RESUMO
BACKGROUND: Extracellular lysophosphatidic acid (LPA) species transmit signals via six different G protein-coupled receptors (LPAR1-6) and are indispensible for brain development and function of the nervous system. However, under neuroinflammatory conditions or brain damage, LPA levels increase, thereby inducing signaling cascades that counteract brain function. We describe a critical role for 1-oleyl-2-hydroxy-sn-glycero-3-phosphate (termed "LPA" throughout our study) in mediating a motile and pro-inflammatory microglial phenotype via LPAR5 that couples to protein kinase D (PKD)-mediated pathways. METHODS: Using the xCELLigence system and time-lapse microscopy, we investigated the migrational response of microglial cells. Different M1 and M2 markers were analyzed by confocal microscopy, flow cytometry, and immunoblotting. Using qPCR and ELISA, we studied the expression of migratory genes and quantitated the secretion of pro-inflammatory cytokines and chemokines, respectively. Different transcription factors that promote the regulation of pro-inflammatory genes were analyzed by western blot. Reactive oxygen species (ROS) and nitric oxide (NO) production, phagocytosis, and microglial cytotoxicity were determined using commercially available assay kits. RESULTS: LPA induces MAPK family and AKT activation and pro-inflammatory transcription factors' phosphorylation (NF-κB, c-Jun, STAT1, and STAT3) that were inhibited by both LPAR5 and PKD family antagonists. LPA increases migratory capacity, induces secretion of pro-inflammatory cytokines and chemokines and expression of M1 markers, enhances production of ROS and NO by microglia, and augments cytotoxicity of microglial cell-conditioned medium towards neurons. The PKD family inhibitor blunted all of these effects. We propose that interference with this signaling axis could aid in the development of new therapeutic approaches to control neuroinflammation under conditions of overshooting LPA production. CONCLUSIONS: In the present study, we show that inflammatory LPA levels increased the migratory response of microglia and promoted a pro-inflammatory phenotype via the LPAR5/PKD axis. Interference with this signaling axis reduced microglial migration, blunted microglial cytotoxicity, and abrogated the expression and secretion of pro-inflammatory mediators.
Assuntos
Movimento Celular/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Microglia/efeitos dos fármacos , Proteína Quinase C/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Ácidos Carboxílicos/farmacologia , Células Cultivadas , Córtex Cerebral/citologia , Citocinas/genética , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Fosforilação , Proteína Quinase C/genética , Pirimidinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidoresRESUMO
BACKGROUND AND PURPOSE: The antioxidant 5-hydroxymethylfurfural (5-HMF) exerts documented beneficial effects in several experimental pathologies and is currently tested as an antisickling drug in clinical trials. In the present study, we examined the cardiovascular effects of 5-HMF and elucidated the mode of action of the drug. EXPERIMENTAL APPROACH: The cardiovascular effects of 5-HMF were studied with pre-contracted porcine coronary arteries and rat isolated normoxic-perfused hearts. Isolated hearts subjected to ischaemia/reperfusion (I/R) injury were used to test for potential cardioprotective effects of the drug. The effects of 5-HMF on action potential and L-type Ca2+ current (ICa,L ) were studied by patch-clamping guinea pig isolated ventricular cardiomyocytes. KEY RESULTS: 5-HMF relaxed coronary arteries in a concentration-dependent manner and exerted negative inotropic, lusitropic and chronotropic effects in rat isolated perfused hearts. On the other hand, 5-HMF improved recovery of inotropic and lusitropic parameters in isolated hearts subjected to I/R. Patch clamp experiments revealed that 5-HMF inhibits L-type Ca2+ channels. Reduced ICa,L density, shift of ICa,L steady-state inactivation curves toward negative membrane potentials and slower recovery of ICa,L from inactivation in response to 5-HMF accounted for the observed cardiovascular effects. CONCLUSIONS AND IMPLICATIONS: Our data revealed a cardioprotective effect of 5-HMF in I/R that is mediated by inhibition of L-type Ca2+ channels. Thus, 5-HMF is suggested as a beneficial additive to cardioplegic solutions, but adverse effects and contraindications of Ca2+ channel blockers have to be considered in therapeutic application of the drug.