RESUMO
BACKGROUND: While our understanding of the single-cell gene expression patterns underlying the transformation of vascular cell types during the progression of atherosclerosis is rapidly improving, the clinical and pathophysiological relevance of these changes remains poorly understood. METHODS: Single-cell RNA sequencing data generated with SmartSeq2 (≈8000 genes/cell) in 16â 588 single cells isolated during atherosclerosis progression in Ldlr-/-Apob100/100 mice with human-like plasma lipoproteins and from humans with asymptomatic and symptomatic carotid plaques was clustered into multiple subtypes. For clinical and pathophysiological context, the advanced-stage and symptomatic subtype clusters were integrated with 135 tissue-specific (atherosclerotic aortic wall, mammary artery, liver, skeletal muscle, and visceral and subcutaneous, fat) gene-regulatory networks (GRNs) inferred from 600 coronary artery disease patients in the STARNET (Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task) study. RESULTS: Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by 3 smooth muscle cells (SMCs), and 3 macrophage subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type proinflammatory/Trem2-high lipid-associated (macrophage) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of 3 arterial wall GRNs: GRN33 (macrophage), GRN39 (SMC), and GRN122 (macrophage) with major contributions to coronary artery disease heritability and strong associations with clinical scores of coronary atherosclerosis severity. The presence and pathophysiological relevance of GRN39 were verified in 5 independent RNAseq data sets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM in cultured human coronary artery SMCs. CONCLUSIONS: By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a coronary artery disease framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced, symptomatic atherosclerosis.
Assuntos
Aterosclerose , Redes Reguladoras de Genes , Análise de Célula Única , Humanos , Animais , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Camundongos , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Masculino , Placa Aterosclerótica , Progressão da Doença , Feminino , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos Knockout , Receptores de LDL/genética , Receptores de LDL/metabolismo , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologiaRESUMO
BACKGROUND: Thromboembolic events, including myocardial infarction (MI) or stroke, caused by the rupture or erosion of unstable atherosclerotic plaques are the leading cause of death worldwide. Although most mouse models of atherosclerosis develop lesions in the aorta and carotid arteries, they do not develop advanced coronary artery lesions. Moreover, they do not undergo spontaneous plaque rupture with MI and stroke or do so at such a low frequency that they are not viable experimental models to study late-stage thrombotic events or to identify novel therapeutic approaches for treating atherosclerotic disease. This has stymied the development of more effective therapeutic approaches for reducing these events beyond what has been achieved with aggressive lipid lowering. Here, we describe a diet-inducible mouse model that develops widespread advanced atherosclerosis in coronary, brachiocephalic, and carotid arteries with plaque rupture, MI, and stroke. METHODS: We characterized a novel mouse model with a C-terminal mutation in the scavenger receptor class B, type 1 (SR-BI), combined with Ldlr knockout (designated SR-BI∆CT/∆CT/Ldlr-/-). Mice were fed Western diet (WD) for 26 weeks and analyzed for MI and stroke. Coronary, brachiocephalic, and carotid arteries were analyzed for atherosclerotic lesions and indices of plaque stability. To validate the utility of this model, SR-BI∆CT/∆CT/Ldlr-/- mice were treated with the drug candidate AZM198, which inhibits myeloperoxidase, an enzyme produced by activated neutrophils that predicts rupture of human atherosclerotic lesions. RESULTS: SR-BI∆CT/∆CT/Ldlr-/- mice show high (>80%) mortality rates after 26 weeks of WD feeding because of major adverse cardiovascular events, including spontaneous plaque rupture with MI and stroke. Moreover, WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice displayed elevated circulating high-sensitivity cardiac troponin I and increased neutrophil extracellular trap formation within lesions compared with control mice. Treatment of WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice with AZM198 showed remarkable benefits, including >90% improvement in survival and >60% decrease in the incidence of plaque rupture, MI, and stroke, in conjunction with decreased circulating high-sensitivity cardiac troponin I and reduced neutrophil extracellular trap formation within lesions. CONCLUSIONS: WD-fed SR-BI∆CT/∆CT/Ldlr-/- mice more closely replicate late-stage clinical events of advanced human atherosclerotic disease than previous models and can be used to identify and test potential new therapeutic agents to prevent major adverse cardiac events.
