RESUMO
Myeloperoxidase (MPO) is one of the most abundant proteins in neutrophil granules. It catalyzes the production of reactive oxygen species, which are important in inflammation and immune defense. MPO also binds to several proteins, lipids, and DNA to alter their function. MPO is present at the feto-maternal interface during pregnancy, where neutrophils are abundant. In this study, we determined the effect of MPO on JEG-3 human choriocarcinoma cells as a model of extravillous trophoblasts (EVTs) during early pregnancy. We found that MPO was internalized by JEG-3 cells and localized to the cytoplasm and nuclei. MPO internalization and activity enhanced JEG-3 cell migration and invasion, whereas this effect was impaired by pre-treating cells with heparin, to block cellular uptake, and MPO-activity inhibitor 4-ABAH. This study identifies a novel mechanism for the effect of MPO on EVT function during normal pregnancy and suggests a potential role of MPO in abnormal pregnancies.
Assuntos
Coriocarcinoma , Trofoblastos , Feminino , Humanos , Gravidez , Linhagem Celular Tumoral , Coriocarcinoma/metabolismo , Coriocarcinoma/patologia , Peroxidase/metabolismo , Proteínas/metabolismo , Trofoblastos/metabolismoRESUMO
BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease is highly prevalent and its underlying pathogenesis involves dyslipidemia including pro-atherogenic high density lipoprotein (HDL) remodeling. Vitamins C and E have been proposed as atheroprotective agents for cardiovascular disease management. However, their effects and benefits on high density lipoprotein function and remodeling are unknown. In this study, we evaluated the role of vitamin C and E on non HDL lipoproteins as well as HDL function and remodeling, along with their effects on inflammation/oxidation biomarkers and atherosclerosis in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice. METHODS AND RESULTS: Mice were pre-treated for 5 weeks before and during atherogenic diet feeding with vitamin C and E added to water and diet, respectively. Compared to a control group, combined vitamin C and E administration reduced serum total cholesterol and triglyceride levels by decreasing apo B-48-containing lipoproteins, remodeled HDL particles by reducing phospholipid as well as increasing PON1 and apo D content, and diminished PLTP activity and levels. Vitamin supplementation improved HDL antioxidant function and lowered serum TNF-α levels. Vitamin C and E combination attenuated atherogenesis and increased lifespan in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice. CONCLUSIONS: Vitamin C and E administration showed significant lipid metabolism regulating effects, including HDL remodeling and decreased levels of apoB-containing lipoproteins, in mice. In addition, this vitamin supplementation generated a cardioprotective effect in a murine model of severe and lethal atherosclerotic ischemic heart disease.
Assuntos
Antioxidantes/farmacologia , Apolipoproteína B-48/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Hiperlipidemias/prevenção & controle , Lipoproteínas HDL/efeitos dos fármacos , Isquemia Miocárdica/prevenção & controle , Vitamina E/farmacologia , Animais , Apolipoproteína B-48/sangue , Cardiotônicos/farmacologia , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/prevenção & controle , Citocinas/sangue , Dieta Aterogênica , Suplementos Nutricionais , Ensaio de Imunoadsorção Enzimática , Feminino , Hiperlipidemias/sangue , Immunoblotting , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas HDL/sangue , Masculino , Camundongos Endogâmicos C57BL , Isquemia Miocárdica/sangue , Proteínas de Transferência de Fosfolipídeos/sangue , Valores de Referência , Reprodutibilidade dos Testes , Receptores Depuradores Classe B/sangue , Receptores Depuradores Classe B/efeitos dos fármacos , Resultado do TratamentoRESUMO
BACKGROUND AND AIMS: Atherosclerotic cardiovascular disease is highly prevalent and its underlying pathogenesis involves dyslipidemia including pro-atherogenic high density lipoprotein (HDL) remodeling. Vitamins C and E have been proposed as atheroprotective agents for cardiovascular disease management. However, their effects and benefits on high density lipoprotein function and remodeling are unknown. In this study, we evaluated the role of vitamin C and E on non HDL lipoproteins as well as HDL function and remodeling, along with their effects on inflammation/ oxidation biomarkers and atherosclerosis in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice. METHODS AND RESULTS: Mice were pre-treated for 5 weeks before and during atherogenic diet feeding with vitamin C and E added to water and diet, respectively. Compared to a control group, combined vitamin C and E administration reduced serum total cholesterol and triglyceride levels by decreasing apo B-48-containing lipoproteins, remodeled HDL particles by reducing phospholipid as well as increasing PON1 and apo D content, and diminished PLTP activity and levels. Vitamin supplementation improved HDL antioxidant function and lowered serum TNF-α levels. Vitamin C and E combination attenuated atherogenesis and increased lifespan in atherogenic diet-fed SR-B1 KO/ApoER61h/h mice. CONCLUSIONS: Vitamin C and E administration showed significant lipid metabolism regulating effects, including HDL remodeling and decreased levels of apoB-containing lipoproteins, in mice. In addition, this vitamin supplementation generated a cardioprotective effect in a murine model of severe and lethal atherosclerotic ischemic heart disease.
