Laura M. DeGraff

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.

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.

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

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.

Transcriptional profiling reveals a role for RORα in regulating gene expression in obesity-associated inflammation and hepatic steatosis

Retinoid-related orphan receptor (ROR)α4 is the major RORα isoform expressed in adipose tissues and liver. In this study we demonstrate that RORα-deficient staggerer mice (RORα(sg/sg)) fed with a high-fat diet (HFD) exhibited reduced adiposity and hepatic triglyceride levels compared with wild-type (WT) littermates and were resistant to the development of hepatic steatosis, adipose-associated inflammation, and insulin resistance. Gene expression profiling showed that many genes involved in triglyceride synthesis and storage, including Cidec, Cidea, and Mogat1, were expressed at much lower levels in liver of RORα(sg/sg) mice. In contrast, overexpression of RORα in mouse hepatoma Hepa1-6 cells significantly increased the expression of genes that were repressed in RORα(sg/sg) liver, including Sult1b1, Adfp, Cidea, and ApoA4. ChIP and promoter analysis suggested that several of these genes were regulated directly by RORα. In addition to reduced lipid accumulation, inflammation was greatly diminished in white adipose tissue (WAT) of RORα(sg/sg) mice fed with an HFD. The infiltration of macrophages and the expression of many immune response and proinflammatory genes, including those encoding various chemo/cytokines, Toll-like receptors, and TNF signaling proteins, were significantly reduced in RORα(sg/sg) WAT. Moreover, RORα(sg/sg) mice fed with an HFD were protected from the development of insulin resistance. RORα(sg/sg) mice consumed more oxygen and produced more carbon dioxide, suggesting increased energy expenditure in this genotype. Our study indicates that RORα plays a critical role in the regulation of several aspects of metabolic syndrome. Therefore, RORα may provide a novel therapeutic target in the management of obesity and associated metabolic diseases.

Increased blood pressure in mice lacking cytochrome P450 2J5

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis‐epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (‐/‐) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17β‐estradiol levels were reduced in female Cyp2j5 (‐/‐) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (‐/‐) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex‐specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen‐dependent mechanism.— Athirakul, K., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Ma, J., Zhao, Y., Couse, J. F., Quigley, R., Harder, D. R., Zhao, X., Imig, J. D., Pedersen, T. L., Newman, J. W., Hammock, B. D., Conley, A. J., Korach, K. S., Coffman, T. M., Zeldin, D. C. Increased blood pressure in mice lacking cytochrome P450 2J5. FASEB J. 22, 4096–4108 (2008)

Prostaglandin E2 protects murine lungs from bleomycin-induced pulmonary fibrosis and lung dysfunction

Prostaglandin E(2) (PGE(2)) is a lipid mediator that is produced via the metabolism of arachidonic acid by cyclooxygenase enzymes. In the lung, PGE(2) acts as an anti-inflammatory factor and plays an important role in tissue repair processes. Although several studies have examined the role of PGE(2) in the pathogenesis of pulmonary fibrosis in rodents, results have generally been conflicting, and few studies have examined the therapeutic effects of PGE(2) on the accompanying lung dysfunction. In this study, an established model of pulmonary fibrosis was used in which 10-12-wk-old male C57BL/6 mice were administered a single dose (1.0 mg/kg) of bleomycin via oropharyngeal aspiration. To test the role of prostaglandins in this model, mice were dosed, via surgically implanted minipumps, with either vehicle, PGE(2) (1.32 μg/h), or the prostacyclin analog iloprost (0.33 μg/h) beginning 7 days before or 14 days after bleomycin administration. Endpoints assessed at 7 days after bleomycin administration included proinflammatory cytokine levels and measurement of cellular infiltration into the lung. Endpoints assessed at 21 days after bleomycin administration included lung function assessment via invasive (FlexiVent) analysis, cellular infiltration, lung collagen content, and semiquantitative histological analysis of the degree of lung fibrosis (Ashcroft method). Seven days after bleomycin administration, lymphocyte numbers and chemokine C-C motif ligand 2 expression were significantly lower in PGE(2)- and iloprost-treated animals compared with vehicle-treated controls (P < 0.05). When administered 7 days before bleomycin challenge, PGE(2) also protected against the decline in lung static compliance, lung fibrosis, and collagen production that is associated with 3 wk of bleomycin exposure. However, PGE(2) had no therapeutic effect on these parameters when administered 14 days after bleomycin challenge. In summary, PGE(2) prevented the decline in lung static compliance and protected against lung fibrosis when it was administered before bleomycin challenge but had no therapeutic effect when administered after bleomycin challenge.

Functional characterization of cytochrome P450-derived epoxyeicosatrienoic acids in adipogenesis and obesity

Adipogenesis plays a critical role in the initiation and progression of obesity. Although cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have emerged as a potential therapeutic target for cardiometabolic disease, the functional contribution of EETs to adipogenesis and the pathogenesis of obesity remain poorly understood. Our studies demonstrated that induction of adipogenesis in differentiated 3T3-L1 cells (in vitro) and obesity-associated adipose expansion in high-fat diet (HFD)-fed mice (in vivo) significantly dysregulate the CYP epoxygenase pathway and evoke a marked suppression of adipose-derived EET levels. Subsequent in vitro experiments demonstrated that exogenous EET analog administration elicits potent anti-adipogenic effects via inhibition of the early phase of adipogenesis. Furthermore, EET analog administration to mice significantly mitigated HFD-induced weight gain, adipose tissue expansion, pro-adipogenic gene expression, and glucose intolerance. Collectively, these findings suggest that suppression of EET bioavailability in adipose tissue is a key pathological consequence of obesity, and strategies that promote the protective effects of EETs in adipose tissue offer enormous therapeutic potential for obesity and its downstream pathological consequences.