John M. Seubert

Contrasting effects of cyclooxygenase-1 (COX-1) and COX-2 deficiency on the host response to influenza A viral infection.

Influenza is a significant cause of morbidity and mortality worldwide despite extensive research and vaccine availability. The cyclooxygenase (COX) pathway is important in modulating immune responses and is also a major target of nonsteroidal anti-inflammatory drugs (NSAIDs) and the newer COX-2 inhibitors. The purpose of the present study was to examine the effect of deficiency of COX-1 or COX-2 on the host response to influenza. We used an influenza A viral infection model in wild type (WT), COX-1−/−, and COX-2−/− mice. Infection induced less severe illness in COX-2−/− mice in comparison to WT and COX-1−/− mice as evidenced by body weight and body temperature changes. Mortality was significantly reduced in COX-2−/− mice. COX-1−/− mice had enhanced inflammation and earlier appearance of proinflammatory cytokines in the BAL fluid, whereas the inflammatory and cytokine responses were blunted in COX-2−/− mice. However, lung viral titers were markedly elevated in COX-2−/− mice relative to WT and COX-1−/− mice on day 4 of infection. Levels of PGE2 were reduced in COX-1−/− airways whereas cysteinyl leukotrienes were elevated in COX-2−/− airways following infection. Thus, deficiency of COX-1 and COX-2 leads to contrasting effects in the host response to influenza infection, and these differences are associated with altered production of prostaglandins and leukotrienes following infection. COX-1 deficiency is detrimental whereas COX-2 deficiency is beneficial to the host during influenza viral infection.

Overexpression of CYP2J2 provides protection against doxorubicin-induced cardiotoxicity

Human cytochrome P-450 (CYP)2J2 is abundant in heart and active in biosynthesis of epoxyeicosatrienoic acids (EETs). Recently, we demonstrated that these eicosanoid products protect myocardium from ischemia-reperfusion injury. The present study utilized transgenic (Tr) mice with cardiomyocyte-specific overexpression of human CYP2J2 to investigate protection toward toxicity resulting from acute (0, 5, or 15 mg/kg daily for 3 days, followed by 24-h recovery) or chronic (0, 1.5, or 3.0 mg/kg biweekly for 5 wk, followed by 2-wk recovery) doxorubicin (Dox) administration. Acute treatment resulted in marked elevations of serum lactate dehydrogenase and creatine kinase levels that were significantly greater in wild-type (WT) than CYP2J2 Tr mice. Acute treatment also resulted in less activation of stress response enzymes in CYP2J2 Tr mice (catalase 750% vs. 300% of baseline, caspase-3 235% vs. 165% of baseline in WT vs. CYP2J2 Tr mice). Moreover, CYP2J2 Tr hearts exhibited less Dox-induced cardiomyocytes apoptosis (measured by TUNEL) compared with WT hearts. After chronic treatment, comparable decreases in body weight were observed in WT and CYP2J2 Tr mice. However, cardiac function, assessed by measurement of fractional shortening with M-mode transthoracic echocardiography, was significantly higher in CYP2J2 Tr than WT hearts after chronic Dox treatment (WT 37 +/- 2%, CYP2J2 Tr 47 +/- 1%). WT mice also had larger increases in beta-myosin heavy chain and cardiac ankryin repeat protein compared with CYP2J2 Tr mice. CYP2J2 Tr hearts had a significantly higher rate of Dox metabolism than WT hearts (2.2 +/- 0.25 vs. 1.6 +/- 0.50 ng.min(-1).100 microg protein(-1)). In vitro data from H9c2 cells demonstrated that EETs attenuated Dox-induced mitochondrial damage. Together, these data suggest that cardiac-specific overexpression of CYP2J2 limited Dox-induced toxicity.

