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1.
Cancers (Basel) ; 15(2)2023 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-36672299

RESUMO

BACKGROUND: Platelet-cancer cell interactions modulate tumor metastasis and thrombosis in cancer. Platelet-derived extracellular vesicles (EVs) can contribute to these outcomes. METHODS: We characterized the medium-sized EVs (mEVs) released by thrombin-stimulated platelets of colorectal cancer (CRC) patients and healthy subjects (HS) on the capacity to induce epithelial-mesenchymal transition (EMT)-related genes and cyclooxygenase (COX)-2(PTGS2), and thromboxane (TX)B2 production in cocultures with four colorectal cancer cell lines. Platelet-derived mEVs were assessed for their size distribution and proteomics signature. RESULTS: The mEV population released from thrombin-activated platelets of CRC patients had a different size distribution vs. HS. Platelet-derived mEVs from CRC patients, but not from HS, upregulated EMT marker genes, such as TWIST1 and VIM, and downregulated CDH1. PTGS2 was also upregulated. In cocultures of platelet-derived mEVs with cancer cells, TXB2 generation was enhanced. The proteomics profile of mEVs released from activated platelets of CRC patients revealed that 119 proteins were downregulated and 89 upregulated vs. HS. CONCLUSIONS: We show that mEVs released from thrombin-activated platelets of CRC patients have distinct features (size distribution and proteomics cargo) vs. HS and promote prometastatic and prothrombotic phenotypes in cancer cells. The analysis of platelet-derived mEVs from CRC patients could provide valuable information for developing an appropriate treatment plan.

2.
Front Endocrinol (Lausanne) ; 12: 706352, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34305819

RESUMO

Polyfluoro- and perfluoro-alkyl substances (PFAS) are organic chemicals extensively used worldwide for industry and consumer products. Due to their chemical stability, PFAS represent a major cause of environmental pollution. PFAS accumulate in animal and human blood and tissues exerting their toxicity. We performed a review of the epidemiological studies exploring the relationship between exposure to PFAS and thromboembolic cardiovascular disease. An increase in cardiovascular disease or death related to PFAS exposure has been reported from cross-sectional and longitudinal observational studies with evidence concerning the relation with early vascular lesions and atherosclerosis. Several studies indicate an alteration in lipid and glucose metabolism disorders and increased blood pressure as a possible link with cardiovascular thromboembolic events. We also examined the recent evidence indicating that legacy and new PFAS can be incorporated in platelet cell membranes giving a solid rationale to the observed increase risk of cardiovascular events in the populations exposed to PFAS by directly promoting thrombus formation. Exposure to PFAS has been related to altered plasma membrane fluidity and associated with altered calcium signal and increased platelet response to agonists, both in vitro and ex vivo in subjects exposed to PFAS. All the functional responses are increased in platelets by incorporation of PFAS: adhesion, aggregation, microvesicles release and experimental thrombus formation. These findings offer mechanistic support the hypothesis that platelet-centred mechanisms may be implicated in the increase in cardiovascular events observed in populations chronically exposed to PFAS.


Assuntos
Plaquetas/patologia , Doenças Cardiovasculares/patologia , Poluentes Ambientais/efeitos adversos , Fluorocarbonos/efeitos adversos , Trombose/patologia , Plaquetas/efeitos dos fármacos , Doenças Cardiovasculares/induzido quimicamente , Doenças Cardiovasculares/epidemiologia , Humanos , Trombose/epidemiologia , Trombose/etiologia
3.
Environ Int ; 154: 106584, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33895438

