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Human P2Y4 is a UTP receptor, while in mice it is activated by both ATP and UTP. P2Y4 knockout (KO) in mice protects against myocardial infarction and is characterized by increased adiponectin secretion by adipocytes, and decreased cardiac inflammation and permeability under ischemic conditions. The relevance of these data has, however, not been explored to date in humans. In a population study comprising 50 patients with coronary artery disease (CAD) and 50 age-matched control individuals, we analyzed P2RY4 mutations and their potential association with CAD severity and fasting plasma parameters. Among the mutations identified, we focused our attention on a coding region polymorphism (rs3745601) that results in replacement of the asparagine at residue 178 with threonine (N178T) located in the second extracellular loop of the P2Y4 receptor. The N178T variant is a loss-of-function mutation of the human P2Y4 receptor and is encountered less frequently in coronary patients than in control individuals. In coronary patients, carriers of the N178T variant had significantly reduced jeopardy and Gensini cardiac severity scores, as well as lower resting heart rates and plasma levels of N-terminal pro-brain natriuretic peptide (NT-proBNP). Regarding fasting plasma parameters, the N178T variant was associated with a lower concentration of glucose. Accordingly, P2Y4 KO mice had significantly improved glucose tolerance and insulin sensitivity compared with their WT littermate controls. The improvement of insulin sensitivity resulting from lack of the P2Y4 receptor was no longer observed in the absence of adiponectin. The present study identifies a frequent loss-of-function P2Y4 variant associated with less severe coronary artery atherosclerosis and lower fasting plasma glucose in coronary patients. The role of the P2Y4 receptor in glucose homeostasis was confirmed in mouse. P2Y4 antagonists could thus have therapeutic applications in the treatment of myocardial infarction and type 2 diabetes.
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[This corrects the article DOI: 10.3389/fimmu.2022.1006934.].
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A better understanding of the immune function of pericardial adipose tissue is essential to adapt treatments after myocardial infarction. We showed previously that inactivation of mouse P2Y4 nucleotide receptor induces adiponectin overexpression and protection against myocardial infarction. We investigated here the inflammatory state of pericardial adipose tissue in ischemic P2Y4-deficient mice. We demonstrated that P2Y4-deficient mice displayed adipocyte beiging with increased PD-L1 expression and a higher number of regulatory leukocytes in their pericardial adipose tissue after left anterior descending artery ligation, compared to wild type mice. Effectively, a higher level of anti-inflammatory M2c macrophages and regulatory T cells was observed in pericardial adipose tissue of P2Y4 KO mice and correlated with reduced post-ischemic expansion of fat-associated lymphoid clusters. Interestingly, the anti-inflammatory effects observed in P2Y4 KO mice, were no more observed in P2Y4/adiponectin double KO ischemic mice. Finally, the reduction of T cell infiltration and cardiac fibrosis observed in P2Y4-deficient heart was lost after injection of anti-PD-L1 blocking antibody in ischemic mice. The present study defines P2Y4 as a regulator of PD-L1 and adiponectin, and as a potential target for anti-inflammatory therapies to improve myocardial infarction outcome. The combined effect of P2Y4 loss on adipocyte beiging and regulatory leukocyte increase highlights this nucleotide receptor as an important player in post-ischemic cardiac response.
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Adiponectina , Antígeno B7-H1/metabolismo , Infarto do Miocárdio , Receptores Purinérgicos P2/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismo , NucleotídeosRESUMO
The ability of cardiac adipose-derived stem cells (cADSC) to differentiate into multiple cell types has opened new perspectives in cardiac cell-based regenerative therapies. P2Y nucleotide receptors have already been described as regulators of adipogenic differentiation of cADSC and bone marrow-derived stem cells. In this study, we defined UTP as a regulator of cADSC endothelial differentiation. A daily UTP stimulation of cADSC during endothelial predifferentiation increased their capacity to form an endothelial network in matrigel. Additionally, pro-angiogenic UTP target genes such as epiregulin and hyaluronan synthase-1 were identified in predifferentiated cADSC by RNA sequencing experiments. Their regulation by UTP was confirmed by qPCR and ELISA experiments. We then evaluated the capacity of UTP-treated predifferentiated cADSC to increase post-ischemic revascularization in mice subjected to left anterior descending artery ligation. Predifferentiated cADSC treated or not with UTP were injected in the periphery of the infarcted zone, 3 days after ligation. We observed a significant increase of capillary density 14 and 30 days after UTP-treated predifferentiated cADSC injection, correlated with a reduction of cardiac fibrosis. This revascularization increase was not observed after injection of UTP-treated cADSC deficient for UTP and ATP nucleotide receptor P2Y2. The present study highlights the P2Y2 receptor as a regulator of cADSC endothelial differentiation and as a potential target for the therapeutic use of cADSC in post-ischemic heart revascularization.