Assuntos
Animais , Masculino , Feminino , Ácido Ascórbico/farmacologia , Vitamina E/farmacologia , Isquemia Miocárdica/prevenção & controle , Apolipoproteína B-48/efeitos dos fármacos , Hiperlipidemias/prevenção & controle , Lipoproteínas HDL/efeitos dos fármacos , Antioxidantes/farmacologia , Valores de Referência , Doença da Artéria Coronariana/prevenção & controle , Doença da Artéria Coronariana/sangue , Ensaio de Imunoadsorção Enzimática , Cardiotônicos/farmacologia , Immunoblotting , Reprodutibilidade dos Testes , Citocinas/sangue , Resultado do Tratamento , Isquemia Miocárdica/sangue , Suplementos Nutricionais , Proteínas de Transferência de Fosfolipídeos/sangue , Dieta Aterogênica , Receptores Depuradores Classe B/efeitos dos fármacos , Receptores Depuradores Classe B/sangue , Metabolismo dos Lipídeos/efeitos dos fármacos , Apolipoproteína B-48/sangue , Hiperlipidemias/sangue , Lipoproteínas HDL/sangue , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: G protein-coupled receptor 55 (GPR55) is a lysophospholipid receptor responsive to certain cannabinoids. The role of GPR55 in inflammatory processes of the gut is largely unknown. Using the recently characterized GPR55 inhibitor CID16020046, we determined the role of GPR55 in experimental intestinal inflammation and explored possible mechanisms of action. METHODS: Colitis was induced by either 2.5% dextran sulfate sodium (DSS) supplemented in the drinking water of C57BL/6 mice or by a single intrarectal application of trinitrobenzene sulfonic acid (TNBS). KEY RESULTS: Daily application of CID16020046 (20 mg/kg) significantly reduced inflammation scores and myeloperoxidase (MPO) activity. In the DSS colitis model, levels of tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß), and the expression of cyclooxygenase (Cox)-2 and signal transducer and activator of transcription 3 (STAT-3) were reduced in colon tissues while in TNBS-induced colitis, levels of Cox-2, IL-1ß and IL-6 were significantly lowered. Evaluation of leukocyte recruitment by flow cytometry indicated reduced presence of lymphocytes and macrophages in the colon following GPR55 inhibition in DSS-induced colitis. In J774A.1 mouse macrophages, inhibition of GPR55 revealed reduced migration of macrophages and decreased CD11b expression, suggesting that direct effects of CID16020046 on macrophages may have contributed to the improvement of colitis. GPR55(-/-) knockout mice showed reduced inflammation scores as compared to wild type mice in the DSS model suggesting a pro-inflammatory role in intestinal inflammation. CONCLUSIONS & INFERENCES: Pharmacological blockade of GPR55 reduces experimental intestinal inflammation by reducing leukocyte migration and activation, in particular that of macrophages. Therefore, CID16020046 represents a possible drug for the treatment of bowel inflammation.
Assuntos
Compostos Azabicíclicos/farmacologia , Benzoatos/farmacologia , Antagonistas de Receptores de Canabinoides/farmacologia , Colite/tratamento farmacológico , Colite/imunologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Animais , Compostos Azabicíclicos/administração & dosagem , Benzoatos/administração & dosagem , Antagonistas de Receptores de Canabinoides/administração & dosagem , Colite/induzido quimicamente , Sulfato de Dextrana/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Canabinoides/efeitos dos fármacos , Ácido Trinitrobenzenossulfônico/farmacologiaRESUMO
BACKGROUND AND PURPOSE: Pulmonary vascular dysfunction is a key event in acute lung injury. We recently demonstrated that PGE2 , via activation of E-prostanoid (EP)4 receptors, strongly enhances microvascular barrier function in vitro. The aim of this study was to investigate the beneficial effects of concomitant EP4 receptor activation in murine models of acute pulmonary inflammation. EXPERIMENTAL APPROACH: Pulmonary inflammation in male BALB/c mice was induced by LPS (20 µg per mouse intranasally) or oleic acid (0.15 µL·g-1 , i.v. ). In-vitro, endothelial barrier function was determined by measuring electrical impedance. KEY RESULTS: PGE2 activation of EP4 receptors reduced neutrophil infiltration, pulmonary vascular leakage and TNF-α concentration in bronchoalveolar lavage fluid from LPS-induced pulmonary inflammation. Similarly, pulmonary vascular hyperpermeability induced by oleic acid was counteracted by EP4 receptor activation. In lung function assays, the EP4 agonist ONO AE1-329 restored the increased resistance and reduced compliance upon methacholine challenge in mice treated with LPS or oleic acid. In agreement with these findings, EP4 receptor activation increased the in vitro vascular barrier function of human and mouse pulmonary microvascular endothelial cells and diminished the barrier disruption induced by LPS. The EP2 agonist ONO AE1-259 likewise reversed LPS-induced lung dysfunction without enhancing vascular barrier function. CONCLUSION AND IMPLICATIONS: Our results show that activation of the EP4 receptor strengthens the microvascular barrier function and thereby ameliorates the pathology of acute lung inflammation, including neutrophil infiltration, vascular oedema formation and airway dysfunction. This suggests a potential benefit for EP4 agonists in acute pulmonary inflammation.
