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NLRP3-inflammasome-mediated signaling is thought to significantly contribute to the extent of myocardial damage after myocardial infarction (MI). The purpose of this study was to investigate the effects of the NLRP3-inflammasome inhibitor IZD334 on cardiac damage in a pig model of myocardial infarction. Prior to in vivo testing, in vitro, porcine peripheral blood mononuclear cells and whole blood were treated with increasing dosages of IZD334, a novel NLRP3-inflammasome inhibitor, and were stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP). After determination of the pharmacological profile in healthy pigs, thirty female Landrace pigs were subjected to 75 min of transluminal balloon occlusion of the LAD coronary artery and treated with placebo or IZD334 (1 mg/kg, 3 mg/kg, or 10 mg/kg once daily) in a blinded randomized fashion. In vitro, NLRP3-inflammasome stimulation showed the pronounced release of interleukin (IL)-1ß that was attenuated by IZD334 (p < 0.001). In vivo, no differences were observed between groups in serological markers of inflammation nor myocardial IL-1ß expression. After 7 days, the ejection fraction did not differ between groups, as assessed with MRI (placebo: 45.1 ± 8.7%, 1 mg/kg: 49.9 ± 6.1%, 3 mg/kg: 42.7 ± 3.8%, 10 mg/kg: 44.9 ± 6.4%, p = 0.26). Infarct size as a percentage of the area at risk was not reduced (placebo: 73.1 ± 3.0%, 1 mg/kg: 75.5 ± 7.3%, 3 mg/kg: 80.3 ± 3.9%, 10 mg/kg: 78.2 ± 8.0%, p = 0.21). In this pig MI model, we did not observe attenuation of the inflammatory response after NLRP3-inflammasome inhibition in vivo. Consecutively, no difference was observed in IS and cardiac function, while in vitro inhibition successfully reduced IL-1ß release from stimulated porcine blood cells.
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Circadian rhythms influence the recruitment of immune cells and the onset of inflammation, which is pivotal in the response to ischemic cardiac injury after a myocardial infarction (MI). The hyperacute immune response that occurs within the first few hours after a MI has not yet been elucidated. Therefore, we characterized the immune response and myocardial damage 3 hours after a MI occurs over a full twenty-four-hour period to investigate the role of the circadian rhythms in this response. MI was induced at Zeitgeber Time (ZT) 2, 8, 14, and 20 by permanent ligation of the left anterior descending coronary artery. Three hours after surgery, animals were terminated and blood and hearts collected to assess the immunological status and cardiac damage. Blood leukocyte numbers varied throughout the day, peaking during the rest-phase (ZT2 and 8). Extravasation of leukocytes was more pronounced during the active-phase (ZT14 and 20) and was associated with greater chemokine release to the blood and expression of adhesion molecules in the heart. Damage to the heart, measured by Troponin-I plasma levels, was elevated during this time frame. Clock gene oscillations remained intact in both MI-induced and sham-operated mice hearts, which could explain the circadian influence of the hyperacute inflammatory response after a MI. These findings are in line with the clinical observation that patients who experience a MI early in the morning (i.e., early active phase) have worse clinical outcomes. This study provides further insight on the immune response occurring shortly after an MI, which may contribute to the development of novel and optimization of current therapeutic approaches.
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RATIONALE: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. OBJECTIVE: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. METHODS AND RESULTS: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4+ and CD8+ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. CONCLUSIONS: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.
Assuntos
Doenças das Artérias Carótidas/genética , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Linfócitos/metabolismo , Células Mieloides/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Análise de Célula Única , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Animais , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Transdiferenciação Celular , Sequenciamento de Cromatina por Imunoprecipitação , Bases de Dados Genéticas , Células Endoteliais/patologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Linfócitos/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Células Mieloides/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , RNA-SeqRESUMO
Heart failure is a growing health issue as a negative consequence of improved survival upon myocardial infarction, unhealthy lifestyle, and the ageing of our population. The large and complex pathology underlying heart failure makes diagnosis and especially treatment very difficult. There is an urgent demand for discriminative biomarkers to aid disease management of heart failure. Studying cellular pathways and pathophysiological mechanisms contributing to disease initiation and progression is crucial for understanding the disease process and will aid to identification of novel biomarkers and potential therapeutic targets. Growth differentiation factor 15 (GDF15) is a proven valuable biomarker for different pathologies, including cancer, type 2 diabetes, and cardiovascular diseases. Although the prognostic value of GDF15 in heart failure is robust, the biological function of GDF15 in adverse cardiac remodelling is not fully understood. GDF15 is a distant member of the transforming growth factor-ß family and involved in various biological processes including inflammation, cell cycle, and apoptosis. However, more research is suggesting a role in fibrosis, hypertrophy, and endothelial dysfunction. As GDF15 is a pleiotropic protein, elucidating the exact role of GDF15 in complex disease processes has proven to be a challenge. In this review, we provide an overview of the role GDF15 plays in various intracellular and extracellular processes underlying heart failure, and we touch upon crucial points that need consideration before GDF15 can be integrated as a biomarker in standard care or when considering GDF15 for therapeutic intervention.