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Diferenciação Celular/efeitos dos fármacos , Células-Tronco Multipotentes/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Uridina Trifosfato/farmacologia , Animais , Epirregulina/genética , Epirregulina/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Multipotentes/metabolismo , Receptores Purinérgicos P2Y2/genética , Receptores Purinérgicos P2Y2/metabolismoRESUMO
The family of P2Y nucleotide receptors is composed of eight members differentiated by their pharmacology and their coupling to specific G-proteins and transduction mechanisms. The laboratory studying these nucleotide receptors at IRIBHM institute (Free University of Brussels) has participated actively in their cloning. We used classical cloning by homology strategies relying on polymerase chain reactions with degenerate primers or on DNA libraries screening with P2Y receptors-related primers or probes, respectively. We identified and characterised four of the eight human P2Y receptors cloned so far: P2Y4, P2Y6, P2Y11 and P2Y13 receptors. These human receptors displayed specific features in terms of pharmacology such as affinity for pyrimidine nucleotides for P2Y4 and P2Y6 receptors and differential G-protein coupling. Their specific and restricted tissue distribution compared to ubiquitous P2Y1 and P2Y2 receptors led us to study their physiological role in chosen cell systems or using mice deficient for these P2Y subtypes. These studies revealed over the years that the P2Y11 receptor was able to confer tolerogenic and tumorigenic properties to human dendritic cells and that P2Y4 and P2Y6 receptors were involved in mouse heart post-natal development and cardioprotection. P2Y receptors and their identified target genes could constitute therapeutic targets to regulate cardiac hypertrophy and regeneration. The multiple roles of P2Y receptors identified in the ischemic heart and cardiac adipose tissue could have multiple innovative clinical applications and present a major interest in the field of cardiovascular diseases. P2Y receptors can induce cardioprotection by the regulation of cardiac inflammation and the modulation of the volume and composition of cardiac adipose tissue. These findings might lead to the pre-clinical validation of P2Y receptors as new targets for the treatment of myocardial ischemia.
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Clonagem Molecular/métodos , Receptores Purinérgicos P2/fisiologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Cardiopatias/tratamento farmacológico , Cardiopatias/fisiopatologia , Humanos , Agonistas do Receptor Purinérgico P2/administração & dosagem , Antagonistas do Receptor Purinérgico P2/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression which act by guiding AGO (argonaute) proteins to target RNA transcripts in the RNA-induced silencing complex (RISC). This macromolecular complex includes multiple additional components (e.g., TNRC6A) that allow for interaction with enzymes mediating inhibition of translation or RNA decay. However, miRNAs also reside in low-molecular weight complexes without being engaged in target repression, and their function in this context is largely unknown. Our recent findings show that endothelial cells exposed to protective high-shear stress or MTORC inhibition activate the macroautophagy/autophagy machinery to sustain viability by promoting differential trafficking of MIR126 strands and by enabling unconventional features of MIR126-5p. Whereas MIR126-3p is degraded upon autophagy activation, MIR126-5p interacts with the RNA-binding protein MEX3A to form a ternary complex with AGO2. This complex forms on the autophagosomal surface and facilitates its nuclear localization. Once in the nucleus, MIR126-5p dissociates from AGO2 and establishes aptamer-like interactions with the effector CASP3 (caspase 3). The binding to MIR126-5p prevents dimerization and proper active site formation of CASP3, thus inhibiting proteolytic activity and limiting apoptosis. Disrupting this pathway in vivo by genetic deletion of Mex3a or by specific deficiency of endothelial autophagy aggravates endothelial apoptosis and exacerbates the progression of atherosclerosis. The direct inhibition of CASP3 by MIR126-5p reveals a non-canonical mechanism by which miRNAs can modulate protein function and mediate the autophagy-apoptosis crosstalk.
