RESUMO
Glucagon-like peptide-1 receptor (GLP-1R) agonists induce weight loss in patients with type 2 diabetes mellitus (T2DM), but the underlying mechanism is unclear. Recently, the mechanism by which metformin induces weight loss could be explained by an increase in growth differentiation factor 15 (GDF15), which suppresses appetite. Therefore, we aimed to investigate whether the GLP-1R agonist liraglutide modifies plasma GDF15 levels in patients with T2DM. GDF15 levels were measured in plasma samples obtained from Dutch Europids and Dutch South Asians with T2DM before and after 26 weeks of treatment with daily liraglutide (n = 44) or placebo (n = 50) added to standard care. At baseline, circulating GDF15 levels did not differ between South Asians and Europids with T2DM. Treatment with liraglutide, compared to placebo, decreased body weight, but did not modify plasma GDF15 levels in all patients, or when data were split by ethnicity. Also, the change in plasma GDF15 levels after treatment with liraglutide did not correlate with changes in body weight or HbA1c levels. In addition, the dose of metformin used did not correlate with baseline plasma GDF15 levels. Compared to placebo, liraglutide treatment for 26 weeks does not modify plasma GDF15 levels in Dutch Europid or South Asian patients with T2DM. Thus, the weight loss induced by liraglutide is likely explained by other mechanisms beyond the GDF15 pathway. HIGHLIGHTS: What is the central question of this study? Growth differentiation factor 15 (GDF15) suppresses appetite and is increased by metformin: does the GLP-1R agonist liraglutide modify plasma GDF15 levels in patients with type 2 diabetes mellitus (T2DM)? What is the main finding and its importance? Plasma GDF15 levels did not differ between South Asians and Europids with T2DM and were not modified by 26 weeks of liraglutide in either ethnicity. Moreover, there was no correlation between the changes in plasma GDF15 levels and dosage of metformin administered, changes in body weight or HbA1c levels. The appetite-suppressing effect of liraglutide is likely exerted via pathways other than GDF15.
Assuntos
Povo Asiático , Diabetes Mellitus Tipo 2 , Fator 15 de Diferenciação de Crescimento , Hipoglicemiantes , Liraglutida , Metformina , Redução de Peso , Humanos , Liraglutida/uso terapêutico , Liraglutida/farmacologia , Fator 15 de Diferenciação de Crescimento/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/sangue , Masculino , Feminino , Redução de Peso/efeitos dos fármacos , Pessoa de Meia-Idade , Metformina/uso terapêutico , Metformina/farmacologia , Hipoglicemiantes/uso terapêutico , Hipoglicemiantes/farmacologia , Idoso , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , População do Sul da ÁsiaRESUMO
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
[Figure: see text].
Assuntos
Estenose das Carótidas/sangue , Quimiocina CCL2/sangue , Placa Aterosclerótica , Idoso , Biomarcadores/sangue , Estenose das Carótidas/mortalidade , Estenose das Carótidas/patologia , Estenose das Carótidas/cirurgia , Estudos Transversais , Endarterectomia das Carótidas/efeitos adversos , Endarterectomia das Carótidas/mortalidade , Feminino , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Medição de Risco , Fatores de Risco , Ruptura Espontânea , Acidente Vascular Cerebral/etiologia , Fatores de TempoRESUMO
The transverse aortic constriction (TAC) model is frequently used to study adverse cardiac remodeling upon pressure overload. We set out to define the most important characteristics that define the degree of cardiac remodeling in this model. A systematic review and meta-analyses were performed on studies using the TAC mouse/rat model and reporting echocardiographic outcome parameters. We included all animal studies in which a constriction around the transverse aorta and at least one of the predefined echocardiography or MRI outcome parameters were assessed. A total of 502 articles and > 3000 wild-type, untreated animals undergoing TAC were included in this study and referenced to a control group. The duration of aortic constriction correlated to the degree of adverse remodeling. However, the mouse data is strongly biased by the preferential use of male C57Bl/6 mice (66% of studies). Furthermore, mostly ketamine/xylazine anesthetics, 27G needle constriction, and silk sutures are used. Nonetheless, despite the homogeneity in experimental design, the model contained a substantial degree of heterogeneity in the functional outcome measures. When looking at study quality, only 12% reported randomization, 23% mentioned any sort of blinding, 25% adequately addressed the outcomes, and an amazingly low percentage (2%) showed sample size calculation. Meta-analyses did not detect specific study characteristics that explained the heterogeneity in the reported outcome measures, however this might be related to the strong bias towards the use of specific mouse lines, sex as well as age or to poor reporting of characteristics of study quality.