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Diabetes Mellitus Tipo 2 , Insuficiência Cardíaca , Biomarcadores , Fator 15 de Diferenciação de Crescimento , Insuficiência Cardíaca/diagnóstico , Humanos , Remodelação VentricularAssuntos
Antígenos CD40/antagonistas & inibidores , Doenças Cardiovasculares/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Animais , Antígenos CD40/metabolismo , Doenças Cardiovasculares/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Nanopartículas/administração & dosagem , Transdução de Sinais/fisiologiaRESUMO
A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creERT2; Leprfl/fl mice reveals that leptin's effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes.
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Aterosclerose/terapia , Doenças Cardiovasculares/terapia , Células-Tronco Hematopoéticas/metabolismo , Inflamação/terapia , Condicionamento Físico Animal , Tecido Adiposo/metabolismo , Animais , Aterosclerose/prevenção & controle , Doenças Cardiovasculares/genética , Doenças Cardiovasculares/fisiopatologia , Doenças Cardiovasculares/prevenção & controle , Epigenoma/genética , Exercício Físico/fisiologia , Hematopoese/genética , Hematopoese/fisiologia , Proteínas de Homeodomínio/genética , Humanos , Inflamação/fisiopatologia , Leucócitos/metabolismo , Leucocitose/fisiopatologia , Leucocitose/terapia , Camundongos , Receptores para Leptina/genética , Comportamento Sedentário , Transcriptoma/genéticaRESUMO
The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1-/-) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1-/- compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1-/- peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1-/- mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque.
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Apoptose , Células Gigantes/citologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Células 3T3 , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Diferenciação Celular , Deleção de Genes , Humanos , Imuno-Histoquímica , Lipídeos/química , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Neutrófilos/metabolismo , Fenótipo , Placa Aterosclerótica/metabolismoRESUMO
Patients with Severe Limb Ischemia (SLI) have a high risk of amputation and mortality. Here, we investigated a panel of serum biomarkers with the aim of identifying biomarkers for major events and mechanisms that contribute to disease progression in established SLI. A panel of biomarkers including GROα, HGF, SCF, SCGFß, SDF1α, TRAIL, IL-6, IL-8, FGFß, GCSF, GMCSF, IP10, MCP1, PDGFbb, RANTES, TNFα, VEGF, sICAM, sVCAM, TM, and E-selectin was measured in serum samples from a subset (n = 108) of the JUVENTAS cohort. The primary outcome was major events, defined as major amputation or death. The inflammatory biomarkers IL-6, IL-8, GROα and IP-10 were significantly elevated in patients who reached a major endpoint. Results were validated in a secondary cohort (n = 146). Cox regression showed that adjusted hazard ratios were 1.40 (95% CI: 1.15-1.70, p = 0.0007) and 1.48 (95% CI 1.16-1.87, p = 0.001) for IL-6 and IP-10 in a fully adjusted model containing both biomarkers. A prediction model using IL-6 and IP-10 showed predictive accuracy with an AUC of ~ 78% in both discovery and validation cohorts, which is higher than previously published models. We conclude that inflammatory biomarkers predict major events in patients with SLI and allow the creation of biomarker-based risk-prediction models.