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Aterosclerose , MicroRNAs , Autofagia/genética , Caspase 3 , Células Endoteliais , Humanos , MicroRNAs/genéticaRESUMO
MicroRNAs (miRNAs) are versatile regulators of gene expression with profound implications for human disease including atherosclerosis, but whether they can exert posttranslational functions to control cell adaptation and whether such noncanonical features harbor pathophysiological relevance is unknown. Here, we show that miR-126-5p sustains endothelial integrity in the context of high shear stress and autophagy. Bound to argonaute-2 (Ago2), miR-126-5p forms a complex with Mex3a, which occurs on the surface of autophagic vesicles and guides its transport into the nucleus. Mutational studies and biophysical measurements demonstrate that Mex3a binds to the central U- and G-rich regions of miR-126-5p with nanomolar affinity via its two K homology domains. In the nucleus, miR-126-5p dissociates from Ago2 and binds to caspase-3 in an aptamer-like fashion with its seed sequence, preventing dimerization of the caspase and inhibiting its activity to limit apoptosis. The antiapoptotic effect of miR-126-5p outside of the RNA-induced silencing complex is important for endothelial integrity under conditions of high shear stress promoting autophagy: ablation of Mex3a or ATG5 in vivo attenuates nuclear import of miR-126-5p, aggravates endothelial apoptosis, and exacerbates atherosclerosis. In human plaques, we found reduced nuclear miR-126-5p and active caspase-3 in areas of disturbed flow. The direct inhibition of caspase-3 by nuclear miR-126-5p reveals a noncanonical mechanism by which miRNAs can modulate protein function.
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Aterosclerose , MicroRNAs , Apoptose , Aterosclerose/genética , Autofagia , Caspase 3 , Humanos , MicroRNAs/genéticaRESUMO
The formation of pericardial adipose tissue (PAT) and its regulatory function in cardiac inflammation are not well understood. We investigated the potential role of the ubiquitous ATP/UTP nucleotide receptor P2Y2 in the PAT by using P2Y2-null mice. We observed that P2Y2-null mice displayed a lower mass of PAT and a reduced density of its fat-associated lymphoid clusters (FALCs) and, more particularly, B cells. Loss of P2Y2 receptor in pericardial preadipocytes decreased their adipogenic differentiation and maturation abilities in vitro. Gene profiling identified P2Y2 target genes in PAT linked to immunomodulation. These data led to the identification of an increase of M2c anti-inflammatory macrophages correlated with increased apoptosis of B lymphocytes in P2Y2-null pericardial fat. In addition, follicular helper T cells, which contribute to B cell expansion in germinal centers, were dramatically decreased. The effect of P2Y2 loss was also investigated after ischemia-mediated expansion of FALCs in a model of myocardial infarct. Loss of P2Y2 led to reduced expansion of B and neutrophil populations in these clusters, whereas density of M2c anti-inflammatory macrophages was increased. Our study defines the P2Y2 nucleotide receptor as a regulator of the formation and inflammatory status of pericardial fat. The P2Y2 receptor could represent a therapeutic target in the regulation of PAT function before and during cardiac ischemia.
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Tecido Adiposo/metabolismo , Gorduras/metabolismo , Linfócitos/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Células-Tronco/metabolismo , Adipogenia/genética , Tecido Adiposo/citologia , Animais , Linfócitos B/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Perfilação da Expressão Gênica/métodos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pericárdio/citologia , Pericárdio/metabolismo , Receptores Purinérgicos P2Y2/genéticaRESUMO
The role of nonclassical monocytes (NCMs) in health and disease is emerging, but their location and function within tissues remain poorly explored. Imaging of NCMs has been limited by the lack of an established single NCM marker. Here, we characterize the immune checkpoint molecule PD-L1 (CD274) as an unequivocal marker for tracking NCMs in circulation and pinpoint their compartmentalized distribution in tissues by two-photon microscopy. Visualization of PD-L1+ NCMs in relation to bone marrow vasculature reveals that conversion of classical monocytes into NCMs requires contact with endosteal vessels. Furthermore, PD-L1+ NCMs are present in tertiary lymphoid organs (TLOs) under inflammatory conditions in both mice and humans, and NCMs exhibit a PD-L1-dependent immunomodulatory function that promotes T cell apoptosis within TLOs. Our findings establish an unambiguous tool for the investigation of NCMs and shed light on their origin and function.