Assuntos
Aorta , Insuficiência Cardíaca , Animais , Constrição , Ecocardiografia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
After myocardial infarction (MI), a strong inflammatory response takes place in the heart to remove the dead tissue resulting from ischemic injury. A growing body of evidence suggests that timely resolution of this inflammatory process may aid in the prevention of adverse cardiac remodeling and heart failure post-MI. The present challenge is to find a way to stimulate this process without interfering with the reparative role of the immune system. Extracellular vesicles (EVs) are natural membrane particles that are released by cells and carry different macromolecules, including proteins and non-coding RNAs. In recent years, EVs derived from various stem and progenitor cells have been demonstrated to possess regenerative properties. They can provide cardioprotection via several mechanisms of action, including immunomodulation. In this review, we summarize the role of the innate immune system in post-MI healing. We then discuss the mechanisms by which EVs modulate cardiac inflammation in preclinical models of myocardial injury through regulation of monocyte influx and macrophage function. Finally, we provide suggestions for further optimization of EV-based therapy to improve its potential for the treatment of MI.
Assuntos
Cardiotônicos/administração & dosagem , Vesículas Extracelulares/transplante , Inflamação/terapia , Infarto do Miocárdio/complicações , Células-Tronco/citologia , Animais , Humanos , Inflamação/etiologia , Inflamação/patologiaRESUMO
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.
Assuntos
Receptores de Lipopolissacarídeos/metabolismo , Monócitos/metabolismo , Placa Aterosclerótica/patologia , Receptores de IgG/metabolismo , Idoso , Feminino , Seguimentos , Humanos , Macrófagos/metabolismo , Masculino , FenótipoRESUMO
The chronic inflammatory response plays an important role in adverse cardiac remodelling and the development of heart failure (HF). There is also evidence that in the pathogenesis of several cardiovascular diseases, chronic inflammation is accompanied by antibody and complement deposits in the heart, suggestive of a true autoimmune response. However, the role of antibody-mediated immune responses in HF progression is less clear. We assessed whether immune cell infiltration and immunoglobulin levels are associated with HF type and disease stage, taking sex differences into account. We found IgG deposits and increased infiltration of immune cells in the affected myocardium of patients with end-stage HF with reduced ejection fraction (HFrEF, n = 20). Circulating levels of IgG1 and IgG3 were elevated in these patients. Furthermore, the percentage of transitional/regulatory B cells was decreased (from 6.9% to 2.4%) compared with healthy controls (n = 5). Similarly, increased levels of circulating IgG1 and IgG3 were observed in men with left ventricular diastolic dysfunction (LVDD, n = 5), possibly an early stage of HF with preserved EF (HFpEF). In conclusion, IgG deposits and infiltrates of immune cells are present in end-stage HFrEF. In addition, both LVDD patients and end-stage HFrEF patients show elevated levels of circulating IgG1 and IgG3, suggesting an antibody-mediated immune response upon cardiac remodelling, which in the early phase of remodelling appear to differ between men and women. These immunoglobulin subclasses might be used as marker for pre-stage HF and its progression. Future identification of auto-antigens might open possibilities for new therapeutic interventions.
