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BACKGROUND: Tissue resident memory T (TRM) cells are a T-cell subset that resides at the site of prior antigen recognition to protect the body against reoccurring encounters. Besides their protective function, TRM cells have also been implicated in inflammatory disorders. TRM cells are characterized by the expression of CD69 and transcription factors Hobit (homolog of Blimp-1 [B lymphocyte-induced maturation protein 1] in T cells) and Blimp-1. As the majority of T cells in the arterial intima expresses CD69, TRM cells may contribute to the pathogenesis of atherosclerosis as well. Here, we aimed to assess the presence and potential role of TRM cells in atherosclerosis. METHODS: To identify TRM cells in human atherosclerotic lesions, a single-cell RNA-sequencing data set was interrogated, and T-cell phenotypes were compared with that of integrated predefined TRM cells. The presence and phenotype of TRM in atherosclerotic lesions was corroborated using a mouse model that enabled tracking of Hobit-expressing TRM cells. To explore the function of TRM cells during atherogenesis, RAG1-/- (recombination activating gene 1 deficient) LDLr-/- (low-density lipoprotein receptor knockout) mice received a bone marrow transplant from HobitKO/CREBlimp-1flox/flox mice, which exhibit abrogated TRM cell formation, whereafter the mice were fed a Western-type diet for 10 weeks. RESULTS: Human atherosclerotic lesions contained T cells that exhibited a TRM cell-associated gene signature. Moreover, a fraction of these T cells clustered together with predefined TRM cells upon integration. The presence of Hobit-expressing TRM cells in the atherosclerotic lesion was confirmed in mice. These lesion-derived TRM cells were characterized by the expression of CD69 and CD49α. Moreover, we demonstrated that this small T-cell subset significantly affects lesion composition, by reducing the amount of intralesional macrophages and increasing collagen content. CONCLUSIONS: TRM cells, characterized by the expression of CD69 and CD49α, constitute a minor population in atherosclerotic lesions and are associated with increased lesion stability in a Hobit and Blimp-1 knockout mouse model.
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Aterosclerose , Modelos Animais de Doenças , Memória Imunológica , Macrófagos , Células T de Memória , Camundongos Endogâmicos C57BL , Placa Aterosclerótica , Receptores de LDL , Animais , Aterosclerose/patologia , Aterosclerose/imunologia , Aterosclerose/metabolismo , Aterosclerose/genética , Humanos , Células T de Memória/imunologia , Células T de Memória/metabolismo , Macrófagos/metabolismo , Macrófagos/imunologia , Macrófagos/patologia , Receptores de LDL/genética , Receptores de LDL/deficiência , Camundongos , Masculino , Camundongos Knockout , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Lectinas Tipo C/metabolismo , Lectinas Tipo C/genética , Fenótipo , Feminino , Antígenos CD/metabolismo , Antígenos CD/genética , Doenças da Aorta/patologia , Doenças da Aorta/imunologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismoRESUMO
INTRODUCTION: Viral infections have been associated with the progression of atherosclerosis and CD8+ T-cells directed against common viruses, such as influenza, Epstein-Barr virus, and cytomegalovirus, have been detected inside human atherosclerotic lesions. These virus-specific CD8+ T-cells have been hypothesized to contribute to the development of atherosclerosis; however, whether they affect disease progression directly remains unclear. In this study, we aimed to characterize the activation status of virus-specific CD8+ T-cells in the atherosclerotic lesion. METHODS: The presence, clonality, tissue enrichment, and phenotype of virus-associated CD8+ T-cells in atherosclerotic lesions were assessed by exploiting bulk T-cell receptor-ß sequencing and single-cell T-cell receptor (α and ß) sequencing datasets on human endarterectomy samples and patient-matched blood samples. To investigate if virus-specific CD8+ T-cells can be activated through T-cell receptor stimulation in the atherosclerotic lesion, the immunopeptidome of human plaques was determined. RESULTS: Virus-associated CD8+ T-cells accumulated more in the atherosclerotic lesion (mean=2.0%), compared with patient-matched blood samples (mean=1.4%; P=0.05), and were more clonally expanded and tissue enriched in the atherosclerotic lesion in comparison with nonassociated CD8+ T-cells from the lesion. Single-cell T-cell receptor sequencing and flow cytometry revealed that these virus-associated CD8+ T-cells were phenotypically highly similar to other CD8+ T-cells in the lesion and that both exhibited a more activated phenotype compared with circulating T-cells. Interestingly, virus-associated CD8+ T-cells are unlikely to be activated through antigen-specific interactions in the atherosclerotic lesion, as no virus-derived peptides were detected on HLA-I in the lesion. CONCLUSIONS: This study suggests that virus-specific CD8+ T-cells are tissue enriched in atherosclerotic lesions; however, their potential contribution to inflammation may involve antigen-independent mechanisms.