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Antígeno B7-H1/imunologia , Monócitos/imunologia , Músculos Abdominais/imunologia , Animais , Anticorpos/farmacologia , Medula Óssea/imunologia , Feminino , Fêmur , Imunoglobulina G/imunologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Linfócitos T/imunologiaRESUMO
AIMS: Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. METHODS AND RESULTS: Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. CONCLUSION: These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function.
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Ácidos Araquidônicos/toxicidade , Quimiotaxia/efeitos dos fármacos , Endocanabinoides/toxicidade , Glicerídeos/toxicidade , Insuficiência Cardíaca/induzido quimicamente , Células Mieloides/efeitos dos fármacos , Infarto do Miocárdio/complicações , Miocárdio/metabolismo , Infiltração de Neutrófilos/efeitos dos fármacos , Administração Intravenosa , Animais , Ácidos Araquidônicos/administração & dosagem , Ácidos Araquidônicos/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Endocanabinoides/administração & dosagem , Endocanabinoides/metabolismo , Feminino , Fibrose , Glicerídeos/administração & dosagem , Glicerídeos/metabolismo , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Mediadores da Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monoacilglicerol Lipases/metabolismo , Células Mieloides/metabolismo , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Receptor CB2 de Canabinoide/genética , Receptor CB2 de Canabinoide/metabolismo , Transdução de Sinais , Remodelação Ventricular/efeitos dos fármacosRESUMO
BACKGROUND: The pericardial adipose tissue (AT) contains a high density of lymphoid clusters. It is unknown whether these clusters play a role in post-myocardial infarction (MI) inflammatory responses and cardiac outcome. METHODS: Lymphoid clusters were examined in epicardial AT of humans with or without coronary artery disease. Murine pericardial lymphoid clusters were visualized in mice subjected to coronary artery ligation. To study the relevance of pericardial clusters during inflammatory responses after MI, we surgically removed the pericardial AT and performed B-cell depletion and granulocyte-macrophage colony-stimulating factor blockade. Leukocytes in murine hearts, pericardial AT, spleen, mediastinal lymph nodes, and bone marrow were quantified by flow cytometry. Cannabinoid receptor CB2 (CB2-/-) mice were used as a model for enhanced B-cell responses. The effect of impaired dendritic cell (DC) trafficking on pericardial AT inflammatory responses was tested in CCR7-/- mice subjected to MI. Cardiac fibrosis and ventricular function were assessed by histology and echocardiography. RESULTS: We identified larger B-cell clusters in epicardial AT of human patients with coronary artery disease in comparison with controls without coronary artery disease. Infarcted mice also had larger pericardial clusters and 3-fold upregulated numbers of granulocyte-macrophage colony-stimulating factor-producing B cells within pericardial AT, but not spleen or lymph nodes. This was associated with higher DC and T-cell counts in pericardial AT, which outnumbered DCs and T cells in lymph nodes. Analysis of DC maturation markers, tracking experiments with fluorescently labeled cells, and use of CCR7-deficient mice suggested that activated DCs migrate from infarcts into pericardial AT via CCR7. B-cell depletion or granulocyte-macrophage colony-stimulating factor neutralization inhibited DC and T-cell expansion within pericardial AT, and translated into reduced bone marrow granulopoiesis and cardiac neutrophil infiltration 3 days after MI. The relevance of the pericardial AT in mediating all these effects was confirmed by removal of pericardial AT and ex vivo coculture with pericardial AT and granulocyte progenitors. Finally, enhanced fibrosis and worsened ejection fraction in CB2-/- mice were limited by pericardial AT removal. CONCLUSIONS: Our findings unveil a new mechanism by which the pericardial AT coordinates immune cell activation, granulopoiesis, and outcome after MI.
