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BACKGROUND: Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants. METHODS: We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4 h during a 24-h period in a light/darkness synchronised environment. FINDINGS: Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P = 0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P = 0.017) with a difference of 56.8 (95% CI 10.3-103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P = 0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients. Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations. INTERPRETATION: Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches. FUNDING: Hartstichting.
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Relógios Circadianos , Insuficiência Cardíaca , Melatonina , Humanos , Masculino , Camundongos , Animais , Pessoa de Meia-Idade , Feminino , Relógios Circadianos/fisiologia , Peixe-Zebra/metabolismo , Hidrocortisona , Ritmo Circadiano/genéticaRESUMO
While immunopathology has been widely studied in patients with severe COVID-19, immune responses in non-hospitalized patients have remained largely elusive. We systematically analyze 484 peripheral cellular or soluble immune features in a longitudinal cohort of 63 mild and 15 hospitalized patients versus 14 asymptomatic and 26 household controls. We observe a transient increase of IP10/CXCL10 and interferon-ß levels, coordinated responses of dominant SARS-CoV-2-specific CD4 and fewer CD8 T cells, and various antigen-presenting and antibody-secreting cells in mild patients within 3 days of PCR diagnosis. The frequency of key innate immune cells and their functional marker expression are impaired in hospitalized patients at day 1 of inclusion. T cell and dendritic cell responses at day 1 are highly predictive for SARS-CoV-2-specific antibody responses after 3 weeks in mild but not hospitalized patients. Our systematic analysis reveals a combinatorial picture and trajectory of various arms of the highly coordinated early-stage immune responses in mild COVID-19 patients.
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Antivirais , COVID-19 , Anticorpos Antivirais , Linfócitos T CD8-Positivos , Humanos , SARS-CoV-2RESUMO
DNA methylation is one of the most important epigenetic modifications and is closely related with several biological processes such as regulation of gene transcription and the development of non-malignant diseases. The prevailing dogma states that DNA methylation in eukaryotes occurs essentially through 5-methylcytosine (5mC) but recently adenine methylation was also found to be present in eukaryotes. In mouse embryonic stem cells, 6-methyladenine (6mA) was associated with the repression and silencing of genes, particularly in the X-chromosome, known to play an important role in cell fate determination. Here, we have demonstrated that 6mA is a ubiquitous eukaryotic epigenetic modification that is put in place during epigenetically sensitive periods such as embryogenesis and fetal development. In somatic cells there are clear tissue specificity in 6mA levels, with the highest 6mA levels being observed in the brain. In zebrafish, during the first 120 h of embryo development, from a single pluripotent cell to an almost fully formed individual, 6mA levels steadily increase. An identical pattern was observed over embryonic days 7-21 in the mouse. Furthermore, exposure to a neurotoxic environmental pollutant during the same early life period may led to a decrease in the levels of this modification in female rats. The identification of the periods during which 6mA epigenetic marks are put in place increases our understanding of this mammalian epigenetic modification, and raises the possibility that it may be associated with developmental processes.
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BACKGROUND: Novel therapeutic targets of heart failure (HF) are needed. Long noncoding RNAs (lncRNAs) are engaged during cardiac regeneration. Unlike in humans, zebrafish naturally undergo cardiac regeneration after HF. We aimed to describe the landscape of lncRNAs during regeneration in a zebrafish model of HF and to investigate their human homologs. METHODS: HF was established in adult zebrafish through thrice-weekly incubations with an anemia-inducing drug, phenylhydrazine hydrochloride (PHZ). After 5 weeks, PHZ treatment ceased and the fish were followed through a regeneration period of 14 days. Total RNA was extracted from the hearts of adult zebrafish after establishment of HF and at 2, 5, and 9 days after treatment cessation (9 hearts per condition at each time point). Gene regulation patterns were characterized with the use of bioinformatics and validated with the use of quantitative polymerase chain reaction. RESULTS: We obtained 14,340 lncRNAs from the reannotated Affymetrix zebrafish microarray. Of these, 187 lncRNAs were found to be differentially expressed (false discovery rate < 0.05 and fold change ≥ 2) at at least 1 time point. 85% of differentially expressed lncRNAs overlapped or were close to (distance < 10 kb) protein-coding genes which were mostly related to muscle development in Gene Ontology analyses. Fifty-seven lncRNAs had human homologs, according to orientation relative to their conserved protein-coding neighbours. CONCLUSIONS: LncRNAs are differentially expressed during regeneration after HF in adult zebrafish and could be potential future therapeutic targets. The extent to which lncRNAs contribute to cardiac regeneration is a worthy avenue for future research.
