RESUMO
(1) Background: Vascular smooth muscle cells (VSMCs) undergo a complex phenotypic switch in response to atherosclerosis environmental triggers, contributing to atherosclerosis disease progression. However, the complex heterogeneity of VSMCs and how VSMC dedifferentiation affects human carotid artery disease (CAD) risk has not been clearly established. (2) Method: A single-cell RNA sequencing analysis of CD45- cells derived from the atherosclerotic aorta of Apolipoprotein E-deficient (Apoe-/-) mice on a normal cholesterol diet (NCD) or a high cholesterol diet (HCD), respecting the site-specific predisposition to atherosclerosis was performed. Growth Differentiation Factor 10 (GDF10) role in VSMCs phenotypic switch was investigated via flow cytometry, immunofluorescence in human atherosclerotic plaques. (3) Results: scRNAseq analysis revealed the transcriptomic profile of seven clusters, five of which showed disease-relevant gene signature of VSMC macrophagic calcific phenotype, VSMC mesenchymal chondrogenic phenotype, VSMC inflammatory and fibro-phenotype and VSMC inflammatory phenotype. Osteoblast factor GDF10 involved in ossification and osteoblast differentiation emerged as a hallmark of VSMCs undergoing phenotypic switch. Under hypercholesteremia, GDF10 triggered VSMC osteogenic switch in vitro. The abundance of GDF10 expressing osteogenic-like VSMCs cells was linked to the occurrence of carotid artery disease (CAD) events. (4) Conclusions: Taken together, these results provide evidence about GDF10-mediated VSMC osteogenic switch, with a likely detrimental role in atherosclerotic plaque stability.
Assuntos
Aterosclerose , Doenças das Artérias Carótidas , Fator 10 de Diferenciação de Crescimento , Placa Aterosclerótica , Animais , Aterosclerose/genética , Doenças das Artérias Carótidas/genética , Proliferação de Células , Células Cultivadas , Fator 10 de Diferenciação de Crescimento/genética , Humanos , Camundongos , Camundongos Knockout para ApoE , Músculo Liso Vascular , Miócitos de Músculo Liso , Osteoblastos , Fenótipo , Placa Aterosclerótica/genética , Análise de Célula ÚnicaRESUMO
(1) Background: Monocytes and nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome orchestrate lipid-driven amplification of vascular inflammation promoting the disruption of the fibrous cap. The components of the NLRP3 inflammasome are expressed in macrophages and foam cells within human carotid atherosclerotic plaques and VSMCs in hypertension. Whether monocytes and NLRP3 inflammasome activation are direct triggers of VSMC phenotypic switch and plaque disruption need to be investigated. (2) Methods: The direct effect of oxLDL-activated monocytes in VSMCs co-cultured system was demonstrated via flow cytometry, qPCR, ELISA, caspase 1, and pyroptosis assay. Aortic roots of VSMCs lineage tracing mice fed normal or high cholesterol diet and human atherosclerotic plaques were used for immunofluorescence quantification of NLRP3 inflammasome activation/VSMCs phenotypic switch. (3) Results: OxLDL-activated monocytes reduced α-SMA, SM22α, Oct-4, and upregulation of KLF-4 and macrophage markers MAC2, F4/80 and CD68 expression as well as caspase 1 activation, IL-1ß secretion, and pyroptosis in VSMCs. Increased caspase 1 and IL-1ß in phenotypically modified VSMCs was detected in the aortic roots of VSMCs lineage tracing mice fed high cholesterol diet and in human atherosclerotic plaques from carotid artery disease patients who experienced a stroke. (4) Conclusions: Taken together, these results provide evidence that monocyte promote VSMC phenotypic switch through VSMC NLRP3 inflammasome activation with a likely detrimental role in atherosclerotic plaque stability in human atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Inflamassomos/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Aterosclerose/complicações , Aterosclerose/genética , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Transdiferenciação Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Hipercolesterolemia/complicações , Hipercolesterolemia/patologia , Interleucina-1beta/metabolismo , Lipoproteínas LDL/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
BACKGROUND: Neutrophils accumulate in atherosclerotic plaques. Neutrophil extracellular traps (NET) were recently identified in experimental atherosclerosis and in complex human lesions. However, not much is known about the NET marker citrullinated histone-3 (H3Cit) expression and functionality in human carotid plaques. Moreover, the association between the proatherosclerotic autoantibody anti-apolipoprotein A-1 (anti-ApoA-1 IgG) and NET has never been investigated. METHODS: Atherosclerotic plaques have been obtained from 36 patients with severe carotid stenosis that underwent carotid endarterectomy for severe carotid stenosis. Samples were sectioned into upstream and downstream regions from the same artery segment. Plaque composition and expression of NET markers neutrophil elastase (NE) and H3Cit were quantified by immunohistochemistry. H3Cit expression and function was evaluated by immunofluorescence and confocal analysis in a subset of patients. RESULTS: Pathological features of vulnerable phenotypes were exacerbated in plaques developed at downstream regions, including higher accumulation of neutrophils and enhanced expression of NE and H3Cit, as compared to plaques from upstream regions. The H3Cit signal was also more intense in downstream regions, with significant extracellular distribution in spaces outside of neutrophils. The percentage of H3Cit colocalization with CD66b (neutrophils) was markedly lower in downstream portions of carotid plaques, confirming the extrusion of NET in this region. In agreement, the maximum distance of the H3Cit signal from neutrophils, extrapolated from vortex distance calculation in all possible directions, was also higher in downstream plaques. The serum anti-ApoA-1index positively correlated with the expression of H3Cit in downstream segments of plaques. Expression of the H3Cit signal outside of neutrophils and H3Cit maximal distance from CD66b-positive cells increased in plaques from serum positive anti-ApoA-1 patients compared with serum negative patients. CONCLUSION: NET elements are differentially expressed in upstream versus downstream regions of human carotid plaques and may be influenced by circulating levels of anti-ApoA-1 IgG. These findings could warrant the investigation of NET elements as potential markers of vulnerability.