Assuntos
Modelos Animais de Doenças , Camundongos Knockout , Infarto do Miocárdio , Peroxidase , Placa Aterosclerótica , Acidente Vascular Cerebral , Animais , Infarto do Miocárdio/patologia , Infarto do Miocárdio/tratamento farmacológico , Placa Aterosclerótica/tratamento farmacológico , Camundongos , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/prevenção & controle , Peroxidase/metabolismo , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Receptores de LDL/genética , Receptores de LDL/deficiência , Camundongos Endogâmicos C57BL , Masculino , Ruptura Espontânea , Dieta Ocidental/efeitos adversos , Inibidores Enzimáticos/uso terapêutico , Inibidores Enzimáticos/farmacologiaRESUMO
BACKGROUND: Thromboembolic events secondary to rupture or erosion of advanced atherosclerotic lesions is the global leading cause of death. The most common and effective means to reduce these major adverse cardiovascular events, including myocardial infarction and stroke, is aggressive lipid lowering via a combination of drugs and dietary modifications. However, we know little regarding the effects of reducing dietary lipids on the composition and stability of advanced atherosclerotic lesions, the mechanisms that regulate these processes, and what therapeutic approaches might augment the benefits of lipid lowering. METHODS: Smooth muscle cell lineage-tracing Apoe-/- mice were fed a high-cholesterol Western diet for 18 weeks and then a zero-cholesterol standard laboratory diet for 12 weeks before treating them with an IL (interleukin)-1ß or control antibody for 8 weeks. We assessed lesion size and remodeling indices, as well as the cellular composition of aortic and brachiocephalic artery lesions, indices of plaque stability, overall plaque burden, and phenotypic transitions of smooth muscle cell and other lesion cells by smooth muscle cell lineage tracing combined with single-cell RNA sequencing, cytometry by time-of-flight, and immunostaining plus high-resolution confocal microscopic z-stack analysis. RESULTS: Lipid lowering by switching Apoe-/- mice from a Western diet to a standard laboratory diet reduced LDL cholesterol levels by 70% and resulted in multiple beneficial effects including reduced overall aortic plaque burden, as well as reduced intraplaque hemorrhage and necrotic core area. However, contrary to expectations, IL-1ß antibody treatment after diet-induced reductions in lipids resulted in multiple detrimental changes including increased plaque burden and brachiocephalic artery lesion size, as well as increasedintraplaque hemorrhage, necrotic core area, and senescence as compared with IgG control antibody-treated mice. Furthermore, IL-1ß antibody treatment upregulated neutrophil degranulation pathways but downregulated smooth muscle cell extracellular matrix pathways likely important for the protective fibrous cap. CONCLUSIONS: Taken together, IL-1ß appears to be required for the maintenance of standard laboratory diet-induced reductions in plaque burden and increases in multiple indices of plaque stability.
Assuntos
Aterosclerose , Modelos Animais de Doenças , Interleucina-1beta , Camundongos Knockout para ApoE , Miócitos de Músculo Liso , Placa Aterosclerótica , Animais , Interleucina-1beta/metabolismo , Aterosclerose/patologia , Aterosclerose/prevenção & controle , Aterosclerose/metabolismo , Aterosclerose/genética , Camundongos , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/efeitos dos fármacos , Masculino , Dieta Ocidental , Camundongos Endogâmicos C57BL , Aorta/patologia , Aorta/metabolismo , Aorta/efeitos dos fármacos , Doenças da Aorta/patologia , Doenças da Aorta/prevenção & controle , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Dieta Hiperlipídica , Músculo Liso Vascular/patologia , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Tronco Braquiocefálico/patologia , Tronco Braquiocefálico/metabolismo , Tronco Braquiocefálico/efeitos dos fármacosRESUMO
BACKGROUND: Smooth muscle cells (SMCs), which make up the medial layer of arteries, are key cell types involved in cardiovascular disease, the leading cause of mortality and morbidity worldwide. In response to microenvironment alterations, SMCs dedifferentiate from a contractile to a synthetic phenotype characterized by an increased proliferation, migration, production of ECM (extracellular matrix) components, and decreased expression of SMC-specific contractile markers. These phenotypic changes result in vascular remodeling and contribute to the pathogenesis of cardiovascular disease, including coronary artery disease, stroke, hypertension, and aortic aneurysms. Here, we aim to identify the genetic variants that regulate ECM secretion in SMCs and predict the causal proteins associated with vascular disease-related loci identified in genome-wide association studies. METHODS: Using human aortic SMCs from 123 multiancestry healthy heart transplant donors, we collected the serum-free media in which the cells were cultured for 24 hours and conducted liquid chromatography-tandem mass spectrometry-based proteomic analysis of the conditioned media. RESULTS: We measured the abundance of 270 ECM and related proteins. Next, we performed protein quantitative trait locus mapping and identified 20 loci associated with secreted protein abundance in SMCs. We functionally annotated these loci using a colocalization approach. This approach prioritized the genetic variant rs6739323-A at the 2p22.3 locus, which is associated with lower expression of LTBP1 (latent-transforming growth factor beta-binding protein 1) in SMCs and atherosclerosis-prone areas of the aorta, and increased risk for SMC calcification. We found that LTBP1 expression is abundant in SMCs, and its expression at mRNA and protein levels was reduced in unstable and advanced atherosclerotic plaque lesions. CONCLUSIONS: Our results unravel the SMC proteome signature associated with vascular disorders, which may help identify potential therapeutic targets to accelerate the pathway to translation.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Humanos , Doenças Cardiovasculares/metabolismo , Estudo de Associação Genômica Ampla , Proteômica , Músculo Liso Vascular/metabolismo , Aorta/metabolismo , Aterosclerose/patologia , Miócitos de Músculo Liso/metabolismo , Células CultivadasRESUMO
