerosis have not been evaluated. We hypothesize that sEH in VSMCs plays a pivotal role in atherosclerosis and injury-induced neointima formation. In this study, sEH expression in human autopsy atherosclerotic plaque was determined by immunohistochemistry. In cultured rat and human VSMCs, the phenotypic switching marker and sEH expression induced by platelet-derived growth factor-BB (PDGF-BB) were examined by Western blot analysis. Carotid-artery balloon injury was performed after adenovirus-mediated overexpression of sEH or oral administration of a potent sEH inhibitor in Sprague-Dawley rats. sEH was highly expressed in VSMCs of the intima and media within human atherosclerotic plaque. In vitro, PDGF-BB upregulated the expression in VSMCs after transcription and promoted cell proliferation and migration; the latter effect could be largely attenuated by an sEH inhibitor. Adenovirus-mediated overexpression of sEH could mimic the effect of PDGF-BB and induce VSMC proliferation and migration. In vivo, the sEH inhibitor led to a significant decrease in injury-induced neointima formation in a rat carotid-artery injury model. These data establish the effect of sEH expression on atherosclerotic progression and vascular remodeling after injury, thus identifying a novel integrative role for sEH in VSMC phenotypic modulation and migration. Blocking sEH activity may be a potential therapeutic approach for ameliorating vascular occlusive disease.

Hyperhomocysteinemia activates the aryl hydrocarbon receptor/CD36 pathway to promote hepatic steatosis in mice

Hyperhomocysteinemia (HHcy) is associated with liver diseases such as fatty liver and hepatic fibrosis; however, the underlying mechanism is still largely unknown. The current study aimed to explore the signaling pathway involved in HHcy‐induced hepatic steatosis (HS). C57BL/6 mice were fed a high‐methionine diet (HMD) for 4 and 8 weeks to establish the HHcy mouse model. Compared to a chow diet, the HMD induced hepatic steatosis and elevated hepatic expression of CD36, a fatty acid transport protein. The increased CD36 expression was associated with activation of the aryl hydrocarbon receptor (AHR). In primary cultured hepatocytes, high levels of homocysteine (Hcy) treatment up‐regulated CD36 and increased subsequent lipid uptake; both were significantly attenuated by small interfering RNA (siRNA) knockdown of CD36 and AHR. Chromatin immunoprecipitation assay revealed that Hcy promoted binding of AHR to the CD36 promoter, and transient transfection assay demonstrated markedly increased activity of the AHR response element by Hcy, which was ligand dependent. Mass spectrometry revealed significantly increased hepatic content of lipoxin A4 (LXA4), a metabolite of arachidonic acid, in HMD‐fed mice. Furthermore, overexpression of 15‐oxoprostaglandin 13‐reductase 1, a LXA4 inactivation enzyme, inhibited Hcy‐induced AHR activation, lipid uptake, and lipid accumulation. Moreover, LXA4‐induced up‐regulation of CD36 and lipid uptake was inhibited by AHR siRNA in vitro in hepatocytes. Finally, treatment with an AHR antagonist reversed HHcy‐induced lipid accumulation by inhibiting the AHR‐CD36 pathway in mice. Conclusion: HHcy activates the AHR‐CD36 pathway by increasing hepatic LXA4 content, which results in hepatic steatosis. (Hepatology 2016;64:92‐105)

Inhibition of soluble epoxide hydrolase alleviated atherosclerosis by reducing monocyte infiltration in Ldlr(-/-) mice.

RATIONALE Circulating monocytes play pivotal roles in chronic inflammatory diseases. Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, are known to have anti-inflammatory effects and are hydrolyzed by soluble epoxide hydrolase (sEH). OBJECTIVE We aimed to investigate the effect of sEH inhibition in atherogenesis. METHODS AND RESULTS Mice with low-density lipoprotein receptor deficiency (Ldlr(-/-)) with or without sEH inhibitor, and Ldlr/sEH double-knockout (DK) mice were fed a Western-type diet (WTD) for 6weeks to induce arteriosclerosis. Both sEH inhibition and gene depletion decreased the WTD-induced hyperlipidemia, plaque area and macrophage infiltration in mice arterial wall. Ly6C(hi) infiltration of monocytes remained similar in blood, spleen and bone marrow of DK mice, but was decreased in aortic lesions. To further assess the role of sEH or EETs in monocyte/macrophage infiltration in atherogenesis, we transplanted DK bone marrow into Ldlr(-/-) recipients, and then fed mice the WTD. Aortic lesions and Ly6C(hi) monocyte infiltration were reduced in mice with transplanted bone marrow of DK mice without diminishing the cholesterol level. Furthermore, sEH inhibition or gene depletion increased the ratio of EETs/DHETs and diminished the expression of P-selectin glycoprotein ligand 1 (PSGL-1) in mice peripheral-blood mononuclear cells. Monocyte adhesion to P-selectin and to tumor necrosis factor α-activated endothelial cells was also diminished by sEH inhibition. CONCLUSION sEH inhibition and gene depletion attenuated atherosclerosis in mice by decreasing the infiltration of monocytes into the artery wall. EET and PSGL-1 may play pivotal roles in monocyte/macrophage infiltration and atherogenesis.

