J. Alyce Bradbury

Enhanced Postischemic Functional Recovery in CYP2J2 Transgenic Hearts Involves Mitochondrial ATP-Sensitive K+ Channels and p42/p44 MAPK Pathway

Human CYP2J2 is abundant in heart and active in the biosynthesis of epoxyeicosatrienoic acids (EETs); however, the functional role of this P450 and its eicosanoid products in the heart remains unknown. Transgenic mice with cardiomyocyte-specific overexpression of CYP2J2 were generated. CYP2J2 transgenic (Tr) mice have normal heart anatomy and basal contractile function. CYP2J2 Tr hearts have improved recovery of left ventricular developed pressure (LVDP) compared with wild-type (WT) hearts after 20 minutes ischemia and 40 minutes reperfusion. Perfusion with the selective P450 epoxygenase inhibitor N-methylsulphonyl-6-(2-proparglyloxyphenyl)hexanamide (MS-PPOH) for 20 minutes before ischemia results in reduced postischemic LVDP recovery in WT hearts and abolishes the improved postischemic LVDP recovery in CYP2J2 Tr hearts. Perfusion with the ATP-sensitive K+ channel (KATP) inhibitor glibenclamide (GLIB) or the mitochondrial KATP (mitoKATP) inhibitor 5-hydroxydecanoate (5-HD) for 20 minutes before ischemia abolishes the cardioprotective effects of CYP2J2 overexpression. Flavoprotein fluorescence, a marker of mitoKATP activity, is higher in cardiomyocytes from CYP2J2 Tr versus WT mice. Moreover, CYP2J2-derived EETs (1 to 5 &mgr;mol/L) increase flavoprotein fluorescence in WT cardiomyocytes. CYP2J2 Tr mice exhibit increased expression of phospho-p42/p44 mitogen-activated protein kinase (MAPK) after ischemia, and addition of the p42/p44 MAPK kinase (MEK) inhibitor PD98059 during reperfusion abolishes the cardioprotective effects of CYP2J2 overexpression. Together, these data suggest that CYP2J2-derived metabolites are cardioprotective after ischemia, and the mechanism for this cardioprotection involves activation of mitoKATP and p42/p44 MAPK.

Inhibition of Vascular Smooth Muscle Cell Migration by Cytochrome P450 Epoxygenase-Derived Eicosanoids

Vascular smooth muscle cell (SMC) migration and proliferation contribute to neointimal hyperplasia and restenosis after vascular injury. The epoxyeicosatrienoic acids (EETs), which are products of cytochrome P450 (CYP) epoxygenases, possess vasodilatory, antiinflammatory, and fibrinolytic properties. To determine whether these compounds also possess antimigratory and antiproliferative properties, we stimulated rat aortic SMCs with either 20% serum or platelet-derived growth factor (PDGF-BB, 20 ng/mL). In a concentration-dependent manner, treatment with EETs, particularly 11,12-EET, inhibited SMC migration through a modified transwell filter by 53% to 60%. EETs, however, have no inhibitory effects on PDGF-stimulated SMC proliferation. Adenoviral-mediated overexpression of the CYP isoform, CYP2J2, in SMCs also inhibited serum- and PDGF-induced SMC migration by 32% and 26%, respectively; both effects of which were reversed by the CYP inhibitors SKF525A or clotrimazole, but not by the KCa channel blocker, charybdotoxin, or the cyclooxygenase inhibitor, diclofenac. The effect of EETs correlated with increases in intracellular cAMP levels. Indeed, forskolin and 8-bromo-cAMP exert similar inhibitory effects on SMC migration as 11,12-EET and the effects of 11,12-EET were blocked by cAMP and protein kinase A (PKA) inhibitors. These findings indicate that EETs possess antimigratory effects on SMCs through the cAMP-PKA pathway and suggest that CYP epoxygenase-derived eicosanoids may play important roles in vascular disease and remodeling.