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Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , RNA Longo não Codificante/genética , Regeneração/fisiologia , Animais , Modelos Animais de Doenças , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , RNA Longo não Codificante/metabolismo , Peixe-Zebra/genéticaRESUMO
INTRODUCTION: A few major clinical factors such as sex, obesity or comorbidities have already been associated with COVID-19 severity, but there is a need to identify new epidemiological, clinical, digital and biological characteristics associated with severity and perform deep phenotyping of patients according to severity. The objectives of the Predi-COVID study are (1) to identify new determinants of COVID-19 severity and (2) to conduct deep phenotyping of patients by stratifying them according to risk of complications, as well as risk factors for infection among household members of Predi-COVID participants (the Predi-COVID-H ancillary study). METHODS AND ANALYSIS: Predi-COVID is a prospective, hybrid cohort study composed of laboratory-confirmed COVID-19 cases in Luxembourg who will be followed up remotely for 1 year to monitor their health status and symptoms. Predi-COVID-H is an ancillary cohort study on household members of index cases included in Predi-COVID to monitor symptoms and household clusters in this high-risk population. A subcohort of up to 200 Predi-COVID and 300 Predi-COVID-H participants with biological samples will be included. Severity of infection will be evaluated by occurrence and duration of hospitalisation, admission and duration of stay in intensive care units or equivalent structures, provision of and duration of supplemental oxygen and ventilation therapy, transfer to another hospital, as well as the impact of infection on daily activities following hospital discharge. ETHICS AND DISSEMINATION: The study has been approved by the National Research Ethics Committee of Luxembourg (study number 202003/07) in April 2020. An informed consent is signed by study participants. Scientific articles will be submitted to international peer-reviewed journals, along with press releases for lay audience for major results. TRIAL REGISTRATION NUMBER: NCT04380987.
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Teste para COVID-19/métodos , COVID-19/diagnóstico , Características da Família , Unidades de Terapia Intensiva , SARS-CoV-2 , Adulto , COVID-19/epidemiologia , Feminino , Seguimentos , Humanos , Incidência , Luxemburgo/epidemiologia , Masculino , Pandemias , Estudos Prospectivos , Fatores de Risco , Índice de Gravidade de Doença , Fatores de TempoRESUMO
Atrial fibrillation (AF) is a common arrhythmia for which the genetic studies mainly focused on the genes involved in electrical remodeling, rather than left atrial muscle remodeling. To identify rare variants involved in atrial myopathy using mutational screening, a high-throughput next-generation sequencing (NGS) workflow was developed based on a custom AmpliSeq™ panel of 55 genes potentially involved in atrial myopathy. This workflow was applied to a cohort of 94 patients with AF, 76 with atrial dilatation and 18 without. Bioinformatic analyses used NextGENe® software and in silico tools for variant interpretation. The AmpliSeq custom-made panel efficiently explored 96.58% of the targeted sequences. Based on in silico analysis, 11 potentially pathogenic missense variants were identified that were not previously associated with AF. These variants were located in genes involved in atrial tissue structural remodeling. Three patients were also carriers of potential variants in prevalent arrhythmia-causing genes, usually associated with AF. Most of the variants were found in patients with atrial dilatation (n=9, 82%). This NGS approach was a sensitive and specific method that identified 11 potentially pathogenic variants, which are likely to play roles in the predisposition to left atrial myopathy. Functional studies are needed to confirm their pathogenicity.
