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Objectives: The aim was to investigate the effects of nicotine on neutrophil extracellular traps (NETs) formation in current and non-smokers and on a murine model of RA. Methods: We compared spontaneous and phorbol 12-myristate 13-acetate-induced NETosis between current and non-smokers by DNA release binding. Nicotine-induced NETosis from non-smokers was assessed by DNA release binding, NET-specific (myeloperoxidase (MPO)-DNA complex) ELISA and real-time fluorescence microscopy. We also used immunofluorescent staining to detect nicotinic acetylcholine receptors (nAChRs) on neutrophils and performed a functional analysis to assess the role of nAChRs in nicotine-induced NETosis. Finally, we investigated the effects of systemic nicotine exposure on arthritis severity and NETosis in the CIA mouse model. Results: Neutrophils derived from current smokers displayed elevated levels of spontaneous and phorbol 12-myristate 13-acetate-induced NETosis. Nicotine induced dose-dependent NETosis in ex vivo neutrophils from healthy non-smokers, and co-incubation with ACPA-immune complexes or TNF-α facilitated a synergistic effect on NETosis. Real-time fluorescence microscopy revealed robust formation of NET-like structures in nicotine-exposed neutrophils. Immunofluorescent staining demonstrated the presence of the α7 subunit of the nAChR on neutrophils. Stimulation of neutrophils with an α7-specific nAChR agonist induced NETosis, whereas pretreatment with an nAChR antagonist attenuated nicotine-induced NETosis. Nicotine administration to mice with CIA exacerbated inflammatory arthritis, with higher plasma levels of NET-associated MPO-DNA complex. Conclusion: We demonstrate that nicotine is a potent inducer of NETosis, which may play an important role in accelerating arthritis in the CIA model. This study generates awareness of and the mechanisms by which nicotine-containing products, including e-cigarettes, may have deleterious effects on patients with RA.
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Artrite Reumatoide/etiologia , Armadilhas Extracelulares/efeitos dos fármacos , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Animais , Artrite Experimental/etiologia , Cartilagem/fisiologia , Relação Dose-Resposta a Droga , Sistemas Eletrônicos de Liberação de Nicotina , Ensaio de Imunoadsorção Enzimática , Humanos , Infusões Subcutâneas , Masculino , Camundongos Endogâmicos DBA , Neutrófilos/efeitos dos fármacos , Nicotina/administração & dosagem , Agonistas Nicotínicos/administração & dosagem , Peroxidase/metabolismo , Fumar/efeitos adversos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
PURPOSE: To compare the inflammatory profile of hip synovial tissue in those with atraumatic microinstability to patients with femoroacetabular impingement (FAI). METHODS: Patients with cam and mixed-type FAI (FAI group) and patients with hip instability underwent sampling of the anterolateral synovium. Demographic data, intraoperative measurements, and functional outcome scores (International Hip Outcomes Tool and Short Form-12) were recorded. Cryosections were stained and examined under light microscopy as well as confocal fluorescent microscopy for anti-CD45 (common leukocyte antigen), anti-CD31 (endothelial), and anti-CD68 (macrophage) cell surface markers. A grading system was used to quantify synovitis under light microscopy whereas digital image analysis was used to quantify immunofluorescence staining area. Comparison were made with Student t test, Mann-Whitney U, χ2, and regression analysis. RESULTS: There were 12 patients in the FAI group and 5 in the instability group. Mean age was not significantly different (P > .05), but there was a significantly greater proportion of females in the instability group versus the FAI group (P < .001). There was a significant correlation (r = 0.653; P = .005) between number of turns needed for 10 mm of distraction and increased synovitis. Synovitis scores also were increased significantly in patients with cam morphology and articular cartilage damage (P = .024) versus those without. Immunohistochemistry did not reveal differences (P > .082) between the instability and FAI groups, but CD68 staining was significantly greater in those with cam morphology and cartilage damage (P < .045). CD45+/CD68- cells were noted in the perivascular area while CD45+/CD68+ cells were noted within the synovial lining in both groups. CONCLUSIONS: Increased synovial inflammation was associated with an increased number of turns to achieve joint distraction. Both instability and FAI groups demonstrated baseline levels of synovial inflammation. Synovitis scores also were increased in patients with cartilage damage. CLINICAL RELEVANCE: An understanding of the molecular and cellular mechanisms behind both hip instability and FAI may lead to novel therapeutic anti-inflammatory therapy, which may serve as an adjunct to treatment of mechanical abnormalities in this conditions.