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Tecido Adiposo/fisiologia , Granulócitos/imunologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Pericárdio/patologia , Animais , Diferenciação Celular , Modelos Animais de Doenças , Feminino , Fibrose , Humanos , Imunidade Inata , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/metabolismo , Receptor CB2 de Canabinoide/genética , Receptores CCR7/genética , CicatrizaçãoRESUMO
OBJECTIVE: Circadian regulation of neutrophil homeostasis affects myocardial infarction (MI) healing. It is unknown whether diurnal variations of monocyte counts exist in the heart and whether this affects their cardiac infiltration in response to MI. APPROACH AND RESULTS: Murine blood and organs were harvested at distinct times of day and analyzed by flow cytometry. Ly6Chigh monocyte surface expression levels of chemokine receptors (CCR) were ≈2-fold higher at the beginning of the active phase, Zeitgeber Time (ZT) 13 compared with ZT5. This was because of enhanced receptor surface expression at ZT13, whereas no significant changes in total cellular protein levels were found. Most blood Ly6Chigh monocytes were CCR2high, whereas only a minority was CCR1high and CCR5high. We also found diurnal changes of classical monocyte blood counts in humans, being higher in the evening, while exhibiting enhanced CCR2 surface expression in the morning. In support of monocyte oscillations between blood and tissue, murine cardiac Ly6Chigh monocyte counts were highest at ZT13, accompanied by an upregulation of cardiac CC chemokine ligand 2 mRNA. Mice subjected to MI at ZT13 had an even higher upregulation of CCR2 surface expression on circulating monocytes compared with noninfarcted mice and more elevated cardiac CC chemokine ligand 2 protein expression and more pronounced Ly6Chigh monocyte infiltration compared with ZT5-infarcted mice. Concomitantly, CCR2 antagonism only inhibited the excessive cardiac Ly6Chigh monocyte infiltration after ZT13 MI but not ZT5 MI. CONCLUSIONS: CCR2 surface expression on Ly6Chigh monocytes changes in a time-of-day-dependent manner, which crucially affects cardiac monocyte recruitment after an acute ischemic event.
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Antígenos Ly/imunologia , Quimiotaxia de Leucócito , Ritmo Circadiano , Monócitos/imunologia , Infarto do Miocárdio/imunologia , Miocárdio/imunologia , Adulto , Animais , Antígenos Ly/metabolismo , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Imunofenotipagem/métodos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Monócitos/metabolismo , Infarto do Miocárdio/genética , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Fenótipo , Receptores CCR1/imunologia , Receptores CCR1/metabolismo , Receptores CCR2/genética , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Receptores CCR5/imunologia , Receptores CCR5/metabolismo , Transdução de Sinais , Fatores de Tempo , Adulto JovemRESUMO
Aims: Acute myocardial infarction (MI) is the leading cause of mortality worldwide. Anti-inflammatory strategies to reduce neutrophil-driven acute post-MI injury have been shown to limit acute cardiac tissue damage. On the other hand, whether neutrophils are required for resolving post-MI inflammation and repair is unknown. Methods and Results: We show that neutrophil-depleted mice subjected to MI had worsened cardiac function, increased fibrosis, and progressively developed heart failure. Flow cytometry of blood, lymphoid organs and digested hearts revealed reduced numbers of Ly6Chigh monocytes in infarcts of neutrophil-depleted mice, whereas the number of macrophages increased, which was paralleled by reduced splenic Ly6Chigh monocyte mobilization but enhanced proliferation of cardiac macrophages. Macrophage subtype analysis revealed reduced cardiac expression of M1 markers, whereas M2 markers were increased in neutrophil-depleted mice. Surprisingly, we found reduced expression of phagocytosis receptor myeloid-epithelial-reproductive tyrosine kinase, a marker of reparative M2c macrophages which mediate clearance of apoptotic cells. In agreement with this finding, neutrophil-depleted mice had increased numbers of TUNEL-positive cells within infarcts. We identified neutrophil gelatinase-associated lipocalin (NGAL) in the neutrophil secretome as a key inducer of macrophages with high capacity to engulf apoptotic cells. The cardiac macrophage phenotype in neutrophil-depleted mice was restored by administration of neutrophil secretome or NGAL. Conclusion: Neutrophils are crucially involved in cardiac repair after MI by polarizing macrophages towards a reparative phenotype. Therapeutic strategies to reduce acute neutrophil-driven inflammation after MI should be carefully balanced as they might interfere with the healing response and cardiac remodelling.
