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BACKGROUND AND AIMS: Tobacco smoking is a known risk factor for atherosclerotic disease, with more elevated risks in women compared to men. We hypothesized that atherosclerotic plaques from smokers show different gene expression patterns compared to non-smokers, in a sex-specific manner. METHODS: Gene expression data of 625 carotid plaques (151 females and 474 males) were analyzed for differential gene expression between current smokers (n = 226) and non-smokers (n = 399). All analyses were stratified by sex and by molecular plaque characteristics. Finally, we projected the activity of gene regulatory networks and utilized single-cell transcriptomics from 38 plaques (26 males and 12 females) to interpret the sex- and plaque-type specific signals. RESULTS: We observed higher expression levels of CRLF1 gene in atherosclerotic plaques from smokers compared to non-smokers (log2FC = 0.48, FDR = 0.012). CRLF1 upregulation was interacting with sex (p = 0.01) and was more pronounced in females (log2FC = 0.93, p = 1.53E-05) compared to males (log2FC = 0.35, p = 0.0018). Through single-cell RNA-seq analysis, we identified the highest CRLF1 expression within the transitioning and synthetic smooth muscle cell populations. CRLF1 expression was increased in fibro-inflammatory and fibro-cellular plaque types. Gene annotations pointed to increased expression of CRLF1 in networks with extracellular matrix related genes. CONCLUSIONS: Atherosclerotic plaques from current smokers show sex-dependent upregulation of smooth muscle cell gene CRLF1. This may explain the different contributions of smoking to cardiovascular risk in females.
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In developing B cells, V(D)J gene recombination is initiated by the RAG1/2 endonuclease complex, introducing double-stranded DNA breaks (DSBs) in V, D, and J genes and resulting in the formation of the hypervariable parts of immunoglobulins (Ig). Persistent or aberrant RAG1/2 targeting is a potential threat to genome integrity. While RAG1 and RAG2 have been shown to bind various regions genome-wide, the in vivo off-target DNA damage instigated by RAG1/2 endonuclease remains less well understood. In the current study, we identified regions containing RAG1/2-induced DNA breaks in mouse pre-B cells on a genome-wide scale using a global DNA DSB detection strategy. We detected 1489 putative RAG1/2-dependent DSBs, most of which were located outside the Ig loci. DNA sequence motif analysis showed a specific enrichment of RAG1/2-induced DNA DSBs at GA- and CA-repeats and GC-rich motifs. These findings provide further insights into RAG1/2 off-target activity. The ability of RAG1/2 to introduce DSBs on the non-Ig loci during the endogenous V(D)J recombination emphasizes its genotoxic potential in developing lymphocytes.
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BACKGROUND: Cell phenotype switching is increasingly being recognized in atherosclerosis. However, our understanding of the exact stimuli for such cellular transformations and their significance for human atherosclerosis is still evolving. Intraplaque hemorrhage is thought to be a major contributor to plaque progression in part by stimulating the influx of CD163+ macrophages. Here, we explored the hypothesis that CD163+ macrophages cause plaque progression through the induction of proapoptotic endothelial-to-mesenchymal transition (EndMT) within the fibrous cap. METHODS: Human coronary artery sections from CVPath's autopsy registry were selected for pathological analysis. Athero-prone ApoE-/- and ApoE-/-/CD163-/- mice were used for in vivo studies. Human peripheral blood mononuclear cell-induced macrophages and human aortic endothelial cells were used for in vitro experiments. RESULTS: In 107 lesions with acute coronary plaque rupture, 55% had pathological evidence of intraplaque hemorrhage in nonculprit vessels/lesions. Thinner fibrous cap, greater CD163+ macrophage accumulation, and a larger number of CD31/FSP-1 (fibroblast specific protein-1) double-positive cells and TUNEL (terminal deoxynucleotidyl transferase-dUTP nick end labeling) positive cells in the fibrous cap were observed in nonculprit intraplaque hemorrhage lesions, as well as in culprit rupture sections versus nonculprit fibroatheroma sections. Human aortic endothelial cells cultured with supernatants from hemoglobin/haptoglobin-exposed macrophages showed that increased mesenchymal marker proteins (transgelin and FSP-1) while endothelial markers (VE-cadherin and CD31) were reduced, suggesting EndMT induction. Activation of NF-κB (nuclear factor kappa ß) signaling by proinflammatory cytokines released from CD163+ macrophages directly regulated the expression of Snail, a critical transcription factor during EndMT induction. Western blot analysis for cleaved caspase-3 and microarray analysis of human aortic endothelial cells indicated that apoptosis was stimulated during CD163+ macrophage-induced EndMT. Additionally, CD163 deletion in athero-prone mice suggested that CD163 is required for EndMT and plaque progression. Using single-cell RNA sequencing from human carotid endarterectomy lesions, a population of EndMT was detected, which demonstrated significant upregulation of apoptosis-related genes. CONCLUSIONS: CD163+ macrophages provoke EndMT, which may promote plaque progression through fibrous cap thinning.