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Impacto Femoroacetabular/patologia , Articulação do Quadril/patologia , Instabilidade Articular/patologia , Sinovite/patologia , Adulto , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Artroscopia , Feminino , Fluoroscopia , Humanos , Antígenos Comuns de Leucócito/metabolismo , Macrófagos/metabolismo , Masculino , Microscopia , TraçãoRESUMO
HYPOTHESIS: We hypothesized that patients with full-thickness rotator cuff tears would have greater synovial inflammation compared with those without rotator cuff tear pathology, with gene expression relating to histologic findings. METHODS: Synovial sampling was performed in 19 patients with full-thickness rotator cuff tears (RTC group) and in 11 patients without rotator cuff pathology (control group). Cryosections were stained and examined under light microscopy and confocal fluorescent microscopy for anti-cluster CD45 (common leukocyte antigen), anti-CD31 (endothelial), and anti-CD68 (macrophage) cell surface markers. A grading system was used to quantitate synovitis under light microscopy, and digital image analysis was used to quantify the immunofluorescence staining area. Quantitative polymerase chain reaction was performed for validated inflammatory markers. Data were analyzed with analysis of covariance, Mann-Whitney U, and Spearman rank order testing, with significance set at α = .05. RESULTS: The synovitis score was significantly increased in the RTC group compared with controls. Immunofluorescence demonstrated significantly increased staining for CD31, CD45, and CD68 in the RTC vs control group. CD45+/68- cells were found perivascularly, with CD45+/68+ cells toward the joint lining edge of the synovium. Levels of matrix metalloproteinase-3 (MMP-3) and interleukin-6 were significantly increased in the RTC group, with a positive correlation between the synovitis score and MMP-3 expression. CONCLUSIONS: Patients with full-thickness rotator cuff tears have greater levels of synovial inflammation, angiogenesis, and MMP-3 upregulation compared with controls. Gene expression of MMP-3 correlates with the degree of synovitis.
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Expressão Gênica , Metaloproteinase 3 da Matriz/genética , Lesões do Manguito Rotador/complicações , Sinovite/genética , Sinovite/metabolismo , Adulto , Idoso , Antígenos CD/análise , Antígenos CD/genética , Antígenos de Diferenciação Mielomonocítica/análise , Antígenos de Diferenciação Mielomonocítica/genética , Biomarcadores/análise , Estudos de Casos e Controles , Feminino , Humanos , Mediadores da Inflamação/análise , Interleucina-6/metabolismo , Antígenos Comuns de Leucócito/análise , Antígenos Comuns de Leucócito/genética , Masculino , Metaloproteinase 3 da Matriz/metabolismo , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Índice de Gravidade de Doença , Membrana Sinovial/química , Membrana Sinovial/patologia , Sinovite/etiologia , Sinovite/patologia , Regulação para CimaRESUMO
Visceral obesity has been defined as an important element of the metabolic syndrome and contributes to the development of insulin resistance and cardiovascular disease. Increasing endogenous levels of epoxyeicosatrienoic acids (EETs) are known for their analgesic, antihypertensive, and antiinflammatory effects. The availability of EETs is limited primarily by the soluble epoxide hydrolase (sEH, EPHX2), which metabolizes EETs to their less active diols. In this study, we tested the hypothesis that EETs are involved in glucose regulation and in retarding the development of insulin resistance. To address the role of EETs in regulating glucose homeostasis and insulin signaling, we used mice with targeted gene deletion of sEH (Ephx2-null mice) and a subsequent study with a selective sEH inhibitor. When wild-type mice are fed a high fat diet, insulin resistance develops. However, knockout or inhibition of sEH activity resulted in a significant decrease in plasma glucose. These findings are characterized by enhancement of tyrosyl phosphorylation of the insulin receptor, insulin receptor substrate 1, and their downstream cascade. In addition, pancreatic islets were larger when sEH was disrupted. This effect was associated with an increase in vasculature. These observations were supported by pharmacological inhibition of sEH. These data suggest that an increase in EETs due to sEH-gene knockout leads to an increase in the size of islets and improved insulin signaling and sensitivity.