Assuntos
Insuficiência Cardíaca/sangue , Insuficiência Cardíaca/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Miócitos Cardíacos/imunologia , Estudos de Casos e Controles , Progressão da Doença , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Miocárdio/imunologia , Volume Sistólico/imunologia , Disfunção Ventricular Esquerda/sangue , Disfunção Ventricular Esquerda/imunologiaRESUMO
AIMS: Myocardial infarction (MI) triggers an intense inflammatory response that is associated with infarct expansion and is detrimental for cardiac function. Interleukin (IL)-1ß and IL-18 are key players in this response and are controlled by the NLRP3-inflammasome. In the current study, we therefore hypothesized that selective inhibition of the NLRP3-inflammasome reduces infarct size and preserves cardiac function in a porcine MI model. METHODS AND RESULTS: Thirty female landrace pigs were subjected to 75 min transluminal balloon occlusion and treated with the NLRP3-inflammasome inhibitor MCC950 (6 or 3 mg/kg) or placebo for 7 days in a randomized, blinded fashion. After 7 days, 3D-echocardiography was performed to assess cardiac function and Evans blue/TTC double staining was executed to assess the area at risk (AAR) and infarct size (IS). The IS/AAR was lower in the 6 mg/kg group (64.6 ± 8.8%, P = 0.004) and 3 mg/kg group (69.7 ± 7.2%, P = 0.038) compared with the control group (77.5 ± 6.3%). MCC950 treatment markedly preserved left ventricular ejection fraction in treated animals (6 mg/kg 47 ± 8%, P = 0.001; 3 mg/kg 45 ± 7%, P = 0.031; control 37 ± 6%). Myocardial neutrophil influx was attenuated in treated compared with non-treated animals (6 mg/kg 132 ± 72 neutrophils/mm2, P = 0.035; 3 mg/kg 207 ± 210 neutrophils/mm2, P = 0.5; control 266 ± 158 neutrophils/mm2). Myocardial IL-1ß levels were dose-dependently reduced in treated animals. CONCLUSIONS: NLRP3-inflammasome inhibition reduces infarct size and preserves cardiac function in a randomized, blinded translational large animal MI model. Hence, NLRP3-inflammasome inhibition may have therapeutic potential in acute MI patients.
Assuntos
Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Infarto do Miocárdio/prevenção & controle , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sulfonas/farmacologia , Animais , Oclusão com Balão , Biomarcadores/metabolismo , Ecocardiografia/métodos , Feminino , Furanos , Hemodinâmica/fisiologia , Indenos , Inflamassomos/antagonistas & inibidores , Leucócitos Mononucleares/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Distribuição Aleatória , Sulfonamidas , SuínosRESUMO
OBJECTIVE: The genetically modified mouse is the most commonly used animal model for studying the pathogenesis of atherosclerotic disease. We aimed to assess if mice atherosclerosis-related genes could be validated in human disease through examination of results from genome-wide association studies. APPROACH AND RESULTS: We performed a systematic review to identify atherosclerosis-causing genes in mice and carried out gene-based association tests of their human orthologs for an association with human coronary artery disease and human large artery ischemic stroke. Moreover, we investigated the association of these genes with human atherosclerotic plaque characteristics. In addition, we assessed the presence of tissue-specific cis-acting expression quantitative trait loci for these genes in humans. Finally, using pathway analyses we show that the putative atherosclerosis-causing genes revealed few associations with human coronary artery disease, large artery ischemic stroke, or atherosclerotic plaque characteristics, despite the fact that the majority of these genes have cis-acting expression quantitative trait loci. CONCLUSIONS: A role for genes that has been observed in mice for atherosclerotic lesion development could scarcely be confirmed by studying associations of disease development with common human genetic variants. The value of murine atherosclerotic models for selection of therapeutic targets in human disease remains unclear.
