RESUMEN
INTRODUCTION: Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) leads to blindness. It has been widely reported that increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) diets reduce CNV. Of the three major pathways metabolizing ω-3 (and ω-6 LCPUFA), the cyclooxygenase and lipoxygenase pathways generally produce pro-angiogenic metabolites from ω-6 LCPUFA and anti-angiogenic ones from ω-3 LCPUFA. Howevehr, cytochrome P450 oxidase (CPY) 2C produces pro-angiogenic metabolites from both ω-6 and ω-3 LCPUFA. The effects of CYP2J2 products on ocular neovascularization are still unknown. Understanding how each metabolic pathway affects the protective effect of ω-3 LCPUFA on retinal neovascularization may lead to therapeutic interventions. OBJECTIVES: To investigate the effects of LCPUFA metabolites through CYP2J2 pathway and CYP2J2 regulation on CNV both in vivo and ex vivo. METHODS: The impact of CYP2J2 overexpression and inhibition on neovascularization in the laser-induced CNV mouse model was assessed. The plasma levels of CYP2J2 metabolites were measured by liquid chromatography and tandem mass spectroscopy. The choroidal explant sprouting assay was used to investigate the effects of CYP2J2 inhibition and specific LCPUFA CYP2J2 metabolites on angiogenesis ex vivo. RESULTS: CNV was exacerbated in Tie2-Cre CYP2J2-overexpressing mice and was associated with increased levels of plasma docosahexaenoic acids. Inhibiting CYP2J2 activity with flunarizine decreased CNV in both ω-6 and ω-3 LCPUFA-fed wild-type mice. In Tie2-Cre CYP2J2-overexpressing mice, flunarizine suppressed CNV by 33â¯% and 36â¯% in ω-6, ω-3 LCPUFA diets, respectively, and reduced plasma levels of CYP2J2 metabolites. The pro-angiogenic role of CYP2J2 was corroborated in the choroidal explant sprouting assay. Flunarizine attenuated ex vivo choroidal sprouting, and 19,20-EDP, a ω-3 LCPUFA CYP2J2 metabolite, increased sprouting. The combined inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast further enhanced CNV suppression via tumor necrosis factor-α suppression. CONCLUSIONS: CYP2J2 inhibition augmented the inhibitory effect of ω-3 LCPUFA on CNV. Flunarizine suppressed pathological choroidal angiogenesis, and co-treatment with montelukast inhibiting CYP2C8 further enhanced the effect. CYP2 inhibition might be a viable approach to suppress CNV in AMD.
Asunto(s)
Neovascularización Coroidal , Ácidos Grasos Omega-3 , Degeneración Macular , Animales , Neovascularización Coroidal/tratamiento farmacológico , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/prevención & control , Citocromo P-450 CYP2C8/metabolismo , Modelos Animales de Enfermedad , Ácidos Docosahexaenoicos , Ácidos Grasos Omega-3/farmacología , Ácidos Grasos Omega-3/uso terapéutico , Ácidos Grasos Insaturados/uso terapéutico , Flunarizina/uso terapéutico , Degeneración Macular/tratamiento farmacológico , Degeneración Macular/metabolismo , Ratones , Ratones Endogámicos C57BL , NADPH-Ferrihemoproteína ReductasaRESUMEN
BACKGROUND: Oxidative stress plays a key role in the pathogenesis of respiratory diseases; however, studies on antioxidant vitamins and respiratory outcomes have been conflicting. We evaluated whether lower serum levels of vitamins A, C, D, and E are associated with respiratory morbidity and mortality in the U.S. adult population. METHODS: We conducted a pooled analysis of data from the 1988-1994 and 1999-2006 National Health and Nutrition Examination Survey (participants aged ≥ 20 years). We estimated covariate-adjusted odds ratios (aOR) per interquartile decrease in each serum vitamin level to quantify associations with respiratory morbidity, and covariate-adjusted hazard ratios (aHR) to quantify associations with respiratory mortality assessed prospectively through 2015. Vitamin supplementation and smoking were evaluated as potential effect modifiers. RESULTS: Lower serum vitamin C increased the odds of wheeze among all participants (overall aOR: 1.08, 95% CI: 1.01-1.16). Among smokers, lower serum α-tocopherol vitamin E increased the odds of wheeze (aOR: 1.11, 95% CI: 1.04-1.19) and chronic bronchitis/emphysema (aOR: 1.13, 95% CI: 1.03-1.24). Conversely, lower serum γ-tocopherol vitamin E was associated with lower odds of wheeze and chronic bronchitis/emphysema (overall aORs: 0.85, 95% CI: 0.79-0.92 and 0.85, 95% CI: 0.76-0.95, respectively). Lower serum vitamin C was associated with increased chronic lower respiratory disease (CLRD) mortality in all participants (overall aHR: 1.27, 95% CI: 1.07-1.51), whereas lower serum 25-hydroxyvitamin D (25-OHD) tended to increase mortality from CLRD and influenza/pneumonia among smokers (aHR range: 1.33-1.75). Mortality from influenza/ pneumonia increased with decreasing serum vitamin A levels in all participants (overall aHR: 1.21, 95% CI: 0.99-1.48). In pooled analysis, vitamin C deficiency and 25-OHD insufficiency were associated with mortality from influenza/pneumonia, increasing mortality risk up to twofold. CONCLUSIONS: Our analysis of nationally representative data on over 34,000 participants showed that lower serum levels of vitamins A, C, D, and α-tocopherol vitamin E are associated with increased respiratory morbidity and/or mortality in U.S. adults. The results underscore the importance of antioxidant vitamins in respiratory health.
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Bronquitis Crónica , Enfisema , Gripe Humana , Adulto , Antioxidantes , Ácido Ascórbico , Humanos , Morbilidad , Encuestas Nutricionales , Vitamina A , Vitaminas , alfa-TocoferolRESUMEN
The incidence of metabolic and chronic diseases including cancer, obesity, inflammation-related diseases sharply increased in the 21st century. Major underlying causes for these diseases are inflammation and oxidative stress. Accordingly, natural products and their bioactive components are obvious therapeutic agents for these diseases, given their antioxidant and anti-inflammatory properties. Research in this area has been significantly expanded to include chemical identification of these compounds using advanced analytical techniques, determining their mechanism of action, food fortification and supplement development, and enhancing their bioavailability and bioactivity using nanotechnology. These timely topics were discussed at the 20th Frontier Scientists Workshop sponsored by the Korean Academy of Science and Technology, held at the University of Hawaii at Manoa on 23 November 2019. Scientists from South Korea and the U.S. shared their recent research under the overarching theme of Bioactive Compounds, Nanoparticles, and Disease Prevention. This review summarizes presentations at the workshop to provide current knowledge of the role of natural products in the prevention and treatment of metabolic diseases.
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Antiinflamatorios , Antioxidantes , Productos Biológicos , Enfermedades Metabólicas , Animales , Suplementos Dietéticos , Humanos , Enfermedades Metabólicas/tratamiento farmacológico , Enfermedades Metabólicas/metabolismo , Ratones , Nanopartículas , Obesidad/tratamiento farmacológico , Obesidad/metabolismo , Estrés Oxidativo/efectos de los fármacos , RatasRESUMEN
Colon cancer is the third most common cancer and the second leading cause of cancer-related death in the United States, emphasizing the need for the discovery of new cellular targets. Using a metabolomics approach, we report here that epoxygenated fatty acids (EpFA), which are eicosanoid metabolites produced by cytochrome P450 (CYP) monooxygenases, were increased in both the plasma and colon of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colon cancer mice. CYP monooxygenases were overexpressed in colon tumor tissues and colon cancer cells. Pharmacologic inhibition or genetic ablation of CYP monooxygenases suppressed AOM/DSS-induced colon tumorigenesis in vivo. In addition, treatment with 12,13-epoxyoctadecenoic acid (EpOME), which is a metabolite of CYP monooxygenase produced from linoleic acid, increased cytokine production and JNK phosphorylation in vitro and exacerbated AOM/DSS-induced colon tumorigenesis in vivo. Together, these results demonstrate that the previously unappreciated CYP monooxygenase pathway is upregulated in colon cancer, contributes to its pathogenesis, and could be therapeutically explored for preventing or treating colon cancer. SIGNIFICANCE: This study finds that the previously unappreciated CYP monooxygenase eicosanoid pathway is deregulated in colon cancer and contributes to colon tumorigenesis.
