RESUMEN
Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EET-induced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes.
Asunto(s)
Miocardio , Miocitos Cardíacos , Ratones , Animales , Miocitos Cardíacos/metabolismo , Miocardio/metabolismo , Isquemia/metabolismo , Eicosanoides/metabolismo , Reperfusión , Hidrolasas/metabolismo , Epóxido Hidrolasas/metabolismoRESUMEN
Human and animal studies support that consuming a high level of linoleic acid (LA, 18:2ω-6), an essential fatty acid and key component of the human diet, increases the risk of colon cancer. However, results from human studies have been inconsistent, making it challenging to establish dietary recommendations for optimal LA intake. Given the importance of LA in the human diet, it is crucial to better understand the molecular mechanisms underlying its potential colon cancer-promoting effects. Using LC-MS/MS-based targeted lipidomics, we find that the cytochrome P450 (CYP) monooxygenase pathway is a major pathway for LA metabolism in vivo. Furthermore, CYP monooxygenase is required for the colon cancer-promoting effects of LA, since the LA-rich diet fails to exacerbate colon cancer in CYP monooxygenase-deficient mice. Finally, CYP monooxygenase mediates the pro-cancer effects of LA by converting LA to epoxy octadecenoic acids (EpOMEs), which have potent effects on promoting colon tumorigenesis via gut microbiota-dependent mechanisms. Overall, these results support that CYP monooxygenase-mediated conversion of LA to EpOMEs plays a crucial role in the health effects of LA, establishing a unique mechanistic link between dietary fatty acid intake and cancer risk. These results could help in developing more effective dietary guidelines for optimal LA intake and identifying subpopulations that may be especially vulnerable to LA's negative effects.
Asunto(s)
Neoplasias del Colon , Ácido Linoleico , Humanos , Ratones , Animales , Ácido Linoleico/farmacología , Ácido Linoleico/metabolismo , Cromatografía Liquida , Espectrometría de Masas en Tándem , Eicosanoides , Sistema Enzimático del Citocromo P-450/metabolismo , Dieta , Neoplasias del Colon/etiologíaRESUMEN
PURPOSE: Over 550 loci have been associated with human pulmonary function in genome-wide association studies (GWAS); however, the causal role of most remains uncertain. Single nucleotide polymorphisms in a disintegrin and metalloprotease domain 19 (ADAM19) are consistently related to pulmonary function in GWAS. Thus, we used a mouse model to investigate the causal link between Adam19 and pulmonary function. METHODS: We created an Adam19 knockout (KO) mouse model and validated the gene targeting using RNA-Seq and RT-qPCR. Mouse body composition was assessed using dual-energy X-ray absorptiometry. Mouse lung function was measured using flexiVent. RESULTS: Contrary to prior publications, the KO was not neonatal lethal. KO mice had lower body weight and shorter tibial length than wild-type (WT) mice. Their body composition revealed lower soft weight, fat weight, and bone mineral content. Adam19 KO had decreased baseline respiratory system elastance, minute work of breathing, tissue damping, tissue elastance, and forced expiratory flow at 50% forced vital capacity but higher FEV0.1 and FVC. Adam19 KO had attenuated tissue damping and tissue elastance in response to methacholine following LPS exposure. Adam19 KO also exhibited attenuated neutrophil extravasation into the airway after LPS administration compared to WT. RNA-Seq analysis of KO and WT lungs identified several differentially expressed genes (Cd300lg, Kpna2, and Pttg1) implicated in lung biology and pathogenesis. Gene set enrichment analysis identified negative enrichment for TNF pathways. CONCLUSION: Our murine findings support a causal role of ADAM19, implicated in human GWAS, in regulating pulmonary function.
RESUMEN
The mammalian epoxide hydrolase (EPHX)3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo. Ephx3-/- mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced. Although the Ephx3-/- pups appear normal, measurements of transepidermal water loss detected a modest and statistically significant increase compared with the wild-type or heterozygote mice, reflecting a skin barrier impairment that was not evident in the knockouts of mouse microsomal (EPHX1/microsomal epoxide hydrolase) or soluble (EPHX2/sEH). This barrier phenotype in the Ephx3-/- pups was associated with a significant decrease in the covalently bound ceramides in the epidermis (40% reduction, p < 0.05), indicating a corresponding structural impairment in the integrity of the water barrier. Quantitative LC-MS analysis of the esterified linoleate-derived triols in the murine epidermis revealed a marked and isomer-specific reduction (â¼85%) in the Ephx3-/- epidermis of the major trihydroxy isomer 9R,10S,13R-trihydroxy-11E-octadecenoate. We conclude that EPHX3 (and not EPHX1 or EPHX2) catalyzes hydrolysis of the 12R-LOX/eLOX3-derived epoxyalcohol esterified in acylceramide and may function to control flux through the alternative and crucial route of metabolism via the dehydrogenation pathway of SDR9C7. Importantly, our findings also identify a functional role for EPHX3 in transformation of a naturally esterified epoxide substrate, pointing to its potential contribution in other tissues.