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Macrófagos/fisiologia , Infarto do Miocárdio/fisiopatologia , Neutrófilos/fisiologia , Animais , Apoptose/fisiologia , Proliferação de Células/fisiologia , Fibrose Endomiocárdica/etiologia , Fibrose Endomiocárdica/fisiopatologia , Feminino , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Ligadura , Lipocalina-2/fisiologia , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Neutropenia/fisiopatologia , Fenótipo , Remodelação Ventricular/fisiologia , Cicatrização/fisiologia , c-Mer Tirosina Quinase/metabolismoRESUMO
Cardiac adipose tissue-derived stem cells (cASCs) have the ability to differentiate into multiple cell lineages giving them a high potential for use in regenerative medicine. Cardiac fat tissue still raises many unsolved questions related to its formation and features. P2Y nucleotide receptors have already been described as regulators of differentiation of bone-marrow derived stem cells, but remain poorly investigated in cASCs. We defined, in this study, the P2Y4 nucleotide receptor as a negative regulator of cardiac fat formation and cASC adipogenic differentiation. Higher expression of P2Y4 receptor in cardiac fat tissue was observed compared to other adipose tissues. P2Y4-null mice displayed a higher mass of cardiac adipose tissue specifically. We therefore examined the role of P2Y4 receptor in cASC adipogenic differentiation. An inhibitory effect of uridine 5'-triphosphate (UTP), ligand of P2Y4, was observed on the maturation state of differentiated cASCs, and on the expression of adipogenesis-linked genes and adiponectin, a cardioprotective adipokine. Higher adiponectin secretion by P2Y4-null adipocytes could be linked with cardioprotection previously observed in the heart of P2Y4-null ischemic mice. We realized here left anterior descending artery ligation on simple and double-knockout mice for P2Y4 and adiponectin. No cardioprotective effect of P2Y4 loss was observed in the absence of adiponectin secretion. In addition, P2Y4 loss was correlated with higher expression of UCP-1 (uncoupling protein-1) and CD137, two markers of brown/beige cardiac adipocytes. Our data highlight the P2Y4 receptor as an inhibitor of cardiac fat formation and cASC adipogenic differentiation, and as a potential therapeutic target in the regulation of cardioprotective function of cardiac fat.
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Tecido Adiposo/citologia , Adiposidade , Diferenciação Celular , Miocárdio/citologia , Receptores Purinérgicos P2/metabolismo , Células-Tronco/citologia , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Adipogenia/efeitos dos fármacos , Adiponectina/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Biomarcadores/metabolismo , Cardiotônicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Feminino , Gotículas Lipídicas/efeitos dos fármacos , Gotículas Lipídicas/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/patologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/metabolismo , Uridina Trifosfato/farmacologiaRESUMO
The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced postnatal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy.
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Cardiomegalia/metabolismo , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Receptores Purinérgicos P2/metabolismo , Animais , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/patologia , Hiperplasia , Isoproterenol/efeitos adversos , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Knockout , Isquemia Miocárdica/induzido quimicamente , Isquemia Miocárdica/genética , Isquemia Miocárdica/patologia , Miócitos Cardíacos/patologia , Receptores Purinérgicos P2/genéticaRESUMO
Myocardial infarction (MI) is the leading cause of death in Western countries. Epidemiological studies show acute MI to be more prevalent in the morning and to be associated with a poorer outcome in terms of mortality and recovery. The mechanisms behind this association are not fully understood. Here, we report that circadian oscillations of neutrophil recruitment to the heart determine infarct size, healing, and cardiac function after MI Preferential cardiac neutrophil recruitment during the active phase (Zeitgeber time, ZT13) was paralleled by enhanced myeloid progenitor production, increased circulating numbers of CXCR2(hi) neutrophils as well as upregulated cardiac adhesion molecule and chemokine expression. MI at ZT13 resulted in significantly higher cardiac neutrophil infiltration compared to ZT5, which was inhibited by CXCR2 antagonism or neutrophil-specific CXCR2 knockout. Limiting exaggerated neutrophilic inflammation at this time point significantly reduced the infarct size and improved cardiac function.