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Carcinogénesis/efectos de los fármacos , Neoplasias del Colon/prevención & control , Sistema Enzimático del Citocromo P-450/química , Eicosanoides/metabolismo , Inhibidores Enzimáticos/farmacología , Metabolómica , Animales , Antifúngicos/farmacología , Apoptosis , Azoximetano/toxicidad , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proliferación Celular , Clotrimazol/farmacología , Neoplasias del Colon/genética , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Sistema Enzimático del Citocromo P-450/fisiología , Sulfato de Dextran/toxicidad , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proadifeno/farmacología , ARN Interferente Pequeño/genética , Células Tumorales CultivadasRESUMEN
Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.
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Neovascularización Coroidal/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Metabolismo de los Lípidos/fisiología , Sistemas de Mensajero Secundario/fisiología , Animales , Citocromo P-450 CYP2C8/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Epóxido Hidrolasas/metabolismo , Ácidos Grasos Insaturados/metabolismo , Leucocitos/metabolismo , Degeneración Macular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
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.
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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
Neovascular eye diseases including retinopathy of prematurity, diabetic retinopathy and age-related-macular-degeneration are major causes of blindness. Fenofibrate treatment in type 2 diabetes patients reduces progression of diabetic retinopathy independent of its peroxisome proliferator-activated receptor (PPAR)α agonist lipid lowering effect. The mechanism is unknown. Fenofibrate binds to and inhibits cytochrome P450 epoxygenase (CYP)2C with higher affinity than to PPARα. CYP2C metabolizes ω-3 long-chain polyunsaturated fatty acids (LCPUFAs). While ω-3 LCPUFA products from other metabolizing pathways decrease retinal and choroidal neovascularization, CYP2C products of both ω-3 and ω-6 LCPUFAs promote angiogenesis. We hypothesized that fenofibrate inhibits retinopathy by reducing CYP2C ω-3 LCPUFA (and ω-6 LCPUFA) pro-angiogenic metabolites. Fenofibrate reduced retinal and choroidal neovascularization in PPARα-/-mice and augmented ω-3 LCPUFA protection via CYP2C inhibition. Fenofibrate suppressed retinal and choroidal neovascularization in mice overexpressing human CYP2C8 in endothelial cells and reduced plasma levels of the pro-angiogenic ω-3 LCPUFA CYP2C8 product, 19,20-epoxydocosapentaenoic acid. 19,20-epoxydocosapentaenoic acid reversed fenofibrate-induced suppression of angiogenesis ex vivo and suppression of endothelial cell functions in vitro. In summary fenofibrate suppressed retinal and choroidal neovascularization via CYP2C inhibition as well as by acting as an agonist of PPARα. Fenofibrate augmented the overall protective effects of ω-3 LCPUFAs on neovascular eye diseases.