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Epóxido Hidrolases/deficiência , Glucose/metabolismo , Resistência à Insulina , Ilhotas Pancreáticas/citologia , Animais , Ácido Araquidônico/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Epóxido Hidrolases/genética , Epóxido Hidrolases/fisiologia , Homeostase , Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Obesidade Abdominal , Pâncreas/metabolismo , Transdução de SinaisRESUMO
Soluble epoxide hydrolase (Ephx2, sEH) is a bifunctional enzyme with COOH-terminal hydrolase and NH(2)-terminal phosphatase activities. sEH converts epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), and the phosphatase activity is suggested to be involved in cholesterol metabolism. EETs participate in a wide range of biological functions, including regulation of vascular tone, renal tubular transport, cardiac contractility, and inflammation. Inhibition of sEH is a potential approach for enhancing the biological activity of EETs. Therefore, disruption of sEH activity is becoming an attractive therapeutic target for both cardiovascular and inflammatory diseases. To define the physiological role of sEH, we characterized a knockout mouse colony lacking expression of the Ephx2 gene. Lack of sEH enzyme is characterized by elevation of EET to DHET ratios in both the linoleate and arachidonate series in plasma and tissues of both female and male mice. In male mice, this lack of expression was also associated with decreased plasma testosterone levels, sperm count, and testicular size. However, this genotype was still able to sire litters. Plasma cholesterol levels also declined in this genotype. Behavior tests such as anxiety-like behavior and hedonic response were also examined in Ephx2-null and WT mice, as all can be related to hormonal changes. Null mice showed a level of anxiety with a decreased hedonic response. In conclusion, this study provides a broad biochemical, physiological, and behavioral characterization of the Ephx2-null mouse colony and suggests a mechanism by which sEH and its substrates may regulate circulating levels of testosterone through cholesterol biosynthesis and metabolism.
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Epóxido Hidrolases/genética , Testosterona/sangue , Animais , Ansiedade/genética , Ansiedade/fisiopatologia , Comportamento Animal/fisiologia , Colesterol/sangue , Colesterol/metabolismo , Eicosanoides/metabolismo , Compostos de Epóxi/metabolismo , Feminino , Fertilidade/genética , Genótipo , Hidrólise , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , SolubilidadeRESUMO
Hypertension and Type 2 diabetes are co-morbid diseases that lead to the development of nephropathy. sEH (soluble epoxide hydrolase) inhibitors are reported to provide protection from renal injury. We hypothesized that the sEH inhibitor AUDA [12-(3-adamantan-1-yl-ureido)-dodecanoic acid] protects the kidney from the development of nephropathy associated with hypertension and Type 2 diabetes. Hypertension was induced in spontaneously diabetic GK (Goto-Kakizaki) rats using AngII (angiotensin II) and a high-salt diet. Hypertensive GK rats were treated for 2 weeks with either AUDA or its vehicle added to drinking water. MAP (mean arterial pressure) increased from 118+/-2 mmHg to 182+/-20 and 187+/-6 mmHg for vehicle and AUDA-treated hypertensive GK rats respectively. AUDA treatment did not alter blood glucose. Hypertension in GK rats resulted in a 17-fold increase in urinary albumin excretion, which was decreased with AUDA treatment. Renal histological evaluation determined that AUDA treatment decreased glomerular and tubular damage. In addition, AUDA treatment attenuated macrophage infiltration and inhibited urinary excretion of MCP-1 (monocyte chemoattractant protein-1) and kidney cortex MCP-1 gene expression. Taken together, these results provide evidence that sEH inhibition with AUDA attenuates the progression of renal damage associated with hypertension and Type 2 diabetes.