Epoxyeicosatrienoic acids limit damage to mitochondrial function following stress in cardiac cells

Epoxyeicosatrienoic acids (EETs) are polyunsaturated fatty acids synthesized from arachidonic acid by CYP2J2 epoxygenase and inactivated by soluble epoxide hydrolase (sEH or Ephx2) to dihydroxyeicosatrienoic acids. Mitochondrial function following ischemic insult is a critical determinant of reperfusion-induced cell death in the myocardium. The objectives of the current study were to investigate the protective role of EETs in mitochondrial function. Mice with the targeted disruption of the Ephx2 gene, cardiomyocyte-specific overexpression of CYP2J2 or perfused with EETs all have improved postischemic LVDP recovery compared to wild-type (WT). Perfusion with the mPTP opener, atractyloside, abolished the improved postischemic functional recovery observed in CYP2J2 Tr, sEH null and EET perfused hearts. Electron micrographs demonstrated WT hearts to have increased mitochondrial fragmentation and T-tubule swelling compared to CYP2J2 Tr hearts following 20 min global ischemia and 20 min reperfusion. Direct effects of EETs on mitochondria were assessed in isolated rat cardiomyocytes and H9c2 cells. Laser-induced loss of mitochondrial membrane potential (DeltaPsi(m)) and mPTP opening was significantly reduced in cells treated with 14, 15-EET (1 microM). The EET protective effect was blocked by the putative EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (1 muM, 14, 15-EEZE), paxilline (10 microM, BK(Ca) inhibitor) and 5HD (100 microM, K(ATP) inhibitor). Our studies show that EETs can limit mitochondrial dysfunction following cellular stress via a K(+) channel-dependent mechanism.

Epoxyeicosatrienoic acid prevents postischemic electrocardiogram abnormalities in an isolated heart model.

Cytochrome P450 epoxygenases metabolize arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) which are in turn converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). The main objective of this study was to investigate the protective effects of EETs following ischemic injury using an ex vivo electrocardiogram (EKG) model. Hearts from C57Bl/6, transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 (Tr) and wildtype (WT) littermates were excised and perfused with constant pressure in a Langendorff apparatus. Electrodes were placed superficially at the right atrium and left ventricle to assess EKG waveforms. In ischemic reperfusion experiments hearts were subjected to 20 min of global no-flow ischemia followed by 20 min of reperfusion (R20). The EKG from C57Bl/6 hearts perfused with 1 microM 14,15-EET showed less QT prolongation (QTc) and ST elevation (STE) (QTc=41+/-3, STE=2.3+/-0.3; R20: QTc=42+/-2 ms, STE=1.2+/-0.2mv) than control hearts (QTc=36+/-2, STE=2.3+/-0.2; R20: QTc=53+/-3 ms; STE=3.6+/-0.4mv). Similar results of reduced QT prolongation and ST elevation were observed in EKG recording from CYP2J2 Tr mice (QTc=35+/-1, STE=1.9+/-0.1; R20: QTc=38+/-4 ms, STE=1.3+/-0.2mv) compared to WT hearts. The putative epoxygenase inhibitor MS-PPOH (50 microM) and EET antagonist 14,15-EEZE (10 microM) both abolished the cardioprotective response, implicating EETs in this process. In addition, separate exposure to the K(ATP) channel blockers glibenclamide (1 microM) and HMR1098 (10 microM), or the PKA protein inhibitor H89 (50 nM) during reperfusion abolished the improved repolarization in both the models. Consistent with a role of PKA, CYP2J2 Tr mice had an enhanced activation of the PKAalpha regulatory II subunit in plasma membrane following IR injury. The present data demonstrate that EETs can enhance the recovery of ventricular repolarization following ischemia, potentially by facilitating activation of K(+) channels and PKA-dependent signaling.