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Inhibidores de la Angiogénesis/farmacología , Neovascularización Coroidal/metabolismo , Neovascularización Coroidal/patología , Inhibidores Enzimáticos del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/metabolismo , Fenofibrato/farmacología , Neovascularización Retiniana/metabolismo , Neovascularización Retiniana/patología , Animales , Neovascularización Coroidal/tratamiento farmacológico , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Ácidos Grasos Omega-3/metabolismo , Humanos , Ratones , Ratones Transgénicos , PPAR alfa/metabolismo , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/etiología , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Neovascularización Retiniana/tratamiento farmacológico , Transducción de SeñalRESUMEN
OBJECTIVE: Pathological ocular neovascularization is a major cause of blindness. Increased dietary intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) reduces retinal neovascularization and choroidal neovascularization (CNV), but ω-3 LCPUFA metabolites of a major metabolizing pathway, cytochrome P450 oxidase (CYP) 2C, promote ocular pathological angiogenesis. We hypothesized that inhibition of CYP2C activity will add to the protective effects of ω-3 LCPUFA on neovascular eye diseases. APPROACH AND RESULTS: The mouse models of oxygen-induced retinopathy and laser-induced CNV were used to investigate pathological angiogenesis in the retina and choroid, respectively. The plasma levels of ω-3 LCPUFA metabolites of CYP2C were determined by mass spectroscopy. Aortic ring and choroidal explant sprouting assays were used to investigate the effects of CYP2C inhibition and ω-3 LCPUFA-derived CYP2C metabolic products on angiogenesis ex vivo. We found that inhibition of CYP2C activity by montelukast added to the protective effects of ω-3 LCPUFA on retinal neovascularization and CNV by 30% and 20%, respectively. In CYP2C8-overexpressing mice fed a ω-3 LCPUFA diet, montelukast suppressed retinal neovascularization and CNV by 36% and 39% and reduced the plasma levels of CYP2C8 products. Soluble epoxide hydrolase inhibition, which blocks breakdown and inactivation of CYP2C ω-3 LCPUFA-derived active metabolites, increased oxygen-induced retinopathy and CNV in vivo. Exposure to selected ω-3 LCPUFA metabolites of CYP2C significantly reversed the suppression of both angiogenesis ex vivo and endothelial cell functions in vitro by the CYP2C inhibitor montelukast. CONCLUSIONS: Inhibition of CYP2C activity adds to the protective effects of ω-3 LCPUFA on pathological retinal neovascularization and CNV.
Asunto(s)
Acetatos/farmacología , Inhibidores de la Angiogénesis/farmacología , Neovascularización Coroidal/prevención & control , Inhibidores del Citocromo P-450 CYP2C8/farmacología , Citocromo P-450 CYP2C8/metabolismo , Ácidos Grasos Omega-3/farmacología , Quinolinas/farmacología , Neovascularización Retiniana/prevención & control , Retinopatía de la Prematuridad/prevención & control , Animales , Aorta/efectos de los fármacos , Aorta/enzimología , Células Cultivadas , Neovascularización Coroidal/enzimología , Neovascularización Coroidal/genética , Neovascularización Coroidal/fisiopatología , Ciclopropanos , Citocromo P-450 CYP2C8/genética , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/enzimología , Ácidos Grasos Omega-3/metabolismo , Genotipo , Humanos , Hiperoxia/complicaciones , Rayos Láser , Ratones Endogámicos C57BL , Ratones Transgénicos , Neovascularización Fisiológica/efectos de los fármacos , Fenotipo , Neovascularización Retiniana/enzimología , Neovascularización Retiniana/genética , Neovascularización Retiniana/fisiopatología , Retinopatía de la Prematuridad/enzimología , Retinopatía de la Prematuridad/genética , Retinopatía de la Prematuridad/fisiopatología , Sulfuros , Técnicas de Cultivo de TejidosRESUMEN
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