RESUMO

BACKGROUND: Health concerns associated with the exposure to legacy perfluoro-alkyl substances (PFAS) led to the development of new-generation PFAS, such as C6O4. Here we investigated the possible effects of C6O4 on the platelet's activation profile, by incubating human platelets from healthy donors with C6O4 at different concentrations and evaluating the effects on activation, production and phenotype of platelets micro-particles (MPV) and aggregation under-flow. Based on the eventual platelet pro-aggregation profile detected, the preventive effect of acetylsalicylic acid (ASA) was also explored. METHODS: Adhesion-induced platelet aggregation of platelet rich plasma (PRP) under flow was evaluated on collagen-coated microchip at a shear stress of 10 Dyne. The turbidimetric method was used to investigate platelet aggregation. Finally, the in vitro generation of pro-coagulant MPV in PRP was evaluated by flow cytometry, as characterized by CD41 and annexin V positive events, under resting conditions and after stimulation with agonists at low shear stress. RESULTS: The generation of platelet aggregates under flow was significantly increased by the pretreatment of PRP with 100-200 ng/mL C6O4, compared to both the control condition and the experiment performed in presence of ASA. Arachidonic acid (AA), ADP and collagen induced an higher maximal aggregation, at turbidimetric evaluation, when PRP was pretreated with 100-500 ng/mL C6O4. In addition, PRP stimulated with AA also showed a steeper slope of the aggregation curve. The aggregation induced by the tested agonists was almost abolished by ASA. Finally, pretreatment with C6O4 increased the number of MPV in resting conditions and in presence of ADP and TRAP. ASA tended to reduce MPV generation. CONCLUSIONS: Exposure to C6O4 associates with an increased platelet response to agonists, translating into a possible increased risk of cardiovascular events. Pending a further clarification on the toxicokinetics of this compound, our results claim the possible prophylactic use of ASA.


Assuntos
Sinais (Psicologia) , Agregação Plaquetária , Aspirina/farmacologia , Plaquetas , Humanos , Testes de Função Plaquetária
4.
J Thromb Haemost ; 19(6): 1558-1571, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33590615

RESUMO

BACKGROUND: Ion channels are transmembrane proteins that play important roles in cell function regulation modulating ionic cell permeability. In megakaryocytes and platelets, regulated ion flows have been demonstrated to modulate platelet production and function. However, a relatively limited characterization of ion channel expression and function is available in the human megakaryocyte-platelet lineage. OBJECTIVE: We analyzed the expression and function of the large-conductance calcium and voltage-activated potassium channel Kca 1.1 (also known as Maxi-K, BK, slo1) in human megakaryocytes and platelets. METHODS: To investigate the functionality of Kca 1.1, we exploited different agonists (BMS-191011, NS1619, NS11021, epoxyeicosatrienoic acid isoforms) and inhibitors (iberiotoxin, penitrem A) of the channel. RESULTS: In megakaryocytes, Kca 1.1 agonists determined a decreased proplatelet formation and altered interaction with the extracellular matrix. Analysis of the actin cytoskeleton demonstrated a significant decrease in megakaryocyte spreading and adhesion to collagen. In platelets, the opening of the channel Kca 1.1 led to a reduced sensitivity to agonists with blunted aggregation in response to ADP, with an inhibitory capacity additive to that of aspirin. The Kca 1.1 agonists, but not the inhibitors, determined a reduction of platelet adhesion and aggregation onto immobilized collagen underflow to an extent similar to that of aspirin and ticagrelor. The opening of the Kca 1.1 resulted in cell hyperpolarization impairing free intracellular calcium in ADP-stimulated platelets and megakaryocytes. CONCLUSIONS: The present study reveals new mechanisms in platelet formation and activation, suggesting that targeting Kca 1.1 channels might be of potential pharmacological interest in hemostasis and thrombosis.


Assuntos
Cálcio , Megacariócitos , Benzimidazóis , Plaquetas , Humanos , Canais de Potássio
5.
Front Pharmacol ; 10: 1256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31780927

RESUMO

Platelet-derived large extracellular vesicles (often referred to as microparticles in the field of cardiovascular disease) have been identified as effector in the atherothrombotic process, therefore representing a target of pharmacological intervention of potential interest. Despite that, limited evidence is so far available concerning the effects of antiplatelet agents on the release of platelet-derived extracellular vesicles. In the present narrative review, the mechanisms leading to vesiculation in platelets and the pathophysiological processes implicated will be discussed. This will be followed by a summary of the present evidence concerning the effects of antiplatelet agents under experimental conditions and in clinical settings.