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Amputação Cirúrgica/estatística & dados numéricos , Extremidades/irrigação sanguínea , Inflamação/sangue , Isquemia/sangue , Idoso , Biomarcadores/sangue , Quimiocina CXCL1/sangue , Quimiocina CXCL10/sangue , Quimiocinas/sangue , Citocinas/sangue , Extremidades/cirurgia , Feminino , Humanos , Interleucina-6/sangue , Interleucina-8/sangue , Isquemia/mortalidade , Isquemia/cirurgia , Masculino , Pessoa de Meia-Idade , Doença Arterial Periférica/sangue , Doença Arterial Periférica/complicações , Doença Arterial Periférica/cirurgia , Valor Preditivo dos Testes , Modelos de Riscos Proporcionais , Análise de SobrevidaRESUMO
AIMS: Mouse studies have established distinct monocyte subtypes that participate in the process of atherosclerotic lesion formation. The pro-inflammatory Ly6Chigh monocyte subtype actively contributes to murine plaque progression and destabilization. Also in humans, different peripheral monocyte subtypes have been identified, of which the CD14+CD16- classical monocyte is suggested to display similar pro-atherosclerotic properties as the murine Ly6Chigh subtype. We aimed to investigate if circulating CD14+CD16- classical monocytes associate with characteristics of a vulnerable carotid atherosclerotic plaque and if they associate with the risk of secondary adverse manifestations of atherosclerotic disease. METHODS AND RESULTS: We enrolled 175 carotid endarterectomy patients of the Athero-Express biobank in our study. Just prior to surgical procedure, blood was collected and peripheral blood mononuclear cells were isolated. Characterization of monocyte subsets was performed by flow cytometry. Plaque characteristics were semi-quantitatively scored for the presence of fat, collagen, intraplaque hemorrhage and calcification. Vessel density, smooth muscle cells and macrophages were assessed quantitatively on a continuous scale. All features of a vulnerable plaque phenotype, including low amounts of collagen and smooth muscle cells, and increased fat content, vessel density, intraplaque hemorrhage and plaque macrophages were not significantly associated with differential levels of peripheral classical CD14+CD16- monocytes or other monocyte subsets. Using Cox regression models to evaluate the prognostic value of circulating monocyte subtypes, we found that total counts of peripheral monocytes, as well as CD14+CD16- classical and other monocyte subtypes were not associated with the risk of secondary cardiovascular events during 3â¯years follow-up. CONCLUSION: Circulating classical CD14+CD16- monocytes do not associate with specific vulnerable plaque characteristics. In addition, they do not predict secondary adverse manifestations. This suggests that in patients with established carotid artery disease, the circulating monocytes do not reflect plaque characteristics and have no value in identifying patients at risk for future cardiovascular events.
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Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Placa Aterosclerótica/patologia , Receptores de IgG/metabolismo , Idoso , Feminino , Seguimentos , Humanos , Macrófagos/metabolismo , Masculino , FenótipoRESUMO
Upon myocardial damage, the release of cardiac proteins induces a strong antibody-mediated immune response, which can lead to adverse cardiac remodeling and eventually heart failure (HF). Stem cell therapy using mesenchymal stromal cells (MSCs) or cardiomyocyte progenitor cells (CPCs) previously showed beneficial effects on cardiac function despite low engraftment in the heart. Paracrine mediators are likely of great importance, where, for example, MSC-derived extracellular vesicles (EVs) also show immunosuppressive properties in vitro. However, the limited capacity of MSCs to differentiate into cardiac cells and the sufficient scaling of MSC-derived EVs remain a challenge to clinical translation. Therefore, we investigated the immunosuppressive actions of endogenous CPCs and CPC-derived EVs on antibody production in vitro, using both healthy controls and end-stage HF patients. Both MSCs and CPCs strongly inhibit lymphocyte proliferation and antibody production in vitro. Furthermore, CPC-derived EVs significantly lowered the levels of IgG1, IgG4, and IgM, especially when administered for longer duration. In line with previous findings, plasma cells of end-stage HF patients showed high production of IgG3, which can be inhibited by MSCs in vitro. MSCs and CPCs inhibit in vitro antibody production of both healthy and end-stage HF-derived immune cells. CPC-derived paracrine factors, such as EVs, show similar effects, but do not provide the complete immunosuppressive capacity of CPCs. The strongest immunosuppressive effects were observed using MSCs, suggesting that MSCs might be the best candidates for therapeutic targeting of B-cell responses in HF.