Assuntos
Apolipoproteína A-I/imunologia , Artérias Carótidas/imunologia , Artérias Carótidas/patologia , Armadilhas Extracelulares/metabolismo , Imunoglobulina G/imunologia , Placa Aterosclerótica/imunologia , Idoso , Apolipoproteína A-I/sangue , Estudos de Coortes , Feminino , Histonas/metabolismo , Humanos , Elastase de Leucócito/metabolismo , Masculino , Placa Aterosclerótica/sangueRESUMO
BACKGROUND: Apelin is an endogenous peptidergic system which modulates cardiovascular function. Recent studies pointed out a fundamental contribution of apelin on atherosclerosis development; however, such reports revealed contradictory data, and to date, it is difficult to accurately define a beneficial or deleterious role. To better understand apelin function on atherosclerosis, we aimed to investigate apelin-13 treatment effects on atherosclerotic plaques composition. DESIGN: Apolipoprotein E gene-deleted mice were fed on Western-type diet for 11 weeks. Atherosclerotic plaque formation was induced in the carotid artery by a shear stress modifier device, which exposes the same vessel to distinct patterns of shear stress enabling the formation of plaques with different composition. Mice were treated with apelin-13 (2 mg kg-1 day-1 ) or vehicle for the last 3 weeks. RESULTS: Apelin-13 treatment did not alter the lipid content of low shear stress- and oscillatory shear stress-induced plaques in the carotid. However, apelin-13 greatly ameliorated plaque stability by increasing intraplaque collagen content and reducing MMP-9 expression. Furthermore, apelin-13 decreased the infiltration of inflammatory cells (neutrophil and macrophage) and intraplaque reactive oxygen species content. Interestingly, apelin-13 treatment reduced total cholesterol, LDL levels and free fatty acid serum levels, while HDL, triglycerides serum levels were not significantly changed. CONCLUSIONS: Apelin-13 treatment for 3 weeks did not alter the lesion size, but it significantly enhanced the stable phenotype of atherosclerotic plaques and improved serum lipid profile. These results indicate that activation of apelin system decreases plaque vulnerability.
Assuntos
Apelina/farmacologia , Doenças das Artérias Carótidas/fisiopatologia , Placa Aterosclerótica/fisiopatologia , Animais , Doenças das Artérias Carótidas/metabolismo , Colágeno/metabolismo , Dieta Ocidental , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/sangue , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout para ApoE , Placa Aterosclerótica/metabolismo , Distribuição Aleatória , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND: Different cut-off values of serum lipoprotein (a) [Lp (a)] were recently identified to better stratify cardiovascular risk categories. Both pathophysiological and prognostic values of Lp (a) remain unclear. MATERIALS AND METHODS: Here, the prognostic value of Lp (a) and its correlation with intraplaque features were assessed in patients with severe carotid artery stenosis undergoing endarterectomy (n = 180). The cut-off value of 10 mg/dL for serum Lp (a) was selected to predict 24-month follow-up acute coronary syndrome (ACS). In addition, the association between serum Lp (a) and intraplaque lipids, collagen, inflammatory and vascular cells was assessed. Serum Lp (a) levels were measured by nephelometric assay. RESULTS: Patients with high Lp (a) had similar comorbidities, medications and laboratory parameters as compared to low Lp (a) levels. At 24-month follow-up, patients with high Lp (a) had more ACS as compared to low levels. Histological parameters within plaques were comparable in the study groups. No significant correlation between Lp (a) serum levels and intraplaque parameters was found, except for a weak positive association with smooth muscle cells in upstream plaque portions. When adjusted for gender, the presence of dyslipidaemia and chronic coronary artery disease, Lp (a) ≥10 mg/dL remained predictive for ACS. CONCLUSIONS: Lp (a) determination could be a useful tool to predict ACS in patients with severe carotid stenosis.