Role of Soluble Epoxide Hydrolase in Postischemic Recovery of Heart Contractile Function

Cytochrome P450 epoxygenases metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (Ephx2, sEH). To examine the functional role of sEH in the heart, mice with targeted disruption of the Ephx2 gene were studied. Hearts from sEH null mice have undetectable levels of sEH mRNA and protein and cannot convert EETs to DHETs. sEH null mice have normal heart anatomy and basal contractile function, but have higher fatty acid epoxide:diol ratios in plasma and cardiomyocyte cell culture media compared with wild type (WT). sEH null hearts have improved recovery of left ventricular developed pressure (LVDP) and less infarction compared with WT hearts after 20 minutes ischemia. Perfusion with the putative EET receptor antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 to 100 nmol/L) before ischemia abolishes this cardioprotective phenotype. Inhibitor studies demonstrate that perfusion with phosphatidylinositol-3 kinase (PI3K) inhibitors wortmannin (200 nmol/L) or LY294002 (5 &mgr;mol/L), the ATP-sensitive K+ channel (KATP) inhibitor glibenclamide (1 &mgr;mol/L), the mitochondrial KATP (mitoKATP) inhibitor 5-hydroxydecanoate (100 to 200 &mgr;mol/L), or the Ca2+-sensitive K+ channel (KCa) inhibitor paxilline (10 &mgr;mol/L) abolishes the cardioprotection in sEH null hearts. Consistent with increased activation of the PI3K cascade, sEH null mice exhibit increased cardiac expression of glycogen synthase kinase-3&bgr; (GSK-3&bgr;) phospho-protein after ischemia. Together, these data suggest that targeted disruption of sEH increases the availability of cardioprotective EETs that work by activating PI3K signaling pathways and K+ channels.

Gender Differences in Murine Airway Responsiveness and Lipopolysaccharide-Induced Inflammation

The roles of gender and sex hormones in lung function and disease are complex and not completely understood. The present study examined the influence of gender on lung function and respiratory mechanics in naive mice and on acute airway inflammation and hyperresponsiveness induced by intratracheal LPS administration. Basal lung function characteristics did not differ between naive males and females, but males demonstrated significantly greater airway responsiveness than females following aerosolized methacholine challenge as evidenced by increased respiratory system resistance and elastance (p < 0.05). Following LPS administration, males developed more severe hypothermia and greater airway hyperresponsiveness than females (p < 0.05). Inflammatory indices including bronchoalveolar lavage fluid total cells, neutrophils, and TNF-α content were greater in males than in females 6 h following LPS administration (p < 0.05), whereas whole-lung TLR-4 protein levels did not differ among treatment groups, suggesting that differential expression of TLR-4 before or after LPS exposure did not underlie the observed inflammatory outcomes. Gonadectomy decreased airway inflammation in males but did not alter inflammation in females, whereas administration of exogenous testosterone to intact females increased their inflammatory responses to levels observed in intact males. LPS-induced airway hyperresponsiveness was also decreased in castrated males and was increased in females administered exogenous testosterone. Collectively, these data indicate that airway responsiveness in naive mice is influenced by gender, and that male mice have exaggerated airway inflammatory and functional responses to LPS compared with females. These gender differences are mediated, at least in part, by effects of androgens.