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Ritmo Circadiano , Infarto do Miocárdio/patologia , Infiltração de Neutrófilos , Animais , Quimiocinas/metabolismo , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/fisiopatologiaRESUMO
In acute and chronic inflammation, neutrophils and platelets, both of which promote monocyte recruitment, are often activated simultaneously. We investigated how secretory products of neutrophils and platelets synergize to enhance the recruitment of monocytes. We found that neutrophil-borne human neutrophil peptide 1 (HNP1, α-defensin) and platelet-derived CCL5 form heteromers. These heteromers stimulate monocyte adhesion through CCR5 ligation. We further determined structural features of HNP1-CCL5 heteromers and designed a stable peptide that could disturb proinflammatory HNP1-CCL5 interactions. This peptide attenuated monocyte and macrophage recruitment in a mouse model of myocardial infarction. These results establish the in vivo relevance of heteromers formed between proteins released from neutrophils and platelets and show the potential of targeting heteromer formation to resolve acute or chronic inflammation.
Assuntos
Plaquetas/metabolismo , Quimiocina CCL5/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Multimerização Proteica , alfa-Defensinas/metabolismo , Adesão Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Monócitos/citologia , Miocárdio/citologia , Neutrófilos/citologia , Ligação ProteicaRESUMO
Nucleotides are released in the heart under pathological conditions, but little is known about their contribution to cardiac inflammation. The present study defines the P2Y4 nucleotide receptor, expressed on cardiac microvascular endothelial cells and involved in postnatal heart development, as an important regulator of the inflammatory response to cardiac ischemia. P2Y4-null mice displayed smaller infarcts in the left descending artery ligation model, as well as reduced neutrophil infiltration and fibrosis. Gene profiling identified inter alia endothelin-1 (ET-1) as one of the target genes of P2Y4 in ischemic heart. The reduced level of ET-1 was correlated with reduction of microvascular hyperpermeability, neutrophil infiltration, and endothelial adhesion molecule expression, and it could be explained by the decreased number of endothelial cells in P2Y4-null mice. Expression analysis of metalloproteinases and their tissue inhibitors in ischemic heart revealed reduced expression of matrix metalloproteinase (MMP)-9, reported to be potentially regulated by ET-1, and MMP-8, considered as neutrophil collagenase, as well as reduction of tissue inhibitor of MMP-1 and tissue inhibitor of MMP-4 in P2Y4-null mice. Reduction of cardiac permeability and neutrophil infiltration was also observed in P2Y4-null mice in LPS-induced inflammation model. Protection against infarction resulting from loss of P2Y4 brings new therapeutic perspectives for cardiac ischemia and remodeling.
Assuntos
Endotelina-1/biossíntese , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Receptores Purinérgicos P2/deficiência , Animais , Modelos Animais de Doenças , Regulação para Baixo , Ensaio de Imunoadsorção Enzimática , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Infarto do Miocárdio/fisiopatologia , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
OBJECTIVE: As adenosine monophosphate (AMP)-activated protein kinase both controls cytoskeleton organization in endothelial cells and exerts anti-inflammatory effects, we here postulated that it could influence vascular permeability and inflammation, thereby counteracting cardiac wall edema during sepsis. DESIGN: Controlled animal study. SETTINGS: University research laboratory. SUBJECTS: C57BL/6J, α1AMPK, and α1AMPK mice. INTERVENTION: Sepsis was triggered in vivo using a sublethal injection of lipopolysaccharide (O55B5, 10 mg/kg), inducing systolic left ventricular dysfunction. Left ventricular function, edema, vascular permeability, and inflammation were assessed in vivo in both wild-type mice (α1AMPK) and α1AMP-activated protein kinase-deficient mice (α1AMPK). The 5-aminoimidazole-4-carboxamide riboside served to study the impact of AMP-activated protein kinase activation on vascular permeability in vivo. The integrity of endothelial cell monolayers was also examined in vitro after lipopolysaccharide challenge in the presence of aminoimidazole-4-carboxamide