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Linfócitos T CD8-Positivos , Ativação Linfocitária , Placa Aterosclerótica , Humanos , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Aterosclerose/imunologia , Aterosclerose/virologia , Aterosclerose/patologia , Masculino , Fenótipo , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Feminino , Pessoa de Meia-Idade , Idoso , Doenças das Artérias Carótidas/imunologia , Doenças das Artérias Carótidas/virologia , Doenças das Artérias Carótidas/patologia , Interações Hospedeiro-PatógenoRESUMO
BACKGROUND: Atherosclerosis is the major underlying pathology of cardiovascular disease and is driven by dyslipidemia and inflammation. Inhibition of the immunoproteasome, a proteasome variant that is predominantly expressed by immune cells and plays an important role in antigen presentation, has been shown to have immunosuppressive effects. METHODS: We assessed the effect of ONX-0914, an inhibitor of the immunoproteasomal catalytic subunits LMP7 (proteasome subunit ß5i/large multifunctional peptidase 7) and LMP2 (proteasome subunit ß1i/large multifunctional peptidase 2), on atherosclerosis and metabolism in LDLr-/- and APOE*3-Leiden.CETP mice. RESULTS: ONX-0914 treatment significantly reduced atherosclerosis, reduced dendritic cell and macrophage levels and their activation, as well as the levels of antigen-experienced T cells during early plaque formation, and Th1 cells in advanced atherosclerosis in young and aged mice in various immune compartments. Additionally, ONX-0914 treatment led to a strong reduction in white adipose tissue mass and adipocyte progenitors, which coincided with neutrophil and macrophage accumulation in white adipose tissue. ONX-0914 reduced intestinal triglyceride uptake and gastric emptying, likely contributing to the reduction in white adipose tissue mass, as ONX-0914 did not increase energy expenditure or reduce total food intake. Concomitant with the reduction in white adipose tissue mass upon ONX-0914 treatment, we observed improvements in markers of metabolic syndrome, including lowered plasma triglyceride levels, insulin levels, and fasting blood glucose. CONCLUSIONS: We propose that immunoproteasomal inhibition reduces 3 major causes underlying cardiovascular disease, dyslipidemia, metabolic syndrome, and inflammation and is a new target in drug development for atherosclerosis treatment.
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Tecido Adiposo Branco , Aterosclerose , Modelos Animais de Doenças , Síndrome Metabólica , Camundongos Endogâmicos C57BL , Complexo de Endopeptidases do Proteassoma , Receptores de LDL , Animais , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Aterosclerose/tratamento farmacológico , Aterosclerose/imunologia , Aterosclerose/genética , Aterosclerose/metabolismo , Síndrome Metabólica/tratamento farmacológico , Síndrome Metabólica/imunologia , Tecido Adiposo Branco/metabolismo , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/patologia , Receptores de LDL/genética , Receptores de LDL/deficiência , Complexo de Endopeptidases do Proteassoma/metabolismo , Masculino , Inibidores de Proteassoma/farmacologia , Apolipoproteína E3/genética , Apolipoproteína E3/metabolismo , Doenças da Aorta/prevenção & controle , Doenças da Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/enzimologia , Doenças da Aorta/imunologia , Doenças da Aorta/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/imunologia , Placa Aterosclerótica , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Camundongos Knockout para ApoE , Camundongos , Metabolismo Energético/efeitos dos fármacos , OligopeptídeosRESUMO