6.
Front Pharmacol ; 9: 1081, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30319416

RESUMO

The thromboxane (TX) A2 elicits TP-dependent different platelet responses. Low amounts activate Src kinases and the Rho-Rho kinase pathway independently of integrin αIIbß3 and ADP secretion and synergize with epinephrine to induce aggregation. Aim of the present study was to investigate the role Src kinases and the interplay with calcium signals in reactive oxygen species (ROS) generation in the activatory pathways engaged by TXA2 in human platelets. All the experiments were performed in vitro or ex vivo. Washed platelets were stimulated with 50-1000 nM U46619 and/or 10 µM epinephrine in the presence of acetylsalicylic acid and the ADP scavenger apyrase. The effects of the ROS scavenger EUK-134, NADPH oxidase (NOX) inhibitor apocynin, Src kinase inhibitor PP2 and calcium chelator BAPTA were tested. Intracellular calcium and ROS generation were measured. Platelet rich plasma from patients treated with dasatinib was used to confirm the data obtained in vitro. We observed that 50 nM U46619 plus epinephrine increase intracellular calcium similarly to 1000 nM U46619. ROS generation was blunted by the NOX inhibitor apocynin. BAPTA inhibited ROS generation in resting and activated platelets. Phosphorylation of Src and MLC proteins were not significantly affected by antioxidants agents. BAPTA and antioxidants reduced P-Selectin expression, activation of integrin αIIbß3and platelet aggregation. TXA2-induced increase in intracellular calcium is required for Src phosphorylation and ROS generation. NADPH oxidase is the source of ROS in TX stimulated platelets. The proposed model helps explain why an incomplete inhibition of TP receptor results in residual platelet activation, and define new targets for antiplatelet treatment.

7.
Clin Pharmacol Ther ; 104(1): 111-119, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29574792

RESUMO

We studied the influence of cardiovascular (CV) risk factors, previous CV events, and cotreatments with preventive medicines, on residual platelet thromboxane (TX)B2 production in 182 patients chronically treated with enteric coated (EC)-aspirin (100 mg/day). The response to aspirin was also verified by assessing arachidonic acid-induced platelet aggregation and urinary 11-dehydro-TXB2 levels. Residual serum TXB2 levels exceeded the upper limit value for an adequate aspirin response in 14% of individuals. This phenomenon was detected at 12 hours after dosing with aspirin. The coadministration of statins (mostly atorvastatin) was an independent predictor of residual serum TXB2 levels, and the percentage of patients with enhanced values was significantly lower in statin users vs. nonusers. We provide evidence in vitro that atorvastatin reduced residual TXB2 generation by increasing the extent of acetylation of platelet COX-1 by aspirin. In conclusion, the coadministration of statins may counter the mechanisms associated with reduced bioavailability of aspirin detected in some individuals with CV disease.


Assuntos
Aspirina/uso terapêutico , Atorvastatina/uso terapêutico , Plaquetas/metabolismo , Doenças Cardiovasculares/prevenção & controle , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Tromboxano B2/biossíntese , Acetilação/efeitos dos fármacos , Idoso , Aspirina/farmacologia , Atorvastatina/farmacologia , Disponibilidade Biológica , Doenças Cardiovasculares/epidemiologia , Ciclo-Oxigenase 1/efeitos dos fármacos , Ciclo-Oxigenase 1/metabolismo , Quimioterapia Combinada , Feminino , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Inibidores da Agregação Plaquetária/farmacologia , Prevenção Primária , Fatores de Risco , Prevenção Secundária , Comprimidos com Revestimento Entérico , Tromboxano B2/análogos & derivados , Tromboxano B2/urina
8.
Clin Sci (Lond) ; 131(16): 2095-2107, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28634189