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Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Macrófagos , Placa Aterosclerótica , Receptores de Superficie Celular , Humanos , Antígenos de Diferenciación Mielomonocítica/metabolismo , Antígenos de Diferenciación Mielomonocítica/genética , Animales , Antígenos CD/metabolismo , Antígenos CD/genética , Macrófagos/metabolismo , Macrófagos/patología , Placa Aterosclerótica/patología , Placa Aterosclerótica/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/genética , Ratones , Células Cultivadas , Células Endoteliales/metabolismo , Células Endoteliales/patología , Masculino , Ratones Noqueados para ApoE , Ratones Endogámicos C57BL , Apoptosis , Femenino , Transición Epitelial-Mesenquimal , Vasos Coronarios/patología , Vasos Coronarios/metabolismoRESUMEN
T cell activation is critical for an effective immune response against pathogens. However, dysregulation contributes to the pathogenesis of autoimmune diseases, including Juvenile Idiopathic Arthritis (JIA). The molecular mechanisms underlying T cell activation are still incompletely understood. T cell activation promotes the acetylation of histone 3 at Lysine 27 (H3K27ac) at enhancer and promoter regions of proinflammatory cytokines, thereby increasing the expression of these genes which is essential for T cell function. Co-activators E1A binding protein P300 (P300) and CREB binding protein (CBP), collectively known as P300/CBP, are essential to facilitate H3K27 acetylation. Presently, the role of P300/CBP in human CD4+ T cells activation remains incompletely understood. To assess the function of P300/CBP in T cell activation and autoimmune disease, we utilized iCBP112, a selective inhibitor of P300/CBP, in T cells obtained from healthy controls and JIA patients. Treatment with iCBP112 suppressed T cell activation and cytokine signaling pathways, leading to reduced expression of many proinflammatory cytokines, including IL-2, IFN-γ, IL-4, and IL-17A. Moreover, P300/CBP inhibition in T cells derived from the inflamed synovium of JIA patients resulted in decreased expression of similar pathways and preferentially suppressed the expression of disease-associated genes. This study underscores the regulatory role of P300/CBP in regulating gene expression during T cell activation while offering potential insights into the pathogenesis of autoimmune diseases. Our findings indicate that P300/CBP inhibition could potentially be leveraged for the treatment of autoimmune diseases such as JIA in the future.