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/fisiologia , Cardiotônicos/farmacologia , Vasos Coronários/efeitos dos fármacos , Furaldeído/análogos & derivados , Miócitos Cardíacos/efeitos dos fármacos , Animais , Vasos Coronários/fisiologia , Feminino , Furaldeído/farmacologia , Cobaias , Ventrículos do Coração/citologia , Masculino , Miócitos Cardíacos/fisiologia , Ratos Sprague-Dawley , Traumatismo por Reperfusão/fisiopatologia , SuínosRESUMO
BACKGROUND: Overexpression the KCNJ3, a gene that encodes subunit 1 of G-protein activated inwardly rectifying K(+) channel (GIRK1) in the primary tumor has been found to be associated with reduced survival times and increased lymph node metastasis in breast cancer patients. METHODS: In order to survey possible tumorigenic properties of GIRK1 overexpression, a range of malignant mammary epithelial cells, based on the MCF-7 cell line that permanently overexpress different splice variants of the KCNJ3 gene (GIRK1a, GIRK1c, GIRK1d and as a control, eYFP) were produced. Subsequently, selected cardinal neoplasia associated cellular parameters were assessed and compared. RESULTS: Adhesion to fibronectin coated surface as well as cell proliferation remained unaffected. Other vital parameters intimately linked to malignancy, i.e. wound healing, chemoinvasion, cellular velocities / motilities and angiogenesis were massively affected by GIRK1 overexpression. Overexpression of different GIRK1 splice variants exerted differential actions. While GIRK1a and GIRK1c overexpression reinforced the affected parameters towards malignancy, overexpression of GIRK1d resulted in the opposite. Single channel recording using the patch clamp technique revealed functional GIRK channels in the plasma membrane of MCF-7 cells albeit at very low frequency. DISCUSSION: We conclude that GIRK1d acts as a dominant negative constituent of functional GIRK complexes present in the plasma membrane of MCF-7 cells, while overexpression of GIRK1a and GIRK1c augmented their activity. The core component responsible for the cancerogenic action of GIRK1 is apparently presented by a segment comprising aminoacids 235-402, that is present exclusively in GIRK1a and GIRK1c, but not GIRK1d (positions according to GIRK1a primary structure). CONCLUSIONS: The current study provides insight into the cellular and molecular consequences of KCNJ3 overexpression in breast cancer cells and the mechanism upon clinical outcome in patients suffering from breast cancer.
Assuntos
Processamento Alternativo , Neoplasias da Mama/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Neoplasias da Mama/genética , Adesão Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Regulação para CimaRESUMO
Trophoblast invasion into uterine tissues represents a hallmark of first trimester placental development. As expression of serum amyloid A4 (SAA4) occurs in tumorigenic and invasive tissues we here investigated whether SAA4 is present in trophoblast-like human AC1-M59/Jeg-3 cells and trophoblast preparations of human first trimester and term placenta. SAA4 mRNA was expressed in non-stimulated and cytokine-treated AC1-M59/Jeg-3 cells. In purified trophoblast cells SAA4 mRNA expression was upregulated at weeks 10 and 12 of pregnancy. Western-blot and immunohistochemical staining of first trimester placental tissue revealed pronounced SAA4 expression in invasive trophoblast cells indicating a potential role of SAA4 during invasion.