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Fibrilação Atrial/genética , Remodelamento Atrial , Análise Mutacional de DNA , Proteínas Musculares/genética , Proteínas de Transporte , Átrios do Coração , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
AIMS: New therapeutic approaches are needed to fight against the growing epidemic of heart failure. Unlike mammals, zebrafish possess the incredible ability to regenerate cardiac tissue after acute trauma such as apical resection. Yet, the ability of zebrafish to recover after a chronic stress leading to heart failure has not been reported. The aim of this study was to test whether zebrafish can recover a normal cardiac function after anaemia-induced heart failure. METHODS AND RESULTS: Eight- to ten-month-old zebrafish were treated with phenylhydrazine hydrochloride, an anaemia inducer, to generate heart failure. Treatment was stopped after 5â¯weeks and fish were followed-up for 3â¯weeks. Assessment of ventricular function by ultrasound at the end of the treatment revealed an increase in ventricle diameter (+47%) and a decrease in heart rate (-36%) and fractional shortening (-30%). A decrease in swim capacity was also observed (-31%). Tissue staining showed a thickening of the ventricular wall (5-fold), cell apoptosis and proliferation but no fibrosis. Expression of foetal genes, angiogenic factor and inflammation markers was increased, and ß-adrenergic receptor-1 was decreased. Three weeks after phenylhydrazine hydrochloride withdrawal, all parameters returned to baseline and the fish recovered a normal cardiac function, tissue morphology and gene expression. CONCLUSIONS: Zebrafish are able to completely recover from anaemia-induced heart failure. This model represents a unique opportunity to investigate the mechanisms of cardiac repair and may lead to the discovery of novel therapeutic targets of heart failure.
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Anemia/fisiopatologia , Insuficiência Cardíaca/fisiopatologia , Coração/fisiopatologia , Regeneração/fisiologia , Anemia/induzido quimicamente , Anemia/complicações , Anemia/genética , Animais , Apoptose/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento/genética , Coração/crescimento & desenvolvimento , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/genética , Ventrículos do Coração/crescimento & desenvolvimento , Ventrículos do Coração/fisiopatologia , Humanos , Fenil-Hidrazinas/toxicidade , Receptores Adrenérgicos beta 1/genética , Função Ventricular/efeitos dos fármacos , Função Ventricular/fisiologia , Peixe-Zebra/genética , Peixe-Zebra/fisiologiaRESUMO
Hypoxia promotes blood vessel growth through up-regulation of pro-angiogenic pathways but its role on the lymphatic system remains unclear. The homeobox transcription factor Prox1 is a master control gene for generating lymphatic endothelial cells (LECs) and is up-regulated by hypoxia-inducible factors in mammals. While vascular endothelial growth factor A (VEGFA) is critical for angiogenesis, VEGFC and its receptor VEGF receptor-3 (VEGFR-3) are essential for the initial sprouting and directed migration as well as for the subsequent survival of LECs. The aim of this study was to determine the effects of hypoxia on the development of the lymphatic system in zebrafish. Zebrafish embryos were obtained from Tg(SAGFF27C; UAS:GFP) animals carrying a lymphatic reporter gene coupled to green fluorescent protein (GFP). Exposure of 1-day old zebrafish embryos to hypoxic conditions (5% O2) for 24 h inhibited thoracic duct formation (-27%, p < 0.0001). Hypoxia inhibited the expression of pro-lymphangiogenic factors prox1a, vegfc and vegfr-3. This inhibition was relieved after re-oxygenation. On the other hand, hypoxia increased the expression of vegfa, a pro-angiogenic factor. In conclusion, hypoxia has opposite effects on vascular development in zebrafish, inhibiting the development of the lymphatic vascular system while promoting the development of the blood vascular system.
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Células Endoteliais/metabolismo , Hipóxia , Linfangiogênese , Vasos Linfáticos/metabolismo , Ducto Torácico/crescimento & desenvolvimento , Peixe-Zebra , Animais , Animais Geneticamente Modificados , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Masculino , Oxigênio/química , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
So far, imaging of the adult zebrafish heart and assessment of heart failure in adult zebrafish have been very limited. Here, we describe a new method for in vivo imaging of the hypertrabeculated heart of the adult zebrafish using miniaturized cardiac ultrasound catheters obtained from the cardiac catheterization laboratory. This method allows the observation of the ventricle of zebrafish and the assessment of ventricular diameters during diastole and systole, as well as heart rate and fractional shortening. Significant changes in these parameters were detected through the use of an adult zebrafish heart failure model induced by chronic anemia. This imaging technique opens the door to detailed in vivo analysis of the adult heart failure phenotype in zebrafish.