BACKGROUND: Atherosclerosis, the main underlying pathology of cardiovascular disease, is a chronic inflammatory disease characterized by lipid accumulation and immune cell responses in the vascular wall, resulting in plaque formation. It is well-known that atherosclerosis prevalence and manifestation vary by sex. However, sexual dimorphism in the immune landscape of atherosclerotic plaques has up to date not been studied at high-resolution. In this study, we investigated sex-specific differences in atherosclerosis development and the immunological landscape of aortas at single-cell level in aged Ldlr-/- mice. METHODS: We compared plaque morphology between aged male and female chow diet-fed Ldlr-/- mice (22 months old) with histological analysis. Using single-cell RNA-sequencing and flow cytometry on CD45+ immune cells from aortas of aged Ldlr-/- mice, we explored the immune landscape in the atherosclerotic environment in males and females. RESULTS: We show that plaque volume is comparable in aged male and female mice, and that plaques in aged female mice contain more collagen and cholesterol crystals, but less necrotic core and macrophage content compared to males. We reveal increased immune cell infiltration in female aortas and found that expression of pro-atherogenic markers and inflammatory signaling pathways was enriched in plaque immune cells of female mice. Particularly, female aortas show enhanced activation of B cells (Egr1, Cd83, Cd180), including age-associated B cells, in addition to an increased M1/M2 macrophage ratio, where Il1b+ M1-like macrophages display a more pro-inflammatory phenotype (Nlrp3, Cxcl2, Mmp9) compared to males. In contrast, increased numbers of age-associated Gzmk+CD8+ T cells, dendritic cells, and Trem2+ macrophages were observed in male aortas. CONCLUSIONS: Altogether, our findings highlight that sex is a variable that contributes to immunological differences in the atherosclerotic plaque environment in mice and provide valuable insights for further preclinical studies into the impact of sex on the pathophysiology of atherosclerosis.
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RATIONALE: Atherosclerotic lesions are known for their cellular heterogeneity, yet the molecular complexity within the cells of human plaques has not been fully assessed. OBJECTIVE: Using single-cell transcriptomics and chromatin accessibility, we gained a better understanding of the pathophysiology underlying human atherosclerosis. METHODS AND RESULTS: We performed single-cell RNA and single-cell ATAC sequencing on human carotid atherosclerotic plaques to define the cells at play and determine their transcriptomic and epigenomic characteristics. We identified 14 distinct cell populations including endothelial cells, smooth muscle cells, mast cells, B cells, myeloid cells, and T cells and identified multiple cellular activation states and suggested cellular interconversions. Within the endothelial cell population, we defined subsets with angiogenic capacity plus clear signs of endothelial to mesenchymal transition. CD4+ and CD8+ T cells showed activation-based subclasses, each with a gradual decline from a cytotoxic to a more quiescent phenotype. Myeloid cells included 2 populations of proinflammatory macrophages showing IL (interleukin) 1B or TNF (tumor necrosis factor) expression as well as a foam cell-like population expressing TREM2 (triggering receptor expressed on myeloid cells 2) and displaying a fibrosis-promoting phenotype. ATACseq data identified specific transcription factors associated with the myeloid subpopulation and T cell cytokine profiles underlying mutual activation between both cell types. Finally, cardiovascular disease susceptibility genes identified using public genome-wide association studies data were particularly enriched in lesional macrophages, endothelial, and smooth muscle cells. CONCLUSIONS: This study provides a transcriptome-based cellular landscape of human atherosclerotic plaques and highlights cellular plasticity and intercellular communication at the site of disease. This detailed definition of cell communities at play in atherosclerosis will facilitate cell-based mapping of novel interventional targets with direct functional relevance for the treatment of human disease.