Assuntos
Doença da Artéria Coronariana/genética , Perfilação da Expressão Gênica , Arteriosclerose Intracraniana/genética , Polimorfismo de Nucleotídeo Único , Acidente Vascular Cerebral/genética , Animais , Biologia Computacional , Doença da Artéria Coronariana/patologia , Bases de Dados Genéticas , Modelos Animais de Doenças , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Marcadores Genéticos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Arteriosclerose Intracraniana/patologia , Camundongos , Fenótipo , Placa Aterosclerótica , Locos de Características Quantitativas , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Risco , Especificidade da Espécie , Acidente Vascular Cerebral/patologiaRESUMO
Modulation of immune responses may form a powerful approach to treat atherosclerosis. It was shown that clearance of apoptotic cells results in tolerance induction to cleared Ags by dendritic cells (DCs); however, this seems impaired in atherosclerosis because Ag-specific tolerance is lacking. This could result, in part, from decreased emigration of DCs from atherosclerotic lesions because of the high-cholesterol environment. Nonetheless, local induction of anti-inflammatory responses by apoptotic cell clearance seems to dampen atherosclerosis, because inhibition of apoptotic cell clearance worsens atherosclerosis. In this study, we assessed whether i.v. administration of oxLDL-induced apoptotic DCs (apop(ox)-DCs) and, as a control, unpulsed apoptotic DCs could modulate atherosclerosis by inducing tolerance. Adoptive transfer of apop(ox)-DCs into low-density lipoprotein receptor knockout mice either before or during feeding of a Western-type diet resulted in increased numbers of CD103(+) tolerogenic splenic DCs, with a concomitant increase in regulatory T cells. Interestingly, both types of apoptotic DCs induced an immediate 40% decrease in Ly-6C(hi) monocyte numbers and a 50% decrease in circulating CCL2 levels, but only apop(ox)-DC treatment resulted in long-term effects on monocytes and CCL2 levels. Although initial lesion development was reduced by 40% in both treatment groups, only apop(ox)-DC treatment prevented lesion progression by 28%. Moreover, progressed lesions of apop(ox)-DC-treated mice showed a robust 45% increase in collagen content, indicating an enhanced stability of lesions. Our findings clearly show that apoptotic DC treatment significantly decreases lesion development, but only apop(ox)-DCs can positively modulate lesion progression and stability. These findings may translate into a safe treatment for patients with established cardiovascular diseases using patient-derived apop(ox)-DCs.
Assuntos
Aterosclerose/terapia , Células Dendríticas/imunologia , Imunoterapia Adotiva , Lipoproteínas LDL/farmacologia , Placa Aterosclerótica/terapia , Imunidade Adaptativa , Animais , Antígenos Ly/genética , Antígenos Ly/imunologia , Apoptose , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/imunologia , Colágeno/genética , Colágeno/imunologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/patologia , Células Dendríticas/transplante , Regulação da Expressão Gênica , Tolerância Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/patologia , Placa Aterosclerótica/genética , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/patologia , Cultura Primária de Células , Receptores de LDL , Transdução de Sinais , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/patologiaRESUMO
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.
Assuntos
Infarto do Miocárdio/cirurgia , Animais , Modelos Animais de Doenças , Ecocardiografia , Feminino , Coração/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica , Miocárdio/patologia , Recuperação de Função Fisiológica , SuínosRESUMO
AIMS: The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. METHODS AND RESULTS: Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4(+) cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr(-/-) mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. CONCLUSION: In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.
Assuntos
Aterosclerose/genética , Hemorragia/genética , Neutrófilos/metabolismo , Placa Aterosclerótica/genética , Receptores CXCR4/genética , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Medula Óssea , Adesão Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Progressão da Doença , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Feminino , Regulação da Expressão Gênica , Vetores Genéticos , Hemorragia/metabolismo , Hemorragia/patologia , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptores CXCR4/antagonistas & inibidores , Receptores CXCR4/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Transdução de SinaisRESUMO
Leukocyte chemotaxis is deemed instrumental in initiation and progression of atherosclerosis. It is mediated by G-protein-coupled receptors (e.g., CCR2 and CCR5), the activity of which is controlled by G-protein-coupled receptor kinases (GRKs). In this study, we analyzed the effect of hematopoietic deficiency of a potent regulator kinase of chemotaxis (GRK2) on atherogenesis. LDL receptor-deficient (LDLr(-/-)) mice with heterozygous hematopoietic GRK2 deficiency, generated by bone marrow transplantation (n=15), displayed a dramatic attenuation of plaque development, with 79% reduction in necrotic core and increased macrophage content. Circulating monocytes decreased and granulocytes increased in GRK2(+/-) chimeras, which could be attributed to diminished granulocyte colony-forming units in bone marrow. Collectively, these data pointed to myeloid cells as major mediators of the impaired atherogenic response in GRK2(+/-) chimeras. LDLr(-/-) mice with macrophage/granulocyte-specific GRK2 deficiency (LysM-Cre GRK2(flox/flox); n=8) failed to mimic the aforementioned phenotype, acquitting these cells as major responsible subsets for GRK2 deficiency-associated atheroprotection. To conclude, even partial hematopoietic GRK2 deficiency prevents atherosclerotic lesion progression beyond the fatty streak stage, identifying hematopoietic GRK2 as a potential target for intervention in atherosclerosis.