RESUMO

Loss-of-function mutations of the the ATP-binding cassette-1 (ABCA1) gene are the cause of Tangier disease (TD) in homozygous subjects and familial HDL deficiency (FHD) in heterozygous subjects. These disorders are characterized by reduced plasma HDL-cholesterol (HDL-C) and altered efflux of cholesterol from cells. Previous studies in TD patients and ABCA1-/- murine models reported defects in platelet count, morphology, and function, but the issue is still controversial. We analyzed three subjects with low to very low HDL-C levels due to the loss-of-function mutations of the ABCA1 gene. Two related patients with FHD were heterozygous carriers of two mutations on the same ABCA1 allele; one, with TD, was homozygous for a different mutation. Mild to moderate thrombocytopenia was observed in all the patients. No morphological platelet abnormalities were detected under optical or EM. History of moderate bleeding tendency was recorded only in one of the FHD patients. Only limited alterations in platelet aggregation and activation of the integrin αIIbß3 were observed in one FHD patient. While α-granule secretion (P-selectin), content, and secretion of platelet δ-granules (serotonin, ATP, and ADP) and thromboxane (TX) A2 synthesis were normal in all the patients, the expression of lysosomal CD63, in response to some agonists, was reduced in TD patients. In conclusion, three patients carrying ABCA1 genetic variants had low platelet count, with the lowest values observed in TD, not associated with major alterations in platelet morphology and response to agonists or bleeding.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/genética , Plaquetas/fisiologia , Mutação , Trombocitopenia/genética , Idoso , Plaquetas/ultraestrutura , Coleta de Amostras Sanguíneas/métodos , Feminino , Humanos , Hipoalfalipoproteinemias/sangue , Hipoalfalipoproteinemias/genética , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Agregação Plaquetária/fisiologia , Contagem de Plaquetas , Testes de Função Plaquetária/métodos , Doença de Tangier/sangue , Doença de Tangier/genética , Trombocitopenia/sangue
9.
Front Pharmacol ; 7: 314, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27695417

RESUMO

Platelet microparticles (PMPs) contribute to thrombogenesis but the effects of antiplatelet drugs on PMPs generation is undefined. The present study investigated the cellular events regulating PMPs shedding, testing in vitro platelet agonists and inhibitors. Platelet-rich plasma from healthy subjects was stimulated with arachidonic acid (AA), U46619, collagen type-I (10 and 1.5 µg/mL), epinephrine, ADP or TRAP-6 and pre-incubated with acetylsalicylic acid (ASA, 100 and 10 µmol/L), SQ-29,548, apyrase, PSB-0739, or eptifibatide. PMPs were detected by flow-cytometry using CD61 and annexin-V as fluorescent markers. Platelet agonists induced annexin V-positive PMPs shedding. The strongest response was to high concentration collagen. ADP-triggered PMPs shedding was dose-independent. ASA reduced PMPs induced by AA- (645, 347-2946 vs. 3061, 446-4901 PMPs/µL; median ad range, n = 9, P < 0.001), collagen 10 µg/mL (5317, 2027-15935 vs. 10252, 4187-46316 PMPs/µL; n = 13, P < 0.001), collagen 1.5 µg/mL (1078, 528-2820 vs. 1465, 582-5948 PMPs/µL; n = 21, P < 0.001) and TRAP-6 (2008, 1621-2495 vs. 2840, 2404-3031 PMPs/µL; n = 3, P < 0.01) but did not affect the response to epinephrine or ADP. The ADP scavenger apyrase reduced PMPs induced by U46619 (1256, 395-2908 vs. 3045, 1119-5494 PMPs/µL, n = 6, P < 0.05), collagen 1.5 µg/mL (1006, 780-1309 vs. 2422, 1839-3494 PMPs/µL, n = 3, P < 0.01) and TRAP-6 (904, 761-1224 vs. 2840, 2404-3031 PMPs/µL, n = 3, P < 0.01). The TP receptor antagonist SQ-29,548 and the P2Y12 receptor antagonist PSB-0739 markedly inhibited PMPs induced by low doses of collagen. Except for high-dose collagen, eptifibatide abolished agonist-induced PMPs release. Both TXA2 generation and ADP secretion are required as amplifiers of PMP shedding. The crucial role of the fibrinogen receptor and the collagen receptor in PMPs generation, independently of platelet aggregation, was identified.