Assuntos
Doenças das Artérias Carótidas/genética , Células Endoteliais/metabolismo , Perfilação da Expressão Gênica , Linfócitos/metabolismo , Células Mieloides/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Análise de Célula Única , Transcriptoma , Idoso , Idoso de 80 Anos ou mais , Animais , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Transdiferenciação Celular , Sequenciamento de Cromatina por Imunoprecipitação , Bases de Dados Genéticas , Células Endoteliais/patologia , Feminino , Estudo de Associação Genômica Ampla , Humanos , Linfócitos/patologia , Masculino , Camundongos , Pessoa de Meia-Idade , Células Mieloides/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , RNA-SeqRESUMO
OBJECTIVE: Mast cells (MCs) are important contributors to atherosclerotic plaque progression. For prospective studies on mast cell contributions to plaque instability, the distribution of intraplaque MCs needs to be elucidated. Plaque stability is generally histologically assessed by dividing the plaque specimen into segments to be scored on an ordinal scale. However, owing to competitive use, studies may have to deviate to adjacent segments, yet intersegment differences of plaque characteristics, especially MCs, are largely unknown. Therefore, the hypothesis that there is no segment to segment difference in MC distribution between atherosclerotic plaque segments was tested, and intersegment associations between MCs and other plaque characteristics was investigated. METHODS: Twenty-six carotid atherosclerotic plaques from patients undergoing carotid endarterectomy included in the Athero-Express Biobank were analysed. The plaque was divided in 5 mm segments, differentiating between the culprit lesion (segment 0), adjacent segments (-1/+1) and more distant segments (-2/+2) for the presence of MCs. The associations between the intersegment distribution of MCs and smooth muscle cells, macrophage content, and microvessel density in the culprit lesion were studied. RESULTS: A statistically significant difference in MCs/mm2 between the different plaque segments (p < .001) was found, with a median of 2.79 (interquartile range [IQR] 1.63 - 7.10) for the culprit lesion, 1.34 (IQR 0.26 - 4.45) for the adjacent segment, and 0.62 (0.14 - 2.07) for the more distant segment. Post hoc analyses showed that intersegment differences were due to differences in MCs/mm2 between the culprit and adjacent segment (p = .037) and between the culprit lesion and the more distant segment (p < .001). MCs/mm2 in multiple different segments were positively correlated with microvessel density and macrophage content in the culprit lesion. CONCLUSION: MC numbers reveal significant intersegment differences in human carotid plaques. Future histological studies on MCs should use a standardised segment for plaque characterisation as plaque segments cannot be used interchangeably for histological MC analyses.
Assuntos
Estenose das Carótidas/patologia , Mastócitos/fisiologia , Placa Aterosclerótica/patologia , Idoso , Estenose das Carótidas/complicações , Estenose das Carótidas/cirurgia , Proliferação de Células , Estudos de Coortes , Endarterectomia das Carótidas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Países Baixos , Placa Aterosclerótica/complicações , Placa Aterosclerótica/cirurgiaRESUMO
Mast cells (MCs) are potent innate immune cells that aggravate atherosclerosis through the release of proinflammatory mediators inside atherosclerotic plaques. Similarly, CD4+ T cells are constituents of the adaptive immune response and accumulate within the plaques following lipid-specific activation by APCs. Recently it has been proposed that these two cell types can interact in a direct manner. However, no indication of such an interaction has been investigated in the context of atherosclerosis. In our study, we aimed to examine whether MCs can act as APCs in atherosclerosis, thereby modulating CD4+ T cell responses. We observed that MCs increased their MHC class II expression under hyperlipidemic conditions both in vivo and in vitro. Furthermore, we showed that MCs can present Ags in vivo via MHC class II molecules. Serum from high-fat diet-fed mice also enhanced the expression of the costimulatory molecule CD86 on cultured MCs, whereas OVA peptide-loaded MCs increased OT-II CD4+ T cell proliferation in vitro. The aortic CD4+ and TH1 cell content of atherosclerotic mice that lack MCs was reduced as compared with their wild-type counterparts. Importantly, we identified MCs that express HLA-DR in advanced human atheromata, indicating that these cells are capable of Ag presentation within human atherosclerotic plaques. Therefore, in this artice, we show that MCs may directly modulate adaptive immunity by acting as APCs in atherosclerosis.
Assuntos
Aterosclerose/imunologia , Linfócitos T CD4-Positivos/imunologia , Hipercolesterolemia/imunologia , Mastócitos/imunologia , Animais , Células Cultivadas , Humanos , Masculino , Camundongos , Camundongos KnockoutRESUMO
RATIONALE: Deficiency of secreted IgM (sIgM-/-) accelerates atherosclerosis in Ldlr-/-mice. Several atheroprotective effects of increased levels of IgM antibodies have been suggested, including preventing inflammation induced by oxidized low-density lipoprotein and promoting apoptotic cell clearance. However, the mechanisms by which the lack of sIgM promotes lesion formation remain unknown. OBJECTIVE: To identify the mechanisms by which sIgM deficiency accelerates atherosclerosis in mice. METHODS AND RESULTS: We here show that both sIgM-/- and Ldlr-/-sIgM-/- mice develop increased plasma IgE titers because of impaired generation of B cells expressing the low-affinity IgE receptor CD23, which mediates the clearance of IgE antibodies. We further report that Ldlr-/-sIgM-/- mice exhibit increased numbers of activated mast cells and neutrophils in the perivascular area of atherosclerotic plaques. Treatment with an anti-IgE-neutralizing antibody fully reversed vascular inflammation and accelerated atherosclerotic lesion formation in cholesterol-fed Ldlr-/-sIgM-/- mice. CONCLUSIONS: Thus, our data identify a previously unsuspected mechanism by which sIgM deficiency aggravates atherosclerosis.