Inhibition of Soluble Epoxide Hydrolase by trans-4- ½4-(3-adamantan-1-yl-ureido)-cyclohexyloxy-benzoic acid Is Protective Against Ischemia-Reperfusion Injury

Arachidonic acid, a polyunsaturated fatty acid, can be metabolized to cardioprotective epoxyeicosatrienoic acids (EETs) by cytochrome P450 epoxygenases, which are subsequently hydrolyzed to less bioactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). To study the effects of pharmacological inhibitor of sEH (sEHi), C57BL6 mice hearts were perfused in Langendorff mode for 40 minutes of baseline and subjected to 30 minutes of global no-flow ischemia followed by 40 minutes of reperfusion. Hearts were perfused with the sEHi, trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 0.05, 0.1, 0.5, and 1 μM). To study the mechanism(s), hearts were perfused with 0.1 μM t-AUCB in the presence or absence of putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM) or phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin (200 nM) or LY294002 (5 μM). Infarct size was determined at the end of 2-hour reperfusion by 2,3,5-triphenyltetrazolium chloride staining. Inhibition of sEH by t-AUCB significantly improved postischemic left ventricular developed pressure (LVDP) recovery and reduced the infarct size after ischemia and reperfusion, as compared with control hearts. Perfusion with 14,15-epoxyeicosa-5(Z)-enoic acid, wortmannin or LY294002 before ischemia abolished the cardioprotective phenotype; however, coperfusion of both t-AUCB and 11,12-EET did not result in an additive effect on improved LVDP recovery. Together, our data suggest that pharmacological inhibition of sEH by t-AUCB is cardioprotective.

Cardioprotective effect of a dual acting epoxyeicosatrienoic acid analogue towards ischaemia reperfusion injury

BACKGROUND AND PURPOSE Epoxyeicosatrienoic acids (EETs) are cytochrome P450 epoxygenase metabolites of arachidonic acid that are metabolized into dihydroxyepoxyeicosatrienoic acids (DHET) by soluble epoxide hydrolase (sEH). The current investigations were performed to examine the cardioprotective effects of UA‐8 (13‐(3‐propylureido)tridec‐8‐enoic acid), a synthetic compound that possesses both EET‐mimetic and sEH inhibitory properties, against ischaemia‐reperfusion injury.

Cytochrome P450 Enzymes and the Heart

The cytochrome P450 monooxygenase system (CYP) is a multigene superfamily of heme‐thiolate enzymes, which are important in the metabolism of foreign and endogenous compounds. Genetic variations, drug interactions, or pathophysiological factors can lead to reduced, absent, or increased enzymatic activity. This altered CYP activity greatly influences an individuals response to therapeutic treatment. What is not known is the impact of these changes on the many functional roles of CYP in physiological and pathophysiological processes of the heart. Many extrahepatic tissues, like heart, contain active P450 enzymes but lack information regarding their role in cellular injury or homeostasis. Much of our current knowledge about cardiac CYP has been limited to studies investigating the role of fatty acid metabolites in heart. Traditional risk factors including diabetes, smoking, and hypertension have well established links to cardiovascular disease. And new evidence strongly suggests exposure to chemicals and other environmental agents has a profound impact on the cardiovascular system. These risk factors can independently affect the expression and activity of CYP enzymes. Therefore, altered CYP activity is important from a detoxification as well as a bioactivation perspective. Considering CYP, interactions are greatly dependent on inherited differences or acquired changes in enzyme activity further research into their potential impact on pathogenesis, risk assessment, and therapy of heart disease is warranted. This review explores the expression of CYP isoforms, their functional roles, and the effects of genetic variation in the heart.

The effect of Nrf2 knockout on the constitutive expression of drug metabolizing enzymes and transporters in C57Bl/6 mice livers.