RESUMO
The structural integrity of apolipoprotein A-I (apo A-I) is critical to the physiological function of high-density lipoprotein (HDL). Oxidized lipoproteins are thought to be of central importance in atherogenesis, and oxidation products characteristic of myeloperoxidase, a heme protein secreted by activated phagocytes, have been detected in human atherosclerotic tissue. At plasma concentrations of halide ion, hypochlorous acid is a major product of the myeloperoxidase-hydrogen peroxide-chloride system. We therefore investigated the effects of activated human neutrophils, a potent source of myeloperoxidase and hydrogen peroxide, on the protein and lipid components of HDL. Both free and HDL-associated apo A-I exposed to activated human neutrophils underwent extensive degradation as monitored by RP-HPLC and Western blotting with a polyclonal antibody to apo A-I. Replacement of the neutrophils with reagent HOCl resulted in comparable damage (at molar oxidant : HDL subclass 3 ratio = 100) as observed in the presence of activated phagocytes. Apo A-I degradation by activated neutrophils was partially inhibited by the HOCl scavenger methionine, by the heme inhibitor azide, by chloride-free conditions, by the peroxide scavenger catalase, and by a combination of superoxide dismutase (SOD)/catalase, implicating HOCl in the cell-mediated reaction. The addition of a protease inhibitor (3,4-dichloroisocoumarin) further reduced the extent of apo A-I damage. In contrast to the protein moiety, there was little evidence for oxidation of unsaturated fatty acids or cholesterol in HDL3 exposed to activated neutrophils, suggesting that HOCl was selectively damaging apo A-I. Our observations indicate that HOCl generated by myeloperoxidase represents one pathway for protein degradation in HDL3 exposed to activated phagocytes.
Assuntos
Cloretos/metabolismo , Peróxido de Hidrogênio/metabolismo , Neutrófilos/metabolismo , Peroxidase/metabolismo , Western Blotting , Catalase/metabolismo , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Humanos , Neutrófilos/enzimologia , Superóxido Dismutase/metabolismoAssuntos
Antígenos CD36/isolamento & purificação , Endotélio Vascular/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de LDL/isolamento & purificação , Receptores de Lipoproteínas , Antígenos CD36/química , Antígenos CD36/metabolismo , Ésteres do Colesterol/metabolismo , Humanos , Ácido Hipocloroso/química , Lipoproteínas HDL/química , Receptores de LDL/química , Receptores de LDL/metabolismo , Receptores de LDL Oxidado , Receptores Depuradores , Receptores Depuradores Classe B , Receptores Depuradores Classe E , Veias Umbilicais/citologiaRESUMO
Hypochlorous acid/hypochlorite, generated by the myeloperoxidase/H(2)O(2)/halide system of activated phagocytes, has been shown to oxidize/modify low density lipoprotein (LDL) in vitro and may be involved in the formation of atherogenic lipoproteins in vivo. Accordingly, hypochlorite-modified (lipo)proteins have been detected in human atherosclerotic lesions where they colocalize with macrophages and endothelial cells. The present study investigates the influence of hypochlorite-modified LDL on endothelial synthesis of nitric oxide (NO) measured as formation of citrulline (coproduct of NO) and cGMP (product of the NO-activated soluble guanylate cyclase) upon cell stimulation with thrombin or ionomycin. Pretreatment of human umbilical vein endothelial cells with hypochlorite-modified LDL led to a time- and concentration-dependent inhibition of agonist-induced citrulline and cGMP synthesis compared with preincubation of cells with native LDL. This inhibition was neither due to a decreased expression of endothelial NO synthase (eNOS) nor to a deficiency of its cofactor tetrahydrobiopterin. Likewise, the uptake of l-arginine, the substrate of eNOS, into the cells was not affected. Hypochlorite-modified LDL caused remarkable changes of intracellular eNOS distribution including translocation from the plasma membrane and disintegration of the Golgi location without altering myristoylation or palmitoylation of the enzyme. In contrast, cyclodextrin known to deplete plasma membrane of cholesterol and to disrupt caveolae induced only a disappearance of eNOS from the plasma membrane that was not associated with decreased agonist-induced citrulline and cGMP formation. The present findings suggest that mislocalization of NOS accounts for the reduced NO formation in human umbilical vein endothelial cells treated with hypochlorite-modified LDL and point to an important role of Golgi-located NOS in these processes. We conclude that inhibition of NO synthesis by hypochlorite-modified LDL may be an important mechanism in the development of endothelial dysfunction and early pathogenesis of atherosclerosis.