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Aterosclerose/sangue , Aterosclerose/patologia , Imunoglobulina E/sangue , Imunoglobulina M/deficiência , Animais , Biomarcadores/sangue , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Mast cells are pluripotent leukocytes that reside in the mucosa and connective tissue. Recent studies show an increased prevalence of cardiovascular disease among patients with mastocytosis, which is a hematological disease that is characterized by the accumulation of mast cells due to clonal proliferation. This association suggests an important role for mast cells in cardiovascular disease. Indeed, the evidence establishing the contribution of mast cells to the development and progression of atherosclerosis is continually increasing. Mast cells may contribute to plaque formation by stimulating the formation of foam cells and causing a pro-inflammatory micro-environment. In addition, these cells are able to promote plaque instability by neo-vessel formation and also by inducing intraplaque hemorrhage. Furthermore, mast cells appear to stimulate the formation of fibrosis after a cardiac infarction. In this review, the available data on the role of mast cells in cardiovascular disease are summarized, containing both in vitro research and animal studies, followed by a discussion of human data on the association between cardiovascular morbidity and diseases in which mast cells are important: Kounis syndrome, mastocytosis and allergy.
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Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Animais , Doenças Cardiovasculares/diagnóstico , Comorbidade , Suscetibilidade a Doenças , Humanos , Hipersensibilidade/complicações , Morbidade , Risco , Pesquisa Translacional BiomédicaRESUMO
OBJECTIVE: Inflammasomes are multiprotein complexes, and their activation has been associated with cardiovascular disease. Inflammasome activation leads to secretion of caspase-1 by innate immune cells, resulting in the activation of interleukin-1ß. Recently, a potent and selective inhibitor of the NLRP3 inflammasome, MCC950, was described. In this study, we investigated the effect of MCC950 on atherosclerotic lesion development in apoE-/- mice. APPROACH AND RESULTS: First, we determined the efficacy of MCC950 in vitro. Bone marrow-derived macrophages and dendritic cells were stimulated with lipopolysaccharide and cholesterol crystals resulting in high levels of interleukin-1ß release, which was inhibited by MCC950. In vivo MCC950 treatment reduced lipopolysaccharide-induced interleukin-1ß secretion, without affecting the tumor necrosis factor-α response. Subsequently, atherosclerotic plaques were induced in Western-type diet fed apoE-/- mice by semiconstrictive perivascular collar placement at the carotid arteries, after which the mice received MCC950 (10 mg/kg) or vehicle control 3× per week intraperitoneally for 4 weeks. After euthanize, atherosclerotic plaque size and volume were quantified in hematoxylin-eosin-stained 10-µm cryosections throughout the artery. MCC950 treatment significantly reduced the development of atherosclerotic lesions as determined by maximal stenosis, average plaque size, and plaque volume. Although the amount of collagen and the necrotic core size were not affected, the number of macrophages in the plaque was significantly reduced on treatment. In addition, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) mRNA expression was significantly reduced in the carotids of MCC950-treated mice. CONCLUSIONS: These findings show that specific inhibition of the NLRP3 inflammasome using MCC950 can be a promising therapeutic approach to inhibit atherosclerotic lesion development.
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Anti-Inflamatórios/farmacologia , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Artérias Carótidas/efeitos dos fármacos , Doenças das Artérias Carótidas/prevenção & controle , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Sulfonas/farmacologia , Animais , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Artérias Carótidas/metabolismo , Artérias Carótidas/patologia , Doenças das Artérias Carótidas/genética , Doenças das Artérias Carótidas/metabolismo , Doenças das Artérias Carótidas/patologia , Células Cultivadas , Colesterol/farmacologia , Cristalização , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Dieta Ocidental , Modelos Animais de Doenças , Feminino , Furanos , Predisposição Genética para Doença , Indenos , Inflamassomos/metabolismo , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Necrose , Fenótipo , Placa Aterosclerótica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfonamidas , Fatores de Tempo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismoAssuntos
Diabetes Mellitus , Mastócitos , Animais , Contagem de Células , Camundongos , Camundongos Obesos , MicrovasosRESUMO
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.