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Linfócitos B/imunologia , Insuficiência Cardíaca/terapia , Imunoglobulina G/imunologia , Imunoglobulina M/imunologia , Transplante de Células-Tronco Mesenquimais , Mioblastos Cardíacos/transplante , Linfócitos B/citologia , Proliferação de Células , Células Cultivadas , Vesículas Extracelulares/imunologia , Insuficiência Cardíaca/imunologia , HumanosRESUMO
BACKGROUND: Tobacco smoking is a major risk factor for atherosclerotic disease and has been associated with DNA methylation (DNAm) changes in blood cells. However, whether smoking influences DNAm in the diseased vascular wall is unknown but may prove crucial in understanding the pathophysiology of atherosclerosis. In this study, we associated current tobacco smoking to epigenome-wide DNAm in atherosclerotic plaques from patients undergoing carotid endarterectomy. METHODS: DNAm at commonly methylated sites (cytosine-guanine nucleotide pairs separated by a phospho-group [CpGs]) was assessed in atherosclerotic plaque samples and peripheral blood samples from 485 carotid endarterectomy patients. We tested the association of current tobacco smoking with DNAm corrected for age and sex. To control for bias and inflation because of cellular heterogeneity, we applied a Bayesian method to estimate an empirical null distribution as implemented by the R package bacon. Replication of the smoking-associated methylated CpGs in atherosclerotic plaques was executed in the second sample of 190 carotid endarterectomy patients, and results were meta-analyzed using a fixed-effects model. RESULTS: Tobacco smoking was significantly associated to differential DNAm in atherosclerotic lesions of 4 CpGs (false discovery rate <0.05) mapped to 2 different genes ( AHRR, ITPK1) and 17 CpGs mapped to 8 genes and RNAs in blood. The strongest associations were found for CpGs mapped to the gene AHRR, a repressor of the aryl hydrocarbon receptor transcription factor involved in xenobiotic detoxification. One of these methylated CpGs were found to be regulated by local genetic variation. CONCLUSIONS: The risk factor tobacco smoking associates with DNAm at multiple loci in carotid atherosclerotic lesions. These observations support further investigation of the relationship between risk factors and epigenetic regulation in atherosclerotic disease.
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Aterosclerose/genética , Doenças das Artérias Carótidas/genética , Metilação de DNA , Epigenômica/métodos , Estudo de Associação Genômica Ampla/métodos , Fumar/efeitos adversos , Idoso , Aterosclerose/etiologia , Doenças das Artérias Carótidas/etiologia , Ilhas de CpG/genética , Endarterectomia das Carótidas/métodos , Endarterectomia das Carótidas/estatística & dados numéricos , Epigênese Genética , Feminino , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Placa Aterosclerótica/etiologia , Placa Aterosclerótica/genéticaRESUMO
BACKGROUND AND AIMS: Neovascularization is associated with atherosclerotic plaque instability and increased chance of myocardial infarction (MI). Patients with chronic inflammatory diseases (CID) have increased risk of atherosclerosis, and evidence demonstrates that NF-κB inducing kinase (NIK)-mediated noncanonical NF-κB signaling in endothelial cells (EC) is linked to inflammation and angiogenesis. Here, we hypothesized NIK may also be activated in EC of atherosclerotic lesion microvessels. METHODS: Using cohorts of atherosclerotic lesions from coronary and carotid arteries, we quantified NIK expression in plaque microvessels and compared it to pathological markers, including inflammatory cell content, plaque characteristics and MI. Differences in gene transcripts were evaluated between stable and ruptured lesions. RESULTS: NIK+EC were present in both coronary and carotid lesions. In CID patients, plaques with stenosis >40% had an increased number of NIK+EC and higher content of immune cells (pâ¯<â¯.05) as compared to controls. Immune cells per NIK+EC were also greater in CID patients (pâ¯<â¯.05), with pronounced differences as stenosis increased. In unstable lesions, NIK+EC were elevated as were EC expressing CXCL12 (pâ¯<â¯.05). NIK+EC were increased in lesions with lipid content >40% (pâ¯<â¯.05) and more abundant in coronary artery lesions implicated in MI (pâ¯<â¯.05). These vessels also associated with atheromatous rather than fibrous plaque morphology (pâ¯<â¯.05). Transcriptomic profiling demonstrated components of noncanonical NF-κB pathway were also upregulated in ruptured plaques (pâ¯<â¯.05). CONCLUSIONS: NIK+EC associate with chronic inflammation in advanced lesions and are linked to markers of local inflammation, lipid content, unstable plaque phenotype and development of MI. Therefore, targeting noncanonical NF-κB signaling may hold therapeutic potential for patients with atherosclerosis and cardiovascular disease.