Previous reports have proposed a cross-talk between the nuclear factor erythroid-2 p45-related factor-2 (Nrf2)/antioxidant response element (ARE) and the aryl hydrocarbon receptor (AhR)/xenobiotic response element (XRE) signaling pathways. Therefore, the aim of the current study was to examine the level of phase I, phase II drug metabolizing enzymes (DMEs), and phase III transporters and their related transcription factors in the Nrf2 knockout model. Our results showed that phase II DMEs that are under the control of Nrf2 typified by NAD(P)H: quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase (Gst) were significantly lower at the mRNA, protein, and catalytic activity levels in the livers of Nrf2 knockout mice compared to wild type. Furthermore, phase I cytochrome P450s (CYPs), Cyp1, and Cyp2b10 at mRNA, protein, and catalytic activity levels were significantly lower in the livers of Nrf2 knockout mice. Interestingly, our results showed that the transcription factors AhR, constitutive androstane receptor (CAR), and pregnane X receptor (PXR) at mRNA, and protein expression levels were significantly lower in the livers of Nrf2 knockout mice compared to wild type. Importantly, phase III drug transporters mRNA levels of the multiple drug resistance associated proteins (Mrp2 and Mrp3), and solute carrier organic anion transporters (Slco1a6 and Slco2b1) were significantly lower in the liver of Nrf2 knockout mice. Co-activators, Ncoa1, Ncoa2, and Ncoa3 mRNA levels were not altered while co-repressors, Ncor1 and Ncor2 were significantly lower in the livers of Nrf2 knockout mice. In conclusion, knockout of Nrf2 causes disruption to the coordination of phase I, phase II drug DMEs, and phase III drug transporters through altering the transcription factors controlling them.

Role of B-type natriuretic peptide in epoxyeicosatrienoic acid-mediated improved post-ischaemic recovery of heart contractile function

AIMS This study examined the functional role of B-type natriuretic peptide (BNP) in epoxyeicosatrienoic acid (EET)-mediated cardioprotection in mice with targeted disruption of the sEH or Ephx2 gene (sEH null). METHODS AND RESULTS Isolated mouse hearts were perfused in the Langendorff mode and subjected to global no-flow ischaemia followed by reperfusion. Hearts were analysed for recovery of left ventricular developed pressure (LVDP), mRNA levels, and protein expression. Naïve hearts from sEH null mice had similar expression of preproBNP (Nppb) mRNA compared with wild-type (WT) hearts. However, significant increases in Nppb mRNA and BNP protein expression occurred during post-ischaemic reperfusion and correlated with improved post-ischaemic recovery of LVDP. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid prior to ischaemia reduced the preproBNP mRNA in sEH null hearts. Inhibitor studies demonstrated that perfusion with the natriuretic peptide receptor type-A (NPR-A) antagonist, A71915, limited the improved recovery in recombinant full-length mouse BNP (rBNP)- and 11,12-EET-perfused hearts as well as in sEH null mice. Increased expression of phosphorylated protein kinase C epsilon and Akt were found in WT hearts perfused with either 11,12-EET or rBNP, while mitochondrial glycogen synthase kinase-3beta was significantly lower in the same samples. Furthermore, treatment with the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin abolished improved LVDP recovery in 11,12-EET-treated hearts but not did significantly inhibit recovery of rBNP-treated hearts. CONCLUSION Taken together, these data indicate that EET-mediated cardioprotection involves BNP and PI3K signalling events.

Epoxyeicosatrienoic acids and cardioprotection: The road to translation

Cardiovascular disease, including acute myocardial infarction (AMI), is the leading cause of morbidity and mortality globally, despite well-established treatments. The discovery and development of novel therapeutics that prevent the progression of devastating consequences following AMI are thus important in reducing the global burden of this devastating disease. Scientific evidence for the protective effects of epoxyeicosatrienoic acids (EETs) in the cardiovascular system is rapidly emerging and suggests that promoting the effects of these cytochrome P450-derived epoxyeicosanoids is a potentially viable clinical therapeutic strategy. Through a translational lens, this review will provide insight into the potential clinical utility of this therapeutic strategy for AMI by 1) outlining the known cardioprotective effects of EETs and underlying mechanisms demonstrated in preclinical models of AMI with a particular focus on myocardial ischemia-reperfusion injury, 2) describing studies in human cohorts that demonstrate a relationship between EETs and associated pathways with coronary artery disease risk, and 3) discussing preclinical and clinical areas that require further investigation in order to increase the probability of successfully translating this rapidly emerging body of evidence into a clinically applicable therapeutic strategy for AMI.