Assuntos
Proteína Amiloide A Sérica/metabolismo , Trofoblastos/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Terceiro Trimestre da GravidezRESUMO
AIMS/HYPOTHESIS: For beta cells, contact with TNF-α triggers signalling cascades that converge on pathways important for cell survival and inflammation, specifically nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase pathways. Here, we investigated the function of baculoviral inhibitors of apoptosis repeat containing (BIRC) proteins in regulating TNF signalling cascades. METHODS: TNF regulation of Birc genes was studied by mRNA expression and promoter analysis. Birc gene control of cell signalling was studied in beta cell lines, and in islets from Birc2(-/-) and Birc3(-/-) mice, and from Birc3(-/-) Birc2Δ beta cell mice that selectively lack Birc2 and Birc3 (double knockout [DKO]). Islet function was tested by intraperitoneal glucose tolerance test and transplantation. RESULTS: TNF-α selectively induced Birc3 in beta cells, which in turn was sufficient to drive and potentiate NF-κB reporter activity. Conversely, Birc3(-/-) islets exhibited delayed TNF-α-induced IκBα degradation with reduced expression of Ccl2 and Cxcl10. DKO islets showed a further delay in IκBα degradation kinetics. Surprisingly, DKO islets exhibited stimulus-independent and TNF-dependent hyperexpression of TNF target genes A20 (also known as Tnfaip3), Icam1, Ccl2 and Cxcl10. DKO islets showed hyperphosphorylation of the JNK-substrate, c-Jun, while a JNK-antagonist prevented increases of Icam1, Ccl2 and Cxcl10 expression. Proteosome blockade of MIN6 cells phenocopied DKO islets. DKO islets showed more rapid loss of glucose homeostasis when challenged with the inflammatory insult of transplantation. CONCLUSIONS/INTERPRETATION: BIRC3 provides a feed-forward loop, which, with BIRC2, is required to moderate the normal speed of NF-κB activation. Paradoxically, BIRC2 and BIRC3 act as a molecular brake to rein in activation of the JNK signalling pathway. Thus BIRC2 and BIRC3 fine-tune NF-κB and JNK signalling to ensure transcriptional responses are appropriately matched to extracellular inputs. This control is critical for the beta cell's stress response.
Assuntos
Proteínas Inibidoras de Apoptose/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Animais , Proteína 3 com Repetições IAP de Baculovírus , Linhagem Celular , Proteínas Inibidoras de Apoptose/genética , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , NF-kappa B/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Ubiquitina-Proteína LigasesRESUMO
The synthesis of acute-phase protein serum amyloid A (SAA) is largely regulated by inflammation- associated cytokines and a high concentration of circulating SAA may represent an ideal marker for acute and chronic inflammatory diseases. However, SAA is also synthesized in extrahepatic tissues, e.g. human carcinoma metastases and cancer cell lines. An increasing body of in vitro data supports the concept of involvement of SAA in carcinogenesis and neoplastic diseases. Accumulating evidence suggests that SAA might be included in a group of biomarkers to detect a pattern of physiological events that reflect the growth of malignancy and host response. This review is meant to provide a broad overview of the many ways that SAA could contribute to tumour development, and accelerate tumour progression and metastasis, and to gain a better understanding of this acute-phase reactant as a possible link between chronic inflammation and neoplasia.
Assuntos
Biomarcadores Tumorais/sangue , Neoplasias/metabolismo , Proteína Amiloide A Sérica/fisiologia , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Biomarcadores , Biomarcadores Tumorais/fisiologia , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Neoplasias/sangue , Neoplasias/etiologia , Receptor para Produtos Finais de Glicação Avançada , Receptores de Formil Peptídeo/metabolismo , Receptores Imunológicos/metabolismo , Receptores de Lipoxinas/metabolismo , Receptores Depuradores Classe B/metabolismo , Proteína Amiloide A Sérica/genética , Proteína Amiloide A Sérica/metabolismoRESUMO
BACKGROUND AND PURPOSE: Endothelins (ETs) and their G protein-coupled receptors exert key physiological functions during normal and aberrant placental development. Trophoblast cells mediate the contact between the embryo and the mother, by establishing a transient organ, the placenta. Choriocarcinoma cells display many of the biochemical and morphological characteristics of in utero invasive trophoblast cells and may therefore be used as a suitable model to study epithelial tumour progression of foetal-derived cells. EXPERIMENTAL APPROACH: The present study aimed at investigating ET receptor-mediated activation of the mitogen-activated protein kinase (MAPK) pathway in human choriocarcinoma. KEY RESULTS: Both JAR and Jeg-3 choriocarcinoma cell lines expressed ET receptor subtype B (ET(B)) but not ET(A) receptor transcripts. ET(B) receptor engagement by ET-1 and ET-3 resulted in a similar time- and concentration-dependent phosphorylation of p42/44 MAPK, also known as extracellular regulated kinase 1/2. Using specific pharmacological antagonists/inhibitors, we showed that ET-1/-3-mediated signal transduction by the ET(B) receptor is transmitted via G(i)- and G(q)-dependent pathways through activation of the Src (G(i)) and protein kinase C (G(q)) axis that converge at Ras/Raf, leading to downstream activation of p42/44. On a functional level, ET(B) engagement and subsequent phosphorylation of p42/44 resulted in enhanced transcription of the immediate early response genes c-fos and c-jun, a process commonly assumed to be mediated by the ET(A) receptor, and increased cell growth and relative cell area. CONCLUSIONS AND IMPLICATIONS: As human choriocarcinoma cells secrete ETs, pharmacological antagonism of ETs and/or ET(B) receptor-mediated signal transduction could represent a likely target therapy for choriocarcinoma.