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Doenças das Artérias Carótidas/metabolismo , Doença da Artéria Coronariana/metabolismo , Células Endoteliais/metabolismo , Mediadores da Inflamação/metabolismo , Inflamação/metabolismo , Microvasos/metabolismo , Infarto do Miocárdio/metabolismo , NF-kappa B/metabolismo , Placa Aterosclerótica , Transdução de Sinais , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/patologia , Doença da Artéria Coronariana/genética , Doença da Artéria Coronariana/patologia , Células Endoteliais/patologia , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Inflamação/patologia , Microvasos/patologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , NF-kappa B/genética , Prognóstico , Proteínas Serina-Treonina Quinases/metabolismo , Ruptura Espontânea , Índice de Gravidade de Doença , Quinase Induzida por NF-kappaBRESUMO
BACKGROUND: Atherosclerosis is an inflammatory lipid disorder and the main underlying pathology of acute ischemic events. Despite a vast amount of data from murine atherosclerosis models, evidence of B-cell involvement in human atherosclerotic disease is limited. We therefore investigated the association of circulating B-cell subtypes with the occurrence of secondary cardiovascular events in advanced atherosclerotic disease. METHODS AND RESULTS: This cohort study consists of 168 patients who were included in the Athero-Express biobank between 2009 and 2011. Before surgery, peripheral blood mononuclear cells were isolated and stored in liquid nitrogen. After gentle thawing of the peripheral blood mononuclear cells, different B-cell subtypes including naïve, (un)switched memory, and CD27+CD43+ B1-like B cells, were analyzed by flow cytometry. Univariable and multivariable Cox proportional hazard models were used to analyze associations between B-cell subtypes, circulating antibodies and secondary cardiovascular manifestations during the 3-year follow-up period. Mean age was 70.1±9.6 years, males represented 62.8% of the population, and 54 patients had secondary manifestations during follow-up. High numbers of unswitched memory cells were protective against secondary outcome (hazard ratio, 0.30 [95% CI, 0.13-0.69]; P<0.01). Similar results were obtained for the switched memory cells that also showed to be protective against secondary outcome (hazard ratio, 0.33 [95% CI, 0.14-0.77]; P=0.01). CONCLUSIONS: A high number of (un)switched memory B cells is associated with better outcome following carotid artery endarterectomy. These findings suggest a potential role for B-cell subsets in prediction and prevention of secondary cardiovascular events in patients with atherosclerosis.
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Linfócitos B/imunologia , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/imunologia , Memória Imunológica , Placa Aterosclerótica , Idoso , Idoso de 80 Anos ou mais , Autoanticorpos/sangue , Linfócitos B/metabolismo , Artérias Carótidas/cirurgia , Doenças das Artérias Carótidas/mortalidade , Doenças das Artérias Carótidas/patologia , Doenças das Artérias Carótidas/cirurgia , Distribuição de Qui-Quadrado , Endarterectomia das Carótidas/efeitos adversos , Endarterectomia das Carótidas/mortalidade , Feminino , Citometria de Fluxo , Humanos , Imunoglobulina M/sangue , Imunofenotipagem/métodos , Lipoproteínas LDL/imunologia , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Infarto do Miocárdio/etiologia , Infarto do Miocárdio/mortalidade , Fenótipo , Modelos de Riscos Proporcionais , Fatores de Risco , Acidente Vascular Cerebral/etiologia , Acidente Vascular Cerebral/mortalidade , Fatores de Tempo , Resultado do TratamentoRESUMO
An involement of Toll-like receptor 2 (TLR2) has been established in cardiac dysfunction after acute myocardial infarction; however, its role in chronic pressure overload is unclear. We sought to evaluate the role of TLR2 in cardiac hypertrophy, fibrosis and dysfunction in sustained pressure overload. We induced pressure overload via transverse aortic constriction (TAC) in TLR2-/- and wild type (WT) mice, and followed temporal changes over 8 weeks. Despite similar increases in heart weight, left ventricular (LV) ejection fraction (EF) and diastolic function (mitral E/A ratio) were preserved in TLR2-/- mice but impaired in WT mice following TAC. TAC produced less LV fibrosis in TLR2-/- mice associated with lower mRNA levels of collagen genes (Col1a1 and Col3a1) and lower protein level of TGFbeta1, compared to WT mice. Following TAC, the influx of macrophages and CD3 T cells into LV was similar between TLR2-/- and WT mice, whereas levels of cyto/chemokines were lower in the heart and plasma in TLR2-/- mice. TLR2-/- bone marrow-derived cells protected against LVEF decline and fibrosis following TAC. Our findings show that leukocytic TLR2 deficiency protects against LV dysfunction and fibrosis probably via a reduction in inflammatory signaling in sustained pressure overload.