Assuntos
Adamantano/análogos & derivados , Anti-Hipertensivos/uso terapêutico , Nefropatias Diabéticas/prevenção & controle , Epóxido Hidrolases/antagonistas & inibidores , Hipertensão/tratamento farmacológico , Ácidos Láuricos/uso terapêutico , Adamantano/uso terapêutico , Adamantano/urina , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/enzimologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/enzimologia , Nefropatias Diabéticas/enzimologia , Nefropatias Diabéticas/patologia , Progressão da Doença , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/uso terapêutico , Hipertensão/complicações , Hipertensão/enzimologia , Hipertensão/patologia , Insulina/sangue , Ácidos Láuricos/urina , Lipídeos/sangue , Masculino , NF-kappa B/metabolismo , Ratos , Ratos EndogâmicosRESUMO
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 micromol/L), the ATP-sensitive K+ channel (K(ATP)) inhibitor glibenclamide (1 micromol/L), the mitochondrial K(ATP) (mitoK(ATP)) inhibitor 5-hydroxydecanoate (100 to 200 micromol/L), or the Ca2+-sensitive K+ channel (K(Ca)) inhibitor paxilline (10 micromol/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-3beta (GSK-3beta) 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.
Assuntos
Epóxido Hidrolases/metabolismo , Coração/fisiopatologia , Contração Miocárdica/fisiologia , Isquemia Miocárdica/enzimologia , Animais , Ecocardiografia Transesofagiana , Glibureto/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitocôndrias Cardíacas/enzimologia , Isquemia Miocárdica/fisiopatologia , Miocárdio/enzimologiaRESUMO
The P450 eicosanoids epoxyeicosatrienoic acids (EETs) are produced in brain and perform important biological functions, including protection from ischemic injury. The beneficial effect of EETs, however, is limited by their metabolism via soluble epoxide hydrolase (sEH). We tested the hypothesis that sEH inhibition is protective against ischemic brain damage in vivo by a mechanism linked to enhanced cerebral blood flow (CBF). We determined expression and distribution of sEH immunoreactivity (IR) in brain, and examined the effect of sEH inhibitor 12-(3-adamantan-1-yl-ureido)-dodecanoic acid butyl ester (AUDA-BE) on CBF and infarct size after experimental stroke in mice. Mice were administered a single intraperitoneal injection of AUDA-BE (10 mg/kg) or vehicle at 30 mins before 2-h middle cerebral artery occlusion (MCAO) or at reperfusion, in the presence and absence of P450 epoxygenase inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MS-PPOH). Immunoreactivity for sEH was detected in vascular and non-vascular brain compartments, with predominant expression in neuronal cell bodies and processes. 12-(3-Adamantan-1-yl-ureido)-dodecanoic acid butyl ester was detected in plasma and brain for up to 24 h after intraperitoneal injection, which was associated with inhibition of sEH activity in brain tissue. Finally, AUDA-BE significantly reduced infarct size at 24 h after MCAO, which was prevented by MS-PPOH. However, regional CBF rates measured by iodoantipyrine (IAP) autoradiography at end ischemia revealed no differences between AUDA-BE- and vehicle-treated mice. The findings suggest that sEH inhibition is protective against ischemic injury by non-vascular mechanisms, and that sEH may serve as a therapeutic target in stroke.
Assuntos
Adamantano/análogos & derivados , Inibidores Enzimáticos/uso terapêutico , Epóxido Hidrolases/antagonistas & inibidores , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/enzimologia , Ureia/análogos & derivados , Adamantano/farmacocinética , Adamantano/uso terapêutico , Animais , Antipirina/análogos & derivados , Autorradiografia , Barreira Hematoencefálica , Western Blotting , Capilares/patologia , Eletroforese em Gel de Poliacrilamida , Inibidores Enzimáticos/farmacocinética , Imuno-Histoquímica , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Artéria Cerebral Média/fisiopatologia , Proteínas do Tecido Nervoso/isolamento & purificação , Ureia/farmacocinética , Ureia/uso terapêuticoRESUMO
The inflammatory process underlying chronic obstructive pulmonary disease (COPD) may be caused by tobacco smoke (TS) exposure. Previous studies show that epoxyeicosatrienoic acids (EETs) possess promising anti-inflammatory properties, therefore stabilization of EETs and other fatty acid epoxides through inhibition of soluble epoxide hydrolase (sEH) was investigated in mouse models of acute and sub-chronic inflammation caused by TS exposure. During the entire TS exposure, the potent sEH inhibitor 1-(1-methylsulfonyl-piperidin-4-yl)-3-(4-trifluoromethoxy-phenyl)-urea (TUPS) was given via drinking water. To assess drug target engagement of TUPS, a tandem mass spectrometry method was used for bioactive lipid profiling of a broad range of fatty acid metabolites, including EETs, and their corresponding diols (DHETs) derived from arachidonic acid, as well as epoxides and diols derived from other fatty acids. Several, but not all, plasma epoxide/diol ratios increased in mice treated with sEH inhibitor, compared to non-treated mice suggesting a wider role for sEH involving more fatty acid precursors besides arachidonic acid. This study supports qualitative use of epoxide/diol ratios explored by bioactive lipid profiling to indicate drug target engagement in mouse models of TS exposure relevant to COPD, which may have ramifications for future therapeutic interventions of sEH.