Assuntos
Coriocarcinoma/genética , Endotelina-1/farmacologia , Endotelina-3/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica/efeitos dos fármacos , Genes fos/efeitos dos fármacos , Genes jun/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Receptor de Endotelina B/genética , Western Blotting , Contagem de Células , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Primers do DNA , Humanos , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Sais de Tetrazólio , TiazóisRESUMO
Myeloperoxidase (MPO), a member of the haem peroxidase-cyclooxygenase superfamily, is abundantly expressed in neutrophils and to a lesser extent in monocytes and certain type of macrophages. MPO participates in innate immune defence mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity of MPO is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, evidence has emerged that MPO-derived oxidants contribute to tissue damage and the initiation and propagation of acute and chronic vascular inflammatory disease. The fact that circulating levels of MPO have been shown to predict risks for major adverse cardiac events and that levels of MPO-derived chlorinated compounds are specific biomarkers for disease progression, has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, detailed information on the structure of ferric MPO and its complexes with low- and high-spin ligands is available. This, together with a thorough understanding of reaction mechanisms including redox properties of intermediates, enables a rationale attempt in developing specific MPO inhibitors that still maintain MPO activity during host defence and bacterial killing but interfere with pathophysiologically persistent activation of MPO. The various approaches to inhibit enzyme activity of MPO and to ameliorate adverse effects of MPO-derived oxidants will be discussed. Emphasis will be put on mechanism-based inhibitors and high-throughput screening of compounds as well as the discussion of physiologically useful HOCl scavengers.
Assuntos
Inibidores Enzimáticos/farmacologia , Peroxidase/antagonistas & inibidores , Peroxidase/fisiologia , Animais , Cloretos/metabolismo , Inibidores Enzimáticos/uso terapêutico , Sequestradores de Radicais Livres/farmacologia , Humanos , Peróxido de Hidrogênio/metabolismo , Modelos Moleculares , Oxidantes/metabolismo , Peroxidase/química , Peroxidase/metabolismoRESUMO
Alpha-tocopheryl-succinate (alphaTS) is a synthetic, anti-neoplastic derivative of alpha-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated alphaTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated alphaTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with alphaTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective alphaTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective alphaTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, beta-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of alphaTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or beta-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.
Assuntos
Carcinoma/tratamento farmacológico , Lipoproteínas HDL/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Vitamina E/análogos & derivados , Vitamina E/farmacologia , Adenoviridae/genética , Animais , Antígenos CD36 , Divisão Celular , Linhagem Celular Tumoral , Centrifugação com Gradiente de Concentração , Fragmentação do DNA , Relação Dose-Resposta a Droga , Endocitose , Humanos , Hidrólise , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Nucleossomos/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Receptores Imunológicos/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Fatores de Tempo , Tocoferóis , Transfecção , beta-Galactosidase/metabolismoRESUMO
Events leading to hyperactivity of human blood platelets are accompanied by an enhanced risk of atherosclerosis and arterial thrombosis. Lipoprotein disorders affect platelet functions, and hypersensitive platelets are observed in various stages of hyperlipidemia. Low-density lipoprotein (LDL), a circulating complex of lipids and proteins that is increased in hypercholesterolemia, enhances platelet function and increases sensitivity of platelets to several naturally occurring agonists. LDL sensitizes platelets via binding of apoB-100 to a receptor on the platelet membrane and via transfer of lipids to the platelet membrane. The receptor that mediates binding of LDL to the platelet and initiates subsequent intracellular signaling cascades has not yet been identified. Modification of native LDL generates a platelet-activating particle, and this interaction might contribute to the development of the atherosclerotic plaque. Lysophosphatidic acid is formed upon mild oxidation of LDL and is responsible for subsequent platelet activation induced by the modified LDL particle. Thus, LDL changes the functions of platelets via a broad spectrum of interactions.