riboside and/or after α1AMP-activated protein kinase silencing. MEASUREMENTS AND MAIN RESULTS: α1AMP-activated protein kinase deficiency dramatically impaired tolerance to lipopolysaccharide challenge. Indeed, α1AMPK exhibited heightened cardiac vascular permeability after lipopolysaccharide challenge compared with α1AMPK. Consequently, an increase in left ventricular mass corresponding to exaggerated wall edema occurred in α1AMPK, without any further decrease in systolic function. Mechanistically, the lipopolysaccharide-induced α1AMPK cardiac phenotype could not be attributed to major changes in the systemic inflammatory response but was due to an increased disruption of interendothelial tight junctions. Accordingly, AMP-activated protein kinase activation by aminoimidazole-4-carboxamide riboside counteracted lipopolysaccharide-induced hyperpermeability in wild-type mice in vivo as well as in endothelial cells in vitro. This effect was associated with a potent protection of zonula occludens-1 linear border pattern in endothelial cells. CONCLUSIONS: Our results demonstrate for the first time the involvement of a signaling pathway in the control of left ventricular wall edema during sepsis. AMP-activated protein kinase exerts a protective action through the preservation of interendothelial tight junctions. Interestingly, exaggerated left ventricular wall edema was not coupled with aggravated systolic dysfunction. However, it could contribute to diastolic dysfunction in patients with sepsis.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Permeabilidade Capilar , Edema/etiologia , Endotoxemia/complicações , Endotoxemia/enzimologia , Cardiopatias/etiologia , Inflamação/etiologia , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Corantes/farmacocinética , Citocinas/sangue , Ecocardiografia , Edema/diagnóstico , Edema/fisiopatologia , Células Endoteliais/efeitos dos fármacos , Endotoxemia/induzido quimicamente , Azul Evans/farmacocinética , Inativação Gênica , Cardiopatias/diagnóstico , Cardiopatias/fisiopatologia , Ventrículos do Coração/fisiopatologia , Humanos , Inflamação/sangue , Lipopolissacarídeos/farmacologia , Pulmão/enzimologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peroxidase/metabolismo , Ribonucleosídeos/farmacologia , Junções Íntimas/efeitos dos fármacosRESUMO
Nucleotides released within the heart under pathological conditions can be involved in cardioprotection or cardiac fibrosis through the activation purinergic P2Y(2) and P2Y(6) receptors, respectively. We previously demonstrated that adult P2Y(4)-null mice display a microcardia phenotype related to a cardiac angiogenic defect. To evaluate the functional consequences of this defect, we performed here a combination of cardiac monitoring and exercise tests. We investigated the exercise capacity of P2Y(4) wild-type and P2Y(4)-null mice in forced swimming and running tests. Analysis of their stress, locomotion, and resignation was realized in open field, black and white box, and tail suspension experiments. Exercise-induced cardiac hypertrophy was evaluated after repeated and prolonged exercise in P2Y(4) wild-type and P2Y(4)-null hearts. We showed that P2Y(4)-null mice have a lower exercise capacity in both swimming and treadmill tests. This was not related to decreased motivation or increased stress, since open field, white and black box, and mouse tail suspension tests gave comparable results in P2Y(4) wild-type and P2Y(4)-null mice. Heart rate and blood pressure rose normally in P2Y(4)-null swimming mice equipped with a telemetric implant. On the contrary, we observed a delayed recovery of postexercise blood pressure after exercise in P2Y(4)-null mice. The heart rate increment in response to catecholamines was also similar in P2Y(4) wild-type and P2Y(4)-null implanted mice, which is consistent with a similar level of cardiac ß-receptor expression. Interestingly, the heart of P2Y(4)-null mice displayed a reduced sympathetic innervation associated with a decreased norepinephrine level. We also demonstrated that exercise-induced cardiac hypertrophy was lower in P2Y(4)-null mice after repeated and prolonged exercise. This was associated with a lower increase in cardiomyocyte size and microvessel density. In conclusion, besides its role in cardiac development, P2Y(4) receptor could constitute an important regulator of acute and chronic response to exercise.