Asunto(s)
Ceramidas/metabolismo , Compuestos Epoxi/metabolismo , Ácido Linoleico/metabolismo , Piel/metabolismo , Animales , Eliminación de Gen , Hidrólisis , Ratones , PermeabilidadRESUMEN
Stimuli such as inflammation or hypoxia induce cytochrome P450 epoxygenase-mediated production of arachidonic acid-derived epoxyeicosatrienoic acids (EETs). EETs have cardioprotective, vasodilatory, angiogenic, anti-inflammatory, and analgesic effects, which are diminished by EET hydrolysis yielding biologically less active dihydroxyeicosatrienoic acids (DHETs). Previous in vitro assays have suggested that epoxide hydrolase 2 (EPHX2) is responsible for nearly all EET hydrolysis. EPHX1, which exhibits slow EET hydrolysis in vitro, is thought to contribute only marginally to EET hydrolysis. Using Ephx1-/-, Ephx2-/-, and Ephx1-/-Ephx2-/- mice, we show here that EPHX1 significantly contributes to EET hydrolysis in vivo Disruption of Ephx1 and/or Ephx2 genes did not induce compensatory changes in expression of other Ephx genes or CYP2 family epoxygenases. Plasma levels of 8,9-, 11,12-, and 14,15-DHET were reduced by 38, 44, and 67% in Ephx2-/- mice compared with wildtype (WT) mice, respectively; however, plasma from Ephx1-/-Ephx2-/- mice exhibited significantly greater reduction (100, 99, and 96%) of those respective DHETs. Kinetic assays and FRET experiments indicated that EPHX1 is a slow EET scavenger, but hydrolyzes EETs in a coupled reaction with cytochrome P450 to limit basal EET levels. Moreover, we also found that EPHX1 activities are biologically relevant, as Ephx1-/-Ephx2-/- hearts had significantly better postischemic functional recovery (71%) than both WT (31%) and Ephx2-/- (51%) hearts. These findings indicate that Ephx1-/-Ephx2-/- mice are a valuable model for assessing EET-mediated effects, uncover a new paradigm for EET metabolism, and suggest that dual EPHX1 and EPHX2 inhibition may represent a therapeutic approach to manage human pathologies such as myocardial infarction.
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Eicosanoides/metabolismo , Epóxido Hidrolasas/metabolismo , Isquemia Miocárdica/metabolismo , Miocardio/metabolismo , Animales , Epóxido Hidrolasas/química , Epóxido Hidrolasas/deficiencia , Hidrólisis , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Isquemia Miocárdica/patología , Miocardio/patología , Oxilipinas/sangre , Conformación ProteicaRESUMEN
Human genome-wide association studies have identified over 50 loci associated with pulmonary function and related phenotypes, yet follow-up studies to determine causal genes or variants are rare. Single nucleotide polymorphisms in serotonin receptor 4 (HTR4) are associated with human pulmonary function in genome-wide association studies and follow-up animal work has demonstrated that Htr4 is causally associated with pulmonary function in mice, although the precise mechanisms were not identified. We sought to elucidate the role of neural innervation and pulmonary architecture in the lung phenotype of Htr4-/- animals. We report here that the Htr4-/- phenotype in mouse is dependent on vagal innervation to the lung. Both ex vivo tracheal ring reactivity and in vivo flexiVent pulmonary functional analyses demonstrate that vagotomy abrogates the Htr4-/- airway hyperresponsiveness phenotype. Hyperpolarized 3He gas magnetic resonance imaging and stereological assessment of wild-type and Htr4-/- mice reveal no observable differences in lung volume, inflation characteristics, or pulmonary microarchitecture. Finally, control of breathing experiments reveal substantive differences in baseline breathing characteristics between mice with/without functional HTR4 in breathing frequency, relaxation time, flow rate, minute volume, time of inspiration and expiration and breathing pauses. These results suggest that HTR4's role in pulmonary function likely relates to neural innervation and control of breathing.