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The intestine is vulnerable to chemotherapy-induced damage due to the high rate of intestinal epithelial cell (IEC) proliferation. We have developed a human intestinal organoid-based 3D model system to study the direct effect of chemotherapy-induced IEC damage on T cell behavior. Exposure of intestinal organoids to busulfan, fludarabine, and clofarabine induced damage-related responses affecting both the capacity to regenerate and transcriptional reprogramming. In ex vivo co-culture assays, prior intestinal organoid damage resulted in increased T cell activation, proliferation, and migration. We identified galectin-9 (Gal-9) as a key molecule released by damaged organoids. The use of anti-Gal-9 blocking antibodies or CRISPR/Cas9-mediated Gal-9 knock-out prevented intestinal organoid damage-induced T cell proliferation, interferon-gamma release, and migration. Increased levels of Gal-9 were found early after HSCT chemotherapeutic conditioning in the plasma of patients who later developed acute GVHD. Taken together, chemotherapy-induced intestinal damage can influence T cell behavior in a Gal-9-dependent manner which may provide novel strategies for therapeutic intervention.
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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.
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Aterosclerosis , Redes Reguladoras de Genes , Análisis de la Célula Individual , Humanos , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Ratones , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Masculino , Placa Aterosclerótica , Progresión de la Enfermedad , Femenino , Macrófagos/metabolismo , Macrófagos/patología , Ratones Noqueados , Receptores de LDL/genética , Receptores de LDL/metabolismo , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologíaRESUMEN
BACKGROUND: Epigenetic age estimators (clocks) are predictive of human mortality risk. However, it is not yet known whether the epigenetic age of atherosclerotic plaques is predictive for the risk of cardiovascular events. METHODS: Whole-genome DNA methylation of human carotid atherosclerotic plaques (n=485) and of blood (n=93) from the Athero-Express endarterectomy cohort was used to calculate epigenetic age acceleration (EAA). EAA was linked to clinical characteristics, plaque histology, and future cardiovascular events (n=136). We studied whole-genome DNA methylation and bulk and single-cell transcriptomics to uncover molecular mechanisms of plaque EAA. We experimentally confirmed our in silico findings using in vitro experiments in primary human coronary endothelial cells. RESULTS: Male and female patients with severe atherosclerosis had a median chronological age of 69 years. The median epigenetic age was 65 years in females (median EAA, -2.2 [interquartile range, -4.3 to 2.2] years) and 68 years in males (median EAA, -0.3 [interquartile range, -2.9 to 3.8] years). Patients with diabetes and a high body mass index had higher plaque EAA. Increased EAA of plaque predicted future events in a 3-year follow-up in a Cox regression model (univariate hazard ratio, 1.7; P=0.0034) and adjusted multivariate model (hazard ratio, 1.56; P=0.02). Plaque EAA predicted outcome independent of blood EAA (hazard ratio, 1.3; P=0.018) and of plaque hemorrhage (hazard ratio, 1.7; P=0.02). Single-cell RNA sequencing in plaque samples from 46 patients in the same cohort revealed smooth muscle and endothelial cells as important cell types in plaque EAA. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally confirmed by TGFß-triggered endothelial-to-mesenchymal transition inducing rapid epigenetic aging in coronary endothelial cells. CONCLUSIONS: Plaque EAA is a strong and independent marker of poor outcome in patients with severe atherosclerosis. Plaque EAA was linked to mesenchymal endothelial and smooth muscle cells. Endothelial-to-mesenchymal transition was associated with EAA, which was experimentally validated. Epigenetic aging mechanisms may provide new targets for treatments that reduce atherosclerosis complications.