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Cardiomiopatias/diagnóstico por imagem , Ventrículos do Coração/diagnóstico por imagem , Ultrassonografia , Função Ventricular , Peixe-Zebra/fisiologia , Animais , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , MasculinoRESUMO
Increase of blood capillary density at the interface between normal and ischemic tissue after acute MI reduces infarct size and improves cardiac function. Cardiac injury triggers the production of the matricellular component TSP-1, but its role in angiogenesis is not clear, as both anti- and proangiogenic properties have been reported. It is unknown whether TSP-1 is modulated by other factors released during cardiac injury. Among these, Ado is a well-known promoter of angiogenesis. This study determined whether Ado modulates TSP-1 expression and the implication on angiogenesis. Ado dose dependently increased the production of TSP-1 by human macrophages. With the use of agonists and antagonists of AdoRs, coupled to RNA interference, we observed that this effect is mediated via A2AR and A2BR. The Ado effect was reproduced by cholera toxin (Gs protein activator) and forskolin (adenylate cyclase activator) and blocked by the PKA inhibitor H89. Conditioned medium from Ado-treated macrophages stimulated microvessel outgrowth from aortic ring explants by 400%, and induced vessel formation in matrigel plugs. Microvessel outgrowth and vessel formation were blocked completely by addition of anti-TSP-1 antibodies to conditioned medium. Chronic administration of Ado to rats after MI maintained long-term expression of TSP-1 in the infarct border zone, and this was associated with enhanced border-zone vascularization. Ado up-regulates TSP-1 production by macrophages, resulting in stimulation of angiogenesis. The mechanism involves A2AR and A2BR and is mediated through the cAMP/PKA pathway. This information may be important when designing Ado-based therapies of angiogenesis.
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Adenosina/metabolismo , Macrófagos/metabolismo , Infarto do Miocárdio/metabolismo , Neovascularização Fisiológica/fisiologia , Trombospondina 1/biossíntese , Animais , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Imunofluorescência , Humanos , Masculino , Microscopia Confocal , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo Real , Regulação para CimaRESUMO
Recent evidence suggests that Toll-like receptor 4 (TLR4) is not only involved in innate immunity but is also an important mediator of adverse left ventricular remodeling and heart failure following acute myocardial infarction (MI). TLR4 is activated by lipopolysaccharide (LPS) but also by products of matrix degradation such as hyaluronic acid and heparan sulfate. Although cardioprotective properties of adenosine (Ado) have been extensively studied, its potential to interfere with TLR4 activation is unknown. We observed that TLR4 pathway is activated in white blood cells from MI patients. TLR4 mRNA expression correlated with troponin T levels (R (2) = 0.75; P = 0.01) but not with levels of white blood cells and C-reactive protein. Ado downregulated TLR4 expression at the surface of human macrophages (-50%, P < 0.05). Tumor necrosis factor-α production induced by the TLR4 ligands LPS, hyaluronic acid, and heparan sulfate was potently inhibited by Ado (-75% for LPS, P < 0.005). This effect was reproduced by the A2A Ado receptor agonist CGS21680 and the non-selective agonist NECA and was inhibited by the A2A antagonist SCH58261 and the A2A/A2B antagonist ZM241,385. In contrast, Ado induced a 3-fold increase of TLR4 mRNA expression (P = 0.008), revealing the existence of a feedback mechanism to compensate for the loss of TLR4 expression at the cell surface. In conclusion, the TLR4 pathway is activated after MI and correlates with infarct severity but not with the extent of inflammation. Reduction of TLR4 expression by Ado may therefore represent an important strategy to limit remodeling post-MI.
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Adenosina/metabolismo , Membrana Celular/efeitos dos fármacos , Heparitina Sulfato/farmacologia , Ácido Hialurônico/farmacologia , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Infarto do Miocárdio/metabolismo , Receptor 4 Toll-Like/agonistas , Agonistas do Receptor A2 de Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Adulto , Idoso , Estudos de Casos e Controles , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Ligantes , Luxemburgo , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/genética , Infarto do Miocárdio/imunologia , RNA Mensageiro/metabolismo , Receptor A2A de Adenosina/efeitos dos fármacos , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/efeitos dos fármacos , Receptor A2B de Adenosina/metabolismo , Sistema de Registros , Fatores de Tempo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
VEGFR-1 (or Flt-1) exists under a sFlt-1 or a mFlt-1 form. sFlt-1 is antiangiogenic, and mFlt-1 is proangiogenic. The cardioprotective nucleoside Ado is proangiogenic, but its effects on Flt-1 are unknown and were tested in this study. In primary human macrophages from healthy volunteers, Ado inhibited sFlt-1 expression induced by LPS (-43%, P=0.006), HS, and IL-1ß but not hypoxia. This effect was also observed in macrophages from patients with acute MI (-33%, P<0.001). It was reproduced by the A(2A) Ado receptor agonist CGS21680 and abrogated by the A(2A) antagonist SCH58261. Conversely, Ado increased mFlt-1 expression, thus switching sFlt-1 from the soluble toward the membrane form. This switch was also present in macrophages from acute MI patients (P<0.001). Assessment of HIF-1α nuclear translocation and activation together with siRNA experiments suggested that the effect of Ado on Flt-1 involves HIF-1α. In conclusion, Ado down-regulates sFlt-1 and up-regulates mFlt-1 production, an effect that indicates that Ado may be used to stimulate angiogenesis in the heart.