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Pulmón/inervación , Pulmón/fisiología , Receptores de Serotonina 5-HT4/deficiencia , Nervio Vago/fisiología , Animales , Genotipo , Técnicas In Vitro , Ratones Endogámicos C57BL , Contracción Muscular/fisiología , Fenotipo , Ventilación Pulmonar/fisiología , Receptores de Serotonina 5-HT4/metabolismo , Respiración , Pruebas de Función Respiratoria , Hipersensibilidad Respiratoria/fisiopatología , Tráquea/fisiología , Vagotomía , Nervio Vago/cirugíaRESUMEN
The CYP2C subfamily of the cytochrome P450 gene superfamily encodes heme-thiolate proteins that have a myriad of biologic functions. CYP2C proteins detoxify xenobiotics and metabolize endogenous lipids such as arachidonic acid to bioactive eicosanoids. We report new methods and results for the quantitative polymerase reaction (qPCR) analysis for the 15 members of the mouse Cyp2c subfamily (Cyp2c29, Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c40, Cyp2c44, Cyp2c50, Cyp2c54, Cyp2c55, Cyp2c65, Cyp2c66, Cyp2c67, Cyp2c68, Cyp2c69, and Cyp2c70). Commercially available TaqMan primer/probe assays were compared with developed SYBR Green primer sets for specificity toward the mouse Cyp2c cDNAs and analysis of their tissue distribution. TaqMan primer/probe assays for 10 of the mouse Cyp2c isoforms were shown to be specific for their intended mouse Cyp2c cDNA; however, there were no TaqMan primer/probe assays specific for the mouse Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, or Cyp2c69 transcripts. Each of the SYBR Green primer sets was specific for its intended mouse Cyp2c cDNA. The two qPCR methods confirmed similar patterns of Cyp2c tissue expression: Cyp2c37, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c50, Cyp2c54, and Cyp2c70 were most highly expressed in liver; Cyp2c55 was highly expressed in large intestine; Cyp2c65 was highly expressed in stomach, duodenum, and large intestine; and Cyp2c66 was highly expressed in both duodenum and jejunum. For isoforms without specific TaqMan primer/probe assays, the SYBR Green primer sets detected high level expression of Cyp2c29, Cyp2c40, Cyp2c67, Cyp2c68, and Cyp2c69 in the liver. Lower expression levels of the mouse Cyp2cs were also detected in other tissues.
Asunto(s)
Sistema Enzimático del Citocromo P-450/biosíntesis , Intestinos/enzimología , Hígado/enzimología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Secuencia de Aminoácidos , Animales , Clonación Molecular , Sistema Enzimático del Citocromo P-450/genética , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Isoenzimas , Masculino , Ratones Endogámicos C57BL , Especificidad de Órganos , ARN Mensajero/biosíntesis , ARN Mensajero/genéticaRESUMEN
Cyclooxygenase (COX)-2 has been shown to be involved in regulating basal airway function, bacterial LPS-induced airway hyperresponsiveness (AHR) and lung inflammation, and bleomycin-induced lung fibrosis; however, the cellular source of COX-2 that underlies these effects is unknown. We generated mice with alveolar type II (ATII) cell-specific knockdown of COX-2 (AT2CC(-/-)), to examine the role of ATII cell-derived prostaglandins (PGs) in these processes. Specific knockdown of COX-2 was confirmed by real-time RT-PCR and Western blot analyses. LC/MS/MS analysis showed that ATII cells produced PGs. Basal airway responsiveness of AT2CC(-/-) mice was decreased compared to that of wild-type (WT) mice. LPS-induced hypothermic response, infiltration of inflammatory cells into the airway, and lung inflammation were enhanced in AT2CC(-/-) mice relative to WT controls; however, LPS-induced AHR and proinflammatory cytokine and chemokine expression were similar between the genotypes. After 21 d of bleomycin administration, AT2CC(-/-) mice behaved in a manner similar to WT mice. Thus, ATII cell-derived COX-2 plays an important role in regulating basal airway function and LPS-induced lung inflammation, but does not play a role in bleomycin-induced fibrosis. These findings provide insight into the cellular source of COX-2 related to these lung phenotypes.