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Pressão Sanguínea , Cardiopatias/etiologia , Cardiopatias/fisiopatologia , Leucócitos/metabolismo , Receptor 2 Toll-Like/deficiência , Animais , Biópsia , Citocinas/metabolismo , Modelos Animais de Doenças , Fibrose , Cardiopatias/metabolismo , Cardiopatias/patologia , Testes de Função Cardíaca , Imuno-Histoquímica , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Knockout , Função Ventricular Esquerda , Remodelação Ventricular/genéticaRESUMO
BACKGROUND: Recent studies found an immune regulatory role for Y chromosome and a relationship between loss of Y chromosome (LOY) in blood cells and a higher risk of cancer and mortality. Given involvement of immune cells in atherosclerosis, we hypothesized that LOY is associated with the severity of atherosclerotic plaque characteristics and outcome in men undergoing carotid endarterectomy. METHODS AND RESULTS: LOY was quantified in blood and plaque from raw intensity genotyping data in men within the Athero-Express biobank study. Plaques were dissected, and the culprit lesions used for histology and the measurement of inflammatory proteins. We tested LOY for association with (inflammatory) atherosclerotic plaque phenotypes and cytokines and assessed the association of LOY with secondary events during 3-year follow-up. Of 366 patients with carotid endarterectomy, 61 exhibited some degree of LOY in blood. LOY was also present in atherosclerotic plaque lesions (n=8/242, 3%). LOY in blood was negatively associated with age (ß=-0.03/10 y; r2=0.07; P=1.6×10-7) but not with cardiovascular disease severity at baseline. LOY in blood was associated with a larger atheroma size (odds ratio, 2.15; 95% confidence interval, 1.06-4.76; P=0.04); however, this association was not significant after correction for multiple testing. LOY was independently associated with secondary major cardiovascular events (hazard ratio=2.28; 95% confidence interval, 1.11-4.67; P=0.02) in blood when corrected for confounders. CONCLUSIONS: In this hypothesis-generating study, LOY in blood is independently associated with secondary major cardiovascular events in a severely atherosclerotic population. Our data could indicate that LOY affects secondary outcome via other mechanisms than inflammation in the atherosclerotic plaque.
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Aterosclerose/patologia , Cromossomos Humanos Y/genética , Idoso , Aterosclerose/sangue , Aterosclerose/genética , Artérias Carótidas/cirurgia , Estudos de Coortes , Endarterectomia das Carótidas , Seguimentos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Razão de Chances , Placa Aterosclerótica/sangue , Placa Aterosclerótica/genética , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo Real , FumarRESUMO
Toll-like receptors (TLR) provide a critical link between innate and adaptive immunity, both important players in atherosclerosis. Since evidence for the role of TLR5 is lacking, we aimed to establish this in the immune axis of atherosclerosis. We assessed the effect of the TLR5-specific ligand Flagellin on macrophage maturation and T-cell polarisation. Next, we generated TLR5-/-LDLr-/- chimeras to study the effect of hematopoietic TLR5 deficiency on atherosclerosis formation. Flagellin stimulation did not influence wildtype or TLR5-/- macrophage maturation. Only in wildtype macrophages, Flagellin exposure increased MCP-1 and IL6 expression. Flagellin alone reduced T-helper 1 proliferation, which was completely overruled in the presence of T-cell receptor activation. In vivo, hematopoietic TLR5 deficiency attenuated atherosclerotic lesion formation by ≈25% (1030*103 ± 63*103 vs. 792*103 ± 61*103 µm2; p = 0.013) and decreased macrophage area (81.3 ± 12.0 vs. 44.2 ± 6.6 µm2; p = 0.011). In TLR5-/- chimeric mice, we observed lower IL6 plasma levels (36.4 ± 5.6 vs. 15.1 ± 2.2 pg/mL; p = 0.003), lower (activated) splenic CD4+ T-cell content (32.3 ± 2.1 vs. 21.0 ± 1.2%; p = 0.0018), accompanied by impaired T-cell proliferative responses. In conclusion, hematopoietic TLR5 deficiency inhibits atherosclerotic lesion formation by attenuated macrophage accumulation and defective T-cell responsiveness.