Detection of Human CYP2C8, CYP2C9 and CYP2J2 in Cardiovascular Tissues

The cytochrome P450 (P450) enzymes CYP2C8, CYP2C9, and CYP2J2 metabolize arachidonic acid to epoxyeicosatrienoic acids, which are known to be vital in regulation of vascular tone and cardiovascular homeostasis. Because there is limited information regarding the relative expression of these P450 enzymes in cardiovascular tissues, this study examined the expression of CYP2C8, CYP2C9, and CYP2J2 mRNA and protein in human heart, aorta, and coronary artery samples by real-time polymerase chain reaction, immunoblotting, and immunohistochemistry. CYP2J2 and CYP2C9 mRNA levels were highly variable in human hearts, whereas CYP2C8 mRNA was present in lower abundance. CYP2J2 mRNA was approximately 103 times higher than CYP2C9 or CYP2C8 in human heart. However, CYP2C9 mRNA was more abundant than CYP2J2 or CYP2C8 in one ischemic heart. In human aorta, mean CYP2C9 mRNA levels were ∼50 times higher than that of CYP2J2 and 5-fold higher than that of CYP2C8. In human coronary artery, mean values for CYP2C9 mRNA were ∼2-fold higher than that of CYP2J2 mRNA and 6-fold higher than that of CYP2C8 mRNA. Immunoblotting results show relatively high levels of CYP2J2 and CYP2C8 protein in human hearts, which was confirmed by immunohistochemistry. CYP2C9 protein was also detected at high levels in one ischemic heart by immunoblotting. CYP2C9 was present at higher levels than CYPJ2 in aorta and coronary artery, whereas CYP2C8 protein was below the limits of detection. The expression of CYP2J2 and CYP2C8 in human heart, and CYPC9 and CYP2J2 in aorta and coronary artery is consistent with a physiological role for these enzymes in these tissues.

Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension-induced renal injury in mice

Renal cytochrome P450 (CYP)‐derived epoxyeicosatrienoic acids (EETs) regulate sodium transport and blood pressure. Although endothelial CYP‐derived EETs are potent vasodilators, their contribution to the regulation of blood pressure remains unclear. Consequently, we developed transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases to increase endothelial EET biosynthesis. Compared to wild‐type littermate controls, an attenuated afferent arteriole constrictor response to endothelin‐1 and enhanced dilator response to acetylcholine was observed in CYP2J2 and CYP2C8 transgenic mice. CYP2J2 and CYP2C8 transgenic mice demonstrated modestly, but not significantly, lower mean arterial pressure under basal conditions compared to wild‐type controls. However, mean arterial pressure was significantly lower in both CYP2J2 and CYP2C8 transgenic mice during coadministration of N‐nitro‐l‐arginine methyl ester and indomethacin. In a separate experiment, a high‐salt diet and subcutaneous angiotensin II was administered over 4 wk. The angiotensin/high‐salt‐induced increase in systolic blood pressure, proteinuria, and glomerular injury was significantly attenuated in CYP2J2 and CYP2C8 transgenic mice compared to wild‐type controls. Collectively, these data demonstrate that increased endothelial CYP epoxygenase expression attenuates afferent arteriolar constrictor reactivity and hypertension‐induced increases in blood pressure and renal injury in mice. We conclude that endothelial CYP epoxygenase function contributes to the regulation of blood pressure.—Lee, C. R., Imig, J. D., Edin, M. E., Foley, J., DeGraff, L. M., Bradbury, J. A., Graves, J. P., Lih, F. B., Clark, J., Myers, P., Perrow, A. L., Lepp, A. N., Kannon, M. A., Ronnekleiv, O. K., Alkayed, N.J., Falck, J. R., Tomer, K B., Zeldin, D. C. Endothelial expression of human cytochrome P450 epoxygenases lowers blood pressure and attenuates hypertension‐induced renal injury in mice. FASEB J. 24, 3770–3781 (2010). www.fasebj.org

Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice

Cytochrome P‐450 (CYP)‐derived epoxyei‐cosatrienoic acids (EETs) possess potent anti‐inflammatory effects in vitro. However, the effect of increased CYP‐mediated EET biosynthesis and decreased soluble epoxide hydrolase (sEH, Ephx2)‐mediated EET hydrolysis on vascular inflammation in vivo has not been rigorously investigated. Consequently, we characterized acute vascular inflammatory responses to endotoxin in transgenic mice with endothelial expression of the human CYP2J2 and CYP2C8 epoxygenases and mice with targeted disruption of Ephx2. Compared to wild‐type controls, CYP2J2 transgenic, CYP2C8 transgenic, and Ephx2−/− mice each exhibited a significant attenuation of endotoxin‐induced activation of nuclear factor (NF)‐κB signaling, cellular adhesion molecule, chemokine and cytokine expression, and neutrophil infiltration in lung in vivo. Furthermore, attenuation of endotoxin‐induced NF‐κB activation and cellular adhesion molecule and chemokine expression was observed in primary pulmonary endothelial cells isolated from CYP2J2 and CYP2C8 transgenic mice. This attenuationwas inhibited bya putative EET receptor antagonist and CYP epoxygenase inhibitor, directly implicating CYP epoxygenase‐derived EETs with the observed anti‐inflammatory phenotype. Collectively, these data demonstrate that potentiation of the CYP epoxygenase pathway by either increased endothelial EET biosynthesis or globally decreased EET hydrolysis attenuates NF‐κB‐dependent vascular inflammatory responses in vivo and may serve as a viable anti‐inflammatory therapeutic strategy.—Deng, Y., Edin, M. L., Theken, K N., Schuck, R N., Flake, G. P., Kannon, M. A., DeGraff, L. M., Lih, F. B., Foley, J., Bradbury, J. A., Graves, J. P., Tomer, K. B., Falck, J. R., Zeldin, D. C., Lee, C. R. Endothelial CYP epoxygenase overexpression and soluble epoxide hydrolase disruption attenuate acute vascular inflammatory responses in mice. FASEB J. 25, 703–713 (2011). www.fasebj.org

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.

Cardiac and vascular KATP channels in rats are activated by endogenous epoxyeicosatrienoic acids through different mechanisms

We have reported that epoxyeicosatrienoic acids (EETs), the cytochrome P450 (CYP) epoxygenase metabolites of arachidonic acid (AA), are potent sarcolemmal ATP‐sensitive K+ (KATP) channel activators. However, activation of cardiac and vascular KATP channels by endogenously produced EETs under physiological intracellular conditions has not been demonstrated and direct comparison of the mechanisms whereby EETs activate the KATP channels in cardiac myocytes versus vascular smooth muscle cells has not been made. In this study, we examined the effects of AA on KATP channels in freshly isolated cardiac myocytes from rats, wild‐type (WT) and transgenic mice overexpressing CYP2J2 cDNA, and mesenteric arterial smooth muscle cells from rats. We also compared the activation of cardiac and vascular KATP channels by extracellularly and intracellularly applied 11,12‐EET. We found that 1 μm AA enhanced KATP channel activities in both cardiac and vascular smooth muscle cells, and the AA effects were inhibited by preincubation with CYP epoxygenase inhibitors. Baseline cardiac KATP current densities in CYP2J2 transgenic mice were 190% higher than those of WT mice, and both were reduced to similar levels by CYP epoxygenase inhibition. Western blot analysis showed that expression of Kir6.2 and SUR2A was similar between WT and CYP2J2 transgenic hearts. 11,12‐EET (5 μm) applied intracellularly enhanced the KATP currents by 850% in cardiac myocytes, but had no effect in vascular smooth muscle cells. In contrast, 11,12‐EET (5 μm) applied extracellularly increased KATP currents by 520% in mesenteric arterial smooth muscle cells, but by only 209% in cardiac myocytes. Preincubation with 100 μmm‐iodobenzylguanidine or 5 μm myristoylated PKI amide did not alter the activation of cardiac KATP channels by 5 μm 11,12‐EET, but significantly inhibited activation of vascular KATP channels. Moreover, EET only enhanced the inward component of cardiac KATP currents, but activated both the inward and outward components of vascular KATP currents. Our results indicate that endogenously derived CYP metabolites of AA potently activate cardiac and vascular KATP channels. EETs regulate cardiac electrophysiology and vascular tone by KATP channel activation, albeit through different mechanisms: the cardiac KATP channels are directly activated by EETs, whereas activation of the vascular KATP channels by EETs is protein kinase A dependent.