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Ciclooxigenasa 2/genética , Neumonía/metabolismo , Fibrosis Pulmonar/metabolismo , Células Epiteliales Alveolares/efectos de los fármacos , Células Epiteliales Alveolares/metabolismo , Animales , Bleomicina/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Ratones Transgénicos , Neumonía/genética , Neumonía/patología , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/patologíaRESUMEN
The hepatic circadian clock plays a key role in the daily regulation of glucose metabolism, but the precise molecular mechanisms that coordinate these two biological processes are not fully understood. In this study, we identify a novel connection between the regulation of RORγ by the clock machinery and the diurnal regulation of glucose metabolic networks. We demonstrate that particularly at daytime, mice deficient in RORγ exhibit improved insulin sensitivity and glucose tolerance due to reduced hepatic gluconeogenesis. This is associated with a reduced peak expression of several glucose metabolic genes critical in the control of gluconeogenesis and glycolysis. Genome-wide cistromic profiling, promoter and mutation analysis support the concept that RORγ regulates the transcription of several glucose metabolic genes directly by binding ROREs in their promoter regulatory region. Similar observations were made in liver-specific RORγ-deficient mice suggesting that the changes in glucose homeostasis were directly related to the loss of hepatic RORγ expression. Altogether, our study shows that RORγ regulates several glucose metabolic genes downstream of the hepatic clock and identifies a novel metabolic function for RORγ in the diurnal regulation of hepatic gluconeogenesis and insulin sensitivity. The inhibition of the activation of several metabolic gene promoters by an RORγ antagonist suggests that antagonists may provide a novel strategy in the management of metabolic diseases, including type 2 diabetes.
Asunto(s)
Ritmo Circadiano/genética , Glucosa/metabolismo , Resistencia a la Insulina , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/biosíntesis , Animales , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Regulación de la Expresión Génica/efectos de los fármacos , Gluconeogénesis/efectos de los fármacos , Gluconeogénesis/genética , Humanos , Insulina/genética , Insulina/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/deficiencia , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Tretinoina/farmacologíaRESUMEN
Human genome-wide association studies (GWASs) have identified numerous associations between single nucleotide polymorphisms (SNPs) and pulmonary function. Proving that there is a causal relationship between GWAS SNPs, many of which are noncoding and without known functional impact, and these traits has been elusive. Furthermore, noncoding GWAS-identified SNPs may exert trans-regulatory effects rather than impact the proximal gene. Noncoding variants in 5-hydroxytryptamine (serotonin) receptor 4 (HTR4) are associated with pulmonary function in human GWASs. To gain insight into whether this association is causal, we tested whether Htr4-null mice have altered pulmonary function. We found that HTR4-deficient mice have 12% higher baseline lung resistance and also increased methacholine-induced airway hyperresponsiveness (AHR) as measured by lung resistance (27%), tissue resistance (48%), and tissue elastance (30%). Furthermore, Htr4-null mice were more sensitive to serotonin-induced AHR. In models of exposure to bacterial lipopolysaccharide, bleomycin, and allergic airway inflammation induced by house dust mites, pulmonary function and cytokine profiles in Htr4-null mice differed little from their wild-type controls. The findings of altered baseline lung function and increased AHR in Htr4-null mice support a causal relationship between genetic variation in HTR4 and pulmonary function identified in human GWAS.
Asunto(s)
Pulmón/fisiología , Receptores de Serotonina 5-HT4/genética , Receptores de Serotonina 5-HT4/fisiología , Resistencia de las Vías Respiratorias/genética , Resistencia de las Vías Respiratorias/fisiología , Alérgenos/administración & dosificación , Animales , Antígenos Dermatofagoides/administración & dosificación , Bleomicina/toxicidad , Hiperreactividad Bronquial/inducido químicamente , Hiperreactividad Bronquial/genética , Hiperreactividad Bronquial/fisiopatología , Estudio de Asociación del Genoma Completo , Humanos , Pulmón/inmunología , Masculino , Ratones , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/genética , Fibrosis Pulmonar/fisiopatología , Receptores de Serotonina 5-HT4/deficiencia , Pruebas de Función Respiratoria , Especificidad de la EspecieRESUMEN
Non-alcoholic steatohepatitis (NASH) is an emerging public health problem without effective therapies. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into bioactive epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory and protective effects. However, the functional relevance of the CYP epoxyeicosanoid metabolism pathway in the pathogenesis of NASH remains poorly understood. Our studies demonstrate that both mice with methionine-choline deficient (MCD) diet-induced NASH and humans with biopsy-confirmed NASH exhibited significantly higher free EET concentrations compared to healthy controls. Targeted disruption of Ephx2 (the gene encoding for soluble epoxide hydrolase) in mice further increased EET levels and significantly attenuated MCD diet-induced hepatic steatosis, inflammation and injury, as well as high fat diet-induced adipose tissue inflammation, systemic glucose intolerance and hepatic steatosis. Collectively, these findings suggest that dysregulation of the CYP epoxyeicosanoid pathway is a key pathological consequence of NASH in vivo, and promoting the anti-inflammatory and protective effects of EETs warrants further investigation as a novel therapeutic strategy for NASH.