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CONTEXT: Articular cartilage has a unique functional architecture capable of providing a lifetime of pain-free joint motion. This tissue, however, undergoes substantial age-related physiologic, mechanical, biochemical, and functional changes that reduce its ability to overcome the effects of mechanical stress and injury. Many factors affect joint function in the maturing athlete-from chondrocyte survival and metabolism to structural composition and genetic/epigenetic factors governing cartilage and synovium. An evaluation of age-related changes for joint homeostasis and risk for osteoarthritis is important to the development of new strategies to rejuvenate aging joints. OBJECTIVE: This review summarizes the current literature on the biochemical, cellular, and physiologic changes occurring in aging articular cartilage. DATA SOURCES: PubMed (1969-2013) and published books in sports health, cartilage biology, and aging. STUDY SELECTION: Keywords included aging, athlete, articular cartilage, epigenetics, and functional performance with age. STUDY DESIGN: Systematic review. LEVEL OF EVIDENCE: Level 3. DATA EXTRACTION: To be included, research questions addressed the effect of age-related changes on performance, articular cartilage biology, molecular mechanism, and morphology. RESULTS: The mature athlete faces challenges in maintaining cartilage health and joint function due to age-related changes to articular cartilage biology, morphology, and physiology. These changes include chondrocyte loss and a decline in metabolic response, alterations to matrix and synovial tissue composition, and dysregulation of reparative responses. CONCLUSION: Although physical decline has been regarded as a normal part of aging, many individuals maintain overall fitness and enjoy targeted improvement to their athletic capacity throughout life. Healthy articular cartilage and joints are needed to maintain athletic performance and general activities. Genetic and potentially reversible epigenetic factors influence cartilage physiology and its response to mechanical and injurious stimuli. Improved understandings of the physical and molecular changes to articular cartilage with aging are important to develop successful strategies for joint rejuvenation.
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In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, providing alternate nonurologic and/or nonautologous tissue sources for bladder grafts. Such a model would also help decipher the mechanisms of urothelial differentiation and would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, perhaps elucidating areas of intervention for improved treatments. Thus far, in vitro derivation of urothelium from human embryonic stem cells (hESCs) or human induced pluripotent stem (hiPS) cells has not been reported. The goal of this work was to develop an efficient in vitro protocol for the induction of hESCs into urothelium through an intermediary definitive endoderm step and free of matrices and cell contact. During directed differentiation in a urothelial-specific medium ("Uromedium"), hESCs produced up to 60% urothelium, as determined by uroplakin expression; subsequent propagation selected for 90% urothelium. Alteration of the epithelial and mesenchymal cell signaling contribution through noncell contact coculture or conditioned media did not enhance the production of urothelium. Temporospatial evaluation of transcription factors known to be involved in urothelial specification showed association of IRF1, GET1, and GATA4 with uroplakin expression. Additional hESC and hiPS cell lines could also be induced into urothelium using this in vitro system. These results demonstrate that derivation and propagation of urothelium from hESCs and hiPS cells can be efficiently accomplished in vitro in the absence of matrices, cell contact, or adult cell signaling and that the induction process appears to mimic normal differentiation.