10.
J Matern Fetal Neonatal Med ; 28(8): 869-75, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25034210

RESUMO

We tested the hypothesis that maternal peripheral blood leukocytes contribute to elevated levels of soluble TNF receptors (sTNFR) in preeclampsia (PE) with concomitant intrauterine growth restriction (IUGR). TNFR1 and TNFR2 were evaluated in a cross-sectional study comparing preeclamptic (n = 15) with or without IUGR versus normotensive pregnant women (PREG, n = 30), and non-pregnant controls (Con; n = 20). Plasma levels of sTNFR1 were higher in PE (1675.0 ± 227.1 pg/mL) compared with PREG (1035.0 ± 101.1 pg/mL) and Con (589.3 ± 82.67 pg/mL), with the highest values observed in PE with IUGR (2624.0 ± 421.4 pg/mL; n = 6). Plasma sTNFR2 was higher during pregnancy (PE: 1836.0 ± 198.7 pg/mL; PREG: 1697.0 ± 95.0 pg/mL) compared with Con (598.3 ± 82.7 pg/mL). Urinary levels of sTNFR1 and sTNFR2 were higher in PE and PREG compared with the Con group. Abundance of TNFR1 mRNA in peripheral blood leukocytes was strongly correlated with plasma levels of sTNFR1 in PE. However, TNFR2 mRNA accumulation in leukocytes did not correlate with sTNFR2 plasma levels. The level of sTNFR1 in plasma was correlated with body weight of the newborn (r = -0.56). The data suggest that maternal leukocytes contribute to sTNFR1 levels in plasma in association with decreasing newborn weight and PE with concomitant IUGR.


Assuntos
Retardo do Crescimento Fetal/imunologia , Leucócitos/metabolismo , Pré-Eclâmpsia/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/sangue , Receptores Tipo I de Fatores de Necrose Tumoral/sangue , Adulto , Biomarcadores/sangue , Biomarcadores/urina , Peso ao Nascer , Estudos de Casos e Controles , Estudos Transversais , Feminino , Retardo do Crescimento Fetal/sangue , Retardo do Crescimento Fetal/urina , Humanos , Recém-Nascido , Pessoa de Meia-Idade , Pré-Eclâmpsia/sangue , Pré-Eclâmpsia/urina , Gravidez , Reação em Cadeia da Polimerase em Tempo Real , Receptores Tipo I de Fatores de Necrose Tumoral/urina , Receptores Tipo II do Fator de Necrose Tumoral/urina , Índice de Gravidade de Doença
11.
Free Radic Biol Med ; 63: 108-14, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23619127

RESUMO

Mitochondrial diseases (MDs) are heterogeneous disorders due to impaired respiratory chain function causing defective ATP production. Although the disruption of oxidative phosphorylation is central to the MD pathophysiology, other factors may contribute to these disorders. We investigated the expression and the cellular localization of TNF-α and its receptors, TNFR1 and TNFR2, in muscle biopsies from 15 patients with mitochondrial respiratory chain dysfunction. Our data unambiguously demonstrate that TNF-α is expressed in muscle fibers with abnormal focal accumulations of mitochondria, so-called ragged red fibers, and is delivered to mitochondria where both receptors are localized. Moreover TNF receptors are differentially regulated in patients' muscle in which the expression levels of TNFR1 mRNA are decreased and those of TNFR2 mRNA are increased compared with controls. These findings suggest for the first time that TNF-α could exert a direct effect on mitochondria via its receptors.