Assuntos
Aterosclerose/etiologia , Aterosclerose/metabolismo , Leucócitos/imunologia , Leucócitos/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Receptor 5 Toll-Like/deficiência , Animais , Aterosclerose/sangue , Aterosclerose/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/metabolismo , Contagem de Leucócitos , Camundongos , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Subpopulações de Linfócitos T/citologiaRESUMO
BACKGROUND: Innate immune responses activated through myeloid cells contribute to the initiation, progression, and complications of atherosclerosis in experimental models. However, the critical upstream pathways that link innate immune activation to foam cell formation are still poorly identified. OBJECTIVES: This study sought to investigate the hypothesis that activation of the triggering receptor expressed on myeloid cells (TREM-1) plays a determinant role in macrophage atherogenic responses. METHODS: After genetically invalidating Trem-1 in chimeric Ldlr-/-Trem-1-/- mice and double knockout ApoE-/-Trem-1-/- mice, we pharmacologically inhibited Trem-1 using LR12 peptide. RESULTS: Ldlr-/- mice reconstituted with bone marrow deficient for Trem-1 (Trem-1-/-) showed a strong reduction of atherosclerotic plaque size in both the aortic sinus and the thoracoabdominal aorta, and were less inflammatory compared to plaques of Trem-1+/+ chimeric mice. Genetic invalidation of Trem-1 led to alteration of monocyte recruitment into atherosclerotic lesions and inhibited toll-like receptor 4 (TLR 4)-initiated proinflammatory macrophage responses. We identified a critical role for Trem-1 in the upregulation of cluster of differentiation 36 (CD36), thereby promoting the formation of inflammatory foam cells. Genetic invalidation of Trem-1 in ApoE-/-/Trem-1-/- mice or pharmacological blockade of Trem-1 in ApoE-/- mice using LR-12 peptide also significantly reduced the development of atherosclerosis throughout the vascular tree, and lessened plaque inflammation. TREM-1 was expressed in human atherosclerotic lesions, mainly in lipid-rich areas with significantly higher levels of expression in atheromatous than in fibrous plaques. CONCLUSIONS: We identified TREM-1 as a major upstream proatherogenic receptor. We propose that TREM-1 activation orchestrates monocyte/macrophage proinflammatory responses and foam cell formation through coordinated and combined activation of CD36 and TLR4. Blockade of TREM-1 signaling may constitute an attractive novel and double-hit approach for the treatment of atherosclerosis.
Assuntos
Artérias Carótidas/patologia , Doenças das Artérias Carótidas/terapia , Terapia Genética/métodos , Imunidade Inata , Ácidos Láuricos/farmacologia , Glicoproteínas de Membrana/biossíntese , Placa Aterosclerótica/terapia , Receptores Imunológicos/biossíntese , Rodaminas/farmacologia , Animais , Apoptose , Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/antagonistas & inibidores , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oligopeptídeos , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Receptores Imunológicos/antagonistas & inibidores , Receptor Gatilho 1 Expresso em Células MieloidesRESUMO
Mesenchymal stem cells (MSCs) have regenerative properties, but recently they were also found to have immunomodulatory capacities. We therefore investigated whether MSCs could reduce atherosclerosis, which is determined by dyslipidaemia and chronic inflammation. We adoptively transferred MSCs into low-density lipoprotein-receptor knockout mice and put these on a Western-type diet to induce atherosclerosis. Initially after treatment, we found higher levels of circulating regulatory T cells. In the long-term, overall numbers of effector T cells were reduced by MSC treatment. Moreover, MSC-treated mice displayed a significant 33% reduction in circulating monocytes and a 77% reduction of serum CCL2 levels. Most strikingly, we found a previously unappreciated effect on lipid metabolism. Serum cholesterol was reduced by 33%, due to reduced very low-density lipoprotein levels, likely a result of reduced de novo hepatic lipogenesis as determined by a reduced expression of Stearoyl-CoA desaturase-1 and lipoprotein lipase. MSCs significantly affected lesion development, which was reduced by 33% in the aortic root. These lesions contained 56% less macrophages and showed a 61% reduction in T cell numbers. We show here for the first time that MSC treatment affects not only inflammatory responses but also significantly reduces dyslipidaemia in mice. This makes MSCs a potent candidate for atherosclerosis therapies.