The role of Hippo/yes‐associated protein signalling in vascular remodelling associated with cardiovascular disease

Vascular remodelling is a vital process of a wide range of cardiovascular diseases and represents the altered structure and arrangement of blood vessels. The Hippo pathway controls organ size by regulating cell survival, proliferation and apoptosis. Yes‐associated protein (YAP), a transcription coactivator, is a downstream effector of the Hippo pathway. There is growing evidence for the importance of the Hippo/YAP pathway in vascular‐remodelling and related cardiovascular diseases. The Hippo/YAP pathway alters extracellular matrix production or degradation and the growth, death and migration of vascular smooth muscle cells and endothelial cells, which contributes to vascular remodelling in cardiovascular diseases such as pulmonary hypertension, atherosclerosis, restenosis, aortic aneurysms and angiogenesis. In this review, we summarize and discuss recent findings about the roles and mechanisms of Hippo/YAP signalling in vascular remodelling and related conditions.

Hydroxyeicosapentaenoic acids and epoxyeicosatetraenoic acids attenuate early occurrence of nonalcoholic fatty liver disease

The ω‐3 polyunsaturated fatty acids (PUFAs) mediate protective effects on several metabolic disorders. However, the functions of their metabolites in the early stage of nonalcoholic fatty liver disease (NAFLD) are largely unknown.

CYP2J2-derived EETs attenuated ethanol-induced myocardial dysfunction through inducing autophagy and reducing apoptosis

ABSTRACT Chronic excessive drinking leads to myocardial contractile dysfunction and dilated cardiomyopathy, where ethanol toxicity plays an essential role. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acids to form epoxyeicosatrienoic acids (EETs), which exert beneficial roles in the cardiovascular system, but their role in alcoholic cardiomyopathy is elusive. This study was designed to evaluate the effects and mechanisms of CYP2J2 gene delivery on ethanol‐induced myocardial dysfunction with focus on autophagy and apoptosis. C57BL/6 J mice were challenged with a 4% Lieber‐DeCarli ethanol liquid diet for 8 weeks, before which rAAV9‐CYP2J2 was injected via the tail vein. Cardiac function was assessed using echocardiography, hemodynamic measurement, and cardiac histology. The results showed that chronic ethanol intake led to cardiac dilation, contractile dysfunction, cardiomyocyte hypertrophy, oxidative stress, and cardiomyocyte apoptosis, while CYP2J2 overexpression ameliorated these effects. Additionally, chronic ethanol consumption triggered myocardial autophagosome formation, but impaired autophagic flux via disrupting autophagosome‐lysosome fusion, as evidenced by increased LC3 II/I, Beclin‐1 and SQSTM1 levels, but reduced LAMP‐2 expression. Interestingly, rAAV9‐CYP2J2 treatment exerted cardioprotection via restoring autophagic flux in the alcoholic myocardium. Similarly, exogenous 11,12‐EET addition significantly restored ethanol‐induced neonatal rat cardiomyocyte autophagic flux impairment and inhibited apoptosis, both of which were mediated by AMPK/mTOR signaling pathway in vitro. In conclusion, our data suggest that CYP2J2‐derived EETs attenuate ethanol‐induced myocardial dysfunction through inducing autophagy and reducing apoptosis. Graphical abstract Figure. No Caption available. HighlightsCYP2J2 gene delivery attenuate ethanol‐induced myocardial dysfunction and cardiac dilation.CYP2J2‐EETs restored ethanol‐induced autophagic flux impairment in cardiomyocyte via AMPK/mTOR signaling pathway.CYP2J2‐EETs inhibited ethanol‐induced apoptosis in cardiomyocyte via AMPK/mTOR signaling pathway.

Spectrum evaluation-assisted eicosanoid metabolomics for global eicosanoid profiling in human vascular endothelial cells

Eicosanoids are hundreds of metabolites derived from poly‐unsaturated fatty acids (PUFAs), which regulate biological processes from multiple angles via a complex metabolic network. Targeted eicosanoid metabolomics is used to study the eicosanoid profile in biological samples but only for eicosanoids with available standards. To expand the coverage of eicosanoids detected, we identified the eicosanoids without available standards by estimation of the retention time and comparison of the MS/MS spectra with the reference ones which was collected in a database from literature. Scheduled multiple reaction monitoring‐ information dependent acquisition‐ enhanced product ion (sMRM‐IDA‐EPI) scan mode was applied in this method, which was called Spectrum Evaluation‐assisted Eicosanoid Metabolomics (SEEM). By using this method, 243 eicosanoids (167 without standards) could be relatively quantified with precision over 90 percent. We applied the method to analyze the global profile of eicosanoids secreted by human umbilical vascular endothelial cells at the basal level and with n‐3 PUFA treatment. 26 putative eicosanoids showed altered levels, despite no available standards. In general, n‐3 PUFA treatment increased most of their own metabolites and decreased the epoxy‐, hydroxyl‐ and keto‐ linoleic acid metabolites. The application of the SEEM method proved its potency of identification and quantification of eicosanoids without standards.