Assuntos
Aterosclerose/patologia , Quinase 2 de Receptor Acoplado a Proteína G/genética , Receptores de LDL/genética , Animais , Apoptose , Aterosclerose/genética , Feminino , Citometria de Fluxo , Camundongos , Camundongos Knockout , FagocitoseRESUMO
OBJECTIVE: Atherosclerosis is a chronic autoimmune-like disease in which lipids and fibrous elements accumulate in the arterial blood vessels. T cells are present within atherosclerotic plaques, and their activation is partially dependent on costimulatory signals, which can either provide positive or negative signals that promote T-cell activation or limit T-cell responses, respectively. T-cell immunoglobulin and mucin domain 3 (Tim-3) is a coinhibitory type 1 transmembrane protein that affects the function of several immune cells involved in atherosclerosis, such as monocytes, macrophages, effector T cells, and regulatory T cells. In the present study, we determined the role of Tim-3 in the development of atherosclerosis. APPROACH AND RESULTS: Western-type diet-fed low-density lipoprotein receptor-deficient (LDLr(-/-)) mice were treated with an anti-Tim-3 antibody for 3 and 8 weeks. Anti-Tim-3 administration increased fatty streak formation with 66% and increased atherosclerotic plaque formation after 8 weeks with 35% in the aortic root and with 50% in the aortic arch. Furthermore, blockade of Tim-3 signaling increased percentages of circulating monocytes with 33% and lesional macrophages with 20%. In addition, anti-Tim-3 administration increased CD4(+) T cells with 17%, enhanced their activation status, and reduced percentages of regulatory T cells with 18% and regulatory B cells with 37%. CONCLUSIONS: It is known that Tim-3 acts as a negative regulator of both innate and adaptive immune responses, and in the present study, we show that anti-Tim-3 treatment augments lesion development, accompanied by an increase in the number of monocytes/macrophages and CD4(+) T cells and by decreased regulatory T cells and regulatory B cells.
Assuntos
Aterosclerose/imunologia , Aterosclerose/metabolismo , Receptores Virais/imunologia , Linfócitos T Reguladores/imunologia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/farmacologia , Aorta/imunologia , Aorta/metabolismo , Apoptose/imunologia , Linfócitos B Reguladores/imunologia , Receptor Celular 2 do Vírus da Hepatite A , Macrófagos/metabolismo , Camundongos , Camundongos Knockout , Monócitos/imunologia , Placa Aterosclerótica/imunologia , Placa Aterosclerótica/metabolismo , Receptores Virais/genética , Receptores Virais/metabolismo , Transdução de Sinais/imunologiaRESUMO
OBJECTIVE: Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis. METHODS AND RESULTS: Irradiated low density lipoprotein receptor(-/-) mice, reconstituted with CCL3(-/-) or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3(-/-) chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3(-/-) neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1. CONCLUSIONS: Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.