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Metilación de ADN , Células Endoteliales , Epigénesis Genética , Placa Aterosclerótica , Humanos , Masculino , Femenino , Anciano , Pronóstico , Persona de Mediana Edad , Células Endoteliales/patología , Células Endoteliales/metabolismo , Factores de Edad , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/patología , Enfermedades de las Arterias Carótidas/cirugía , Células Cultivadas , Factores de Riesgo , Medición de RiesgoRESUMEN
N6-methyladenosine (m6A) is a RNA modification that can regulate post-transcriptional processes including RNA stability, translation, splicing and nuclear export. In CD4+ lymphocytes, m6A modifications have been demonstrated to play a role in early differentiation processes. The role of m6A in CD4+ T cell activation and effector function remains incompletely understood. To assess the role of m6A in CD4+ T lymphocyte activation and function, we assessed the transcriptome-wide m6A landscape of human primary CD4+ T cells by methylated RNA immunoprecipitation (meRIP) sequencing. Stimulation of the T cells impacted the m6A pattern of hundreds of transcripts including tumor necrosis factor (TNF). m6A methylation was increased on TNF mRNA after activation, predominantly in the 3' untranslated region (UTR) of the transcript. Manipulation of m6A levels in primary human T cells, the directly affected the expression of TNF. Furthermore, we identified that the m6A reader protein YT521-B homology domain family-2 (YTHDF2) binds m6A-methylated TNF mRNA, and promotes its degradation. Taken together, this study demonstrates that TNF expression in CD4+ T lymphocytes is regulated via m6A and YTHDF2, thereby providing novel insight into the regulation of T cell effector functions.
T helper cells are immune cells of the adaptive immune system. These cells are activated by antigen presenting cells that have engulfed invading pathogens. When the T helper cell is activated, it will produce and excrete signaling molecules (cytokines) that activate other immune cells in order to eradicate these pathogens. Cytokines are formed after translation of RNA molecules that encode for these cytokines. In this study it was found that a modification (m6A) on RNA molecules is involved in the regulation of the life cycle of these RNA molecules. It was found that the degradation of RNA encoding for cytokine TNF was mediated through m6A and its 'reader' protein YTHDF2 in activated T helper cells. As TNF promotes inflammation, reduction of TNF production through this mechanism dampens the immune response and therefore prevents chronic inflammation.
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The cytokine interferon-gamma (IFNγ) plays a multifaceted role in intestinal immune responses ranging from anti-to pro-inflammatory depending on the setting. Here, using a 3D co-culture system based on human intestinal epithelial organoids, we explore the capacity of IFNγ-exposure to reprogram intestinal epithelia and thereby directly modulate lymphocyte responses. IFNγ treatment of organoids led to transcriptional reprogramming, marked by a switch to a pro-inflammatory gene expression profile, including transcriptional upregulation of the chemokines CXCL9, CXCL10, and CXCL11. Proteomic analysis of organoid-conditioned medium post-treatment confirmed chemokine secretion. Furthermore, IFNγ-treatment of organoids led to enhanced T cell migration in a CXCL11-dependent manner without affecting T cell activation status. Taken together, our results suggest a specific role for CXCL11 in T cell recruitment that can be targeted to prevent T cell trafficking to the inflamed intestine.
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BACKGROUND: After myocardial infarction, the lost myocardium is replaced by fibrotic tissue, eventually progressively leading to myocardial dysfunction. Direct reprogramming of fibroblasts into cardiomyocytes via the forced overexpression of cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) offers a promising strategy for cardiac repair. The limited reprogramming efficiency of this approach, however, remains a significant challenge. METHODS: We screened seven factors capable of improving direct cardiac reprogramming of both mice and human fibroblasts by evaluating small molecules known to be involved in cardiomyocyte differentiation or promoting human-induced pluripotent stem cell reprogramming. RESULTS: We found that vitamin C (VitC) significantly increased cardiac reprogramming efficiency when added to GMT-overexpressing fibroblasts from human and mice in 2D and 3D model. We observed a significant increase in reactive oxygen species (ROS) generation in human and mice fibroblasts upon Doxy induction, and ROS generation was subsequently reduced upon VitC treatment, associated with increased reprogramming efficiency. However, upon treatment with dehydroascorbic acid, a structural analog of VitC but lacking antioxidant properties, no difference in reprogramming efficiency was observed, suggesting that the effect of VitC in enhancing cardiac reprogramming is partly dependent of its antioxidant properties. CONCLUSIONS: Our findings demonstrate that VitC supplementation significantly enhances the efficiency of cardiac reprogramming, partially by suppressing ROS production in the presence of GMT.