OBJECTIVE: Regulation of angiogenesis is critical for many diseases. Specifically, pathological retinal neovascularization, a major cause of blindness, is suppressed with dietary ω3-long-chain polyunsaturated fatty acids (ω3LCPUFAs) through antiangiogenic metabolites of cyclooxygenase and lipoxygenase. Cytochrome P450 epoxygenases (CYP2C8) also metabolize LCPUFAs, producing bioactive epoxides, which are inactivated by soluble epoxide hydrolase (sEH) to transdihydrodiols. The effect of these enzymes and their metabolites on neovascularization is unknown. APPROACH AND RESULTS: The mouse model of oxygen-induced retinopathy was used to investigate retinal neovascularization. We found that CYP2C (localized in wild-type monocytes/macrophages) is upregulated in oxygen-induced retinopathy, whereas sEH is suppressed, resulting in an increased retinal epoxide:diol ratio. With a ω3LCPUFA-enriched diet, retinal neovascularization increases in Tie2-driven human-CYP2C8-overexpressing mice (Tie2-CYP2C8-Tg), associated with increased plasma 19,20-epoxydocosapentaenoic acid and retinal epoxide:diol ratio. 19,20-Epoxydocosapentaenoic acids and the epoxide:diol ratio are decreased with overexpression of sEH (Tie2-sEH-Tg). Overexpression of CYP2C8 or sEH in mice does not change normal retinal vascular development compared with their wild-type littermate controls. The proangiogenic role in retina of CYP2C8 with both ω3LCPUFA and ω6LCPUFA and antiangiogenic role of sEH in ω3LCPUFA metabolism were corroborated in aortic ring assays. CONCLUSIONS: Our results suggest that CYP2C ω3LCPUFA metabolites promote retinal pathological angiogenesis. CYP2C8 is part of a novel lipid metabolic pathway influencing retinal neovascularization.
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Hidrocarburo de Aril Hidroxilasas/metabolismo , Ácidos Grasos Omega-3/toxicidad , Macrófagos/enzimología , Monocitos/enzimología , Neovascularización Retiniana/inducido químicamente , Animales , Ácido Araquidónico/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Biotransformación , Hipoxia de la Célula , Citocromo P-450 CYP2C8 , Grasas de la Dieta/farmacocinética , Ácidos Docosahexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Epóxido Hidrolasas/deficiencia , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/fisiología , Proteínas del Ojo/metabolismo , Ácidos Grasos Omega-3/administración & dosificación , Ácidos Grasos Omega-3/clasificación , Ácidos Grasos Omega-3/farmacocinética , Ácidos Grasos Insaturados/administración & dosificación , Ácidos Grasos Insaturados/farmacocinética , Humanos , Lipooxigenasa/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxígeno/toxicidad , Prostaglandina-Endoperóxido Sintasas/metabolismo , ARN Mensajero/biosíntesis , Receptor TIE-2/genética , Proteínas Recombinantes de Fusión/metabolismo , Neovascularización Retiniana/prevención & controlRESUMEN
Recently, specific oxidized linoleic acid metabolites (OLAMs) have been identified as transient receptor potential vanilloid 1 (TRPV1) channel agonists that contribute to inflammatory and heat hyperalgesia mechanisms, yet the specific mechanism responsible for OLAM synthesis in sensory neurons is unknown. Here, we use molecular, anatomical, calcium imaging, and perforated patch electrophysiology methods to demonstrate the specific involvement of cytochrome P450 enzymes (CYPs) in the oxidation of linoleic acid leading to neuronal activation and show that this is enhanced under inflammatory conditions. Additional studies evaluated CYP expressions in the native rat trigeminal ganglia (TG) tissue and cultures as well as changes in their expression pattern following the induction of peripheral inflammation. Fourteen of 20 candidate transcripts were detected in native TG, and 7 of these displayed altered expression under cultured conditions. Moreover, complete Freund's adjuvant-induced inflammation of vibrissal pad selectively increased expression of CYP3A23/3A1 and CYP2J4 transcripts in TG. In situ hybridization studies demonstrated broad expression pattern of CYP3A23/3A1 and CYP2J4 within TG neurons. Anatomical studies characterized the expression of CYP3A1 and the CYP2J families within TG sensory neurons, including those with TRPV1, with about half of all TRPV1-positive neurons showing more prominent CYP3A1 and CYP2J expression. Together, these findings show that CYP enzymes play a primary role in mediating linoleic acid-evoked activation of sensory neurons and furthermore, implicate the involvement of specific CYPs as contributing to the formation of OLAMs that act as TRPV1 agonists within this subpopulation of nociceptors.