Assuntos
Plaquetas/metabolismo , Lipoproteínas HDL/metabolismo , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Metabolismo dos Lipídeos , Lipoproteínas LDL/metabolismo , Receptores de LDL/metabolismo , Transdução de SinaisRESUMO
As villous trophoblast does represent the contact zone between foetal and maternal tissues, the present in vitro study was aimed at investigating cholesterol supply from human high density lipoprotein subclass 3 (HDL(3)) to trophoblast cells isolated from human first trimester and term placenta. Binding of (125)I-HDL(3) was specific and saturable with similar K(d)-values for first trimester (54 microg HDL(3)-protein/ml) and term villous trophoblast cells (29 microg HDL(3)-protein/ml). The cell-association of (125)I-HDL(3) was 3-fold higher for term trophoblast cells while the specific cell-association of [(3)H]cholesterol ester(CE)-labelled HDL(3) was higher for first trimester trophoblast preparations. As a consequence, first trimester trophoblast cells have a pronounced capacity for selective CE-uptake from HDL(3). Competition experiments with native and oxidized low-density lipoprotein as well as cAMP-mediated stimulation of cell-association of [(3)H]CE-HDL(3) in both trophoblast preparations suggested the scavenger receptor class B, type I (SR-BI) as a likely receptor mediating this pathway. SR-BI m RNA could be identified by RT-PCR and Northern blot experiments in both trophoblast preparations. Western blot analysis and immunocytochemistry revealed high expression of SR-BI in first trimester trophoblast. A polyclonal antiserum raised against murine SR-BI significantly decreased cell-association of [(3)H]CE-HDL(3) in trophoblast cells. We conclude that human first trimester and term trophoblast cells express SR-BI which could serve as an efficient route for supplying cholesterol esters from maternal lipoproteins to foetal tissues.
Assuntos
Ésteres do Colesterol/metabolismo , Vilosidades Coriônicas/metabolismo , Proteínas de Membrana , Receptores Imunológicos , Receptores de Lipoproteínas , Trofoblastos/metabolismo , Adulto , Animais , Ligação Competitiva , Western Blotting , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células CHO , Adesão Celular/efeitos dos fármacos , Vilosidades Coriônicas/efeitos dos fármacos , Cricetinae , AMP Cíclico/farmacologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Trabalho de Parto , Lipoproteínas HDL/metabolismo , Lipoproteínas HDL3 , Microscopia Confocal , Gravidez , Primeiro Trimestre da Gravidez , RNA Mensageiro/metabolismo , Receptores Depuradores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Receptores Depuradores Classe B , Trofoblastos/citologia , Trofoblastos/efeitos dos fármacosRESUMO
As in other phagocytic cells, the NADPH-oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2(-.), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH-oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH-oxidase subunit p67(phox) translocates from the cytosol to the plasma membrane upon stimulation. Pre-incubation of microglia in alpha-tocopherol (alphaTocH) containing medium decreased O2(-.) production in a time- and concentration-dependent manner, findings attributed to attenuated p67(phox) translocation to the plasma membrane. Moreover, alphaTocH-supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC-inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to alphaTocH. The addition of okadaic acid or calyculin A completely restored O2(-.) production in alphaTocH-supplemented cells. The present findings suggest that alphaTocH inactivates PKC via a PP1 or PP2A-mediated pathway and, as a consequence, blocks the phosphorylation-dependent translocation of p67(phox) to the plasma membrane. As a result, O2(-.) production by the microglial NADPH-oxidase system is substantially inhibited.