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Sistema Enzimático del Citocromo P-450/metabolismo , Enfermedad del Hígado Graso no Alcohólico/enzimología , Ácido 8,11,14-Eicosatrienoico/metabolismo , Adulto , Animales , Citocromo P-450 CYP2J2 , Dieta/efectos adversos , Progresión de la Enfermedad , Epóxido Hidrolasas/química , Epóxido Hidrolasas/metabolismo , Femenino , Humanos , Hidrólisis , Hígado/enzimología , Masculino , Síndrome Metabólico/complicaciones , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Enfermedad del Hígado Graso no Alcohólico/complicaciones , Enfermedad del Hígado Graso no Alcohólico/metabolismo , SolubilidadRESUMEN
Members of the cytochrome P450 CYP2J subfamily are expressed in multiple tissues in mice and humans. These enzymes are active in the metabolism of fatty acids to generate bioactive compounds. Herein we report new methods and results for quantitative polymerase chain reaction (qPCR) analysis for the seven genes (Cyp2j5, Cyp2j6, Cyp2j8, Cyp2j9, Cyp2j11, Cyp2j12, and Cyp2j13) of the mouse Cyp2j subfamily. SYBR Green primer sets were developed and compared with commercially available TaqMan primer/probe assays for specificity toward mouse Cyp2j cDNA, and analysis of tissue distribution and regulation of Cyp2j genes. Each TaqMan primer/probe set and SYBR Green primer set were shown to be specific for their intended mouse Cyp2j cDNA. Tissue distribution of the mouse Cyp2j isoforms confirmed similar patterns of expression between the two qPCR methods. Cyp2j5 and Cyp2j13 were highly expressed in male kidneys, and Cyp2j11 was highly expressed in both male and female kidneys. Cyp2j6 was expressed in multiple tissues, with the highest expression in the small intestine and duodenum. Cyp2j8 was detected in various tissues, with highest expression found in the skin. Cyp2j9 was highly expressed in the brain, liver, and lung. Cyp2j12 was predominately expressed in the brain. We also determined the Cyp2j isoform expression in Cyp2j5 knockout mice to determine whether there was compensatory regulation of other Cyp2j isoforms, and we assessed Cyp2j isoform regulation during various inflammatory models, including influenza A, bacterial lipopolysaccharide, house dust mite allergen, and corn pollen. Both qPCR methods detected similar suppression of Cyp2j6 and Cyp2j9 during inflammation in the lung.
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Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Animales , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/biosíntesis , Cartilla de ADN , ADN Complementario/biosíntesis , ADN Complementario/genética , Femenino , Regulación Enzimológica de la Expresión Génica/genética , Hipersensibilidad/enzimología , Hipersensibilidad/genética , Riñón/enzimología , Pulmón/enzimología , Masculino , Ratones , Ratones Endogámicos C57BL , Infecciones por Orthomyxoviridae/enzimología , Polen/inmunología , Reacción en Cadena de la Polimerasa , Distribución Tisular , Zea mays/inmunologíaRESUMEN
Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the role of CYP4F2 in vascular disease, we generated mice with endothelial expression of human CYP4F2 (Tie2-CYP4F2-Tr). LC/MS/MS analysis revealed 2-foldincreases in 20-HETE levels in tissues and endothelial cells (ECs), relative to wild-type (WT) controls. Tie2-CYP4F2-Tr ECs demonstrated increases in growth (267.1 ± 33.4 vs. 205.0 ± 13% at 48 h) and tube formation (7.7 ± 1.1 vs. 1.6 ± 0.5 tubes/field) that were 20-HETE dependent and associated with up-regulation of prooxidant NADPH oxidase and proangiogenic VEGF. Increases in VEGF and NADPH oxidase levels were abrogated by inhibitors of NADPH oxidase and MAPK, respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-CYP4F2-Tr mice (18.6 ± 2.7 vs. 7.9 ± 2.7 pg/ml) were up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and blood pressure were unchanged. Our findings indicate that human CYP4F2 significantly increases 20-HETE production, CYP4F2-derived 20-HETE mediates EC proliferation and angiogenesis via VEGF- and NADPH oxidase-dependent manners, and the Tie2-CYP4F2-Tr mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the vasculature.-Cheng, J., Edin, M. L., Hoopes, S. L., Li, H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih, F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake, G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.
