Basal and inducible anti-inflammatory epoxygenase activity in endothelial cells

The roles of CYP lipid-metabolizing pathways in endothelial cells are poorly understood. Human endothelial cells expressed CYP2J2 and soluble epoxide hydrolase (sEH) mRNA and protein. The TLR-4 agonist LPS (1 μg/ml; 24 h) induced CYP2J2 but not sEH mRNA and protein. LC–MS/MS analysis of the stable commonly used human endothelial cell line EA.Hy926 showed active epoxygenase and epoxide hydrolase activity: with arachidonic acid (stable epoxide products 5,6-DHET, and 14,15-DHET), linoleic acid (9,10-EPOME and 12,13-EPOME and their stable epoxide hydrolase products 9,10-DHOME and 12,13-DHOME), docosahexaenoic acid (stable epoxide hydrolase product 19,20-DiHDPA) and eicosapentaenoic acid (stable epoxide hydrolase product 17,18-DHET) being formed. Inhibition of epoxygenases using either SKF525A or MS-PPOH induced TNFα release, but did not affect LPS, IL-1β, or phorbol-12-myristate-13-acetate (PMA)-induced TNFα release. In contrast, inhibition of soluble epoxide hydrolase by AUDA or TPPU inhibited basal, LPS, IL-1β and PMA induced TNFα release, and LPS-induced NFκB p65 nuclear translocation. In conclusion, human endothelial cells contain a TLR-4 regulated epoxygenase CYP2J2 and metabolize linoleic acid > eicosapentaenoic acid > arachidonic acid > docosahexaenoic acid to products with anti-inflammatory activity

Effect of Soluble Epoxide Hydrolase on the Modulation of Coronary Reactive Hyperemia: Role of Oxylipins and PPARγ

Coronary reactive hyperemia (CRH) is a physiological response to ischemic insult that prevents the potential harm associated with an interruption of blood supply. The relationship between the pharmacologic inhibition of soluble epoxide hydrolase (sEH) and CRH response to a brief ischemia is not known. sEH is involved in the main catabolic pathway of epoxyeicosatrienoic acids (EETs), which are converted into dihydroxyeicosatrienoic acids (DHETs). EETs protect against ischemia/reperfusion injury and have numerous beneficial physiological effects. We hypothesized that inhibition of sEH by t-AUCB enhances CRH in isolated mouse hearts through changing the oxylipin profiles, including an increase in EETs/DHETs ratio. Compared to controls, t-AUCB–treated mice had increased CRH, including repayment volume (RV), repayment duration, and repayment/debt ratio (p \u3c 0.05). Treatment with t-AUCB significantly changed oxylipin profiles, including an increase in EET/DHET ratio, increase in EpOME/DiHOME ratio, increase in the levels of HODEs, decrease in the levels of mid-chain HETEs, and decrease in prostanoids (p \u3c 0.05). Treatment with MS-PPOH (CYP epoxygenase inhibitor) reduced CRH, including RV (p \u3c 0.05). Involvement of PPARγ in the modulation of CRH was demonstrated using a PPARγ-antagonist (T0070907) and a PPARγ-agonist (rosiglitazone). T0070907 reduced CRH (p \u3c 0.05), whereas rosiglitazone enhanced CRH (p \u3c 0.05) in isolated mouse hearts compared to the non-treated. These data demonstrate that sEH inhibition enhances, whereas CYP epoxygenases-inhibition attenuates CRH, PPARγ mediate CRH downstream of the CYP epoxygenases-EET pathway, and the changes in oxylipin profiles associated with sEH-inhibition collectively contributed to the enhanced CRH

Vascular Endothelial Over-Expression of Human Soluble Epoxide Hydrolase (Tie2-sEH Tr) Attenuates Coronary Reactive Hyperemia in Mice: Role of Oxylipins and ω-Hydroxylases