Assuntos
Síndrome Coronariana Aguda/diagnóstico , Estenose das Carótidas/complicações , Lipoproteína(a)/metabolismo , Síndrome Coronariana Aguda/sangue , Assistência ao Convalescente , Idoso , Biomarcadores/metabolismo , Estenose das Carótidas/sangue , Feminino , Humanos , Masculino , Projetos Piloto , Placa Aterosclerótica/sangue , Placa Aterosclerótica/diagnósticoRESUMO
BACKGROUND: Neutrophil-mediated inflammation was recently identified as an active contributor to athero-progression. Therapeutic strategies inhibiting neutrophil degranulation or recruitment were hypothesized to positively impact on plaque vulnerability. In this study, we investigated whether treatment with the recently discovered agonist of the Mas-related G-coupled receptor type D (MrgD) alamandine would impact on neutrophil degranulation in vivo and in vitro. MATERIALS AND METHODS: Fifteen-week-old ApoE-/- mice were fed with a Western-type diet for an additional 11 weeks. After the first 2 weeks of diet, mice were surgically implanted with a carotid 'cast' device that alters the blood shear stress and induces different carotid plaque phenotypes. During the last 4 weeks before euthanasia, mice were randomly assigned to subcutaneously receive vehicle (NaCl 0·15 M) or alamandine (24 µg/kg/h) by micropump. For in vitro experiments, neutrophils were obtained after thioglycollate intraperitoneal injection in ApoE-/- mice. RESULTS: Treatment with alamandine was well-tolerated, but failed to affect lipid, macrophage, neutrophil or collagen content within carotid and aortic root plaques. Also, treatment with alamandine did not affect Th-cell polarization in lymphoid organs. Conversely, alamandine administration was associated with a reduction in serum levels of neutrophil granule enzymes, such as MMP-9 and MPO as well as MMP-9 content within aortic root plaques. In vitro, preincubation with alamandine dose-dependently abrogated PMA-induced neutrophil degranulation of MMP-9 and MPO. CONCLUSION: These results suggest that treatment with the MrgD agonist alamandine led to a reduced release of neutrophil granule products, potentially interfering with pro-atherosclerotic neutrophil activation.
Assuntos
Aterosclerose/fisiopatologia , Degranulação Celular/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Oligopeptídeos/farmacologia , Placa Aterosclerótica/fisiopatologia , Animais , Aorta Torácica/efeitos dos fármacos , Aterosclerose/tratamento farmacológico , Artérias Carótidas/efeitos dos fármacos , Progressão da Doença , Técnicas In Vitro , Metabolismo dos Lipídeos/efeitos dos fármacos , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Knockout , Neutrófilos/fisiologia , Peroxidase/metabolismo , Placa Aterosclerótica/tratamento farmacológico , Distribuição Aleatória , Receptores Acoplados a Proteínas G/agonistasRESUMO
Selective pharmacological treatments targeting reperfusion injury produced modest protective effects and might be associated with immunosuppression. In order to identify novel and better-tolerated approaches, we focused on the neutralization of receptor activator of nuclear factor kappa-B ligand [RANKL], a cytokine recently shown to activate inflammatory cells (i.e. neutrophils) orchestrating post-infarction injury and repair. Myocardial ischemia (60min) and reperfusion injury was surgically induced in C57Bl/6 mice. In hearts and serum, RANKL was early upregulated during reperfusion. A "one-shot" injection with neutralizing anti-RANKL IgG during ischemia ameliorated myocardial infarct size and function, but not adverse remodeling (determined by Magnetic Resonance Imaging [MRI]) as compared to Vehicle or control IgG. These beneficial effects were accompanied in vivo by reduction in cardiac neutrophil infiltration, reactive oxygen species (ROS) and MMP-9 release. Anti-RANKL IgG treatment suppressed sudden peak of neutrophil granule products in mouse serum early after reperfusion onset. In vitro, RANK mRNA expression was detected in isolated mouse neutrophils. Co-incubation with neutralizing anti-RANKL IgG abrogated RANKL-induced mouse neutrophil degranulation and migration, suggesting a critical role of RANKL in neutrophil-mediated injury. Conversely, anti-RANKL IgG did not affect salvage pathways in cardiac cells (i.e. ERK p42/p44, Akt and STAT-3) or macrophage cardiac infiltration. Finally, treatment with anti-RANKL IgG showed no effect on B and T lymphocyte polarization (in serum, spleen and infarcted myocardium) and circulating chemokines as compared with Vehicle or control IgG. In conclusion, acute treatment with anti-RANKL IgG improved cardiac infarct size and function by potentially impacting on neutrophil-mediated injury and repair.
Assuntos
Anticorpos Monoclonais/farmacologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Neutrófilos/efeitos dos fármacos , Ligante RANK/antagonistas & inibidores , Disfunção Ventricular/tratamento farmacológico , Animais , Biomarcadores , Degranulação Celular , Citocinas/sangue , Citocinas/metabolismo , Modelos Animais de Doenças , Mediadores da Inflamação/sangue , Mediadores da Inflamação/metabolismo , Subpopulações de Linfócitos/patologia , Macrófagos/patologia , Imageamento por Ressonância Magnética , Masculino , Camundongos , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/etiologia , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ligante RANK/metabolismo , Troponina I/sangue , Troponina I/metabolismoRESUMO
BACKGROUND: Autoantibodies to apolipoprotein A-1 (anti-ApoA-1 IgG) were shown to predict major adverse cardiovascular events and promote atherogenesis. However, their potential relationship with clinical disability and ischaemic lesion volume after acute ischaemic stroke (AIS) remains unexplored. MATERIALS AND METHODS: We included n = 76 patients admitted for AIS and we investigated whether baseline serum anti-ApoA-1 IgG levels could predict (i) AIS-induced clinical disability [assessed by the modified Rankin Scale (mRS)], and (ii) AIS-related ischaemic lesion volume [assessed by Computed Tomography (CT)]. We also evaluated the possible pro-apoptotic and pro-necrotic effects of anti-ApoA-1 IgG on human astrocytoma cell line (U251) using flow cytometry. RESULTS: High levels of anti-ApoA-1 IgG were retrieved in 15·8% (12/76) of patients. Increased baseline levels of anti-ApoA-1 IgG were independently correlated with worse mRS [ß = 0·364; P = 0·002; adjusted odds ratio (OR): 1·05 (95% CI 1·01-1·09); P = 0·017] and CT-assessed ischaemic lesion volume [ß = 0·333; P < 0·001; adjusted OR: 1·06 (95% CI 1·01-1·12); P = 0·048] at 3 months. No difference in baseline clinical, biochemical and radiological characteristics was observed between patients with high vs. low levels of anti-ApoA-1 IgG. Incubating human astrocytoma cells with anti-ApoA-1 IgG dose dependently induced necrosis and apoptosis of U251 cells in vitro. CONCLUSION: Anti-ApoA-1 IgG serum levels at AIS onset are associated with poorer clinical recovery and worse brain lesion volume 3 months after AIS. These observations could be partly explained by the deleterious effect of anti-ApoA-1 IgG on human brain cell survival in vitro and may have clinical implication in the prediction of poor outcome in AIS.