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Adenosina/metabolismo , Macrófagos/metabolismo , Infarto do Miocárdio/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Expressão Gênica/fisiologia , Regulação da Expressão Gênica/fisiologia , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Immunoblotting , Neovascularização Fisiológica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , SolubilidadeRESUMO
It is known from animal models that the cardioprotective nucleoside adenosine stimulates angiogenesis mainly through up-regulation of vascular endothelial growth factor (VEGF). Since macrophages infiltrate the heart after infarction and because adenosine receptors behave differently across species, we evaluated the effect of adenosine on VEGF in human macrophages. Adenosine dose-dependently up-regulated VEGF expression and secretion by macrophages from healthy volunteers. VEGF production was also increased by blockade of extracellular adenosine uptake with dipyridamole. This effect was exacerbated by the toll-like receptor-4 ligands heparan sulfate, hyaluronic acid and lipopolysaccharide, and was associated with an increase of hypoxia inducible factor-1alpha expression, the main transcriptional inducer of VEGF in hypoxic conditions. The agonist of the adenosine A2A receptor CGS21680 reproduced the increase of VEGF and the antagonist SCH58261 blunted it. In conclusion, these results provide evidence that activation of adenosine A2A receptor stimulates VEGF production in human macrophages. This study suggests that adenosine is a unique pro-angiogenic molecule that may be used to stimulate cardiac repair.
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Adenosina/farmacologia , Indutores da Angiogênese/farmacologia , Cardiotônicos/farmacologia , Macrófagos/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Adenosina/análogos & derivados , Agonistas do Receptor A2 de Adenosina , Expressão Gênica/efeitos dos fármacos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/efeitos dos fármacos , Fenetilaminas/farmacologia , Pirimidinas/farmacologia , Receptor 4 Toll-Like/metabolismo , Triazóis/farmacologia , Regulação para CimaRESUMO
AIMS: Matrix metalloproteinase-9 (MMP-9) plays an important role in ventricular remodelling after acute myocardial infarction (MI). The cardioprotectant adenosine (Ado) may be involved in ventricular remodelling. We have shown that Ado inhibits the secretion of MMP-9 by human neutrophils. This study investigated the effect of Ado on MMP-9 production by human macrophages. METHODS AND RESULTS: Cells used in this study were monocytes of healthy volunteers, a human monocyte cell line, and leukocytes from patients following MI. Monocytes were differentiated into macrophages and treated with Ado. Ado enhanced MMP-9 secretion by human macrophages in a time- and dose-dependent manner. Increasing the level of endogenous Ado by inhibition of Ado deaminase or Ado transferase also increased MMP-9 secretion. Ado enhanced MMP-9 production when macrophages were activated by hypoxia or Toll-like receptor-4 ligands such as lipopolysaccharide, hyaluronan, and heparan sulfate. The effect of Ado was replicated by the A3 agonist IB-MECA and inhibited by silencing the A3 receptor. Ado improved monocyte capacity to migrate through a matrix of gelatin B, and this effect was blocked by inhibition of MMP-9 activity. The chemotactic capacity of macrophages was reduced by Ado through a loss of expression of the monocyte chemotactic protein-1 receptor. Finally, MMP-9 expression was higher in blood cells from patients with acute MI compared with healthy volunteers. CONCLUSION: Adenosine activates MMP-9 secretion by macrophages through its A3 receptor. The effect is in contrast to that observed in neutrophils, where Ado inhibits MMP-9 secretion by the A2a receptor. These observations may have important implications for therapeutic strategies targeting Ado receptors in the setting of MI.