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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
BACKGROUND: Leukocyte migration is critical for the infiltration of monocytes and accumulation of monocyte-derived macrophages in inflammation. Considering that Hck and Fgr are instrumental in this process, their impact on atherosclerosis and on lesion inflammation and stability was evaluated. METHODS AND RESULTS: Hematopoietic Hck/Fgr-deficient, LDLr(-/-) chimeras, obtained by bone marrow transplantation, had smaller but, paradoxically, less stable lesions with reduced macrophage content, overt cap thinning, and necrotic core expansion as the most prominent features. Despite a Ly6C(high)-skewed proinflammatory monocyte phenotype, Hck/Fgr deficiency led to disrupted adhesion of myeloid cells to and transmigration across endothelial monolayers in vitro and atherosclerotic plaques in vivo, as assessed by intravital microscopy, flow cytometry, and histological examination of atherosclerotic arteries. Moreover, Hck/Fgr-deficient macrophages showed blunted podosome formation and mesenchymal migration capacity. In consequence, transmigrated double-knockout macrophages were seen to accumulate in the fibrous cap, potentially promoting its focal erosion, as observed for double-knockout chimeras. CONCLUSIONS: The hematopoietic deficiency of Hck and Fgr led to attenuated atherosclerotic plaque formation by abrogating endothelial adhesion and transmigration; paradoxically, it also promoted plaque instability by causing monocyte subset imbalance and subendothelial accumulation, raising a note of caution regarding src kinase-targeted intervention in plaque inflammation.
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Quimiotaxia de Leucócito/fisiologia , Macrófagos Peritoneais/patologia , Monócitos/patologia , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-hck/deficiência , Proteínas Proto-Oncogênicas/deficiência , Quinases da Família src/deficiência , Animais , Apoptose , Adesão Celular , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Células Endoteliais , Proteínas da Matriz Extracelular/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Migração e Rolagem de Leucócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Placa Aterosclerótica/enzimologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-hck/genética , Proteínas Proto-Oncogênicas c-hck/fisiologia , Quimera por Radiação , Receptores de LDL/deficiência , Receptores de LDL/genética , Receptores de LDL/fisiologia , Migração Transendotelial e Transepitelial , Quinases da Família src/genética , Quinases da Família src/fisiologiaRESUMO
The mast cell is a potent immune cell known for its functions in host defense responses and diseases, such as asthma and allergies. In the past years, accumulating evidence established the contribution of the mast cell to cardiovascular diseases as well, in particular, by its effects on atherosclerotic plaque progression and destabilization. Through its release not only of mediators, such as the mast cell-specific proteases chymase and tryptase, but also of growth factors, histamine, and chemokines, activated mast cells can have detrimental effects on its immediate surroundings in the vessel wall. This results in matrix degradation, apoptosis, and enhanced recruitment of inflammatory cells, thereby actively contributing to cardiovascular diseases. In this review, we will discuss the current knowledge on mast cell function in cardiovascular diseases and speculate on potential novel therapeutic strategies to prevent acute cardiovascular syndromes via targeting of mast cells.