BACKGROUND: The Myh11 promoter is extensively used as a smooth muscle cell (SMC) Cre-driver and is regarded as the most restrictive and specific promoter available to study SMCs. Unfortunately, in the existing Myh11-CreERT2 mouse, the transgene was inserted on the Y chromosome precluding the study of female mice. Given the importance of including sex as a biological variable and that numerous SMC-based diseases have a sex-dependent bias, the field has been tremendously limited by the lack of a model to study both sexes. Here, we describe a new autosomal Myh11-CreERT2 mouse (referred to as Myh11-CreERT2-RAD), which allows for SMC-specific lineage tracing and gene knockout studies in vivo using both male and female mice. METHODS: A Myh11-CreERT2-RAD transgenic C57BL/6 mouse line was generated using bacterial artificial chromosome clone RP23-151J22 modified to contain a Cre-ERT2 after the Myh11 start codon. Myh11-CreERT2-RAD mice were crossed with 2 different fluorescent reporter mice and tested for SMC-specific labeling by flow cytometric and immunofluorescence analyses. RESULTS: Myh11-CreERT2-RAD transgene insertion was determined to be on mouse chromosome 2. Myh11-CreERT2-RAD fluorescent reporter mice showed Cre-dependent, tamoxifen-inducible labeling of SMCs equivalent to the widely used Myh11-CreERT2 mice. Labeling was equivalent in both male and female Cre+ mice and was limited to vascular and visceral SMCs and pericytes in various tissues as assessed by immunofluorescence. CONCLUSIONS: We generated and validated the function of an autosomal Myh11-CreERT2-RAD mouse that can be used to assess sex as a biological variable with respect to the normal and pathophysiological functions of SMCs.
Assuntos
Integrases , Miócitos de Músculo Liso , Camundongos , Animais , Masculino , Feminino , Camundongos Transgênicos , Técnicas de Inativação de Genes , Integrases/genética , Integrases/metabolismo , Camundongos Knockout , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/metabolismo , Linhagem da Célula , TamoxifenoRESUMO
BACKGROUND: Women presenting with coronary artery disease more often present with fibrous atherosclerotic plaques, which are currently understudied. Phenotypically modulated smooth muscle cells (SMCs) contribute to atherosclerosis in women. How these phenotypically modulated SMCs shape female versus male plaques is unknown. METHODS: Gene regulatory networks were created using RNAseq gene expression data from human carotid atherosclerotic plaques. The networks were prioritized based on sex bias, relevance for smooth muscle biology, and coronary artery disease genetic enrichment. Network expression was linked to histologically determined plaque phenotypes. In addition, their expression in plaque cell types was studied at single-cell resolution using single-cell RNAseq. Finally, their relevance for disease progression was studied in female and male Apoe-/- mice fed a Western diet for 18 and 30 weeks. RESULTS: Here, we identify multiple sex-stratified gene regulatory networks from human carotid atherosclerotic plaques. Prioritization of the female networks identified 2 main SMC gene regulatory networks in late-stage atherosclerosis. Single-cell RNA sequencing mapped these female networks to 2 SMC phenotypes: a phenotypically modulated myofibroblast-like SMC network and a contractile SMC network. The myofibroblast-like network was mostly expressed in plaques that were vulnerable in women. Finally, the mice ortholog of key driver gene MFGE8 (milk fat globule EGF and factor V/VIII domain containing) showed retained expression in advanced plaques from female mice but was downregulated in male mice during atherosclerosis progression. CONCLUSIONS: Female atherosclerosis is characterized by gene regulatory networks that are active in fibrous vulnerable plaques rich in myofibroblast-like SMCs.
Assuntos
Aterosclerose , Doença da Artéria Coronariana , Placa Aterosclerótica , Feminino , Masculino , Humanos , Camundongos , Animais , Placa Aterosclerótica/patologia , Redes Reguladoras de Genes , Miofibroblastos/metabolismo , Doença da Artéria Coronariana/patologia , Aterosclerose/patologia , Miócitos de Músculo Liso/metabolismoRESUMO
[Figure: see text].
Assuntos
Hialuronan Sintases/metabolismo , Ácido Hialurônico/metabolismo , Músculo Liso Vascular/metabolismo , Placa Aterosclerótica/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Colágeno/genética , Colágeno/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Deleção de Genes , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases/genética , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , FenótipoRESUMO
BACKGROUND: Smooth muscle cells (SMCs) in atherosclerotic plaque take on multiple nonclassical phenotypes that may affect plaque stability and, therefore, the likelihood of myocardial infarction or stroke. However, the mechanisms by which these cells affect stability are only beginning to be explored. METHODS: In this study, we investigated the contribution of inflammatory MCP1 (monocyte chemoattractant protein 1) produced by both classical Myh11 (myosin heavy chain 11)+ SMCs and SMCs that have transitioned through an Lgals3 (galectin 3)+ state in atherosclerosis using smooth muscle lineage tracing mice that label all Myh11+ cells and a dual lineage tracing system that targets Lgals3-transitioned SMC only. RESULTS: We show that loss of MCP1 in all Myh11+ smooth muscle results in a paradoxical increase in plaque size and macrophage content, driven by a baseline systemic monocytosis early in atherosclerosis pathogenesis. In contrast, knockout of MCP1 in Lgals3-transitioned SMCs using a complex dual lineage tracing system resulted in lesions with an increased Acta2 (actin alpha 2, smooth muscle)+ fibrous cap and decreased investment of Lgals3-transitioned SMCs, consistent with increased plaque stability. Finally, using flow cytometry and single-cell RNA sequencing, we show that MCP1 produced by Lgals3-transitioned SMCs influences multiple populations of inflammatory cells in late-stage plaques. CONCLUSIONS: MCP1 produced by classical SMCs influences monocyte levels beginning early in disease and was atheroprotective, while MCP1 produced by the Lgals3-transitioned subset of SMCs exacerbated plaque pathogenesis in late-stage disease. Results are the first to determine the function of Lgals3-transitioned inflammatory SMCs in atherosclerosis and highlight the need for caution when considering therapeutic interventions involving MCP1.