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Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Células Endoteliales/metabolismo , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Animales , Presión Sanguínea/genética , Células Cultivadas , Familia 4 del Citocromo P450 , Citocinas/genética , Citocinas/metabolismo , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Inflamación/genética , Inflamación/metabolismo , Masculino , Ratones , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Estrés Oxidativo/genética , Receptores de Factores de Crecimiento Endotelial Vascular/genética , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Regulación hacia Arriba/genética , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
RATIONALE: Abdominal aortic aneurysms (AAAs) are a chronic inflammatory vascular disease for which pharmacological treatments are not available. A mouse model of AAA formation involves chronic infusion of angiotensin II (AngII), and previous studies indicated a primary role for the AngII type 1a receptor in AAA formation. ß-arrestin (ßarr)-2 is a multifunctional scaffolding protein that binds G-protein-coupled receptors such as AngII type 1a and regulates numerous signaling pathways and pathophysiological processes. However, a role for ßarr2 in AngII-induced AAA formation is currently unknown. OBJECTIVE: To determine whether ßarr2 played a role in AngII-induced AAA formation in mice. METHODS AND RESULTS: Treatment of ßarr2(+/+) and ßarr2(-/-) mice on the hyperlipidemic apolipoprotein E-deficient (apoE(-/-)) background or on normolipidemic C57BL/6 background with AngII for 28 days indicated that ßarr2 deficiency significantly attenuated AAA formation. ßarr2 deficiency attenuated AngII-induced expression of cyclooxygenase-2, monocyte chemoattractant protein-1, macrophage inflammatory protein 1α, and macrophage infiltration. AngII also increased the levels of phosphorylated extracellular signal-regulated kinase 1/2 in apoE(-/-)/ßarr2(+/+) aortas, whereas ßarr2 deficiency diminished this increase. Furthermore, inhibition of extracellular signal-regulated kinase 1/2 activation with CI1040 (100 mg/kg per day) reduced the level of AngII-induced cyclooxygenase-2 expression in apoE(-/-)/ßarr2(+/+) mice to the level observed in apoE(-/-)/ßarr2(-/-) mice. AngII treatment also increased matrix metalloproteinase expression and disruption of the elastic layer in apoE(-/-)/ßarr2(+/+) aortas, and ßarr2 deficiency reduced these effects. CONCLUSIONS: ßarr2 contributes to AngII-induced AAA formation in mice by phosphorylated extracellular signal-regulated kinase 1/2-mediated cyclooxygenase-2 induction and increased inflammation. These studies suggest that for the AngII type 1a receptor, G-protein-independent, ßarr2-dependent signaling plays a major role in AngII-induced AAA formation.
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Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/prevención & control , Arrestinas/deficiencia , Angiotensina II , Animales , Aorta Abdominal/efectos de los fármacos , Aorta Abdominal/patología , Aneurisma de la Aorta Abdominal/inducido químicamente , Aneurisma de la Aorta Abdominal/genética , Aneurisma de la Aorta Abdominal/metabolismo , Aneurisma de la Aorta Abdominal/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Arrestinas/genética , Benzamidas/farmacología , Presión Sanguínea , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Quimiocina CCL2/metabolismo , Quimiocina CCL3/metabolismo , Ciclooxigenasa 2/metabolismo , Modelos Animales de Enfermedad , Tejido Elástico/metabolismo , Tejido Elástico/patología , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Factores de Tiempo , Arrestina beta 2 , beta-ArrestinasRESUMEN
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.