Assuntos
Antioxidantes/farmacologia , Microglia/efeitos dos fármacos , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidases , Proteínas do Tecido Nervoso/fisiologia , Fosfoproteínas Fosfatases/fisiologia , Fosfoproteínas/antagonistas & inibidores , Explosão Respiratória/efeitos dos fármacos , Superóxidos/metabolismo , alfa-Tocoferol/farmacologia , Animais , Membrana Celular/enzimologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Citosol/enzimologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Toxinas Marinhas , Camundongos , Microglia/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Explosão Respiratória/fisiologia , Estaurosporina/farmacologia , SuínosRESUMO
Myeloperoxidase (MPO), a heme enzyme secreted by activated phagocytes, generates an array of oxidants proposed to play critical roles in host defense, tissues damage, and foam cell formation. Although neutrophils are the major source for MPO, the enzyme could be identified abundantly in circulating monocytes and monocytes/macrophages in rabbit lesions. MPO is the only enzyme known to generate hypochlorous acid (HOCl) and HOCl-modified lipoproteins have pronounced atherogenic and/or proinflammatory features in vivo and in vitro. Using specific monoclonal antibodies, HOCl-modified (lipo)proteins were detected in atherosclerotic plaques of heterozygous Watanabe heritable hyperlipidemic rabbits and to a lesser extent in a specific strain of New Zealand White rabbits with a high atherosclerotic response to hypercholesterolemia. Colocalization of immunoreactive MPO and HOCl-modified-epitopes in serial sections of rabbit lesions provides convincing evidence for MPO-H2O2-chloride system-mediated oxidation of (lipo)proteins under in vivo conditions. We propose that monocyte-derived MPO could connect chronic inflammatory conditions with arterial lipid/lipoprotein deposition during diet-induced atherogenesis in rabbits.
Assuntos
Arteriosclerose/etiologia , Arteriosclerose/metabolismo , Dieta Aterogênica , Ácido Hipocloroso/metabolismo , Lipoproteínas/metabolismo , Animais , Colesterol na Dieta/administração & dosagem , Modelos Animais de Doenças , Humanos , Hiperlipidemias/genética , Hiperlipidemias/metabolismo , Inflamação/metabolismo , Lipoproteínas/química , Monócitos/metabolismo , Oxidantes/metabolismo , Peroxidase/metabolismo , Proteínas/química , Proteínas/metabolismo , CoelhosRESUMO
During reverse cholesterol transport plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and pre-beta-HDL. The acute-phase response is accompanied with dramatic changes in lipid metabolism including alterations in HDL concentration, composition, and thereby its function as a substrate for HDL remodeling proteins in circulation. To evaluate how acute-phase HDL (AP-HDL) functions in PLTP-mediated HDL conversion, we collected plasma samples from patients with severe acute-phase response (n=17), and from healthy controls (n=30). Subsequently, total HDL (1.063Assuntos
Reação de Fase Aguda/sangue
, Proteínas de Transporte/sangue
, Glicoproteínas
, Lipoproteínas HDL/metabolismo
, Proteínas de Membrana/sangue
, Proteínas de Transferência de Fosfolipídeos
, Transporte Biológico
, Proteínas de Transporte/química
, Proteínas de Transporte/isolamento & purificação
, Proteínas de Transferência de Ésteres de Colesterol
, Humanos
, Lipoproteínas HDL/química
, Lipoproteínas HDL/isolamento & purificação
, Proteínas de Membrana/química
, Fosfatidilcolina-Esterol O-Aciltransferase/sangue
, Ultracentrifugação
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
Myeloperoxidase (MPO), which is released from cytoplasmic granules of activated phagocytes by a degranulation process, reacts with H(2)O(2) (generated during the oxidative burst) and chloride ions to generate hypochlorous acid/hypochlorite (HOCl/OCl(-)). HOCl, a strong oxidant, in turn reacts with proteins to form HOCl-modified proteins. The presence of these cytotoxic chloramines during inflammatory conditions, eg, atherosclerosis and glomerular and tubulointerstitial injury, suggested that chloramines are powerful oxidants that can have profound biologic effects. In the present study, immunoreactive MPO was identified in fetal membranes and the basal plate and in maternal and fetal blood cells of human placental tissues. Monocytes/macrophages represent the major cell source for MPO in human placental tissues. Immunohistochemical findings revealed that HOCl-modified proteins are present in normal human term placenta but not during the first trimester of pregnancy (Weeks 7 to 12). HOCl-modified proteins were localized in areas formed by fetally derived cells as well as maternal decidual tissues, ie, areas where fetal extravillous trophoblast cells invade the maternal tissue and stimulate the maternal immune system. HOCl-modified proteins, products of the MPO-H(2)O(2)-chloride system in vivo, were not present intracellularly, but immunoreactivity for HOCl-modified proteins was cell-associated and/or present in the extracellular matrix. Extravillous trophoblast cells, which may also exert phagocytic activities, showed no intracellular immunoreactivity for MPO or HOCl-modified proteins. The present findings indicate that the generation of HOCl-modified proteins during normal pregnancy is a physiologic rather than a pathophysiologic process.