Assuntos
Aterosclerose , Quimiocina CCL2 , Placa Aterosclerótica , Animais , Aterosclerose/patologia , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Camundongos , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica/patologiaRESUMO
Atherosclerosis is the process underlying heart attack and stroke. Despite decades of research, its pathogenesis remains unclear. Dogma suggests that atherosclerotic plaques expand primarily via the accumulation of cholesterol and inflammatory cells. However, recent evidence suggests that a substantial portion of the plaque may arise from a subset of "dedifferentiated" vascular smooth muscle cells (SMCs) which proliferate in a clonal fashion. Herein we use multicolor lineage-tracing models to confirm that the mature SMC can give rise to a hyperproliferative cell which appears to promote inflammation via elaboration of complement-dependent anaphylatoxins. Despite being extensively opsonized with prophagocytic complement fragments, we find that this cell also escapes immune surveillance by neighboring macrophages, thereby exacerbating its relative survival advantage. Mechanistic studies indicate this phenomenon results from a generalized opsonin-sensing defect acquired by macrophages during polarization. This defect coincides with the noncanonical up-regulation of so-called don't eat me molecules on inflamed phagocytes, which reduces their capacity for programmed cell removal (PrCR). Knockdown or knockout of the key antiphagocytic molecule CD47 restores the ability of macrophages to sense and clear opsonized targets in vitro, allowing for potent and targeted suppression of clonal SMC expansion in the plaque in vivo. Because integrated clinical and genomic analyses indicate that similar pathways are active in humans with cardiovascular disease, these studies suggest that the clonally expanding SMC may represent a translational target for treating atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Clonagem Molecular , Ativação do Complemento , Miócitos de Músculo Liso/metabolismo , Fagocitose/fisiologia , Animais , Antígeno CD47/metabolismo , Linhagem da Célula , Proliferação de Células , Complemento C3/genética , Complemento C3/metabolismo , Feminino , Humanos , Inflamação , Macrófagos/metabolismo , Masculino , Camundongos Knockout para ApoE , Miócitos de Músculo Liso/citologia , Placa Aterosclerótica/metabolismo , Análise de Sequência de RNA , Regulação para CimaRESUMO
RATIONALE: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies revealed 163 loci associated with CAD. However, the precise molecular mechanisms by which the majority of these loci increase CAD risk are not known. Vascular smooth muscle cells (VSMCs) are critical in the development of CAD. They can play either beneficial or detrimental roles in lesion pathogenesis, depending on the nature of their phenotypic changes. OBJECTIVE: To identify genetic variants associated with atherosclerosis-relevant phenotypes in VSMCs. METHODS AND RESULTS: We quantified 12 atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multiethnic heart transplant donors. After genotyping and imputation, we performed association mapping using 6.3 million genetic variants. We demonstrated significant variations in calcification, proliferation, and migration. These phenotypes were not correlated with each other. We performed genome-wide association studies for 12 atherosclerosis-relevant phenotypes and identified 4 genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD loci with our data set and found nominally significant associations at 79 loci. One of them was the chromosome 1q41 locus, which harbors MIA3. The G allele of the lead risk single nucleotide polymorphism (SNP) rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 (melanoma inhibitory activity protein 3) in VSMCs resulted in lower proliferation, consistent with human genetics findings. Furthermore, we observed a significant reduction of MIA3 protein in VSMCs in thin fibrous caps of late-stage atherosclerotic plaques compared to early fibroatheroma with thick and protective fibrous caps in mice and humans. CONCLUSIONS: Our data demonstrate that genetic variants have significant influences on VSMC function relevant to the development of atherosclerosis. Furthermore, high MIA3 expression may promote atheroprotective VSMC phenotypic transitions, including increased proliferation, which is essential in the formation or maintenance of a protective fibrous cap.