Assuntos
Antígenos de Diferenciação/biossíntese , Diferenciação Celular , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Células-Tronco Pluripotentes Induzidas/metabolismo , Urotélio/metabolismo , Animais , Linhagem Celular , Células-Tronco Embrionárias/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Urotélio/citologiaRESUMO
Epoxyeicosatrienoic acids (EETs) are small molecules produced by cytochrome P450 epoxygenases. They are lipid mediators that act as autocrine or paracrine factors to regulate inflammation and vascular tone. As a result, drugs that raise EET levels are in clinical trials for the treatment of hypertension and many other diseases. However, despite their pleiotropic effects on cells, little is known about the role of these epoxyeicosanoids in cancer. Here, using genetic and pharmacological manipulation of endogenous EET levels, we demonstrate that EETs are critical for primary tumor growth and metastasis in a variety of mouse models of cancer. Remarkably, we found that EETs stimulated extensive multiorgan metastasis and escape from tumor dormancy in several tumor models. This systemic metastasis was not caused by excessive primary tumor growth but depended on endothelium-derived EETs at the site of metastasis. Administration of synthetic EETs recapitulated these results, while EET antagonists suppressed tumor growth and metastasis, demonstrating in vivo that pharmacological modulation of EETs can affect cancer growth. Furthermore, inhibitors of soluble epoxide hydrolase (sEH), the enzyme that metabolizes EETs, elevated endogenous EET levels and promoted primary tumor growth and metastasis. Thus, our data indicate a central role for EETs in tumorigenesis, offering a mechanistic link between lipid signaling and cancer and emphasizing the critical importance of considering possible effects of EET-modulating drugs on cancer.
Assuntos
Eicosanoides/metabolismo , Metástase Neoplásica/fisiopatologia , Neoplasias Experimentais/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/genética , Hidrocarboneto de Aril Hidroxilases/metabolismo , Citocromo P-450 CYP2C8 , Citocromo P-450 CYP2J2 , Inibidores das Enzimas do Citocromo P-450 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Endotélio Vascular/metabolismo , Compostos de Epóxi/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Metástase Neoplásica/patologia , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Neovascularização Patológica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Obesity is an increasingly important public health issue reaching epidemic proportions. Visceral obesity has been defined as an important element of the metabolic syndrome and expansion of the visceral fat mass has been shown to contribute to the development of insulin resistance and cardiovascular disease. To identify novel contributors to cardiovascular and metabolic abnormalities in obesity, we analyzed the adipose proteome and identified soluble epoxide hydrolase (sEH) in the epididymal fat pad from C57BL/6J mice that received either a regular diet or a "western diet." sEH was synthesized in adipocytes and expression levels increased upon differentiation of 3T3-L1 preadipocytes. Although normalized sEH mRNA and protein levels did not differ in the fat pads from mice receiving a regular or a "western diet," total adipose sEH activity was higher in the obese mice, even after normalization for body weight. Furthermore, peroxisome proliferator-activated receptor gamma (PPARgamma) agonists increased the expression of sEH in mature 3T3-L1 adipocytes in vitro and in adipose tissue in vivo. Considering the established role for sEH in inflammation, cardiovascular diseases, and lipid metabolism, and the suggested involvement of sEH in the development of type 2 diabetes, our study has identified adipose sEH as a potential novel therapeutic target that might affect the development of metabolic and cardiovascular abnormalities in obesity.
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Adipócitos/enzimologia , Tecido Adiposo/enzimologia , Dieta , Epóxido Hidrolases/metabolismo , Obesidade/enzimologia , Células 3T3 , Animais , Epididimo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , PPAR gama/agonistas , ProteomaRESUMO
Soluble epoxide hydrolase (sEH) is a bifunctional enzyme with two catalytic domains: a C-terminal epoxide hydrolase domain and an N-terminal phosphatase domain. Epidemiology and animal studies have attributed a variety of cardiovascular and anti-inflammatory effects to the C-terminal epoxide hydrolase domain. The recent association of sEH with cholesterol-related disorders, peroxisome proliferator-activated receptor activity, and the isoprenoid/cholesterol biosynthesis pathway additionally suggest a role of sEH in regulating cholesterol metabolism. Here we used sEH knock-out (sEH-KO) mice and transfected HepG2 cells to evaluate the phosphatase and hydrolase domains in regulating cholesterol levels. In sEH-KO male mice we found a approximately 25% decrease in plasma total cholesterol as compared with wild type (sEH-WT) male mice. Consistent with plasma cholesterol levels, liver expression of HMG-CoA reductase was found to be approximately 2-fold lower in sEH-KO male mice. Additionally, HepG2 cells stably expressing human sEH with phosphatase only or hydrolase only activity demonstrate independent and opposite roles of the two sEH domains. Whereas the phosphatase domain elevated cholesterol levels, the hydrolase domain lowered cholesterol levels. Hydrolase inhibitor treatment in sEH-WT male and female mice as well as HepG2 cells expressing human sEH resulted in higher cholesterol levels, thus mimicking the effect of expressing the phosphatase domain in HepG2 cells. In conclusion, we show that sEH regulates cholesterol levels in vivo and in vitro, and we propose the phosphatase domain as a potential therapeutic target in hypercholesterolemia-related disorders.