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Antioxidantes , Ácido Ascórbico , Humanos , Ratones , Animales , Especies Reactivas de Oxígeno , Ácido Ascórbico/farmacología , Antioxidantes/farmacología , Reprogramación Celular , Proteínas de Dominio T Box/genética , Factores de Transcripción MEF2/genética , Miocitos Cardíacos , Vitaminas , FibroblastosRESUMEN
Background Plaque myofibroblasts are critical players in the initiation and advancement of atherosclerotic disease. They are involved in the production of extracellular matrix, the formation of the fibrous cap, and the underlying lipidic core via modulation processes in response to different environmental cues. Despite clear phenotypic differences between myofibroblast cells and healthy vascular smooth muscle cells, smooth muscle cells are still widely used as a cellular model in atherosclerotic research. Methods and Results Here, we present a conditioned outgrowth method to isolate and culture myofibroblast cells from plaques. We obtained these cells from 27 donors (24 carotid and 3 femoral endarterectomies). We show that they keep their proliferative capacity for 8 passages, are transcriptionally stable, retain donor-specific gene expression programs, and express extracellular matrix proteins (FN1, COL1A1, and DCN) and smooth muscle cell markers (ACTA2, MYH11, and CNN1). Single-cell transcriptomics reveals that the cells in culture closely resemble the plaque myofibroblasts. Chromatin immunoprecipitation sequencing shows the presence of histone H3 lysine 4 dimethylation at the MYH11 promoter, pointing to their smooth muscle cell origin. Finally, we demonstrated that plaque myofibroblasts can be efficiently transduced (>97%) and are capable of taking up oxidized low-density lipoprotein and undergoing calcification. Conclusions In conclusion, we present a method to isolate and culture cells that retain plaque myofibroblast phenotypical and functional capabilities, making them a suitable in vitro model for studying selected mechanisms of atherosclerosis.
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Aterosclerosis , Placa Aterosclerótica , Humanos , Miofibroblastos/metabolismo , Aterosclerosis/metabolismo , Placa Aterosclerótica/metabolismo , Arterias Carótidas/metabolismo , Miocitos del Músculo Liso/metabolismoRESUMEN
Sex differences in coronary artery disease (CAD) presentation, risk factors and prognosis have been widely studied. Similarly, studies on atherosclerosis have shown prominent sex differences in plaque biology. Our understanding of the underlying genetic and molecular mechanisms that drive these differences remains fragmented and largely understudied. Through reviewing genetic and epigenetic studies, we identified more than 40 sex-differential candidate genes (13 within known CAD loci) that may explain, at least in part, sex differences in vascular remodeling, lipid metabolism and endothelial dysfunction. Studies with transcriptomic and single-cell RNA sequencing data from atherosclerotic plaques highlight potential sex differences in smooth muscle cell and endothelial cell biology. Especially, phenotypic switching of smooth muscle cells seems to play a crucial role in female atherosclerosis. This matches the known sex differences in atherosclerotic phenotypes, with men being more prone to lipid-rich plaques, while women are more likely to develop fibrous plaques with endothelial dysfunction. To unravel the complex mechanisms that drive sex differences in CAD, increased statistical power and adjustments to study designs and analysis strategies are required. This entails increasing inclusion rates of women, performing well-defined sex-stratified analyses and the integration of multi-omics data.