Assuntos
Apolipoproteína A-I/imunologia , Autoanticorpos/imunologia , Imunoglobulina G/imunologia , Acidente Vascular Cerebral/imunologia , Idoso , Apoptose/efeitos dos fármacos , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Autoanticorpos/farmacologia , Linhagem Celular Tumoral , Feminino , Citometria de Fluxo , Seguimentos , Proteína Glial Fibrilar Ácida/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Técnicas In Vitro , Receptores de Lipopolissacarídeos/efeitos dos fármacos , Receptores de Lipopolissacarídeos/metabolismo , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Necrose , Razão de Chances , Projetos Piloto , Prognóstico , Estudos Prospectivos , Recuperação de Função Fisiológica , Índice de Gravidade de Doença , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/fisiopatologia , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Tomografia Computadorizada por Raios XRESUMO
Serum c-reactive protein (CRP) was suggested for the assessment of intermediate cardiovascular (CV) risk. Here, systemic or intraplaque CRP levels were investigated as predictors of major adverse cardiovascular events (MACEs) in patients with severe carotid stenosis. CRP levels were assessed in the serum and within different portions (upstream and downstream) of carotid plaques of 217 patients undergoing endarterectomy. The association between CRP and intraplaque lipids, collagen, neutrophils, smooth muscle cells (SMC), and macrophage subsets was determined. No correlation between serum CRP and intraplaque biomarkers was observed. In upstream portions, CRP content was directly correlated with intraplaque neutrophils, total macrophages, and M1 macrophages and inversely correlated with SMC content. In downstream portions, intraplaque CRP correlated with M1 and M2 macrophages. According to the cut-off point (CRP > 2.9%) identified by ROC analysis in upstream portions, Kaplan-Meier analysis showed that patients with high CRP levels had a greater rate of MACEs. This risk of MACEs increased independently of age, male gender, serum CRP, and statin use. In conclusion, in patients with severe carotid artery stenosis, high CRP levels within upstream portions of carotid plaques directly and positively correlate with intraplaque inflammatory cells and predict MACEs at an 18-month follow-up period.
Assuntos
Proteína C-Reativa/metabolismo , Estenose das Carótidas/metabolismo , Fatores Etários , Idoso , Biomarcadores/sangue , Biomarcadores/metabolismo , Estenose das Carótidas/sangue , Feminino , Humanos , Estimativa de Kaplan-Meier , Macrófagos/metabolismo , Masculino , Miócitos de Músculo Liso/metabolismo , Neutrófilos/metabolismo , Curva ROC , Fatores de Risco , Fatores SexuaisRESUMO
AIMS: Several intracellular mediators have been implicated as new therapeutic targets against myocardial ischaemia and reperfusion injury. However, clinically effective salvage pathways remain undiscovered. Here, we focused on the potential role of the adaptor protein p66(Shc) as a regulator of myocardial injury in a mouse model of cardiac ischaemia and reperfusion. METHODS AND RESULTS: Adult male p66(Shc) deficient (p66(Shc) (-/-)) and C57Bl/6 wild-type (WT) mice were exposed to 30, 45, or 60 min of ischaemia and reperfusion (5, 15 min, or 24 h). Infarct size, systemic and intracardiac inflammation and oxidants, as well as cytosolic and mitochondrial apoptotic pathways were investigated. Following 30, but not 45 or 60 min of ischaemia, genetic p66(Shc) deficiency was associated with larger infarcts. In WT mice, in vivo p66(Shc) knock down by siRNA with transient protein deficiency confirmed these findings. P66(Shc) inhibition was not associated with any modification in post-infarction inflammation, oxidative burst nor cardiac vessel density or structure. However, in p66(Shc) (-/-) mice activation of the protective and anti-apoptotic Reperfusion Injury Salvage Kinases and Survivor Activating Factor Enhancement pathways were blunted and mitochondrial swelling and cellular apoptosis via the caspase-3 pathway increased compared with WT. CONCLUSIONS: Genetic deletion of p66(Shc) increased susceptibility to myocardial injury in response to short-term ischaemia and reperfusion in mice. Still, additional studies are needed for assessing the role of this pathway in acute coronary syndrome patients.
Assuntos
Deleção de Genes , Traumatismo por Reperfusão Miocárdica/genética , Proteínas Adaptadoras da Sinalização Shc/genética , Animais , Apoptose/genética , Biomarcadores/metabolismo , Técnicas de Silenciamento de Genes , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos Endogâmicos C57BL , Dilatação Mitocondrial/genética , Reperfusão Miocárdica/métodos , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/metabolismo , Fator de Transcrição STAT3/genética , Proteínas Adaptadoras da Sinalização Shc/deficiência , Proteínas Adaptadoras da Sinalização Shc/metabolismo , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Troponina I/metabolismoRESUMO
BACKGROUND AND AIMS: Different adipokines have been associated with atherosclerotic plaque rupture and cardiovascular events, such as acute ischaemic stroke (AIS). However, the potential role of these molecules in postischaemic brain injury remains largely unknown. METHODS AND METHODS: We performed a substudy analysis on nonobese patients with first atherothrombotic stroke (n = 35) from a recently published prospective cohort. Primary endpoint was to investigate the predictive value of serum leptin/adiponectin ratio on neurological recovery at 90 days after AIS. The secondary endpoint was the predictive value of serum adipokine levels of clinical and radiological outcomes at a shorter follow-up (at days 1 and 7 after AIS). The radiological evaluation included ischaemic lesion volume and haemorrhagic transformation (HT). The clinical examination was based on National Institutes of Health Stroke Scale (NIHSS) and modified Rankin Scale (mRS). RESULTS: At day 1 after AIS, serum leptin and leptin/adiponectin ratio were increased and inversely correlated with both radiological and clinical parameters at all follow-up time points. Once identified the best cut-off points by receiver operating characteristic (ROC) analysis, risk analysis showed that higher circulating leptin improved neurological recovery at day 90. In addition, leptin/adiponectin ratio maintained statistical significance after adjustment for age, gender and thrombolysis, also predicting the occurrence of HT in the first 7 days after AIS (adjusted OR 0·15 [95% CI 0·03-0·83); P = 0·030]). CONCLUSIONS: Higher leptin/adiponectin ratio at day 1 predicted better neurological outcomes in patients with atherothrombotic AIS and might be potentially useful as a prognostic biomarker of the disease.
Assuntos
Adiponectina/sangue , Isquemia Encefálica/sangue , Leptina/sangue , Recuperação de Função Fisiológica , Acidente Vascular Cerebral/sangue , Idoso , Área Sob a Curva , Isquemia Encefálica/complicações , Estudos de Coortes , Progressão da Doença , Feminino , Humanos , Modelos Logísticos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Prognóstico , Estudos Prospectivos , Resistina/sangue , Índice de Gravidade de Doença , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/etiologia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: After an acute ischaemic stroke (AIS), several inflammatory biomarkers have been investigated, but their predictive role on functional recovery remains to be validated. Here, we investigated the prognostic relevance of biomarkers related to atherosclerotic plaque calcification, such as osteopontin (OPN), osteoprotegerin (OPG) and the receptor activator of nuclear factor kappa-B ligand (RANKL) in a cohort of patients with AIS (n = 90) during 90-day follow-up. MATERIALS AND METHODS: Radiological and clinical examinations as well as blood sampling were performed at admission and at days 1, 7 and 90 from the event. Validated scores [such as modified Rankin scale (mRS) and the National Institutes of Health Stroke Scale (NIHSS)] were used to assess poststroke outcome. Serum levels of OPN, OPG and RANKL were measured by colorimetric enzyme-linked immunosorbent assay (ELISA). RESULTS: When compared to the admission, OPN serum levels increased at day 7. Serum OPN levels at this time point were positively correlated with both ischaemic lesion volume and NIHSS at days 7 and 90. A cut-off of 30.53 ng/mL was identified for serum OPN by receiver operating characteristic (ROC) curve analysis. Adjusted logistic regression showed that serum OPN levels at day 7 predicted worse mRS at day 90 [OR 4.13 (95% CI 1.64-10.36); P = 0.002] and NIHSS [1.49 (95% CI 1.16-1.99); P = 0.007], independently of age, gender, hypertension and thrombolysis. CONCLUSIONS: Serum levels of OPN, but not OPG and RANKL, peaked at day 7 after AIS and predicted worse neurological scores. Therefore, OPN might have a pathophysiological and clinical relevance after AIS.
Assuntos
Isquemia Encefálica/sangue , Pessoas com Deficiência , Osteopontina/metabolismo , Acidente Vascular Cerebral/sangue , Idoso , Biomarcadores/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoprotegerina/metabolismo , Prognóstico , Estudos Prospectivos , Ligante RANK/metabolismo , Regulação para Cima/fisiologiaRESUMO
BACKGROUND: Acute pancreatitis is characterized by inflammatory processes affecting not only the pancreas, but also the lung. Here, we investigated timing of leucocyte infiltration and chemokine expression within lung and pancreas during pancreatitis and whether treatments selectively inhibiting chemokines (using Evasins) could improve organ injury. MATERIAL AND METHODS: C57Bl/6 mice were submitted in vivo to 10-h intraperitoneal injections of cerulein and followed for up to 168 h. Five minutes after the first cerulein injection, a single intraperitoneal injection of 10 µg Evasin-3, 1 µg Evasin-4 or an equal volume of vehicle (PBS) was performed. Leucocytes, reactive oxygen species (ROS), necrosis and chemokine/cytokine mRNA expression were assessed in different organs by immunohistology and real-time RT-PCR, respectively. RESULTS: In the lung, neutrophil infiltration and macrophage infiltration peaked at 12 h and were accompanied by increased CXCL2 mRNA expression. CCL2, CXCL1 and TNF-alpha significantly increased after 24 h as compared to baseline. No increase in CCL3 and CCL5 was observed. In the pancreas, neutrophil infiltration peaked at 6 h, while macrophages increased only after 72 h. Treatment with Evasin-3 decreased neutrophil infiltration, ROS production and apoptosis in the lung and reduced neutrophils, macrophages apoptosis and necrosis in the pancreas. Evasin-4 only reduced macrophage content in the lung and did not provide any benefit at the pancreas level. CONCLUSION: Chemokine production and leucocyte infiltration are timely regulated in lung and pancreas during pancreatitis. CXC chemokine inhibition with Evasin-3 improved neutrophil inflammation and injury, potentially interfering with damages in acute pancreatitis and related pulmonary complications.
Assuntos
Anti-Inflamatórios/uso terapêutico , Neutrófilos/efeitos dos fármacos , Pancreatite/tratamento farmacológico , Receptores CXCR/uso terapêutico , Animais , Proteínas de Artrópodes , Ceruletídeo/toxicidade , Quimiocina CXCL1/antagonistas & inibidores , Quimiocina CXCL2/antagonistas & inibidores , Modelos Animais de Doenças , Leucócitos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Necrose , Infiltração de Neutrófilos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Pâncreas/patologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas e Peptídeos SalivaresRESUMO
OBJECTIVE: Endocannabinoid levels are elevated in human and mouse atherosclerosis, but their causal role is not well understood. Therefore, we studied the involvement of fatty acid amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in atherosclerotic plaque vulnerability. METHODS AND RESULTS: We assessed atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) and ApoE(-/-)FAAH(-/-) mice. Before and after 5, 10, and 15 weeks on high-cholesterol diet, we analyzed weight, serum cholesterol, and endocannabinoid levels, and atherosclerotic lesions in thoracoabdominal aortas and aortic sinuses. Serum levels of FAAH substrates anandamide, palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) were 1.4- to 2-fold higher in case of FAAH deficiency. ApoE(-/-)FAAH(-/-) mice had smaller plaques with significantly lower content of smooth muscle cells, increased matrix metalloproteinase-9 expression, and neutrophil content. Circulating and bone marrow neutrophil counts were comparable between both genotypes, whereas CXC ligand1 levels were locally elevated in aortas of FAAH-deficient mice. We observed enhanced recruitment of neutrophils, but not monocytes, to large arteries of ApoE(-/-) mice treated with FAAH inhibitor URB597. Spleens of ApoE(-/-)FAAH(-/-) mice had reduced CD4+FoxP3+regulatory T-cell content, and in vitro stimulation of splenocytes revealed significantly elevated interferon-γ and tumor necrosis factor-α production in case of FAAH deficiency. CONCLUSIONS: Increased anandamide and related FAAH substrate levels are associated with the development of smaller atherosclerotic plaques with high neutrophil content, accompanied by an increased proinflammatory immune response.
Assuntos
Amidoidrolases/deficiência , Aorta/enzimologia , Doenças da Aorta/enzimologia , Aterosclerose/enzimologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Amidas , Amidoidrolases/antagonistas & inibidores , Amidoidrolases/genética , Animais , Aorta/efeitos dos fármacos , Aorta/imunologia , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/imunologia , Doenças da Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Ácidos Araquidônicos/sangue , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Benzamidas/farmacologia , Carbamatos/farmacologia , Células Cultivadas , Quimiocina CXCL1/metabolismo , Colesterol/sangue , Modelos Animais de Doenças , Endocanabinoides/sangue , Inibidores Enzimáticos/farmacologia , Etanolaminas/sangue , Genótipo , Mediadores da Inflamação/metabolismo , Interferon gama/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/patologia , Infiltração de Neutrófilos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Ácidos Oleicos/sangue , Ácidos Palmíticos/sangue , Fenótipo , Placa Aterosclerótica , Alcamidas Poli-Insaturadas/sangue , Baço/imunologia , Linfócitos T Reguladores/imunologia , Fatores de Tempo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Myocardial reperfusion injury is mediated by several processes including increase of reactive oxygen species (ROS). The aim of the study is to identify potential sources of ROS contributing to myocardial ischemia-reperfusion injury. For this purpose, we investigated myocardial ischemia/reperfusion pathology in mice deficient in various NADPH oxidase isoforms (Nox1, Nox2, Nox4, as well as Nox1/2 double knockout). Following 30min of ischemia and 24h of reperfusion, a significant decrease in the size of myocardial infarct was observed in Nox1-, Nox2- and Nox1/Nox2-, but not in Nox4-deficient mice. However, no protection was observed in a model of chronic ischemia, suggesting that NOX1 and NOX2-mediated oxidative damage occurs during reperfusion. Cardioprotective effect of Nox1 and Nox2 deficiencies was associated with decrease of neutrophil invasion, but, on the other hand an improved reperfusion injury was also observed in isolated perfused hearts (Langendorff model) suggesting that inflammatory cells were not the major source of oxidative damage. A decrease in global post-reperfusion oxidative stress was clearly detected in Nox2-, but not in Nox1-deficient hearts. Analysis of key signaling pathways during reperfusion suggests distinct cardioprotective patterns: increased phosphorylation was seen for Akt and Erk in Nox1-deficient mice and for Stat3 and Erk in Nox2-deficient mice. Consequently, NOX1 and NOX2 represent interesting drug targets for controlling reperfusion damage associated with revascularization in coronary disease.