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Colesterol/química , Epóxido Hidrolases/química , Regulação Enzimológica da Expressão Gênica , Animais , Linhagem Celular , Colesterol/sangue , Colesterol/metabolismo , Epóxido Hidrolases/genética , Feminino , Expressão Gênica , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Estrutura Terciária de ProteínaRESUMO
Arachidonic acid-derived epoxides, epoxyeicosatrienoic acids, are important regulators of vascular homeostasis and inflammation, and therefore manipulation of their levels is a potentially useful pharmacological strategy. Soluble epoxide hydrolase converts epoxyeicosatrienoic acids to their corresponding diols, dihydroxyeicosatrienoic acids, modifying or eliminating the function of these oxylipins. To better understand the phenotypic impact of Ephx2 disruption, two independently derived colonies of soluble epoxide hydrolase-null mice were compared. We examined this genotype evaluating protein expression, biofluid oxylipin profile, tissue oxylipin production capacity, and blood pressure. Ephx2 gene disruption eliminated soluble epoxide hydrolase protein expression and activity in liver, kidney, and heart from each colony. Plasma levels of epoxy fatty acids were increased, and fatty acid diols levels were decreased, while measured levels of lipoxygenase- and cyclooxygenase-dependent oxylipins were unchanged. Liver and kidney homogenates also show elevated epoxide fatty acids. However, in whole kidney homogenate a 4-fold increase in the formation of 20-hydroxyeicosatetraenoic acid was measured along with a 3-fold increase in lipoxygenase-derived hydroxylation and prostanoid production. Unlike previous reports, however, neither Ephx2-null colony showed alterations in basal blood pressure. Finally, the soluble epoxide hydrolase-null mice show a survival advantage following acute systemic inflammation. The data suggest that blood pressure homeostasis may be achieved by increasing production of the vasoconstrictor, 20-hydroxyeicosatetraenoic acid in the kidney of the Ephx2-null mice. This shift in renal metabolism is likely a metabolic compensation for the loss of the soluble epoxide hydrolase gene.
Assuntos
Pressão Sanguínea/fisiologia , Epóxido Hidrolases/deficiência , Epóxido Hidrolases/genética , Animais , Pressão Sanguínea/genética , Cruzamentos Genéticos , Compostos de Epóxi/metabolismo , Éxons , Ácidos Graxos não Esterificados/metabolismo , Feminino , Homeostase , Humanos , Rim/fisiologia , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos KnockoutRESUMO
Most studies on the nuclear retinoid-X receptor (RXR) have focused on its role as a heterodimeric partner but less about its own activation pattern during development and the distribution of potential endogenous ligands. The aim of this study is to visualize the distribution of activated RXRalpha in live transgenic Xenopus laevis embryos across a wide range of developmental stages. We adopted a nuclear receptor-Gal4 fusion/upstream activation sequence-based reporter system for our assay. Strong activation of the RXRalpha ligand-binding domain was observed in a segment of the spinal cord just posterior to the hindbrain. This activation is first detected in neurula stage embryos and persists up to swimming tadpole stages, after which activation strongly declines. Addition of exogenous ligands, such as 9-cis retinoic acid or all-trans retinoic acid, expands the activation of RXR throughout the spinal cord but not in the brain, whereas the RXR-specific ligand LG268 expanded the Gal4-RXR activation into the brain and olfactory epithelia. Treatment with the RAR-specific ligand 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid or thyroid hormone had no effect on Gal4-RXR activation, whereas these compounds significantly increased their corresponding Gal4/receptor fusion proteins under similar conditions. Embryos expressing a Gal4-RXR fusion protein with a deletion in the ligand-dependent activation domain (AF2) show no reporter gene activation. The results shown in this paper reveal a specific activation pattern for Gal4-RXRalpha specifically in the developing spinal cord and suggest the existence of RXR ligand "hot-spots" in this region.