Assuntos
Biopterinas/análogos & derivados , Endotélio Vascular/enzimologia , Ácido Hipocloroso/metabolismo , Lipoproteínas LDL/metabolismo , Óxido Nítrico Sintase/metabolismo , Óxido Nítrico/biossíntese , Antioxidantes/farmacologia , Arginina/farmacocinética , Biopterinas/biossíntese , Western Blotting , Membrana Celular/metabolismo , Células Cultivadas , Centrifugação com Gradiente de Concentração , Citrulina/biossíntese , GMP Cíclico/metabolismo , Ciclodextrinas/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/citologia , Complexo de Golgi/metabolismo , Humanos , Immunoblotting , Imuno-Histoquímica , Ionomicina/farmacologia , Ionóforos/farmacologia , Microscopia de Fluorescência , Ácidos Mirísticos/metabolismo , Óxido Nítrico Sintase Tipo III , Ácidos Palmíticos/metabolismo , Testes de Precipitina , RNA Mensageiro/metabolismo , Frações Subcelulares/metabolismo , Sacarose/metabolismo , Fatores de Tempo , Veias Umbilicais/citologiaRESUMO
The serum amyloid A (SAA) family of proteins is encoded by multiple genes that display allelic variation and a high degree of homology in mammals. Triggered by inflammation after stimulation of hepatocytes by lymphokine-mediated processes, the concentrations of SAA may increase during the acute-phase reaction to levels 1000-fold greater than those found in the noninflammatory state. In addition to its role as an acute-phase reactant, SAA (104 amino acids, 12 kDa) is considered to be the precursor protein of secondary reactive amyloidosis, in which the N-terminal portion is incorporated into the bulk of amyloid fibrils. However, the association with lipoproteins of the high-density range and subsequent modulation of the metabolic properties of its physiological carrier appear to be the principal role of SAA. Because SAA may displace apolipoprotein A-I, the major protein component of native high density lipoprotein (HDL), during the acute-phase reaction, the present study was aimed at (1) investigating binding properties of native and acute-phase (SAA-enriched) HDL by J774 macrophages, (2) elucidating whether the presence of SAA on HDL particles affects selective uptake of HDL-associated cholesteryl esters, and (3) comparing cellular cholesterol efflux mediated by native and acute-phase HDL. Both the total and the specific binding at 4 degrees C of rabbit acute-phase HDL were approximately 2-fold higher than for native HDL. Nonlinear regression analysis revealed K(d) values of 7.0 x 10(-7) mol/L (native HDL) and 3.1 x 10(-7) mol/L (acute-phase HDL), respectively. The corresponding B(max) values were 203 ng of total lipoprotein per milligram of cell protein (native HDL) and 250 ng of total lipoprotein per milligram of cell protein (acute-phase HDL). At 37 degrees C, holoparticle turnover was slightly enhanced for acute-phase HDL, a fact reflected by 2-fold higher degradation rates. In contrast, the presence of SAA on HDL specifically increased (1. 7-fold) the selective uptake of HDL cholesteryl esters from acute-phase HDL by J774 macrophages, a widely used in vitro model to study foam cell formation and cholesterol efflux properties. Although ligand blotting experiments with solubilized J774 membrane proteins failed to identify the scavenger receptor-BI as a binding protein for both native and acute-phase HDL, 2 binding proteins with molecular masses of 100 and 72 kDa, the latter comigrating with CD55 (also termed decay-accelerating factor), were identified. During cholesterol efflux studies, it became apparent that the ability of acute-phase HDL with regard to cellular cholesterol removal was considerably lower than that for native HDL. This was reflected by a 1.7-fold increase in tau/2 values (22 versus 36 hours; native versus acute-phase HDL). Our observations of increased HDL cholesteryl ester uptake and reduced cellular cholesterol efflux (acute-phase versus native HDL) suggest that displacement of apolipoprotein A-I by SAA results in considerable altered metabolic properties of its main physiological carrier. These changes in the apolipoprotein moieties appear (at least in the in vitro system tested) to transform an originally antiatherogenic into a proatherogenic lipoprotein particle.