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Aterosclerosis , Enfermedad de la Arteria Coronaria , Placa Aterosclerótica , Femenino , Humanos , Masculino , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Caracteres Sexuales , Placa Aterosclerótica/genética , Aterosclerosis/genéticaRESUMEN
BACKGROUND: Studies in humans and mice using the expression of an X-linked gene or lineage tracing, respectively, have suggested that clones of smooth muscle cells (SMCs) exist in human atherosclerotic lesions but are limited by either spatial resolution or translatability of the model. METHODS: Phenotypic clonality can be detected by X-chromosome inactivation patterns. We investigated whether clones of SMCs exist in unstable human atheroma using RNA in situ hybridization (BaseScope) to identify a naturally occurring 24-nucleotide deletion in the 3'UTR of the X-linked BGN (biglycan) gene, a proteoglycan highly expressed by SMCs. BGN-specific BaseScope probes were designed to target the wild-type or deletion mRNA. Three different coronary artery plaque types (erosion, rupture, and adaptive intimal thickening) were selected from heterozygous females for the deletion BGN. Hybridization of target RNA-specific probes was used to visualize the spatial distribution of mutants. A clonality index was calculated from the percentage of each probe in each region of interest. Spatial transcriptomics were used to identify differentially expressed transcripts within clonal and nonclonal regions. RESULTS: Less than one-half of regions of interest in the intimal plaque were considered clonal with the mean percent regions of interest with clonality higher in the intimal plaque than in the media. This was consistent for all plaque types. The relationship of the dominant clone in the intimal plaque and media showed significant concordance. In comparison with the nonclonal lesions, the regions with SMC clonality had lower expression of genes encoding cell growth suppressors such as CD74, SERF-2 (small EDRK-rich factor 2), CTSB (cathepsin B), and HLA-DPA1 (major histocompatibility complex, class II, DP alpha 1), among others. CONCLUSIONS: Our novel approach to examine clonality suggests atherosclerosis is primarily a disease of polyclonally and to a lesser extent clonally expanded SMCs and may have implications for the development of antiatherosclerotic therapies.
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Aterosclerosis , Placa Aterosclerótica , Femenino , Humanos , Ratones , Animales , Músculo Liso Vascular/metabolismo , Aterosclerosis/patología , Placa Aterosclerótica/patología , Células Clonales/patología , Proliferación Celular , Miocitos del Músculo Liso/metabolismo , ARNRESUMEN
Atherosclerosis is a chronic inflammatory disease which is driven in part by the aberrant trans -differentiation of vascular smooth muscle cells (SMCs). No therapeutic drug has been shown to reverse detrimental SMC-derived cell phenotypes into protective phenotypes, a hypothesized enabler of plaque regression and improved patient outcome. Herein, we describe a novel function of colchicine in the beneficial modulation of SMC-derived cell phenotype, independent of its conventional anti-inflammatory effects. Using SMC fate mapping in an advanced atherosclerotic lesion model, colchicine induced plaque regression by converting pathogenic SMC-derived macrophage-like and osteoblast-like cells into protective myofibroblast-like cells which thickened, and thereby stabilized, the fibrous cap. This was dependent on Notch3 signaling in SMC-derived plaque cells. These findings may help explain the success of colchicine in clinical trials relative to other anti-inflammatory drugs. Thus, we demonstrate the potential of regulating SMC phenotype in advanced plaque regression through Notch3 signaling, in addition to the canonical anti-inflammatory actions of drugs to treat atherosclerosis.