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Adenosina/metabolismo , Ativação de Macrófagos , Macrófagos/enzimologia , Metaloproteinase 9 da Matriz/metabolismo , Infarto do Miocárdio/enzimologia , Receptor A3 de Adenosina/metabolismo , Agonistas do Receptor A3 de Adenosina , Adenosina Desaminase/metabolismo , Inibidores de Adenosina Desaminase , Movimento Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Gelatina/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Macrófagos/efeitos dos fármacos , Metaloproteinase 9 da Matriz/genética , Inibidores de Metaloproteinases de Matriz , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/genética , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Receptor A3 de Adenosina/genética , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Receptor 4 Toll-Like/metabolismo , Regulação para CimaRESUMO
Matrix metalloproteinases (MMPs), and in particular MMP-9 secreted by neutrophils, are capable of degrading the matrix components of the heart and are thought to be the driving force behind myocardial matrix remodeling after infarction. Adenosine, a naturally produced nucleoside, has been shown to have cardioprotective effects and to inhibit secretion of various cytokines. The aim of our study was to determine the effect of adenosine on the secretion of MMP-9 by neutrophils. Neutrophils were isolated from healthy volunteers through Ficoll and Dextran sedimentation. Neutrophils were activated by N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence or absence of adenosine or adenosine analogs. Zymography and enzyme linked immunosorbent assay were used to measure MMP-9 secretion. Adenosine (1 micromol/L) decreased the fMLP-induced MMP-9 secretion by 30+/-2% (n=8, P<0.001). The effect was dose-dependent and was not specific to fMLP because adenosine also inhibited MMP-9 secretion by LPS- or H(2)O(2)-stimulated neutrophils. The effect of adenosine was mimicked by the adenosine A2a receptor agonist CGS21680 and was inhibited by both the A2a antagonist SCH5826 and A2a RNA silencing. The A3 agonist IB-MECA moderately decreased fMLP-induced MMP-9 secretion. Agonists and antagonists of the other types of adenosine receptors had no significant effect. Adenosine increased intracellular cAMP concentration and accelerated the return to baseline of the intracytoplasmic calcium peak. The inhibition of MMP-9 secretion by adenosine, as well as the calcium effect, was prevented by the protein kinase A inhibitor H-89. In conclusion, we show here that adenosine inhibits MMP-9 secretion by neutrophils. Our results suggest that this effect implies the A2a receptor and is mediated through the cAMP/PKA/Ca(2+) pathway. Therefore, adenosine may represent a new approach to prevent matrix degradation and remodeling after myocardial injury.
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Adenosina/farmacologia , Inibidores de Metaloproteinases de Matriz , Neutrófilos/enzimologia , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais , Cálcio/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/metabolismo , Remodelação VentricularRESUMO
BACKGROUND: Matrix metalloproteinases (MMPs) have been associated with the development of left ventricular remodeling after myocardial infarction (MI). We sought to determine whether peripheral levels of MMPs can be used as a risk marker for the development of congestive heart failure (CHF) after acute MI. METHODS AND RESULTS: Plasma levels of MMP-2, MMP-9, C-reactive protein (hs-CRP), tumor necrosis factor-alpha (TNF-alpha), and pro-brain natriuretic peptide (pro-BNP) were measured in 109 consecutive patients with acute MI treated with primary mechanical reperfusion. Echocardiographic assessment of left ventricular wall motion index was performed during admission. Patients were followed for the development of CHF. Left ventricular function and volumes were determined after 2 years with radionuclide ventriculography. During 2-year follow-up, 15 patients developed congestive heart failure (CHF). Using multivariate analysis, MMP-9 levels were the only circulating factor predictive of late onset CHF. Patients who had high MMP-9 levels had a significant risk of late onset CHF (OR of 6.5, P < or = .006) and left ventricular remodeling (DeltaEF = -9%, P = .03, and Deltaend-diastolic volume = +13 mL, P = .03). MMP-2, TNF-alpha, hs-CRP, creatine kinase, and pro-BNP were not predictive of late onset CHF. CONCLUSION: MMP-9 levels may hold prognostic significance in MI patients.