Assuntos
Aterosclerose/genética , Aterosclerose/patologia , Variação Genética , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Placa Aterosclerótica , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/genética , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Aterosclerose/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibrose , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Humanos , Masculino , Camundongos Knockout para ApoE , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
OBJECTIVE: Smooth muscle cells and pericytes display remarkable plasticity during injury and disease progression. Here, we tested the hypothesis that perivascular cells give rise to Klf4-dependent macrophage-like cells that augment adipose tissue (AT) inflammation and metabolic dysfunction associated with diet-induced obesity (DIO). Approach and Results: Using Myh11-CreERT2 eYFP (enhanced yellow fluorescent protein) mice and flow cytometry of the stromovascular fraction of epididymal AT, we observed a large fraction of smooth muscle cells and pericytes lineage traced eYFP+ cells expressing macrophage markers. Subsequent single-cell RNA sequencing, however, showed that the majority of these cells had no detectable eYFP transcript. Further exploration revealed that intraperitoneal injection of tamoxifen in peanut oil, used for generating conditional knockout or reporter mice in thousands of previous studies, resulted in large increase in the autofluorescence and false identification of macrophages within epididymal AT as being eYFP+; and unintended proinflammatory consequences. Using newly generated Myh11-DreERT2tdTomato mice given oral tamoxifen, we virtually eliminated the problem with autofluorescence and identified 8 perivascular cell dominated clusters, half of which were altered upon DIO. Given that perivascular cell KLF4 (kruppel-like factor 4) can have beneficial or detrimental effects, we tested its role in obesity-associated AT inflammation. While smooth muscle cells and pericytes-specific Klf4 knockout (smooth muscle cells and pericytes Klf4Δ/Δ) mice were not protected from DIO, they displayed improved glucose tolerance upon DIO, and showed marked decreases in proinflammatory macrophages and increases in LYVE1+ lymphatic endothelial cells in the epididymal AT. CONCLUSIONS: Perivascular cells within the AT microvasculature dynamically respond to DIO and modulate tissue inflammation and metabolism in a KLF4-dependent manner.
Assuntos
Tecido Adiposo/metabolismo , Plasticidade Celular , Fatores de Transcrição Kruppel-Like/metabolismo , Miócitos de Músculo Liso/metabolismo , Obesidade/metabolismo , Paniculite/metabolismo , Pericitos/metabolismo , Tecido Adiposo/patologia , Animais , Glicemia/metabolismo , Linhagem da Célula , Dieta Hiperlipídica , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Mediadores da Inflamação/metabolismo , Resistência à Insulina , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Knockout , Miócitos de Músculo Liso/patologia , Obesidade/etiologia , Obesidade/genética , Obesidade/patologia , Paniculite/etiologia , Paniculite/genética , Paniculite/patologia , Pericitos/patologiaRESUMO
BACKGROUND: Rupture and erosion of advanced atherosclerotic lesions with a resultant myocardial infarction or stroke are the leading worldwide cause of death. However, we have a limited understanding of the identity, origin, and function of many cells that make up late-stage atherosclerotic lesions, as well as the mechanisms by which they control plaque stability. METHODS: We conducted a comprehensive single-cell RNA sequencing of advanced human carotid endarterectomy samples and compared these with single-cell RNA sequencing from murine microdissected advanced atherosclerotic lesions with smooth muscle cell (SMC) and endothelial lineage tracing to survey all plaque cell types and rigorously determine their origin. We further used chromatin immunoprecipitation sequencing (ChIP-seq), bulk RNA sequencing, and an innovative dual lineage tracing mouse to understand the mechanism by which SMC phenotypic transitions affect lesion pathogenesis. RESULTS: We provide evidence that SMC-specific Klf4- versus Oct4-knockout showed virtually opposite genomic signatures, and their putative target genes play an important role regulating SMC phenotypic changes. Single-cell RNA sequencing revealed remarkable similarity of transcriptomic clusters between mouse and human lesions and extensive plasticity of SMC- and endothelial cell-derived cells including 7 distinct clusters, most negative for traditional markers. In particular, SMC contributed to a Myh11-, Lgals3+ population with a chondrocyte-like gene signature that was markedly reduced with SMC-Klf4 knockout. We observed that SMCs that activate Lgals3 compose up to two thirds of all SMC in lesions. However, initial activation of Lgals3 in these cells does not represent conversion to a terminally differentiated state, but rather represents transition of these cells to a unique stem cell marker gene-positive, extracellular matrix-remodeling, "pioneer" cell phenotype that is the first to invest within lesions and subsequently gives rise to at least 3 other SMC phenotypes within advanced lesions, including Klf4-dependent osteogenic phenotypes likely to contribute to plaque calcification and plaque destabilization. CONCLUSIONS: Taken together, these results provide evidence that SMC-derived cells within advanced mouse and human atherosclerotic lesions exhibit far greater phenotypic plasticity than generally believed, with Klf4 regulating transition to multiple phenotypes including Lgals3+ osteogenic cells likely to be detrimental for late-stage atherosclerosis plaque pathogenesis.