Assuntos
Doenças das Artérias Carótidas/prevenção & controle , Artéria Carótida Primitiva/imunologia , Quimiocina CCL3/deficiência , Quimiotaxia de Leucócito , Leucócitos/imunologia , Infiltração de Neutrófilos , Neutrófilos/imunologia , Animais , Apoptose , Transplante de Medula Óssea , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/patologia , Artéria Carótida Primitiva/patologia , Adesão Celular , Células Cultivadas , Quimiocina CCL3/genética , Quimiocina CXCL1/metabolismo , Ciclofosfamida , Gorduras na Dieta , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Lipopolissacarídeos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutropenia/induzido quimicamente , Neutropenia/imunologia , Placa Aterosclerótica , RNA Mensageiro/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Fatores de Tempo , Irradiação Corporal TotalRESUMO
The aim was to optimize the perfusate composition by including a hemofiltrator to the PhysioHeartplatform for ex situ heart perfusion of porcine slaughterhouse hearts. Fourteen hearts were harvested from Dutch Landrace pigs and slaughtered for human consumption. All hearts were preserved for 4 hours using static cold storage before reperfusion for 4 hours on the PhysioHeart platform. Seven hearts were assigned to the hemofiltration group, where a hemofiltrator was added to the perfusion circuit, while the control group did not receive hemofiltration. In the hemofiltration group, the perfusion fluid was filtrated for 1 hour with a flow of 1 L/hour before reperfusion. After mounting the heart, hemofiltration was maintained at 1 L/hour, and cardiac function and blood samples were analyzed at multiple time points. Preserved cardiac function was defined as a cardiac output >3.0 L/min with a mean aortic pressure >60 mm Hg and a left atrial pressure <15 mm Hg. Hemofiltration resulted in a significantly reduced potassium concentration at all time points ( p < 0.001), while sodium levels remained at baseline values ( p < 0.004). Furthermore, creatinine and ammonia levels decreased over time. Functional assessment demonstrated a reduced left atrial pressure ( p < 0.04) and a reduction of the required dobutamine dose to support myocardial function ( p < 0.003) in the hemofiltration group. Preserved cardiac function did not differ between groups. Hemofiltration results in an improved biochemical composition of the whole blood perfusate and preserves cardiac function better during normothermic perfusion based on a reduced left atrial pressure (LAP) and dobutamine requirement to support function.
Assuntos
Transplante de Coração , Hemofiltração , Humanos , Suínos , Animais , Dobutamina , Coração , Perfusão/métodos , Miocárdio , Preservação de Órgãos/métodosRESUMO
The loss-of-function of the proprotein convertase subtilisin-kexin type 9 (Pcsk9) gene has been associated with significant reductions in plasma serum low-density lipoprotein cholesterol (LDL-C) levels. Both CRISPR/Cas9 and CRISPR-based editor-mediated Pcsk9 inactivation have successfully lowered plasma LDL-C and PCSK9 levels in preclinical models. Despite the promising preclinical results, these studies did not report how vehicle-mediated CRISPR delivery inactivating Pcsk9 affected low-density lipoprotein receptor recycling in vitro or ex vivo. Extracellular vesicles (EVs) have shown promise as a biocompatible delivery vehicle, and CRISPR/Cas9 ribonucleoprotein (RNP) has been demonstrated to mediate safe genome editing. Therefore, we investigated EV-mediated RNP targeting of the Pcsk9 gene ex vivo in primary mouse hepatocytes. We engineered EVs with the rapamycin-interacting heterodimer FK506-binding protein (FKBP12) to contain its binding partner, the T82L mutant FKBP12-rapamycin binding (FRB) domain, fused to the Cas9 protein. By integrating the vesicular stomatitis virus glycoprotein on the EV membrane, the engineered Cas9 EVs were used for intracellular CRISPR/Cas9 RNP delivery, achieving genome editing with an efficacy of ±28.1% in Cas9 stoplight reporter cells. Administration of Cas9 EVs in mouse hepatocytes successfully inactivated the Pcsk9 gene, leading to a reduction in Pcsk9 mRNA and increased uptake of the low-density lipoprotein receptor and LDL-C. These readouts can be used in future experiments to assess the efficacy of vehicle-mediated delivery of genome editing technologies targeting Pcsk9. The ex vivo data could be a step towards reducing animal testing and serve as a precursor to future in vivo studies for EV-mediated CRISPR/Cas9 RNP delivery targeting Pcsk9.