Assuntos
Doenças Mitocondriais/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Biópsia , DNA Mitocondrial/genética , Feminino , Expressão Gênica , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doenças Mitocondriais/genética , Mutação , Fosforilação Oxidativa , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo II do Fator de Necrose Tumoral/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética
12.
Clin Sci (Lond) ; 122(6): 289-97, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21970465

RESUMO

MDs (mitochondrial diseases) are a clinically heterogeneous group of disorders characterized by impairment of the respiratory chain function with altered oxidative phosphorylation. We tested the hypothesis that the function of vascular endothelium is affected by increased oxidative stress in MDs. A total of 12 patients with MDs and pair-matched controls were studied. Endothelial function was assessed by measuring FMD (flow-mediated vasodilation) of brachial and common femoral arteries. The test was repeated after vitamin C (500 mg, twice a day) and E (400 mg, once a day) supplementation for 30 days and 90 days after vitamin withdrawal. FMD was reduced in patients compared with controls [AUC/τ (time-averaged area under the curve) for the brachial artery, 1.05±0.24 compared with 4.19±0.59% respectively, P<0.001; AUC/τ for the femoral artery, 0.98±0.19 compared with 2.36±0.29% respectively, P=0.001; values are means±S.E.M.] and correlated (brachial artery) with plasma lactate (r=-0.63, P<0.01). Urinary 8-iso-PGF2α (8-iso-prostaglandin F2α) was higher in patients than controls (505.6±85.9 compared with 302.5±38.7 pg/mg of creatinine; P<0.05) and correlated with plasma lactate (r=0.70, P<0.05). Immunohistochemical analysis showed 8-iso-PGF2α staining in MD-affected striated muscle cells and in blood vessels in muscle biopsies of patients. Antioxidant vitamins transiently restored FMD in patients [ΔAUC/τ (change in AUC/τ) for the brachial artery, +1.38±0.49%, P<0.05; ΔAUC/τ for the femoral artery, +0.98±0.24%, P<0.01] but had no effect on FMD in controls (brachial artery, -1.3±0.63%; and common femoral artery, -0.58±0.30%), thus abolishing the differences between patients and controls. The results of the present study indicate that oxidative stress is increased and is, at least partly, responsible for endothelial dysfunction in MDs.


Assuntos
Endotélio Vascular/fisiopatologia , Doenças Mitocondriais/fisiopatologia , Estresse Oxidativo , Adulto , Dinoprosta/análogos & derivados , Dinoprosta/urina , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Óxido Nítrico/fisiologia , Espécies Reativas de Oxigênio , Vasodilatação
13.
Mol Cell Proteomics ; 10(4): M110.002964, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21156839

RESUMO

Mitochondrial diseases (MD) are heterogeneous disorders because of impairment of respiratory chain function leading to oxidative stress. We hypothesized that in MD the vascular endothelium may be affected by increased oxidative/nitrative stress causing a reduction of nitric oxide availability. We therefore, investigated the pathobiology of vasculature in MD patients by assaying the presence of 3-nitrotyrosine in muscle biopsies followed by the proteomic identification of proteins which undergo tyrosine nitration. We then measured the flow-mediated vasodilatation as a proof of altered nitric oxide generation/bioactivity. Here, we show that 3-nitrotyrosine staining is specifically located in the small vessels of muscle tissue and that the reaction is stronger and more evident in a significant percentage of vessels from MD patients as compared with controls. Eleven specific proteins which are nitrated under pathological conditions were identified; most of them are involved in energy metabolism and are located mainly in mitochondria. In MD patients the flow-mediated vasodilatation was reduced whereas baseline arterial diameters, blood flow velocity and endothelium-independent vasodilatation were similar to controls. The present results provide evidence that in MD the vessel wall is a target of increased oxidative/nitrative stress.


Assuntos
Síndrome MELAS/metabolismo , Síndrome MERRF/metabolismo , Músculo Esquelético/irrigação sanguínea , Tirosina/análogos & derivados , Adolescente , Adulto , Idoso , Sequência de Bases , Artéria Braquial/fisiopatologia , Estudos de Casos e Controles , Surdez/genética , Surdez/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Endotélio Vascular/metabolismo , Feminino , Artéria Femoral/fisiopatologia , Humanos , Síndrome de Kearns-Sayre/genética , Síndrome de Kearns-Sayre/metabolismo , Síndrome MELAS/genética , Síndrome MERRF/genética , Masculino , Pessoa de Meia-Idade , Doenças Mitocondriais , Músculo Esquelético/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Mutação Puntual , Deleção de Sequência , Tirosina/metabolismo , Vasodilatação
14.
J Am Coll Cardiol ; 44(3): 635-41, 2004 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-15358033