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Sistema Enzimático del Citocromo P-450/metabolismo , Hiperalgesia/metabolismo , Inflamación/metabolismo , Ácido Linoleico/metabolismo , Plasticidad Neuronal , Células Receptoras Sensoriales/metabolismo , Ganglio del Trigémino/enzimología , Animales , Regulación Enzimológica de la Expresión Génica , Hiperalgesia/complicaciones , Inflamación/etiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismo , Hiperhomocisteinemia/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Oxigenasas/genética , Oxigenasas/metabolismo , Factor de Transcripción ReIA/antagonistas & inhibidores , Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido 8,11,14-Eicosatrienoico/farmacología , Animales , Células Cultivadas , Citocromo P-450 CYP2J2 , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Homocisteína/farmacología , Hiperhomocisteinemia/enzimología , Proteínas I-kappa B/metabolismo , Metaloproteinasa 9 de la Matriz/biosíntesis , Ratones , Inhibidor NF-kappaB alfa , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factor de Transcripción ReIA/metabolismo , TransfecciónRESUMEN
Cytochrome P450 arachidonic acid (AA) epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), dilate arteries via hyperpolarization of smooth muscle cells and also have nonvasodilatory effects within the vasculature. The present study investigated the angiogenic effects of endogenous and exogenous EETs and the relevant signaling mechanisms involved. Bovine aortic endothelial cells (BAECs) were incubated with synthetic EETs or infected with recombinant adeno-associated viruses (rAAVs) containing CYP2C11-NADPH-cytochrome P450 oxidoreductase (CYPOR), CYP2J2, or CYP102 F87V mutant to increase endogenous levels of EETs. The following endpoints were measured: BAEC proliferation, migration, capillary formation, and in vivo angiogenesis. The potential involvement of various signaling pathways was explored using selective inhibitors. The results showed that transfection with either rAAV-CYP2C11-CYPOR, rAAV-CYP2J2, or rAAV-CYP102 F87V, or incubation with EETs promoted BAEC proliferation, increased migration of BAECs as assessed by Transwell analysis and wound healing assays, and enhanced capillary tubule formation as determined by chicken embryo chorioallantoic membrane assays and tube formation tests on matrigel. The effects of EETs on proliferation, migration, and capillary tubule formation were attenuated by inhibitors of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3 (PI3)-kinase/Akt pathways and partially attenuated by an endothelial nitric-oxide synthase (eNOS) inhibitor but not by a protein kinase C inhibitor. In a rat ischemic hind limb model, rAAV-mediated AA epoxygenase transfection induced angiogenesis. We conclude that AA epoxygenase metabolites can promote angiogenesis, which may provide protection to ischemic tissues. The results also suggest that the angiogenic effects of EETs involve the MAPK and PI3-kinase/Akt signaling pathways, and to some extent, the eNOS pathway.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Células Endoteliales/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Oxigenasas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal/fisiología , Adenoviridae/genética , Alantoína/metabolismo , Animales , Western Blotting , Bovinos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Embrión de Pollo , Corion/metabolismo , Citocromo P-450 CYP2J2 , ADN Complementario/biosíntesis , ADN Complementario/genética , Citometría de Flujo , Miembro Posterior/irrigación sanguínea , Isquemia/enzimología , Proteínas Proto-Oncogénicas c-akt , Flujo Sanguíneo Regional/fisiología , Estimulación Química , TransfecciónRESUMEN