Assuntos
Aterosclerose/genética , Aterosclerose/patologia , Fatores de Transcrição Kruppel-Like/genética , Miócitos de Músculo Liso/patologia , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/patologia , Animais , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Análise de Sequência de RNA/métodosRESUMO
OBJECTIVE: Oxidized phospholipids (OxPL), such as the oxidized derivatives of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine, 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine, and 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, have been shown to be the principal biologically active components of minimally oxidized LDL (low-density lipoprotein). The role of OxPL in cardiovascular diseases is well recognized, including activation of inflammation within vascular cells. Atherosclerotic Apoe-/- mice fed a high-fat diet develop antibodies to OxPL, and hybridoma B-cell lines producing natural anti-OxPL autoantibodies have been successfully generated and characterized. However, as yet, no studies have been reported demonstrating that treatment with OxPL neutralizing antibodies can be used to prevent or reverse advanced atherosclerosis. Approach and Results: Here, using a screening against 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphorylcholine/1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphorylcholine, we generated a novel IgM autoantibody, 10C12, from the spleens of Apoe-/- mice fed a long-term Western diet, that demonstrated potent OxPL neutralizing activity in vitro and the ability to inhibit macrophage accumulation within arteries of Apoe-/- mice fed a Western diet for 4 weeks. Of interest, 10C12 failed to inhibit atherosclerosis progression in Apoe-/- mice treated between 18 and 26 weeks of Western diet feeding likely due at least in part to high levels of endogenous anti-OxPL antibodies. However, 10C12 treatment caused a 40% decrease in lipid accumulation within aortas of secreted IgM deficient, sIgM-/-Apoe-/-, mice fed a low-fat diet, when the antibody was administrated between 32-40 weeks of age. CONCLUSIONS: Taken together, these results provide direct evidence showing that treatment with a single autoimmune anti-OxPL IgM antibody during advanced disease stages can have an atheroprotective outcome.
Assuntos
Aterosclerose/dietoterapia , Autoanticorpos/imunologia , Dieta com Restrição de Gorduras/métodos , Dieta Ocidental , Imunoglobulina M/imunologia , Animais , Apolipoproteínas E/metabolismo , Aterosclerose/imunologia , Aterosclerose/metabolismo , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Humanos , Imuno-Histoquímica , Masculino , Camundongos , OxirreduçãoRESUMO
Objective- The goal of this study was to determine the role of ZFP148 (zinc-finger protein 148) in aneurysm formation. Approach and Results- ZFP148 mRNA expression increased at day 3, 7, 14, 21, and 28 after during abdominal aortic aneurysm formation in C57BL/6 mice. Loss of ZFP148 conferred abdominal aortic aneurysm protection using ERTCre+ ZFP148 flx/flx mice. In a third set of experiments, smooth muscle-specific loss of ZFP148 alleles resulted in progressively greater protection using novel transgenic mice (MYH [myosin heavy chain 11] Cre+ flx/flx, flx/wt, and wt/wt). Elastin degradation, LGAL3, and neutrophil staining were significantly attenuated, while α-actin staining was increased in ZFP148 knockout mice. Results were verified in total cell ZFP148 and smooth muscle-specific knockout mice using an angiotensin II model. ZFP148 smooth muscle-specific conditional mice demonstrated increased proliferation and ZFP148 was shown to bind to the p21 promoter during abdominal aortic aneurysm formation. ZFP148 smooth muscle-specific conditional knockout mice also demonstrated decreased apoptosis as measured by decreased cleaved caspase-3 staining. ZFP148 bound smooth muscle marker genes via chromatin immunoprecipitation analysis mediated by NF-1 (neurofibromin 1) promote histone H3K4 deacetylation via histone deacetylase 5. Transient transfections and chromatin immunoprecipitation analyses demonstrated that NF-1 was required for ZFP148 protein binding to smooth muscle marker genes promoters during aneurysm formation. Elimination of NF-1 using shRNA approaches demonstrated that NF-1 is required for binding and elimination of NF-1 increased BRG1 recruitment, the ATPase subunit of the SWI/SWF complex, and increased histone acetylation. Conclusions- ZFP148 plays a critical role in multiple murine models of aneurysm formation. These results suggest that ZFP148 is important in the regulation of proliferation, smooth muscle gene downregulation, and apoptosis in aneurysm development.
Assuntos
Aneurisma da Aorta Abdominal/etiologia , Proteínas de Ligação a DNA/metabolismo , Miócitos de Músculo Liso/metabolismo , Neurofibromina 1/metabolismo , Fatores de Transcrição/metabolismo , Acetilação , Angiotensina II/farmacologia , Animais , Aneurisma da Aorta Abdominal/metabolismo , Apoptose , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p21/genética , Feminino , Histonas/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Killer-Antagonista Homóloga a bcl-2/genéticaRESUMO
OBJECTIVE: Cigarette smoke exposure (CSE) is a risk factor for cerebral aneurysm (CA) formation, but the molecular mechanisms are unclear. Although CSE is known to contribute to excess reactive oxygen species generation, the role of oxidative stress on vascular smooth muscle cell (VSMC) phenotypic modulation and pathogenesis of CAs is unknown. The goal of this study was to investigate whether CSE activates a NOX (NADPH oxidase)-dependent pathway leading to VSMC phenotypic modulation and CA formation and rupture. APPROACH AND RESULTS: In cultured cerebral VSMCs, CSE increased expression of NOX1 and reactive oxygen species which preceded upregulation of proinflammatory/matrix remodeling genes (MCP-1, MMPs [matrix metalloproteinase], TNF-α, IL-1ß, NF-κB, KLF4 [Kruppel-like factor 4]) and downregulation of contractile genes (SM-α-actin [smooth muscle α actin], SM-22α [smooth muscle 22α], SM-MHC [smooth muscle myosin heavy chain]) and myocardin. Inhibition of reactive oxygen species production and knockdown of NOX1 with siRNA or antisense decreased CSE-induced upregulation of NOX1 and inflammatory genes and downregulation of VSMC contractile genes and myocardin. p47phox-/- NOX knockout mice, or pretreatment with the NOX inhibitor, apocynin, significantly decreased CA formation and rupture compared with controls. NOX1 protein and mRNA expression were similar in p47phox-/- mice and those pretreated with apocynin but were elevated in unruptured and ruptured CAs. CSE increased CA formation and rupture, which was diminished with apocynin pretreatment. Similarly, NOX1 protein and mRNA and reactive oxygen species were elevated by CSE, and in unruptured and ruptured CAs. CONCLUSIONS: CSE initiates oxidative stress-induced phenotypic modulation of VSMCs and CA formation and rupture. These molecular changes implicate oxidative stress in the pathogenesis of CAs and may provide a potential target for future therapeutic strategies.