Assuntos
Receptores do Ácido Retinoico/metabolismo , Fatores de Transcrição/metabolismo , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Anticolesterolemiantes/farmacologia , Benzoatos/farmacologia , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Ligantes , Ácidos Nicotínicos/farmacologia , Compostos Orgânicos , Receptores do Ácido Retinoico/efeitos dos fármacos , Receptores do Ácido Retinoico/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Receptores X de Retinoides , Retinoides/farmacologia , Proteínas de Saccharomyces cerevisiae/genética , Medula Espinal/embriologia , Medula Espinal/metabolismo , Tetra-Hidronaftalenos/farmacologia , Fatores de Transcrição/efeitos dos fármacos , Fatores de Transcrição/genética , Transcrição Gênica , Ativação Transcricional , Transfecção , Tretinoína/análogos & derivados , Tretinoína/farmacologia , Xenopus laevis/genéticaRESUMO
The presence of ATP in the genital tract fluid of mammals provokes questions regarding its function in the fertilization process. We investigated the effect of extracellular ATP (ATPe) on the activation of bovine spermatozoa. A signal transduction mechanism for ATP involving the receptor-mediated release of second messengers is described. Treatment of spermatozoa with ATP, uridine triphosphate (UTP), or 2-methylthio-ATP resulted in a concentration-dependent increase of acrosomal exocytosis, whereas treatment with either AMP or adenosine induced little exocytosis. This suggested that the receptor involved is of the P2 and not the P1 type. Several lines of evidence also suggest that the ATP purinoceptor is of the P2y and not the P2x type. First, the acrosome reaction was induced by the P2y-agonists ATP, UTP, or 2-methylthio-ATP, but no effects were shown by the P2x-agonists alpha,beta-methylene-ATP or beta,gamma-methylene-ATP. Second, ATP-induced acrosomal exocytosis was inhibited by the P2y antagonists, but not by the P2x antagonists. Third, enhanced Ca2+ uptake into the cells was observed with ATP and 2-methylthio-ATP, but not with beta,gamma-methylene-ATP. Additionally, ATP induced elevation of intracellular Ca2+ and cAMP, and the effect on cAMP was predominantly enhanced by including Ca2+ and the Ca2+-ionophore A23187 in the incubation medium. Extracellular ATP also activates protein kinase Calpha (PKCalpha), and the acrosome reaction, stimulated by ATPe, is inhibited by a PKC-specific inhibitor. In summary, we suggest that ATPe activates the P2 purinoceptor that elevates [Ca2+]i, which leads to PKCalpha activation and culminates in acrosomal exocytosis.
Assuntos
Reação Acrossômica/efeitos dos fármacos , Acrossomo/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Exocitose/efeitos dos fármacos , Receptores Purinérgicos P2/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Animais , Cálcio/metabolismo , Bovinos , AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Immunoblotting , Técnicas In Vitro , Isoenzimas/metabolismo , Masculino , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa , Capacitação Espermática/efeitos dos fármacos , Estimulação Química , Frações Subcelulares/efeitos dos fármacosRESUMO
Microdomains known as "rafts" have been isolated from many cell types as detergent-resistant membranes (DRMs) and are enriched in sphingolipids and cholesterol. However, there has been considerable controversy over whether such domains are found in native membranes or are artificially generated by the purification procedure. This controversy is based at least in part on the fact that raft membranes were first detected following detergent extraction in the cold. We isolated two plasma membrane fractions, without detergent treatment, using a discontinuous sucrose density gradient. One fraction was designated "light" and the other "heavy." These fractions were compared with DRMs, which were isolated in the presence of 1% Triton X-100. We found that Xenopus DRMs are enriched with sphingomyelin and cholesterol and exhibit a phase state similar to the liquid-ordered phase. Comparison of DRM complexes with the light and heavy plasma membrane fractions revealed some physical and biochemical similarities between the light fraction of the plasma membrane and the DRM complexes, based on (1) the phosphatidylcholine/sphingomyelin ratio and (2) the protein composition visualized on a two-dimensional gel. These two fractions are also quite similar in their thermotropic phase behavior, and their high levels of ganglioside GM1. We conclude that the light membrane fraction isolated in a detergent-free environment has many of the characteristics normally associated with DRMs.