Assuntos
Ácido Hipocloroso/metabolismo , Peroxidase/fisiologia , Placenta/enzimologia , Gravidez/metabolismo , Vilosidades Coriônicas/química , Vilosidades Coriônicas/enzimologia , Decídua/química , Decídua/enzimologia , Epitopos/imunologia , Membranas Extraembrionárias/química , Membranas Extraembrionárias/enzimologia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Macrófagos/enzimologia , Monócitos/enzimologia , Oxirredução , Peroxidase/análise , Peroxidase/imunologia , Placenta/química , Proteínas/metabolismoRESUMO
In reverse cholesterol transport, plasma phospholipid transfer protein (PLTP) converts high density lipoprotein(3) (HDL(3)) into two new subpopulations, HDL(2)-like particles and prebeta-HDL. During the acute-phase reaction (APR), serum amyloid A (SAA) becomes the predominant apolipoprotein on HDL. Displacement of apo A-I by SAA and subsequent remodeling of HDL during the APR impairs cholesterol efflux from peripheral tissues, and might thereby change substrate properties of HDL for lipid transfer proteins. Therefore, the aim of this work was to study the properties of SAA-containing HDL in PLTP-mediated conversion. Enrichment of HDL by SAA was performed in vitro and in vivo and the SAA content in HDL varied between 32 and 58 mass%. These HDLs were incubated with PLTP, and the conversion products were analyzed for their size, composition, mobility in agarose gels, and apo A-I degradation. Despite decreased apo A-I concentrations, PLTP facilitated the conversion of acute-phase HDL (AP-HDL) more effectively than the conversion of native HDL(3), and large fusion particles with diameters of 10.5, 12.0, and 13.8 nm were generated. The ability of PLTP to release prebeta from AP-HDL was more profound than from native HDL(3). Prebeta-HDL formed contained fragmented apo A-I with a molecular mass of about 23 kDa. The present findings suggest that PLTP-mediated conversion of AP-HDL is not impaired, indicating that the production of prebeta-HDL is functional during the ARP. However, PLTP-mediated in vitro degradation of apo A-I in AP-HDL was more effective than that of native HDL, which may be associated with a faster catabolism of inflammatory HDL.
Assuntos
Reação de Fase Aguda/metabolismo , Proteínas de Transporte/metabolismo , Lipoproteínas HDL/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Transferência de Fosfolipídeos , Animais , Colesterol/metabolismo , Lipoproteínas de Alta Densidade Pré-beta , Humanos , Tamanho da Partícula , Coelhos , Proteína Amiloide A Sérica/metabolismoRESUMO
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
It is clearly established that an efficient supply to the brain of alpha-tocopherol (alphaTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of alphaTocH into cells constituting the blood-brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of alphaTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated alphaTocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of alphaTocH uptake by pBCECs occurred by selective uptake, exceeding HDL3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated alphaTocH uptake. In accordance with the proposed function of SR-BI, selective HDL-CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14C]alphaTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL3-associated alphaTocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDL-associated alphaTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.