Cytochromes P450 metabolize arachidonic acid (AA) into two vasoactive oxylipins with opposing biologic effects: epoxyeicosatrienoic acids (EETs) and omega-(ω)-terminal hydroxyeicosatetraenoic acids (HETEs). EETs have numerous beneficial physiological effects, including vasodilation and protection against ischemia/reperfusion injury, whereas ω-terminal HETEs induce vasoconstriction and vascular dysfunction. We evaluated the effect of these oxylipins on post-ischemic vasodilation known as coronary reactive hyperemia (CRH). CRH prevents the potential harm associated with transient ischemia. The beneficial effects of EETs are reduced after their hydrolysis to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). ω-terminal HETEs are formed by ω-hydroxylase family members. The relationship among endothelial over-expression of sEH (Tie2-sEH Tr), the changes in oxylipins it may produce, the pharmacologic inhibition of ω-hydroxylases, activation of PPARγ, and CRH response to a brief ischemia is not known. We hypothesized that CRH is attenuated in isolated mouse hearts with endothelial sEH over-expression through modulation of oxylipin profiles, whereas both inhibition of ω-hydroxylases and activation of PPARγ enhance CRH. Compared to WT mice, Tie2-sEH Tr mice had decreased CRH, including repayment volume, repayment duration, and repayment/debt ratio (P \u3c 0.05), whereas inhibition of ω-hydroxylases increased these same CRH parameters in Tie2-sEH Tr mice. Inhibition of sEH with t-AUCB reversed the decreased CRH in Tie2-sEH Tr mice. Endothelial over-expression of sEH significantly changed oxylipin profiles, including decreases in DHETs, mid-chain HETEs, and prostaglandins (P \u3c 0.05). Treatment with rosiglitazone, PPARγ-agonist, enhanced CRH (P \u3c 0.05) in both Tie2-sEH Tr and wild type (WT) mice. These data demonstrate that endothelial over-expression of sEH (through changing the oxylipin profiles) attenuates CRH, whereas inhibition of ω-hydroxylases and activation of PPARγ enhance it

Intimal smooth muscle cells are a source but not a sensor of anti-inflammatory CYP450 derived oxylipins

AbstractVascular pathologies are associated with changes in the presence and expression of morphologically distinct vascular smooth muscle cells. In particular, in complex human vascular lesions and models of disease in pigs and rodents, an intimal smooth muscle cell (iSMC) which exhibits a stable epithelioid or rhomboid phenotype in culture is often found to be present in high numbers, and may represent the reemergence of a distinct developmental vascular smooth muscle cell phenotype. The CYP450-oxylipin - soluble epoxide hydrolase (sEH) pathway is currently of great interest in targeting for cardiovascular disease. sEH inhibitors limit the development of hypertension, diabetes, atherosclerosis and aneurysm formation in animal models. We have investigated the expression of CYP450-oxylipin-sEH pathway enzymes and their metabolites in paired intimal (iSMC) and medial (mSMC) cells isolated from rat aorta. iSMC basally released significantly larger amounts of epoxy-oxylipin CYP450 products from eicosapentaenoic acid > docosahexaenoic acid > arachidonic acid > linoleic acid, and expressed higher levels of CYP2C12, CYP2B1, but not CYP2J mRNA compared to mSMC. When stimulated with the pro-inflammatory TLR4 ligand LPS, epoxy-oxylipin production did not change greatly in iSMC. In contrast, LPS induced epoxy-oxylipin products in mSMC and induced CYP2J4. iSMC and mSMC express sEH which metabolizes primary epoxy-oxylipins to their dihydroxy-counterparts. The sEH inhibitors TPPU or AUDA inhibited LPS-induced NFκB activation and iNOS induction in mSMC, but had no effect on NFκB nuclear localization or inducible nitric oxide synthase in iSMC; effects which were recapitulated in part by addition of authentic epoxy-oxylipins. iSMCs are a rich source but not a sensor of anti-inflammatory epoxy-oxylipins. Complex lesions that contain high levels of iSMCs may be more resistant to the protective effects of sEH inhibitors

The cytochrome p450 epoxygenase pathway regulates the hepatic inflammatory response in fatty liver disease

Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted