RESUMO
OBJECTIVES: Trace elements (TEs) are ubiquitous. TE concentrations vary among individuals and countries, depending on factors such as living area, workplaces and diet. Deficit or excessive TEs concentrations have consequences on the proper functioning of human organism so their biomonitoring is important. The aim of this project was to provide reference values for TEs concentrations in the Swiss population. METHODS: The 1,078 participants to the SKiPOGH cohort included in this study were aged 18-90 years. Their 24-h urine and/or plasma samples were analyzed by inductively coupled plasma mass spectrometry (ICP-MS) to determine 24 TEs concentrations: Ag, Al, As, Be, Bi, Cd, Co, Cr, Cu, Hg, I, Li, Mn, Mo, Ni, Pb, Pd, Pt, Sb, Se, Sn, Tl, V and Zn. Statistical tests were performed to evaluate the influence of covariates (sex, age, BMI, smoking) on these results. Reference intervals for the Swiss adult population were also defined. RESULTS: TEs concentrations were obtained for respectively 994 and 903 persons in plasma and urine matrices. It was possible to define percentiles of interest (P50 and P95) for almost all the TEs. Differences in TEs distribution between men and women were noticed in both matrices; age was also a cofactor. CONCLUSIONS: This first Swiss biomonitoring of a large TEs-panel offers reference values in plasma and in urine for the Swiss population. The results obtained in this study were generally in line with clinical recommendations and comparable to levels reported in other population-based surveys.
RESUMO
Cobalt, chromium, and nickel are used in orthopedic prostheses. They can be released, accumulate in many organs, and be toxic. The aim of this study is to evaluate the cytotoxicity of these metals on human hepatocytes and to improve our knowledge of their cellular toxicity mechanisms by metabolomic analysis. HepaRG cells were incubated for 48 h with increasing concentrations of metals to determine their IC50. Then, a nontargeted metabolomic study using liquid chromatography-high-resolution mass spectrometry (LC-HRMS) was done at IC50 and at a lower concentration (100 nM), near to those found in the blood and liver of patients with prostheses. IC50 were defined at 940, 2, and 1380 µM for Co, Cr, and Ni, respectively. In vitro, Cr appears to be much more toxic than Co and Ni. Metabolomic analysis revealed the disruption of metabolic pathways from the low concentration of 100 nM, in particular tryptophan metabolism and lipid metabolism illustrated by an increase in phenylacetylglycine, a marker of phospholipidosis, for all three metals. They also appear to be responsible for oxidative stress. Dysregulation of these pathways impacts hepatocyte metabolism and may result in hepatotoxicity. Further investigations on accessible biological matrices should be conducted to correlate our in vitro results with the clinical data of prostheses-bearing patients.
Assuntos
Cromo , Cobalto , Cromo/química , Cromo/toxicidade , Cobalto/toxicidade , Hepatócitos/química , Humanos , Metais , Níquel/toxicidadeRESUMO
BACKGROUND: Zinc deficiency is commonly encountered in chronic kidney disease (CKD). The aims of this study were to assess whether zinc deficiency was related to increased renal excretion of zinc and to the progression of CKD. METHODS: Plasma and 24-h urinary zinc levels, urinary electrolytes and uromodulin were measured in 108 CKD patients and 81 individuals without CKD. Serum creatinine values were collected for 3 years to calculate the yearly change in estimated glomerular filtration rate (eGFR). Multivariable regression analysis was performed to assess the association between baseline zinc levels and yearly change in eGFR. RESULTS: CKD patients had lower circulating zinc levels and higher 24-h urinary zinc excretion than non-CKD participants (612.4 ± 425.9 versus 479.2 ± 293.0 µg/day; P = 0.02). Fractional excretion (FE) of zinc was higher and it significantly increased at more advanced CKD stages. Zinc FE was correlated negatively with 24-h urinary uromodulin excretion (r=-0.29; P < 0.01). Lower baseline plasma zinc levels were associated with a faster yearly decline of renal function in age, gender, diabetes and hypertension adjusted models, but this relationship was no longer significant when baseline eGFR or proteinuria were included. CONCLUSIONS: Zinc levels are lower in CKD, and not compensated by reduced renal zinc excretion. The inverse association between urinary zinc excretion and uromodulin possibly points to an impaired tubular activity, which could partly account for zinc imbalance in CKD. These data suggest that zinc status is associated with renal function decline, but further studies elucidating the underlying mechanisms and the potential role of zinc supplements in CKD are needed.
Assuntos
Insuficiência Renal Crônica/fisiopatologia , Zinco/sangue , Zinco/deficiência , Estudos de Casos e Controles , Creatinina/sangue , Progressão da Doença , Feminino , Seguimentos , Taxa de Filtração Glomerular , Humanos , Testes de Função Renal , Masculino , Pessoa de Meia-Idade , Insuficiência Renal Crônica/sangue , Insuficiência Renal Crônica/urina , Uromodulina/urinaRESUMO
Simultaneous assessment of a panel of protein markers is becoming essential in order to enhance biomarker research and improve diagnostics. Specifically, postmortem diagnostics of early myocardial ischemia in sudden cardiac death cases could benefit from a multiplex marker assessment in the same tissue section. Current analytical antibody-based techniques (immunohistochemistry and immunofluorescence) limit multiplex analysis usually to not more than three antibodies. In this study, mass spectrometry-immunohistochemistry (MS-IHC) was performed by combining laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) with rare-metal-isotope-tagged antibodies as a technique for multiplex analysis of human postmortem myocardial tissue samples. Tissue sections with myocardial infarction were simultaneously analyzed for seven primary, rare-metal-isotope-tagged antibodies (troponin T, myoglobin, fibronectin, C5b-9, unphosphorylated connexin 43, VEGF-B, and JunB). Comparison between the MS-IHC approach and chromogenic IHC showed similar patterns in ionic and optical images. In addition, absolute quantification was performed by MS-IHC, providing a proportional relationship between the signal intensity and the local marker concentration in tissue sections. These data demonstrated that LA-ICP-MS combined with rare-metal-isotope-tagged antibodies is an efficient strategy for simultaneous testing of multiple markers and allows not only visualization of molecules within the tissue but also quantification of the signal. Such imaging approach has a great potential in both diagnostics and pathology-related research.
Assuntos
Imuno-Histoquímica , Espectrometria de Massas , Infarto do Miocárdio/metabolismo , Biomarcadores/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Conexina 43/metabolismo , Feminino , Patologia Legal , Humanos , Isótopos , Masculino , Espectrometria de Massas/métodos , Pessoa de Meia-Idade , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Mioglobina , Fatores de Transcrição , Troponina T/metabolismo , Fator B de Crescimento do Endotélio VascularRESUMO
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
Three-dimensional MALDI imaging MS (IMS) is a growing branch of IMS still requiring developments in methodology and technology to make the technique routinely accessible. Many challenges are simply a matter of producing 3D reconstructions and interpreting them in a timely fashion. In this aim and using analysis of lipids from atherosclerotic plaques from a human carotid and mouse aortic sinuses, we describe 3D reconstruction methods using open-source software that provides high-quality visualization and rapid interpretation through multivariate segmentation of the 3D IMS data. Multiple datasets were generated for each sample and we provide insight into simple means to correlate the separate datasets.
Assuntos
Aterosclerose/diagnóstico por imagem , Imageamento Tridimensional/métodos , Lipídeos/isolamento & purificação , Placa Aterosclerótica/diagnóstico por imagem , Animais , Aterosclerose/diagnóstico , Aterosclerose/patologia , Seio Carotídeo/diagnóstico por imagem , Seio Carotídeo/patologia , Humanos , Camundongos , Placa Aterosclerótica/diagnóstico , Placa Aterosclerótica/patologia , Seio Aórtico/diagnóstico por imagem , Seio Aórtico/patologiaRESUMO
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
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
BACKGROUND: Recent experimental data indicate that volatile anaesthetics can induce a neuroinflammatory response in the central nervous system. The questions of to what extent this occurs in the developing brain and whether nonvolatile anaesthetics are also involved remain unanswered. OBJECTIVES: The objective of this study is to investigate the impact of propofol anaesthesia on cytokine mRNA expression profiles in the neonatal brain at defined stages of the brain growth spurt. DESIGN: A randomised placebo-controlled experimental in-vivo study. SETTING: Translational research laboratories at the University of Geneva Medical School. METHODS: Wistar rats received 6-h propofol anaesthesia at postnatal day 10 or 20. A quantitative real-time PCR was used to evaluate the impact of this treatment paradigm on mRNA expression profiles of selected members of the cytokine family in the prefrontal cortex and hippocampus. RESULTS: Propofol anaesthesia induced a transient 1.8-fold (interquartile range, IQR 1.7 to 2.2) increase (Pâ=â0.004) in prefrontal but not hippocampal tumour necrosis factor mRNA concentrations in 10-day-old animals. No such effect was detected in 20-day-old animals. No changes in mRNA concentrations of two other pro-inflammatory cytokines, interleukins IL-6 and IL-1ß, were detected following drug exposure at any developmental stages or in any studied brain regions. In contrast, propofol anaesthesia at postnatal day 10 induced a transient increase in the mRNA expression patterns of two chemokines: Ccl2 and Ccl3 [for Ccl2 mRNA: 4.4-fold (3.8 to 5.6) increase in the prefrontal cortex, Pâ=â0.0002 and a 3.5-fold (2.8 to 5.3) increase in the hippocampus, Pâ=â0.0001; for Ccl3 mRNA: 2.9-fold (2.6 to 4.31) increase in the prefrontal cortex, Pâ=â0.0001, and a 2.7-fold (2.2 to 3.6) increase in the hippocampus, Pâ=â0.0003]. Propofol did not affect Ccl2 and Ccl3 mRNA concentrations in 20-day-old animals. In addition, it did not impact on two other members of the chemokine family, Cxcl1 and Cx3cl1, at any time points or in any brain regions investigated. CONCLUSION: This study suggests that propofol anaesthesia does not have a major impact on pro-inflammatory cytokine expression profiles in the developing central nervous system during the brain growth spurt. These results raise arguments against the involvement of neuroinflammatory pathways in propofol-related neurotoxicity observed following the administration of this drug in the early postnatal period.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Citocinas/biossíntese , Propofol/farmacologia , Transcriptoma/efeitos dos fármacos , Anestésicos Intravenosos , Animais , Citocinas/genética , Ratos , Ratos Wistar , Transcriptoma/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
Systemic and intraplaque biomarkers have been widely investigated in clinical cohorts as promising surrogate parameters of cardiovascular vulnerability. In this pilot study, we investigated if systemic and intraplaque levels of calcification biomarkers were affected by treatment with a statin in a cohort of patients with severe carotid stenosis and being asymptomatic for ischemic stroke. Patients on statin therapy had reduced serum osteopontin (OPN), RANKL/osteoprotegerin (OPG) ratio, and MMP-9/pro-MMP-9 activity as compared to untreated patients. Statin-treated patients exhibited increased levels of collagen and reduced neutrophil infiltration in downstream portions of carotid plaques as compared to untreated controls. In upstream plaque portions, OPG content was increased in statin-treated patients as compared to controls. Other histological parameters (such as lipid, macrophage, smooth muscle cell, and MMP-9 content) as well as RANKL, RANK, and OPG mRNA levels did not differ between the two patient groups. Serum RANKL/OPG ratio positively correlated with serum levels of neutrophilic products, intraplaque neutrophil, and MMP-9 content within downstream portions of carotid plaques. In conclusion, statin treatment was associated with improvement in serum RANKL levels and reduced neutrophil activity both systemically and in atherosclerotic plaques.
Assuntos
Estenose das Carótidas/patologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , NF-kappa B/metabolismo , Neutrófilos/citologia , Ligante RANK/sangue , Idoso , Aterosclerose/tratamento farmacológico , Atorvastatina , Estudos de Coortes , Feminino , Fluorbenzenos/uso terapêutico , Ácidos Heptanoicos/uso terapêutico , Humanos , Masculino , Metaloproteinase 9 da Matriz/metabolismo , Pessoa de Meia-Idade , Ativação de Neutrófilo , Neutrófilos/efeitos dos fármacos , Osteopontina/metabolismo , Osteoprotegerina/metabolismo , Pirimidinas/uso terapêutico , Pirróis/uso terapêutico , RNA Mensageiro/metabolismo , Fatores de Risco , Rosuvastatina Cálcica , Sinvastatina/uso terapêutico , Sulfonamidas/uso terapêuticoRESUMO
BACKGROUND: Toll-like receptors (TLRs) have long been considered to be major culprits in the development of atherosclerosis, contributing both to its progression and clinical complications. However, evidence for most TLRs beyond TLR2 and TLR4 is lacking. METHODS AND RESULTS: We used experimental mouse models, human atheroma cultures, and well-established human biobanks to investigate the role of TLR7 in atherosclerosis. We report the unexpected finding that TLR7, a receptor recognizing self-nucleic acid complexes, is protective in atherosclerosis. In Apoe(-/-) mice, functional inactivation of TLR7 resulted in accelerated lesion development, increased stenosis, and enhanced plaque vulnerability as revealed by Doppler ultrasound and/or histopathology. Mechanistically, TLR7 interfered with macrophage proinflammatory responses to TLR2 and TLR4 ligands, reduced monocyte chemoattractant protein-1 production, and prevented expansion of Ly6C(hi) inflammatory monocytes and accumulation of inflammatory M1 macrophages into developing atherosclerotic lesions. In human carotid endarterectomy specimens TLR7 levels were consistently associated with an M2 anti-inflammatory macrophage signature (interleukin [IL]-10, IL-1RA, CD163, scavenger and C-type lectin receptors) and collagen genes, whereas they were inversely related or unrelated to proinflammatory mediators (IL-12/IL-23, interferon beta, interferon gamma, CD40L) and platelet markers. Moreover, in human atheroma cultures, TLR7 activation selectively suppressed the production of key proatherogenic factors such as monocyte chemoattractant protein-1 and tumor necrosis factor without affecting IL-10. CONCLUSIONS: These findings provide evidence for a beneficial role of TLR7 in atherosclerosis by constraining inflammatory macrophage activation and cytokine production. This challenges the prevailing concept that all TLRs are pathogenic and supports the exploitation of the TLR7 pathway for therapy.
Assuntos
Doenças das Artérias Carótidas/imunologia , Macrófagos Peritoneais/imunologia , Glicoproteínas de Membrana/imunologia , Placa Aterosclerótica/imunologia , Receptor 7 Toll-Like/imunologia , Animais , Aorta/imunologia , Aorta/patologia , Apolipoproteínas E/genética , Biomarcadores/metabolismo , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Humanos , Macrófagos Peritoneais/patologia , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Placa Aterosclerótica/patologia , RNA Mensageiro/metabolismo , Receptor 7 Toll-Like/genéticaRESUMO
The classical renin-angiotensin system pathway has been recently updated with the identification of additional molecules [such as angiotensin converting enzyme 2, ANG-(1-7), and Mas receptor] that might improve some pathophysiological processes in chronic inflammatory diseases. In the present study, we focused on the potential protective role of Mas receptor activation on mouse lipid profile, liver steatosis, and atherogenesis. Mas/apolipoprotein E (ApoE)-double-knockout (DKO) mice (based on C57BL/6 strain of 20 wk of age) were fed under normal diet and compared with aged-matched Mas and ApoE-single-knockout (KO), as well as wild-type mice. Mas/ApoE double deficiency was associated with increased serum levels of atherogenic fractions of cholesterol, triglycerides, and fasting glucose compared with wild-type or single KO. Serum levels of HDL or leptin in DKO were lower than in other groups. Hepatic lipid content as well as alanine aminotransferase serum levels were increased in DKO compared with wild-type or single-KO animals. Accordingly, the hepatic protein content of mediators related to atherosclerotic inflammation, such as peroxisome proliferator-activated receptor-α and liver X receptor, was altered in an adverse way in DKO compared with ApoE-KO. On the other hand, DKO mice did not display increased atherogenesis and intraplaque inflammation compared with ApoE-KO group. In conclusion, Mas deletion in ApoE-KO mice was associated with development of severe liver steatosis and dyslipidemia without affecting concomitant atherosclerosis. Mas receptor activation might represent promising strategies for future treatments targeting both hepatic and metabolic alterations in chronic conditions clustering these disorders.
Assuntos
Apolipoproteínas E/metabolismo , Fígado Gorduroso/metabolismo , Lipídeos/sangue , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Colesterol/metabolismo , Fígado Gorduroso/genética , Técnicas de Inativação de Genes , Genótipo , Lipídeos/genética , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismoRESUMO
Hypercholesterolemia is one of the most important risk factors for erectile dysfunction, mostly due to the impairment of oxidative stress and endothelial function in the penis. The cannabinoid system might regulate peripheral mechanisms of sexual function; however, its role is still poorly understood. We investigated the effects of CB2 activation on oxidative stress and fibrosis within the corpus cavernosum of hypercholesterolemic mice. Apolipoprotein-E-knockout mice were fed with a western-type diet for 11 weeks and treated with JWH-133 (selective CB2 agonist) or vehicle during the last 3 weeks. CB2 receptor expression, total collagen content, and reactive oxygen species (ROS) production within the penis were assessed. In vitro corpus cavernosum strips preparation was performed to evaluate the nitric oxide (NO) bioavailability. CB2 protein expression was shown in cavernosal endothelial and smooth muscle cells of wild type and hypercholesterolemic mice. Treatment with JWH-133 reduced ROS production and NADPH-oxidase expression in hypercholesterolemic mice penis. Furthermore, JWH-133 increased endothelial NO synthase expression in the corpus cavernosum and augmented NO bioavailability. The decrease in oxidative stress levels was accompanied with a reduction in corpus cavernosum collagen content. In summary, CB2 activation decreased histological features, which were associated with erectile dysfunction in hypercholesterolemic mice.
Assuntos
Canabinoides/farmacologia , Disfunção Erétil/complicações , Disfunção Erétil/tratamento farmacológico , Hipercolesterolemia/complicações , Receptor CB2 de Canabinoide/agonistas , Animais , Canabinoides/administração & dosagem , Modelos Animais de Doenças , Disfunção Erétil/metabolismo , Fibrose , Hipercolesterolemia/metabolismo , Lipídeos/sangue , Masculino , Camundongos , Camundongos Knockout , NADPH Oxidases/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Pênis/efeitos dos fármacos , Pênis/metabolismo , Pênis/patologia , Espécies Reativas de Oxigênio/metabolismo , Receptor CB2 de Canabinoide/metabolismoRESUMO
Plaque rupture is the main cause of acute myocardial infarction and stroke. Atherosclerotic plaques have been described to be vulnerable and more prone to rupture when they are characterized by thin, highly inflamed, and collagen-poor fibrous caps and contain elevated levels of proteases, including metalloproteinases (MMPs). Initiation of collagen breakdown in plaques requires interstitial collagenases, a MMP subfamily consisting of MMP-1, MMP-8, and MMP-13. Previous reports demonstrated that MMP-1 and MMP-13 might be overexpressed in both human and experimental atherosclerosis. Since neutrophils have been only recently reported in atherosclerotic plaques, the role of MMP-8 (formerly known as "neutrophil collagenase") was only marginally evaluated. In this paper, we will update and comment on evidence of the most relevant regulatory pathways and activities mediated by MMP-8 in atherogenesis.
Assuntos
Aterosclerose/enzimologia , Metaloproteinase 8 da Matriz/metabolismo , Animais , Aterosclerose/sangue , Aterosclerose/metabolismo , Aterosclerose/patologia , Humanos , Metaloproteinase 8 da Matriz/sangue , Metaloproteinase 8 da Matriz/genéticaRESUMO
AIMS: The chemokine CCL5 plays a critical role as neutrophil and macrophage activator do in atherosclerosis and myocardial infarction. Thus, we investigated whether the treatment with a neutralizing monoclonal antibody (mAb) to mouse CCL5 would provide therapeutic benefit when provoking a coronary-associated ischaemic event. METHODS AND RESULTS: C57Bl/6 mice were submitted to left coronary artery permanent ligature. Then, various parameters were monitored for up to 21 days. At5 min and 3 days after coronary occlusion, mice received one intravenous injection of the rat anti-mouse CCL5 mAb or isotype IgG control. Infarct size was assessed histologically and by measuring serum cardiac troponin I levels. Kinetics of CCL5 tissue expression, leucocyte infiltration, matrix metalloproteinase (MMP) levels, and collagen deposition were histologically assessed. Serum chemokine levels were measured by enzyme-linked immunosorbent assay. Cardiac function and dimensions were assessed by magnetic resonance imaging (MRI). Chronic ischaemia increased both circulating and intracardiac levels of CCL5. At 24 h, treatment with the anti-CCL5 mAb resulted in a smaller infarct size and reduced circulating levels of chemokines. This effect was associated with reduction of neutrophil and macrophage infiltration within the infarcted myocardium. After 3 days of chronic ischaemia, anti-CCL5 mAb treatment reduced cardiac MMP-9. At 7 days, collagen content was significantly lower. At 21 days, neutralizing CCL5 improved mouse survival, cardiac myocyte size, and cardiac function. CONCLUSION: Treatment with anti-CCL5 mAb significantly reduced both infarct size and post-infarction heart failure in a mouse model of chronic cardiac ischaemia. Cardioprotective effects were associated with the reduction of leucocyte recruitment within infarcted hearts.
Assuntos
Anticorpos Monoclonais/farmacologia , Quimiocina CCL5/fisiologia , Insuficiência Cardíaca/patologia , Infarto do Miocárdio/patologia , Animais , Peso Corporal/fisiologia , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/imunologia , Quimiotaxia de Leucócito/fisiologia , Doença Crônica , Colágeno/metabolismo , Ligadura , Macrófagos/fisiologia , Metaloproteinase 8 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/fisiologia , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica/métodos , Infiltração de Neutrófilos/fisiologia , Neutrófilos/fisiologia , Tamanho do Órgão/fisiologia , Taxa de SobrevidaRESUMO
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
Microsampling techniques became more popular in the last decades, and their use for common analyses such as trace element quantification by inductively coupled plasma mass spectrometry (ICP-MS) has been investigated. We decided to compare two of these techniques (dried blood spots and microtubes) to evaluate their potential for the analysis of 12 trace elements in human whole blood: aluminum (Al), total arsenic (As), cadmium (Cd), cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), nickel (Ni), lead (Pb), selenium (Se) and zinc (Zn). Signal contributions from blank filter paper and instability at room temperature for several elements in the dried blood spot samples restrained our enthusiasm for the use of this technique. Conversely, microtube samples presented low background contamination and good stability under different temperature conditions. Therefore, our results demonstrate that the use of microtubes is more suitable than dried blood spots for trace element quantification in human blood, both in research and routine analysis.
Assuntos
Selênio , Oligoelementos , Humanos , Análise Espectral , Selênio/análise , Cobre/análise , ZincoRESUMO
Human lifetime exposure to arsenic through drinking water, food supply or industrial pollution leads to its accumulation in many organs such as liver, kidneys, lungs or pancreas but also adipose tissue. Recently, population-based studies revealed the association between arsenic exposure and the development of metabolic diseases such as obesity and type 2 diabetes. To shed light on the molecular bases of such association, we determined the concentration that inhibited 17% of cell viability and investigated the effects of arsenic acute exposure on adipose-derived human mesenchymal stem cells differentiated in vitro into mature adipocytes and treated with sodium arsenite (NaAsO2, 10 nM to 10 µM). Untargeted metabolomics and gene expression analyses revealed a strong dose-dependent inhibition of lipogenesis and lipolysis induction, reducing the cellular ability to store lipids. These dysregulations were emphasized by the inhibition of the cellular response to insulin, as shown by the perturbation of several genes and metabolites involved in the mentioned biological pathways. Our study highlighted the activation of an adaptive oxidative stress response with the strong induction of metallothioneins and increased glutathione levels in response to arsenic accumulation that could exacerbate the decreased insulin sensitivity of the adipocytes. Arsenic exposure strongly affected the expression of arsenic transporters, responsible for arsenic influx and efflux, and induced a pro-inflammatory state in adipocytes by enhancing the expression of the inflammatory interleukin 6 (IL6). Collectively, our data showed that an acute exposure to low levels of arsenic concentrations alters key adipocyte functions, highlighting its contribution to the development of insulin resistance and the pathogenesis of metabolic disorders.