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Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/etiologia , Metaloproteinase 9 da Matriz/sangue , Infarto do Miocárdio/complicações , Idoso , Biomarcadores/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Medição de RiscoRESUMO
RNA polymerase (pol) III transcription decreases when primary cultures of rat neonatal cardiomyocytes are exposed to low oxygen tension. Previous studies in fibroblasts have shown that the pol III-specific transcription factor IIIB (TFIIIB) is bound and regulated by the proto-oncogene product c-Myc, the mitogen-activated protein kinase ERK and the retinoblastoma tumour suppressor protein, RB. The principal function of TFIIIB is to recruit pol III to its cognate gene template, an activity that is known to be inhibited by RB and stimulated by ERK. We demonstrate by chromatin immunoprecipitation (ChIP) that c-Myc also stimulates pol III recruitment by TFIIIB. However, hypoxic conditions cause TFIIIB dissociation from c-Myc and ERK, at the same time as increasing its interaction with RB. Consistent with this, ChIP assays indicate that the occupancy of tRNA genes by pol III is significantly reduced, whereas promoter binding by TFIIIB is undiminished. The data suggest that hypoxia can inhibit pol III transcription by altering the interactions between TFIIIB and its regulators and thus compromising its ability to recruit the polymerase. These effects are independent of cell cycle changes.
Assuntos
Regulação da Expressão Gênica , Miócitos Cardíacos/metabolismo , RNA Polimerase III/antagonistas & inibidores , RNA de Transferência/genética , Transcrição Gênica , Animais , Hipóxia Celular , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Polimerase III/metabolismo , Ratos , Ratos Sprague-Dawley , Proteína do Retinoblastoma/metabolismoRESUMO
Recently, matrix metalloproteinase-9 (MMP-9) has been identified as a cardiovascular risk marker and is increasingly being determined in clinical studies. Among other matrix metalloproteinases, MMP-9 is known to be self-activable, as the cleavage of the propeptide leads to the formation of an active enzyme. In such a case the issue of storage of biological samples such as plasmas is of outstanding importance, as an enzymatic activity, although minimal, may remain at common storage temperature, i.e., -80 degrees C. Since 2000 our institute has created a plasma library from patients presenting with acute myocardial infarction. Recently, the evaluation of the MMP-9 led to the surprise of finding a dramatically low level of detectable enzyme in the oldest plasma samples. By using zymography, enzyme-linked immunosorbent assay and Western blots, we evaluated new and old samples and found that MMP-9 degrades over time. After 2 years, the detectable total MMP-9 dropped by 65%, and the asymptotic profile of the curve reached a residual 1% level after 43 months. These results were confirmed by zymography and western blotting. TIMP-1, the natural inhibitor of MMP-9 and MMP-2, remained rather stable over time. The results suggest that human plasma MMP-9 levels should be determined as soon as possible after sampling.
Assuntos
Preservação de Sangue , Criopreservação , Metaloproteinase 9 da Matriz/sangue , Western Blotting , Ensaio de Imunoadsorção Enzimática , Humanos , Metaloproteinase 9 da Matriz/química , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/sangue , Inibidor Tecidual de Metaloproteinase-1/químicaRESUMO
Using EPAC1 (exchange protein directly activated by cAMP 1) as bait in two-hybrid screens of foetal and adult human brain libraries, we identified the LC2 (light chain 2) of MAP1A (microtubule-associated protein 1A) as a protein capable of interaction with EPAC1. We applied an immunoprecipitation assay to demonstrate protein interaction between EPAC1 and LC2 in co-transfected human embryonic kidney 293 cells. EPAC2 also co-immunoprecipitated with LC2 from extracts of rat cerebellum. Immunolocalization in co-transfected human embryonic kidney 293 cells revealed that EPAC1 co-localizes with LC2 throughout the cell body. We found that endogenous EPAC2 is also immunolocalized with LC2 in PC12 cells. Immunolocalization of EPAC1 in transfected COS1 cells showed that EPAC1 is associated with the perinuclear region surrounding the nucleus and filamentous structures throughout the cell. Removal of the cAMP-binding domain of EPAC1 (DeltacAMP-EPAC1) appeared to disrupt targeting of EPAC1 in cells resulting in a more dispersed staining pattern. Using two-hybrid assay, we tested the ability of LC2 to interact with DeltacAMP-EPAC1 and DeltaDEP-EPAC1, which lacks a DEP domain (dishevelled, Egl-10 and pleckstrin homology domain). We found that deletion of the cAMP-binding domain inhibited interaction between EPAC1 and LC2 in a two-hybrid assay, but removal of the DEP domain had little effect. LC2 was found to interact with a glutathione-S-transferase-fusion protein of the cAMP-binding domain of EPAC1 in a pull-down assay, but not the DEP, REM (Ras exchange motif) or CAT (catalytic) domains. Together with our two-hybrid results, this suggests that the cAMP-binding domain of EPAC1 mediates interaction with LC2.