The mammalian CYP2C subfamily is one of the largest and most complicated in the cytochrome P450 superfamily. In this report, we describe the organization of the mouse Cyp2c locus, which contains 15 genes and four pseudogenes, all of which are located in a 5.5-megabase region on chromosome 19. We cloned three novel mouse CYP2C cDNAs (designated CYP2C50, CYP2C54, and CYP2C55) from mouse heart, liver, and colon, respectively. All three cDNAs contain open reading frames that encode 490 amino acid polypeptides that are 57 to 95% identical to other CYP2Cs. The recombinant CYP2C proteins were expressed in Escherichia coli after N-terminal modification, partially purified, and shown to be active in the metabolism of both arachidonic acid (AA) and linoleic acid, albeit with different catalytic efficiencies and profiles. CYP2C50 and CYP2C54 metabolize AA to epoxyeicosatrienoic acids (EETs) primarily, and linoleic acid to epoxyoctadecenoic acids (EOAs) primarily, whereas CYP2C55 metabolizes AA to EETs and hydroxyeicosatetraenoic acids and linoleic acid to EOAs and hydroxyoctadecadienoic acids. Northern blotting and reverse transcription-polymerase chain reaction analysis reveal that CYP2C50 transcripts are abundant in liver and heart; CYP2C54 transcripts are present in liver, kidney, and stomach; and CYP2C55 transcripts are abundant in liver, colon, and kidney. Immunoblotting studies demonstrate that CYP2C50 protein is expressed in liver and heart, CYP2C54 protein is detected primarily in liver, and CYP2C55 protein is present primarily in colon. Immunohistochemistry reveals that CYP2C55 is most abundant in surface columnar epithelium in the cecum. We conclude that these new CYP2C enzymes are probably involved in AA and linoleic acid metabolism in mouse hepatic and extrahepatic tissues.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Ácidos Grasos/metabolismo , Secuencia de Aminoácidos , Animales , Mapeo Cromosómico , Cromosomas , Clonación Molecular , Sistema Enzimático del Citocromo P-450/metabolismo , Familia 2 del Citocromo P450 , ADN Complementario/análisis , Femenino , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Oxidación-Reducción , ARN Mensajero/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Distribución TisularRESUMEN
The human CYP2Cs have been studied extensively with respect to the metabolism of clinically important drugs and endogenous chemicals such as arachidonic acid (AA). Five members of the mouse CYP2C family have previously been described that metabolize arachidonic acid into regio- and stereospecific epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids, which have many important physiological roles. Herein, we describe the cloning and characterization of a new mouse cytochrome P450 (P450), CYP2C44, which has the lowest homology with other known mouse CYP2Cs. Western blotting and real-time polymerase chain reaction detected CYP2C44 mRNA and protein in liver >> kidney > adrenals. Kidney contained approximately 10% of the CYP2C44 mRNA content of liver. CYP2C44 metabolized AA to unique stereospecific products, 11R,12S-EET and 8R, 9S-EET, which are similar to those produced by rat CYP2C23. CY2C23 is highly expressed in rat kidney and has been suggested to be important in producing compensatory renal artery vasodilation in response to salt-loading in this species. Immunohistochemistry showed the presence of CYP2C44 in hepatocytes, biliary cells of the liver, and the proximal tubules of the kidney. Unlike mouse CYP2C29, CYP2C38, and CYP2C39, CYP2C44 did not metabolize the common CYP2C substrate tolbutamide. CYP2C44 was not induced by phenobarbital or pregnenolone-16alpha-carbonitrile, two prototypical inducers of hepatic P450s. The presence of CYP2C44 in mouse liver, kidney, and adrenals and the unique stereospecificity of its arachidonic acid metabolites are consistent with the possibility that it may have unique physiological roles within these tissues, such as modulation of electrolyte transport or vascular tone.