Asunto(s)
Adipogénesis/efectos de los fármacos , Sistema Enzimático del Citocromo P-450 , Eicosanoides/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Intolerancia a la Glucosa/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Células 3T3-L1 , Adipogénesis/genética , Animales , Grasas de la Dieta/administración & dosificación , Grasas de la Dieta/efectos adversos , Intolerancia a la Glucosa/inducido químicamente , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/genética , Obesidad/metabolismo , Obesidad/patologíaRESUMEN
RATIONALE: Helper CD4(+) T cell subsets, including IL-9- and IL-10-producing T helper cell type 9 (Th9) cells, exist under certain inflammatory conditions. Cyclooxygenase (COX)-1 and COX-2 play important roles in allergic lung inflammation and asthma. It is unknown whether COX-derived eicosanoids regulate Th9 cells during allergic lung inflammation. OBJECTIVES: To determine the role of COX metabolites in regulating Th9 cell differentiation and function during allergic lung inflammation. METHODS: COX-1(-/-), COX-2(-/-), and wild-type (WT) mice were studied in an in vivo model of ovalbumin-induced allergic inflammation and an in vitro model of Th9 differentiation using flow cytometry, cytokine assays, confocal microscopy, real-time PCR, and immunoblotting. In addition, the role of specific eicosanoids and their receptors was examined using synthetic prostaglandins (PGs), selective inhibitors, and siRNA knockdown. MEASUREMENTS AND MAIN RESULTS: Experimental endpoints were not different between COX-1(-/-) and WT mice; however, the percentage of IL-9(+) CD4(+) T cells was increased in lung, bronchoalveolar lavage fluid, lymph nodes, and blood of allergic COX-2(-/-) mice relative to WT. Bronchoalveolar lavage fluid IL-9 and IL-10, serum IL-9, and lung IL-17RB levels were significantly increased in allergic COX-2(-/-) mice or in WT mice treated with COX-2 inhibitors. IL-9, IL-10, and IL-17RB expression in vivo was inhibited by PGD2 and PGE2, which also reduced Th9 cell differentiation of murine and human naive CD4(+) T cells in vitro. Inhibition of protein kinase A significantly increased Th9 cell differentiation of naive CD4(+) T cells isolated from WT mice in vitro. CONCLUSIONS: COX-2-derived PGD2 and PGE2 regulate Th9 cell differentiation by suppressing IL-17RB expression via a protein kinase A-dependent mechanism.
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Asma/inmunología , Inhibidores de la Ciclooxigenasa 2/inmunología , Pulmón/inmunología , Receptores de Interleucina-17/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Citocinas/análisis , Eicosanoides/inmunología , Eicosanoides/fisiología , Citometría de Flujo , Humanos , Immunoblotting , Inflamación/inmunología , Masculino , Ratones , Microscopía Confocal , Modelos Animales , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Cytochromes P450 can metabolize endogenous fatty acids, such as arachidonic acid, to bioactive lipids such as epoxyeicosatrienoic acids (EETs) that have beneficial effects. EETs protect hearts against ischemic damage, heart failure or fibrosis; however, their effects are limited by hydrolysis to less active dihydroxy oxylipins by soluble epoxide hydrolase (sEH), encoded by the epoxide hydrolase 2 gene (EPHX2, EC 3.3.2.10). Pharmacological inhibition or genetic disruption of sEH/EPHX2 have been widely studied for their impact on cardiovascular diseases. Less well studied is the role of increased EPHX2 expression, which occurs in a substantial human population that carries the EPHX2 K55R polymorphism or after induction by inflammatory stimuli. Herein, we developed a mouse model with cardiomyocyte-selective expression of human EPHX2 (Myh6-EPHX2) that has significantly increased total EPHX2 expression and activity. Myh6-EPHX2 hearts exhibit strong, cardiomyocyte-selective expression of EPHX2. EPHX2 mRNA, protein, and epoxide hydrolysis measurements suggest that Myh6-EPHX2 hearts have 12-fold increase in epoxide hydrolase activity relative to wild type (WT) hearts. This increased activity significantly decreased epoxide:diol ratios in vivo. Isolated, perfused Myh6-EPHX2 hearts were not significantly different from WT hearts in basal parameters of cardiac function; however, compared to WT hearts, Myh6-EPHX2 hearts demonstrated reduced recovery of heart contractile function after ischemia and reperfusion (I/R). This impaired recovery after I/R correlated with reduced activation of PI3K/AKT and GSK3ß signaling pathways in Myh6-EPHX2 hearts compared to WT hearts. In summary, the Myh6-EPHX2 mouse line represents a novel model of cardiomyocyte-selective overexpression of EPHX2 that has detrimental effects on cardiac function.