RESUMO

OBJECTIVES: The goal of this study was to test the hypothesis that NCX-4016 may have broader anti-inflammatory and antithrombotic effects as well as better gastric tolerability than aspirin in humans. BACKGROUND: NCX-4016 is an aspirin derivative containing a nitric oxide-releasing moiety that prevents platelet activation and modulates tissue factor (TF) expression and cytokine release from lipopolysaccharide (LPS)-stimulated monocytes. METHODS: This was a blind-observer, placebo-controlled, parallel-group study in which 48 healthy subjects were randomized to receive NCX-4016 800 mg twice a day, NCX-4016 800 mg twice a day plus aspirin 325 mg, aspirin 325 mg, or placebo for 21 days. RESULTS: Similar to aspirin alone, NCX-4016 effectively inhibited platelet aggregation induced by 0.6 mmol/ arachidonic acid, clot-stimulated thromboxane (TX) B2 generation in whole blood, and urinary excretion of 11-dehydro-TXB2. Unlike aspirin alone, the administration of NCX-4016 significantly inhibited TF expression in monocytes stimulated ex vivo with 10 micromol/l LPS (determined by flow-cytometry analysis of TF on CD14 positive cells). NCX-4016 also inhibited the rapid TF expression induced in monocytes by a proteinase activated receptor agonist (thrombin receptor activator protein, 2 micromol/l) as well as LPS-induced expression of CD11b . Ex vivo, release of MCP-1 and interleukin-6 were significantly inhibited by NCX-4016, but not by aspirin. NCX-4016 was not associated with gastric damage, and significantly reduced gastric injury when co-administered with aspirin, although both drugs reduced gastric PGE2 production to the same extent. CONCLUSIONS: NCX-4016 is equally effective as aspirin in inhibiting cyclooxygenase activity. However, NCX-4016 causes less gastric damage and prevents monocyte activation. Larger multicenter trials are warranted to establish clinical efficacy and safety of NCX-4016.


Assuntos
Aspirina/administração & dosagem , Aspirina/farmacologia , Monócitos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Estômago/efeitos dos fármacos , Administração Oral , Adulto , Aspirina/efeitos adversos , Aspirina/análogos & derivados , Esquema de Medicação , Combinação de Medicamentos , Quimioterapia Combinada , Duodeno/efeitos dos fármacos , Citometria de Fluxo , Mucosa Gástrica/efeitos dos fármacos , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Inibidores da Agregação Plaquetária/administração & dosagem , Inibidores da Agregação Plaquetária/efeitos adversos , Inibidores da Agregação Plaquetária/farmacologia , Valores de Referência , Método Simples-Cego , Tromboxano A2/metabolismo , Tromboxano B2/urina
15.
Circulation ; 106(24): 3120-5, 2002 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-12473561