Assuntos
Aterosclerose/imunologia , Inflamação/imunologia , Mastócitos/imunologia , Animais , Anti-Inflamatórios/uso terapêutico , Aterosclerose/tratamento farmacológico , Aterosclerose/metabolismo , Aterosclerose/patologia , Desenho de Fármacos , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Inflamação/patologia , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Mastócitos/patologia , Terapia de Alvo Molecular , Transdução de SinaisRESUMO
OBJECTIVE: Atherosclerosis, the cause of 50% of deaths in westernized societies, is widely regarded as a chronic vascular inflammatory disease. Vascular smooth muscle cell (VSMC) inflammatory activation in response to local proinflammatory stimuli contributes to disease progression and is a pervasive feature in developing atherosclerotic plaques. Therefore, it is of considerable therapeutic importance to identify mechanisms that regulate the VSMC inflammatory response. APPROACH AND RESULTS: We report that myocardin, a powerful myogenic transcriptional coactivator, negatively regulates VSMC inflammatory activation and vascular disease. Myocardin levels are reduced during atherosclerosis, in association with phenotypic switching of smooth muscle cells. Myocardin deficiency accelerates atherogenesis in hypercholesterolemic apolipoprotein E(-/-) mice. Conversely, increased myocardin expression potently abrogates the induction of an array of inflammatory cytokines, chemokines, and adhesion molecules in VSMCs. Expression of myocardin in VSMCs reduces lipid uptake, macrophage interaction, chemotaxis, and macrophage-endothelial tethering in vitro, and attenuates monocyte accumulation within developing lesions in vivo. These results demonstrate that endogenous levels of myocardin are a critical regulator of vessel inflammation. CONCLUSIONS: We propose myocardin as a guardian of the contractile, noninflammatory VSMC phenotype, with loss of myocardin representing a critical permissive step in the process of phenotypic transition and inflammatory activation, at the onset of vascular disease.
Assuntos
Aterosclerose/metabolismo , Lesões das Artérias Carótidas/metabolismo , Inflamação/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas Nucleares/metabolismo , Transativadores/metabolismo , Animais , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/imunologia , Aterosclerose/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/imunologia , Lesões das Artérias Carótidas/patologia , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Células Cultivadas , Quimiocinas/metabolismo , Quimiotaxia , Citocinas/metabolismo , Modelos Animais de Doenças , Genótipo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Metabolismo dos Lipídeos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/imunologia , Monócitos/metabolismo , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/patologia , Neointima , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Fenótipo , Interferência de RNA , Ratos Wistar , Fatores de Tempo , Transativadores/deficiência , Transativadores/genética , TransfecçãoRESUMO
OBJECTIVES: Unstable atherosclerotic lesions in carotid arteries require surgical endarterectomy to reduce the risk of ischemic stroke. We aimed to identify microRNAs that exert a broad effect on atherosclerotic plaque formation and stability in the carotid artery. BACKGROUND: We made a selection of 164 genes involved in atherosclerosis. Using www.targetscan.org, we determined which microRNAs potentially regulate expression of these genes. We identified multiple microRNAs from the 14q32 microRNA cluster, which is highly involved in vascular remodeling. In human plaques, collected during carotid endarterectomy surgery, we found that 14q32 microRNA (miR-494) was abundantly expressed in unstable lesions. METHODS: We induced atherosclerotic plaque formation in hypercholesterolemic ApoE mice by placing semiconstrictive collars around both carotid arteries. We injected "Gene Silencing Oligonucleotides" against miR-494 (GSO-494) or negative control (GSO-control). Using fluorescently labeled GSOs, we confirmed uptake of GSOs in affected areas of the carotids, but not elsewhere in the vasculature. RESULTS: After injection of GSO-494, we observed significant downregulation of miR-494 expression in the carotid arteries, although miR-494 target genes were upregulated. Further analyses revealed a 65% decrease in plaque size after GSO-494 treatment. Plaque stability was increased in GSO-494-treated mice, determined by an 80% decrease in necrotic core size and a 50% increase in plaque collagen content. Inhibition of miR-494 also resulted in decreased cholesterol levels and decreased very low-density lipoprotein (VLDL) fractions. CONCLUSIONS: Treatment with GSO-494 results in smaller atherosclerotic lesions with increased plaque stability. Inhibition of miR-494 may decrease the risk of surgical complications or even avert endarterectomy surgery in some cases.
Assuntos
Aterosclerose/genética , DNA/genética , Regulação da Expressão Gênica , MicroRNAs/genética , Placa Aterosclerótica/genética , Animais , Aterosclerose/metabolismo , Western Blotting , Artérias Carótidas , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , MicroRNAs/biossíntese , Placa Aterosclerótica/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cardiovascular disease (CVD) remains a leading cause of death worldwide and identification and therapeutic modulation of all its risk factors is necessary to ensure a lower burden on the patient and on society. The physiological response to acute and chronic stress exposure has long been recognized as a potent modulator of immune, endocrine and metabolic pathways, however its direct implications for cardiovascular disease development, progression and as a therapeutic target are not completely understood. More and more attention is given to the bidirectional interaction between psychological and physical health in relation to cardiovascular disease. With atherosclerosis being a chronic disease starting already at an early age the contribution of adverse early life events in affecting adult health risk behavior, health status and disease development is receiving increased attention. In addition, experimental research into the biological pathways involved in stress-induced cardiovascular complications show important roles for metabolic and immunologic maladaptation, resulting in increased disease development and progression. Here we provide a concise overview of human and experimental animal data linking chronic and acute stress to CVD risk and increased progression of the underlying disease atherosclerosis.