Assuntos
Vesículas Extracelulares , Animais , Camundongos , LDL-Colesterol , Sistemas CRISPR-Cas , Hepatócitos , Pró-Proteína Convertase 9/genética , Subtilisinas , Proteína 1A de Ligação a TacrolimoRESUMO
Objectives: Cold exposure is linked to cardiometabolic benefits. Cold activates brown adipose tissue (BAT), increases energy expenditure, and induces secretion of the hormones fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15). The cold-induced increase in energy expenditure exhibits a diurnal rhythm in men. Therefore, we aimed to investigate the effect of cold exposure on serum FGF21 and GDF15 levels in humans and whether cold-induced changes in FGF21 and GDF15 levels differ between morning and evening in males and females. Method: In this randomized cross-over study, serum FGF21 and GDF15 levels were measured in healthy lean males (n = 12) and females (n = 12) before, during, and after 90 min of stable cold exposure in the morning (07:45 h) and evening (19:45 h) with a 1-day washout period in between. Results: Cold exposure increased FGF21 levels in the evening compared to the morning both in males (+61% vs -13%; P < 0.001) and in females (+58% vs +8%; P < 0.001). In contrast, cold exposure did not significantly modify serum GDF15 levels, and no diurnal variation was found. Changes in FGF21 and GDF15 levels did not correlate with changes in cold-induced energy expenditure in the morning and evening. Conclusion: Cold exposure increased serum FGF21 levels in the evening, but not in the morning, in both males and females. GDF15 levels were not affected by cold exposure. Thus, this study suggests that the timing of cold exposure may influence cold-induced changes in FGF21 levels but not GDF15 levels and seems to be independent of changes in energy expenditure.
RESUMO
Introduction: The CC chemokine ligand 18 (CCL18) is a chemokine highly expressed in chronic inflammation in humans. Recent observations of elevated CCL18 plasma levels in patients with acute cardiovascular syndromes prompted an investigation into the role of CCL18 in the pathogenesis of human and mouse atherosclerosis. Methods and results: CCL18 was profoundly upregulated in ruptured human atherosclerotic plaque, particularly within macrophages. Repeated administration of CCL18 in Western-type diet-fed ApoE -/- mice or PCSK9mut-overexpressing wild type (WT) mice led to increased plaque burden, enriched in CD3+ T cells. In subsequent experimental and molecular modeling studies, we identified CCR6 as a functional receptor mediating CCL18 chemotaxis, intracellular Ca2+ flux, and downstream signaling in human Jurkat and mouse T cells. CCL18 failed to induce these effects in vitro in murine spleen T cells with CCR6 deficiency. The ability of CCR6 to act as CCL18 receptor was confirmed in vivo in an inflammation model, where subcutaneous CCL18 injection induced profound focal skin inflammation in WT but not in CCR6-/- mice. This inflammation featured edema and marked infiltration of various leukocyte subsets, including T cells with a Th17 signature, supporting CCR6's role as a Th17 chemotactic receptor. Notably, focal overexpression of CCL18 in plaques was associated with an increased presence of CCR6+ (T) cells. Discussion: Our studies are the first to identify the CCL18/CCR6 axis as a regulator of immune responses in advanced murine and human atherosclerosis.
Assuntos
Aterosclerose , Quimiocinas CC , Receptores CCR6 , Animais , Humanos , Aterosclerose/imunologia , Aterosclerose/metabolismo , Camundongos , Receptores CCR6/metabolismo , Receptores CCR6/genética , Quimiocinas CC/metabolismo , Quimiocinas CC/genética , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Células Jurkat , Placa Aterosclerótica/imunologia , Camundongos Knockout , Masculino , Linfócitos T/imunologia , Linfócitos T/metabolismo , Células Th17/imunologia , Células Th17/metabolismo , Feminino , Camundongos Knockout para ApoERESUMO
Lysophosphatidic acid (LPA), a bioactive lysophospholipid, accumulates in the atherosclerotic plaque. It has the capacity to activate mast cells, which potentially exacerbates plaque progression. In this study, we thus aimed to investigate whether LPA contributes to plaque destabilization by modulating mast cell function. We here show by an imaging mass spectrometry approach that several LPA species are present in atherosclerotic plaques. Subsequently, we demonstrate that LPA is a potent mast cell activator which, unlike other triggers, favors release of tryptase. Local perivascular administration of LPA to an atherosclerotic carotid artery segment increases the activation status of perivascular mast cells and promotes intraplaque hemorrhage and macrophage recruitment without impacting plaque cell apoptosis. The mast cell stabilizer cromolyn could prevent intraplaque hemorrhage elicited by LPA-mediated mast cell activation. Finally, the involvement of mast cells in these events was further emphasized by the lack of effect of perivascular LPA administration in mast cell deficient animals. We demonstrate that increased accumulation of LPA in plaques induces perivascular mast cell activation and in this way contributes to plaque destabilization in vivo. This study points to local LPA availability as an important factor in atherosclerotic plaque stability.