RESUMO

BACKGROUND: NCX-4016 is an acetylsalicylic acid (ASA) derivative containing a nitric oxide-releasing moiety. Compared with ASA, NCX-4016 has a broader spectrum of antithrombotic and antiinflammatory activities. We hypothesized that NCX-4016 might inhibit in vivo lipopolysaccharide (LPS)-induced expression of tissue factor (TF). METHODS AND RESULTS: Rats were administered 90 mg/kg NCX-4016 orally for 5 days. Placebo, 50 mg/kg ASA, and 80 mg/kg isosorbide-5-mononitrate (ISMN) were used in control groups. On day 5, rats were injected intraperitoneally with 100 microg/kg LPS and killed 6 hours later. The expression of TF in monocytes was measured by flow cytometry and Western blot analysis. Reverse transcriptase-polymerase chain reaction was performed to assess expression of TF and cyclooxygenase-2 (COX-2) genes. Plasma concentrations of interleukin-1beta and tumor necrosis factor-alpha were measured. Urine samples were collected to evaluate the excretion of the thromboxane metabolite 11-dehydro-thromboxane (TX)B2. Gastric mucosa was inspected. LPS injection was followed by synthesis TF and COX-2 mRNAs in circulating monocytes, which were blunted by NCX-4016 but not by ASA or ISMN. Both NCX-4016 and ISMN reduced TF expression on surface of circulating monocyte. LPS increased the excretion 11-dehydro-TXB2, and this was prevented by NCX-4016 and ASA. Unlike ASA, NCX-4016 reduced plasma interleukin-1beta and tumor necrosis factor-alpha. In addition, NCX-4016 almost completely prevented mucosal damage, whereas ASA increased the extension of gastric lesions in LPS-injected rats. CONCLUSIONS: NCX-4016 prevents monocyte TF expression; this is accompanied by inhibition of TX and cytokine biosynthesis. These additive effects of nitric oxide release and COX inhibition may help explain efficacy and tolerability of NCX-4016.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Aspirina/análogos & derivados , Aspirina/farmacologia , Dinitrato de Isossorbida/análogos & derivados , Óxido Nítrico/metabolismo , Inibidores da Agregação Plaquetária/farmacologia , Tromboplastina/antagonistas & inibidores , Tromboxano B2/análogos & derivados , Administração Oral , Animais , Western Blotting , Ciclo-Oxigenase 2 , Inibidores de Cisteína Proteinase/farmacologia , Esquema de Medicação , Citometria de Fluxo , Mucosa Gástrica/efeitos dos fármacos , Interleucina-1/sangue , Isoenzimas/biossíntese , Isoenzimas/genética , Dinitrato de Isossorbida/farmacologia , Masculino , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Prostaglandina-Endoperóxido Sintases/biossíntese , Prostaglandina-Endoperóxido Sintases/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tromboplastina/biossíntese , Tromboplastina/genética , Tromboxano B2/urina , Fator de Necrose Tumoral alfa/análise
16.
J Hypertens ; 20(7): 1393-9, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12131536

RESUMO

OBJECTIVES: Despite the claimed disregulation of extracellular matrix synthesis and the increased proliferation rate of different cell types in experimental models of hypertension, very few data are available on collagen synthesis and the proliferation rate of fibroblasts in essential hypertensive patients. DESIGN: We measured collagen I, collagen III, histone H3 mRNA gene expression, collagen protein concentration and thymidine incorporation in fibroblasts from 17 essential hypertensive patients (EH) and 13 healthy normotensive control subjects (NC). METHODS: A Northern blot analysis was performed on fibroblasts in culture obtained from skin biopsies. Collagen protein concentration and DNA synthesis were measured by means of incorporation of tritiated proline and tritiated thymidine, respectively. RESULTS: In cultivated fibroblasts from hypertensives, the expression of collagen III mRNA after addition of fetal calf serum was significantly increased in comparison with that of normotensive-derived cells. After addition of fetal calf serum, collagen protein was statistically increased in cultures from EH patients as compared to NC. In hypertensives, the expression of histone H3 mRNA as well as tritiated thymidine incorporation were both increased as compared to normotensives. CONCLUSIONS: Our data suggest that cultivated fibroblasts from essential hypertensive patients are characterized by an increased expression of type III collagen mRNA and collagen protein synthesis in response to fetal serum, as compared to normotensive-derived cells. Cells from hypertensives are characterized by an increased rate of proliferation after addition of fetal serum, as ascertained by increased thymidine incorporation and increased histone H3 mRNA gene expression, as compared to normotensive-derived cells. This phenotype could be genetically determined and may have an important role in the pathogenesis of essential hypertension.


Assuntos
Divisão Celular/fisiologia , Colágeno Tipo III/genética , Colágeno Tipo I/genética , Fibroblastos/fisiologia , Expressão Gênica/genética , Hipertensão/genética , Hipertensão/fisiopatologia , Pele/irrigação sanguínea , Pele/citologia , Adulto , Índice de Massa Corporal , Feminino , Marcadores Genéticos/genética , Ventrículos do Coração/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Estatística como Assunto
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