Assuntos
Aterosclerose/terapia , Inflamação/terapia , Transplante de Células-Tronco Mesenquimais , Receptores de LDL/genética , Animais , Aorta/metabolismo , Aorta/patologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Dieta , Modelos Animais de Doenças , Humanos , Inflamação/imunologia , Inflamação/patologia , Macrófagos , Células-Tronco Mesenquimais/imunologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Camundongos Knockout , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismoRESUMO
AIMS: Restoration of coronary blood flow is crucial in the treatment of acute myocardial infarction. Reperfusion, however, induces ischaemia-reperfusion (IR) injury, which further deteriorates myocardial function. The innate immune system plays an important role in this process, mediating rapid influx of immune cells into the reperfused myocardium. Leukotriene B4 is an important leucocyte chemoattractant, performing its actions through binding to its specific receptor BLT1. We hypothesized that treatment with LSN2792613, a selective BLT1 antagonist, reduces infarct size (IS) in a mouse model of myocardial IR injury. METHODS AND RESULTS: Male C57Bl/6J mice were subjected to myocardial ischaemia for 30 min by surgical coronary artery ligation, followed by reperfusion. Mice received either LSN2792613 or vehicle, three times daily (orally) for up to 72 h after reperfusion. BLT1 inhibition with LSN2792613 reduced IS compared with vehicle treatment (26.9 ± 2.7 vs. 34.9 ± 2.2%, P = 0.030) at 24 h after reperfusion. The levels of IL-6 and keratinocyte chemoattractant were reduced in the infarcted tissue of LSN2792613-treated mice. Reduced apoptosis in LSN2792613-treated mice was suggested by increased levels of phosphorylated JNK and GSK3α/ß, and confirmed by flow cytometric analysis showing less apoptotic and necrotic cardiomyocytes in the infarcted myocardium. Echocardiography at 4 weeks after myocardial IR showed a slightly higher ejection fraction and stroke volume in mice treated with LSN2792613 compared with vehicle-treated mice, whereas left ventricular volumes were comparable. CONCLUSION: Selective BLT1 inhibition with LSN2792613 reduces inflammation and apoptosis following IR, resulting in reduced IS, and therefore might be a promising strategy to prevent myocardial IR injury.
Assuntos
Anti-Inflamatórios/farmacologia , Cardiotônicos/farmacologia , Antagonistas de Leucotrienos/farmacologia , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/efeitos dos fármacos , Receptores do Leucotrieno B4/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Colágeno/metabolismo , Citoproteção , Modelos Animais de Doenças , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Necrose , Fosforilação , Receptores do Leucotrieno B4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Volume Sistólico/efeitos dos fármacos , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Reperfusion injury following myocardial infarction (MI) increases infarct size (IS) and deteriorates cardiac function. Cardioprotective strategies in large animal MI models often failed in clinical trials, suggesting translational failure. Experimentally, MI is induced artificially and the effect of the experimental procedures may influence outcome and thus clinical applicability. The aim of this study was to investigate if invasive surgery, as in the common open chest MI model affects IS and cardiac function. Twenty female landrace pigs were subjected to MI by transluminal balloon occlusion. In 10 of 20 pigs, balloon occlusion was preceded by invasive surgery (medial sternotomy). After 72 hrs, pigs were subjected to echocardiography and Evans blue/triphenyl tetrazoliumchloride double staining to determine IS and area at risk. Quantification of IS showed a significant IS reduction in the open chest group compared to the closed chest group (IS versus area at risk: 50.9 ± 5.4% versus 69.9 ± 3.4%, P = 0.007). End systolic LV volume and LV ejection fraction measured by echocardiography at follow-up differed significantly between both groups (51 ± 5 ml versus 65 ± 3 ml, P = 0.033; 47.5 ± 2.6% versus 38.8 ± 1.2%, P = 0.005). The inflammatory response in the damaged myocardium did not differ between groups. This study indicates that invasive surgery reduces IS and preserves cardiac function in a porcine MI model. Future studies need to elucidate the effect of infarct induction technique on the efficacy of pharmacological therapies in large animal cardioprotection studies.