Assuntos
Aneurisma Roto/enzimologia , Fumar Cigarros/efeitos adversos , Aneurisma Intracraniano/enzimologia , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , NADPH Oxidases/metabolismo , Estresse Oxidativo , Fumaça , Acetofenonas/farmacologia , Aneurisma Roto/genética , Aneurisma Roto/patologia , Aneurisma Roto/prevenção & controle , Animais , Antioxidantes/farmacologia , Células Cultivadas , Artérias Cerebrais/enzimologia , Artérias Cerebrais/patologia , Dilatação Patológica , Modelos Animais de Doenças , Aneurisma Intracraniano/genética , Aneurisma Intracraniano/patologia , Aneurisma Intracraniano/prevenção & controle , Fator 4 Semelhante a Kruppel , Masculino , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , NADPH Oxidase 1/genética , NADPH Oxidase 1/metabolismo , NADPH Oxidases/antagonistas & inibidores , NADPH Oxidases/genética , Fenótipo , Ratos Sprague-Dawley , Transdução de Sinais , Remodelação VascularRESUMO
Recent smooth muscle cell (SMC) lineage-tracing studies have revealed that SMCs undergo remarkable changes in phenotype during development of atherosclerosis. Of major interest, we demonstrated that Kruppel-like factor 4 (KLF4) in SMCs is detrimental for overall lesion pathogenesis, in that SMC-specific conditional knockout of the KLF4 gene ( Klf4) resulted in smaller, more-stable lesions that exhibited marked reductions in the numbers of SMC-derived macrophage- and mesenchymal stem cell-like cells. However, since the clinical consequences of atherosclerosis typically occur well after our reproductive years, we sought to identify beneficial KLF4-dependent SMC functions that were likely to be evolutionarily conserved. We tested the hypothesis that KLF4-dependent SMC transitions play an important role in the tissue injury-repair process. Using SMC-specific lineage-tracing mice positive and negative for simultaneous SMC-specific conditional knockout of Klf4, we demonstrate that SMCs in the remodeling heart after ischemia-reperfusion injury (IRI) express KLF4 and transition to a KLF4-dependent macrophage-like state and a KLF4-independent myofibroblast-like state. Moreover, heart failure after IRI was exacerbated in SMC Klf4 knockout mice. Surprisingly, we observed a significant cardiac dilation in SMC Klf4 knockout mice before IRI as well as a reduction in peripheral resistance. KLF4 chromatin immunoprecipitation-sequencing analysis on mesenteric vascular beds identified potential baseline SMC KLF4 target genes in numerous pathways, including PDGF and FGF. Moreover, microvascular tissue beds in SMC Klf4 knockout mice had gaps in lineage-traced SMC coverage along the resistance arteries and exhibited increased permeability. Together, these results provide novel evidence that Klf4 has a critical maintenance role within microvascular SMCs: it is required for normal SMC function and coverage of resistance arteries. NEW & NOTEWORTHY We report novel evidence that the Kruppel-like factor 4 gene ( Klf4) has a critical maintenance role within microvascular smooth muscle cells (SMCs). SMC-specific Klf4 knockout at baseline resulted in a loss of lineage-traced SMC coverage of resistance arteries, dilation of resistance arteries, increased blood flow, and cardiac dilation.