RESUMEN
In lung, thromboxane A2 (TXA2) activates the TP receptor to induce proinflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased approximately 2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naive CD4+ T cell differentiation to Th9 cells and IL-9 production were inhibited dose-dependently by TXA2 in vitro. TP receptor-deficient mice had an approximately 2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared with wild-type mice. Naive CD4+ T cells from TP-deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared with CD4+ T cells from wild-type mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, proinflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.
Asunto(s)
Asma , Diferenciación Celular , Linfocitos T Reguladores , Células Th2 , Tromboxano A2 , Animales , Ratones , Células Th2/inmunología , Células Th2/patología , Tromboxano A2/metabolismo , Tromboxano A2/inmunología , Linfocitos T Reguladores/inmunología , Asma/inmunología , Asma/patología , Asma/tratamiento farmacológico , Asma/genética , Ratones Noqueados , Interleucina-9/inmunología , Interleucina-9/genética , Interleucina-9/metabolismo , Neumonía/inmunología , Neumonía/patología , Ratones Endogámicos C57BL , Ratones Endogámicos BALB C , Pulmón/inmunología , Pulmón/patología , Ovalbúmina/inmunología , Femenino , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Cyclooxygenases and their metabolites are important regulators of inflammatory responses and play critical roles in regulating the differentiation of T helper cell subsets in inflammatory diseases. In this review, we highlight new information on regulation of T helper cell subsets by cyclooxygenases and their metabolites. Prostanoids influence cytokine production by both antigen presenting cells and T cells to regulate the differentiation of naïve CD4(+) T cells to Th1, Th2 and Th17 cell phenotypes. Cyclooxygenases and PGE2 generally exacerbate Th2 and Th17 phenotypes, while suppressing Th1 differentiation. Thus, cycloxygenases may play a critical role in diseases that involve immune cell dysfunction. Targeting of cyclooxygenases and their eicosanoid products may represent a new approach for treatment of inflammatory diseases, tumors and autoimmune disorders.
Asunto(s)
Enfermedades Autoinmunes/metabolismo , Ciclooxigenasa 2/metabolismo , Dinoprostona/metabolismo , Células TH1/efectos de los fármacos , Células Th17/efectos de los fármacos , Células Th2/efectos de los fármacos , Enfermedades Autoinmunes/tratamiento farmacológico , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Diferenciación Celular , Inhibidores de la Ciclooxigenasa/farmacología , Citocinas/inmunología , Citocinas/metabolismo , Dinoprostona/farmacología , Humanos , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Inflamación/metabolismo , Inflamación/patología , Células TH1/inmunología , Células TH1/patología , Células Th17/inmunología , Células Th17/patología , Células Th2/inmunología , Células Th2/patologíaRESUMEN
Cytochrome P450 (CYP) epoxygenases CYP2C8 and CYP2J2 generate epoxyeicosatrienoic acids (EETs) from arachidonic acid. Mice with expression of CYP2J2 in cardiomyocytes (αMHC-CYP2J2 Tr) or treated with synthetic EETs have increased functional recovery after ischemia/reperfusion (I/R); however, no studies have examined the role of cardiomyocyte- vs. endothelial-derived EETs or compared the effects of different CYP epoxygenase isoforms in the ischemic heart. We generated transgenic mice with increased endothelial EET biosynthesis (Tie2-CYP2C8 Tr and Tie2-CYP2J2 Tr) or EET hydrolysis (Tie2-sEH Tr). Compared to wild-type (WT), αMHC-CYP2J2 Tr hearts showed increased recovery of left ventricular developed pressure (LVDP) and decreased infarct size after I/R. In contrast, LVDP recovery and infarct size were unchanged in Tie2-CYP2J2 Tr and Tie2-sEH Tr hearts. Surprisingly, compared to WT, Tie2-CYP2C8 Tr hearts had significantly reduced LVDP recovery (from 21 to 14%) and increased infarct size after I/R (from 51 to 61%). Tie2-CYP2C8 Tr hearts also exhibited increased reactive oxygen species (ROS) generation, dihydroxyoctadecenoic acid (DiHOME) formation, and coronary resistance after I/R. ROS scavengers and CYP2C8 inhibition reversed the detrimental effects of CYP2C8 expression in Tie2-CYP2C8 Tr hearts. Treatment of WT hearts with 250 nM 9,10-DiHOME decreased LVDP recovery compared to vehicle (16 vs. 31%, respectively) and increased coronary resistance after I/R. These data demonstrate that increased ROS generation and enhanced DiHOME synthesis by endothelial CYP2C8 impair functional recovery and mask the beneficial effects of increased EET production following I/R.