Assuntos
Glicoproteínas de Membrana/genética , Traumatismo por Reperfusão Miocárdica/genética , NADH NADPH Oxirredutases/genética , NADPH Oxidases/genética , Animais , Citocinas/sangue , Modelos Animais de Doenças , Isoenzimas , Macrófagos/patologia , Masculino , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/metabolismo , Miocárdio/patologia , NADH NADPH Oxirredutases/metabolismo , NADPH Oxidase 1 , NADPH Oxidase 2 , NADPH Oxidase 4 , NADPH Oxidases/metabolismo , Infiltração de Neutrófilos/genética , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Percutaneous transluminal angioplasty is frequently used in patients with severe arterial narrowing due to atherosclerosis. However, it induces severe arterial injury and an inflammatory response leading to restenosis. Here, we studied a potential activation of the endocannabinoid system and the effect of FA amide hydrolase (FAAH) deficiency, the major enzyme responsible for endocannabinoid anandamide degradation, in arterial injury. We performed carotid balloon injury in atherosclerosis-prone apoE knockout (apoE(-/-)) and apoE(-/-)FAAH(-/-) mice. Anandamide levels were systemically elevated in apoE(-/-) mice after balloon injury. ApoE(-/-)FAAH(-/-) mice had significantly higher baseline anandamide levels and enhanced neointima formation compared with apoE(-/-) controls. The latter effect was inhibited by treatment with CB1 antagonist AM281. Similarly, apoE(-/-) mice treated with AM281 had reduced neointimal areas, reduced lesional vascular smooth-muscle cell (SMC) content, and proliferating cell counts. The lesional macrophage content was unchanged. In vitro proliferation rates were significantly reduced in CB1(-/-) SMCs or when treating apoE(-/-) or apoE(-/-)FAAH(-/-) SMCs with AM281. Macrophage in vitro adhesion and migration were marginally affected by CB1 deficiency. Reendothelialization was not inhibited by treatment with AM281. In conclusion, endogenous CB1 activation contributes to vascular SMC proliferation and neointima formation in response to arterial injury.
Assuntos
Aterosclerose/metabolismo , Músculo Liso Vascular/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Amidoidrolases/genética , Amidoidrolases/metabolismo , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/patologia , Proliferação de Células , Humanos , Camundongos , Camundongos Knockout , Morfolinas/farmacologia , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Neointima/genética , Neointima/metabolismo , Pirazóis/farmacologia , Receptor CB1 de Canabinoide/deficiência , Receptor CB1 de Canabinoide/genética , Túnica Íntima/efeitos dos fármacos , Túnica Íntima/lesões , Túnica Íntima/patologiaRESUMO
AIMS: The activation of cannabinoid receptor type 2 (CB(2))-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB(2) pharmacological activation on markers of plaque vulnerability in vivo and in vitro. METHODS AND RESULTS: The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB(1) (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB(2) protein expression was reduced when compared with asymptomatic patients. In these portions, CB(2) levels were inversely correlated (r = -0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB(2) co-localized with neutrophils and MMP-9. Treatment with the selective CB(2) agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils. CONCLUSION: Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB(2) activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.
Assuntos
Artéria Carótida Interna/metabolismo , Estenose das Carótidas/metabolismo , Metaloproteinase 9 da Matriz/fisiologia , Placa Aterosclerótica/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Idoso , Animais , Aorta Torácica/metabolismo , Canabinoides/farmacologia , Estudos de Casos e Controles , Feminino , Flavonoides/farmacologia , Humanos , Indóis/farmacologia , Sistema de Sinalização das MAP Quinases/fisiologia , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Fosforilação/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Receptor CB2 de Canabinoide/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismoRESUMO
AIMS: The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a transforming growth factor-ß superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. METHODS AND RESULTS: In contrast to endogenous Mstn, myocardial Gdf11 declined during the course of ageing and was particularly reduced following ischaemia/reperfusion (I/R) injury, suggesting a therapeutic potential of GDF11 signalling in MI. Unexpectedly, boosting systemic Gdf11 by recombinant GDF11 delivery (0.1â mg/kg body weight over 30 days) prior to myocardial I/R augmented myocardial infarct size in C57BL/6 mice irrespective of their age, predominantly by accelerating pro-apoptotic signalling. While intrinsic cardioprotective signalling pathways remained unaffected by high circulating GDF11, targeted transcriptomics and immunomapping studies focusing on GDF11-associated downstream targets revealed attenuated Nkx2-5 expression confined to CD105-expressing cells, with pro-apoptotic activity, as assessed by caspase-3 levels, being particularly pronounced in adjacent cells, suggesting an indirect effect. By harnessing a highly specific and validated liquid chromatography-tandem mass spectrometry-based assay, we show that in prospectively recruited patients with MI circulating GDF11 but not MSTN levels incline with age. Moreover, GDF11 levels were particularly elevated in those at high risk for adverse outcomes following the acute event, with circulating GDF11 emerging as an independent predictor of myocardial infarct size, as estimated by standardized peak creatine kinase-MB levels. CONCLUSION: Our data challenge the initially reported heart rejuvenating effects of circulating GDF11 and suggest that high levels of systemic GDF11 exacerbate myocardial injury in mice and humans alike. Persistently high GDF11 levels during ageing may contribute to the age-dependent loss of cardioprotective mechanisms and thus poor outcomes of elderly patients following acute MI.