Assuntos
Fatores de Transcrição Kruppel-Like/metabolismo , Microvasos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Macrófagos/metabolismo , Camundongos , Microvasos/citologia , Miofibroblastos/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , RegeneraçãoRESUMO
The historical view of vascular smooth muscle cells (VSMCs) in atherosclerosis is that aberrant proliferation of VSMCs promotes plaque formation, but that VSMCs in advanced plaques are entirely beneficial, for example preventing rupture of the fibrous cap. However, this view has been based on ideas that there is a homogenous population of VSMCs within the plaque, that can be identified separate from other plaque cells (particularly macrophages) using standard VSMC and macrophage immunohistochemical markers. More recent genetic lineage tracing studies have shown that VSMC phenotypic switching results in less-differentiated forms that lack VSMC markers including macrophage-like cells, and this switching directly promotes atherosclerosis. In addition, VSMC proliferation may be beneficial throughout atherogenesis, and not just in advanced lesions, whereas VSMC apoptosis, cell senescence, and VSMC-derived macrophage-like cells may promote inflammation. We review the effect of embryological origin on VSMC behavior in atherosclerosis, the role, regulation and consequences of phenotypic switching, the evidence for different origins of VSMCs, and the role of individual processes that VSMCs undergo in atherosclerosis in regard to plaque formation and the structure of advanced lesions. We think there is now compelling evidence that a full understanding of VSMC behavior in atherosclerosis is critical to identify therapeutic targets to both prevent and treat atherosclerosis.
Assuntos
Aterosclerose/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Apoptose , Aterosclerose/patologia , Diferenciação Celular , Linhagem da Célula , Movimento Celular , Proliferação de Células , Senescência Celular , Humanos , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Fenótipo , Placa Aterosclerótica , Transdução de SinaisRESUMO
Recent genome wide association studies have identified a number of genes that contribute to the risk for coronary heart disease. One such gene, TCF21, encodes a basic-helix-loop-helix transcription factor believed to serve a critical role in the development of epicardial progenitor cells that give rise to coronary artery smooth muscle cells (SMC) and cardiac fibroblasts. Using reporter gene and immunolocalization studies with mouse and human tissues we have found that vascular TCF21 expression in the adult is restricted primarily to adventitial cells associated with coronary arteries and also medial SMC in the proximal aorta of mouse. Genome wide RNA-Seq studies in human coronary artery SMC (HCASMC) with siRNA knockdown found a number of putative TCF21 downstream pathways identified by enrichment of terms related to CAD, including "vascular disease," "disorder of artery," and "occlusion of artery," as well as disease-related cellular functions including "cellular movement" and "cellular growth and proliferation." In vitro studies in HCASMC demonstrated that TCF21 expression promotes proliferation and migration and inhibits SMC lineage marker expression. Detailed in situ expression studies with reporter gene and lineage tracing revealed that vascular wall cells expressing Tcf21 before disease initiation migrate into vascular lesions of ApoE-/- and Ldlr-/- mice. While Tcf21 lineage traced cells are distributed throughout the early lesions, in mature lesions they contribute to the formation of a subcapsular layer of cells, and others become associated with the fibrous cap. The lineage traced fibrous cap cells activate expression of SMC markers and growth factor receptor genes. Taken together, these data suggest that TCF21 may have a role regulating the differentiation state of SMC precursor cells that migrate into vascular lesions and contribute to the fibrous cap and more broadly, in view of the association of this gene with human CAD, provide evidence that these processes may be a mechanism for CAD risk attributable to the vascular wall.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Doença da Artéria Coronariana/genética , Miócitos de Músculo Liso/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Linhagem da Célula/genética , Doença da Artéria Coronariana/patologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , Mioblastos/metabolismo , Mioblastos/patologia , Miócitos de Músculo Liso/metabolismo , Células-TroncoRESUMO
Atherosclerotic plaque rupture with subsequent embolic events is a major cause of sudden death from myocardial infarction or stroke. Although smooth muscle cells (SMCs) produce and respond to collagens in vitro, there is no direct evidence in vivo that SMCs are a crucial source of collagens and that this impacts lesion development or fibrous cap formation. We sought to determine how conditional SMC-specific knockout of collagen type XV (COL15A1) in SMC lineage tracing mice affects advanced lesion formation given that 1) we have previously identified a Col15a1 sequence variant associated with age-related atherosclerosis, 2) COL15A1 is a matrix organizer enhancing tissue structural integrity, and 3) small interfering RNA-mediated Col15a1 knockdown increased migration and decreased proliferation of cultured human SMCs. We hypothesized that SMC-derived COL15A1 is critical in advanced lesions, specifically in fibrous cap formation. Surprisingly, we demonstrated that SMC-specific Col15a1 knockout mice fed a Western diet for 18 wk failed to form advanced lesions. SMC-specific Col15a1 knockout resulted in lesions reduced in size by 78%, with marked reductions in numbers and proliferating SMCs, and lacked a SMC and extracellular matrix-rich lesion or fibrous cap. In vivo RNA-seq analyses on SMC Col15a1 knockout and wild-type lesions suggested that a mechanism for these effects is through global repression of multiple proatherogenic inflammatory pathways involved in lesion development. These results provide the first direct evidence that a SMC-derived collagen, COL15A1, is critical during lesion pathogenesis, but, contrary to expectations, its loss resulted in marked attenuation rather than exacerbation of lesion pathogenesis.NEW & NOTEWORTHY We report the first direct in vivo evidence that a smooth muscle cell (SMC)-produced collagen, collagen type XV (COL15A1), is critical for atherosclerotic lesion development. SMC Col15a1 knockout markedly attenuated advanced lesion formation, likely through reducing SMC proliferation and impairing multiple proatherogenic inflammatory processes.