Asunto(s)
Ácido Araquidónico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Secuencia de Aminoácidos , Animales , Clonación Molecular , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/genética , Familia 2 del Citocromo P450 , ADN Complementario/análisis , Femenino , Immunoblotting , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Conformación Molecular , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Tolbutamida/metabolismoRESUMEN
A cDNA encoding a new cytochrome P450 was cloned from a mouse liver library. Sequence analysis revealed that this 2046-bp cDNA encodes a 501-amino acid polypeptide that is 72-94% identical to other CYP2J subfamily P450s and is designated CYP2J6. Northern analysis demonstrated that CYP2J6 transcripts are abundant in the small intestine and present at lower levels in other mouse tissues. In situ hybridization revealed that CYP2J6 mRNAs are present in luminal epithelial cells of the gastrointestinal mucosa. The CYP2J6 cDNA was expressed in Sf9 cells using baculovirus. The heterologously expressed CYP2J6 protein displayed a typical P450 CO-difference spectrum; however, the protein was unstable as evidenced by the loss of the Soret maxima at 450nm and the appearance of a 420nm peak when CYP2J6-expressing cells were disrupted by mechanical homogenization, sonication, or freeze-thaw. Immunoblotting of mouse microsomes with the anti-human CYP2J2 IgG, which cross-reacts with rodent CYP2Js, demonstrated the presence of multiple distinct murine CYP2J immunoreactive proteins in various tissues. Immunoblotting with an antibody to a CYP2J6-specific peptide detected a prominent 55-57kDa protein in Sf9 cell extracts expressing recombinant CYP2J6 but did not detect a protein of similar molecular mass in mouse small intestinal microsomes. Mixing experiments demonstrated that recombinant CYP2J6 is degraded rapidly in the presence of small intestinal microsomes consistent with proteolysis at highly sensitive sites. Sf9 cells, which express both CYP2J6 and NADPH-P450 oxidoreductase, metabolized benzphetamine but not arachidonic acid. We conclude that CYP2J6 is an unstable P450 that is active in the metabolism of benzphetamine, but not arachidonic acid.
Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Isoenzimas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Citocromo P-450 CYP2J2 , Sistema Enzimático del Citocromo P-450/metabolismo , ADN Complementario/análisis , Estabilidad de Enzimas , Femenino , Hibridación in Situ , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , ARN Mensajero/metabolismo , Proteínas Recombinantes/metabolismo , Fracciones Subcelulares , Distribución TisularRESUMEN
CYP2J2 is abundant in cardiovascular tissue and active in the metabolism of arachidonic acid to eicosanoids that possess potent anti-inflammatory, vasodilatory, and fibrinolytic properties. We cloned and sequenced the entire CYP2J2 gene (approximately 40.3 kb), which contains nine exons and eight introns. We then sequenced the CYP2J2 exons and intron-exon boundaries in 72 healthy persons representing African, Asian, and European/white populations as part of the National Institutes of Health/National Institute of Environmental Health Sciences Environmental Genome Single Nucleotide Polymorphism Program. A variety of polymorphisms were found, four of which resulted in coding changes (Arg158Cys, Ile192Asn, Asp342Asn, and Asn404Tyr). A fifth variant (Thr143Ala) was identified by screening a human heart cDNA library. All five variant cDNAs of CYP2J2 were generated by site-directed mutagenesis and expressed in Sf9 insect cells by using a baculovirus system. The recombinant wild-type and variant CYP2J2 proteins immunoreacted with peptide-based antibodies to CYP2J2 and displayed typical cytochrome P450 (P450) CO-difference spectra; however, the Asn404Tyr and Ile192Asn variants also had prominent spectral peaks at 420 nm. The ability of these variants to metabolize arachidonic acid and linoleic acid was compared with that of wild-type CYP2J2. Three variants (Asn404Tyr, Arg158Cys, and Thr143Ala) showed significantly reduced metabolism of both arachidonic acid and linoleic acid. The Ile192Asn variant showed significantly reduced activity toward arachidonic acid only. The Asp342Asn variant showed similar metabolism to wild-type CYP2J2 for both endogenous substrates. Based on these data, we conclude that allelic variants of the human CYP2J2 gene exist and that some of these variants result in a P450 protein that has reduced catalytic function. Insofar as CYP2J2 products have effects in the cardiovascular system, we speculate that these variants may be functionally relevant.