Assuntos
Aterosclerose/etiologia , Doenças Cardiovasculares/etiologia , Estresse Psicológico/complicações , Animais , Aterosclerose/imunologia , Aterosclerose/fisiopatologia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/fisiopatologia , Doença Crônica , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Ratos , Fatores de Risco , Estresse Psicológico/imunologia , Estresse Psicológico/fisiopatologiaRESUMO
RATIONALE: RNA-binding proteins are critical post-transcriptional regulators of RNA and can influence pre-mRNA splicing, RNA localization, and stability. The RNA-binding protein Quaking (QKI) is essential for embryonic blood vessel development. However, the role of QKI in the adult vasculature, and in particular in vascular smooth muscle cells (VSMCs), is currently unknown. OBJECTIVE: We sought to determine the role of QKI in regulating adult VSMC function and plasticity. METHODS AND RESULTS: We identified that QKI is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Quaking viable mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering noncontractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome, where it interacts with the myocardin pre-mRNA and regulates the splicing of alternative exon 2a. This post-transcriptional event impacts the Myocd_v3/Myocd_v1 mRNA balance and can be modulated by mutating the quaking response element in exon 2a of myocardin. Furthermore, we identified that arterial damage triggers myocardin alternative splicing and is tightly coupled with changes in the expression levels of distinct QKI isoforms. CONCLUSIONS: We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.
Assuntos
Proliferação de Células , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Lesões das Artérias Carótidas/metabolismo , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Movimento Celular , Reestenose Coronária/metabolismo , Reestenose Coronária/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Modelos Animais de Doenças , Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica , Células HEK293 , Humanos , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Quaking , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Neointima , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Interferência de RNA , Proteínas de Ligação a RNA/genética , Transativadores/genética , Transativadores/metabolismo , TransfecçãoRESUMO
Patients suffering from cardiovascular disease have well-established atherosclerotic lesions, rendering lesion regression of therapeutic interest. The OX40 (TNFRSF4)-OX40 ligand (OX40L; TNFSF4) pathway is important for the proliferation and survival of T cells, stimulates B cells, and is associated with cardiovascular disease. We hypothesized that interference with the OX40-OX40L pathway, in combination with decreases in cholesterol, may induce regression of atherosclerosis. LDLr(-/-) mice were fed a Western-type diet for 10 wk, after which they received chow diet and were treated with anti-OX40L or PBS for 10 wk. A significant regression of lesions was observed in the aorta and aortic arch of anti-OX40L-treated mice compared with control mice. Interference of the OX40-OX40L pathway reduced Th2 responses, as shown by decreases in GATA-3 and IL-4 levels. Also, IgE levels were decreased, as demonstrated by reduced mast cell presence and activation. Notably, IL-5 production by T and B1 cells was increased, thus enhancing atheroprotective oxidized low-density lipoprotein-specific IgM production. The increase in IL-5 production and IgM was mediated by IL-33 production by APCs upon OX40L blockade. We conclude that interruption of the OX40-OX40L signaling pathway, combined with decreases in dietary cholesterol, induces the regression of atherosclerosis through induction of IL-5-producing T cells and oxidized low-density lipoprotein-specific IgM and reductions in Th2 and mast cells.
Assuntos
Aterosclerose/metabolismo , Colesterol/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de LDL/deficiência , Receptores OX40/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Células Apresentadoras de Antígenos/metabolismo , Proliferação de Células , Células Cultivadas , Fator de Transcrição GATA3/metabolismo , Imunoglobulina E/sangue , Imunoglobulina M/imunologia , Interleucina-33 , Interleucina-4/metabolismo , Interleucina-5/biossíntese , Interleucinas/biossíntese , Ativação Linfocitária/imunologia , Masculino , Mastócitos/imunologia , Glicoproteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligante OX40 , Receptores de LDL/genética , Células Th2/imunologia , Fatores de Necrose Tumoral/imunologiaRESUMO
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.