Assuntos
Fatores de Diferenciação de Crescimento , Traumatismos Cardíacos , Infarto do Miocárdio , Idoso , Animais , Humanos , Camundongos , Envelhecimento/metabolismo , Proteínas Morfogenéticas Ósseas , Fatores de Diferenciação de Crescimento/genética , Fatores de Diferenciação de Crescimento/metabolismo , Coração , Traumatismos Cardíacos/complicações , Traumatismos Cardíacos/metabolismo , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismoRESUMO
Cannabinoid receptor CB(2) activation inhibits inflammatory proliferation and migration of vascular smooth muscle cells in vitro. The potential in vivo relevance of these findings is unclear. We performed carotid balloon distension injury in hypercholesterolemic apolipoprotein E knockout (ApoE(-/-)) mice receiving daily intraperitoneal injection of the CB(2) agonist JWH133 (5 mg/kg) or vehicle, with the first injection given 30 min before injury. Alternatively, we subjected CB(2)(-/-) and wild-type (WT) mice to balloon injury. We determined CB(2) mRNA and protein expression in dilated arteries of ApoE(-/-) mice. Neointima formation was assessed histologically. We used bone marrow-derived murine CB(2)(-/-) and WT macrophages to study adhesion to plastic, fibronectin, or collagen, and migration was assayed by modified Boyden chamber. Aortic smooth muscle cells were isolated to determine in vitro proliferation rates. We found increased vascular CB(2) expression in ApoE(-/-) mice in response to balloon injury. Seven to twenty-one days after dilatation, injured vessels of JWH133-treated mice had less intimal nuclei numbers as well as intimal and medial areas, associated with less staining for proliferating cells, smooth muscle cells, and macrophages. Complete endothelial repair was observed after 14 days in both JWH133- and vehicle-treated mice. CB(2) deficiency resulted in increased intima formation compared with WT, whereas JWH133 did not affect intimal formation in CB(2)(-/-) mice. Apoptosis rates assessed by in situ terminal deoxynucleotidyl transferase dUTP-mediated nick-end labeling staining 1 h postballooning were significantly higher in the CB(2) knockouts. In vitro, bone marrow-derived CB(2)(-/-) macrophages showed enhanced adherence and migration compared with WT cells and elevated mRNA levels of adhesion molecules, chemokine receptors CCR1 and 5, and chemokine CCL2. Proliferation rates were significantly increased in CB(2)(-/-) smooth muscle cells compared with WT. In conclusion, pharmacological activation or genetic deletion of CB(2) receptors modulate neointima formation via protective effects in macrophages and smooth muscle cells.
Assuntos
Angioplastia com Balão/efeitos adversos , Neointima/fisiopatologia , Receptor CB2 de Canabinoide/fisiologia , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/fisiologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Lesões das Artérias Carótidas/tratamento farmacológico , Lesões das Artérias Carótidas/patologia , Lesões das Artérias Carótidas/fisiopatologia , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Colesterol na Dieta/metabolismo , Colesterol na Dieta/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Neointima/tratamento farmacológico , Neointima/patologia , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/biossínteseRESUMO
RATIONALE: The highly prevalent obstructive sleep apnea syndrome (OSA) with its main component intermittent hypoxia (IH) is a risk factor for cardiovascular mortality. The poor knowledge of its pathophysiology has limited the development of specific treatments, whereas the gold standard treatment, continuous positive airway pressure, may not fully reverse the chronic consequences of OSA and has limited acceptance in some patients. OBJECTIVES: To examine the contribution of IH-induced inflammation to the cardiovascular complications of OSA. METHODS: We investigated systemic and vascular inflammatory changes in C57BL6 mice exposed to IH (21-5% Fi(O(2)), 60-s cycle) or normoxia 8 hours per day up to 14 days. Vascular alterations were reassessed in mice treated with a blocking antibody of regulated upon activation, normal T-cell expressed and secreted (RANTES)/CC chemokine ligand 5 (CCL5) signaling pathway, or with the IgG isotype control throughout the IH exposure. MEASUREMENTS AND MAIN RESULTS: IH induced systemic inflammation combining increased splenic lymphocyte proliferation and chemokine expression, with early and predominant RANTES/CCL5 alterations, and enhanced splenocyte migration toward RANTES/CCL5. IH also induced structural and inflammatory vascular alterations. Leukocyte-endothelium adhesive interactions were increased, attested by leukocyte rolling and intercellular adhesion molecule-1 expression in mesenteric vessels. Aortas had increased intima-media thickness with elastic fiber alterations, mucoid depositions, nuclear factor-κB-p50 and intercellular adhesion molecule-1 overexpression, hypertrophy of smooth-muscle cells overexpressing RANTES/CCL5, and adventitial-periadventitial T-lymphocyte infiltration. RANTES/CCL5 neutralization prevented both intima-media thickening and inflammatory alterations, independently of the IH-associated proatherogenic dyslipidemia. CONCLUSIONS: Inflammation is a determinant mechanism for IH-induced preatherosclerotic remodeling involving RANTES/CCL5, a key chemokine in atherogenesis. Characterization of the inflammatory response could allow identifying at-risk patients for complications, and its pharmacologic manipulation may represent a potential complementary treatment of sleep apnea consequences.