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The accumulation of erythrocyte membranes within an atherosclerotic plaque may contribute to the deposition of free cholesterol and thereby the enlargement of the necrotic core. Erythrocyte membranes can be visualized and quantified in the plaque by immunostaining for the erythrocyte marker glycophorin C. Hence, we theorized that the accumulation of erythrocytes quantified by glycophorin C could function as a marker for plaque vulnerability, possibly reflecting intraplaque hemorrhage (IPH), and offering predictive value for pre-procedural neurological symptoms. We employed the CellProfiler-integrated slideToolKit workflow to visualize and quantify glycophorin C, defined as the total plaque area that is positive for glycophorin C, in single slides of culprit lesions obtained from the Athero-Express Biobank of 1819 consecutive asymptomatic and symptomatic patients who underwent carotid endarterectomy. Our assessment included the evaluation of various parameters such as lipid core, calcifications, collagen content, SMC content, and macrophage burden. These parameters were evaluated using a semi-quantitative scoring method, and the resulting data was dichotomized as predefined criteria into categories of no/minor or moderate/heavy staining. In addition, the presence or absence of IPH was also scored. The prevalence of IPH and pre-procedural neurological symptoms were 62.4% and 87.1%, respectively. The amount of glycophorin staining was significantly higher in samples from men compared to samples of women (median 7.15 (IQR:3.37, 13.41) versus median 4.06 (IQR:1.98, 8.32), p < 0.001). Glycophorin C was associated with IPH adjusted for clinical confounders (OR 1.90; 95% CI 1.63, 2.21; p = < 0.001). Glycophorin C was significantly associated with ipsilateral pre-procedural neurological symptoms (OR:1.27, 95%CI:1.06-1.41, p = 0.005). Sex-stratified analysis, showed that this was also the case for men (OR 1.37; 95%CI 1.12, 1.69; p = 0.003), but not for women (OR 1.15; 95%CI 0.77, 1.73; p = 0.27). Glycophorin C was associated with classical features of a vulnerable plaque, such as a larger lipid core, a higher macrophage burden, less calcifications, a lower collagen and SMC content. There were marked sex differences, in men, glycophorin C was associated with calcifications and collagen while these associations were not found in women. To conclude, the accumulation of erythrocytes in atherosclerotic plaque quantified and visualized by glycophorin C was independently associated with the presence of IPH, preprocedural symptoms in men, and with a more vulnerable plaque composition in both men and women. These results strengthen the notion that the accumulation of erythrocytes quantified by glycophorin C can be used as a marker for plaque vulnerability.
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Calcinosis , Estenosis Carotídea , Placa Aterosclerótica , Humanos , Femenino , Masculino , Placa Aterosclerótica/patología , Glicoforinas , Arterias Carótidas/patología , Hemorragia/patología , Calcinosis/patología , Membrana Eritrocítica/patología , Colágeno , Lípidos , Estenosis Carotídea/patología , Imagen por Resonancia MagnéticaRESUMEN
Coronary artery calcification (CAC), a measure of subclinical atherosclerosis, predicts future symptomatic coronary artery disease (CAD). Identifying genetic risk factors for CAC may point to new therapeutic avenues for prevention. Currently, there are only four known risk loci for CAC identified from genome-wide association studies (GWAS) in the general population. Here we conducted the largest multi-ancestry GWAS meta-analysis of CAC to date, which comprised 26,909 individuals of European ancestry and 8,867 individuals of African ancestry. We identified 11 independent risk loci, of which eight were new for CAC and five had not been reported for CAD. These new CAC loci are related to bone mineralization, phosphate catabolism and hormone metabolic pathways. Several new loci harbor candidate causal genes supported by multiple lines of functional evidence and are regulators of smooth muscle cell-mediated calcification ex vivo and in vitro. Together, these findings help refine the genetic architecture of CAC and extend our understanding of the biological and potential druggable pathways underlying CAC.
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Aterosclerosis , Enfermedad de la Arteria Coronaria , Humanos , Aterosclerosis/genética , Población Negra/genética , Enfermedad de la Arteria Coronaria/genética , Estudio de Asociación del Genoma Completo , Factores de Riesgo , Pueblo Europeo/genéticaRESUMEN
Caspase recruitment-domain containing protein 9 (CARD9) is a key signaling pathway in macrophages but its role in atherosclerosis is still poorly understood. Global deletion of Card9 in Apoe-/- mice as well as hematopoietic deletion in Ldlr-/- mice increases atherosclerosis. The acceleration of atherosclerosis is also observed in Apoe-/-Rag2-/-Card9-/- mice, ruling out a role for the adaptive immune system in the vascular phenotype of Card9 deficient mice. Card9 deficiency alters macrophage phenotype through CD36 overexpression with increased IL-1ß production, increased lipid uptake, higher cell death susceptibility and defective autophagy. Rapamycin or metformin, two autophagy inducers, abolish intracellular lipid overload, restore macrophage survival and autophagy flux in vitro and finally abolish the pro-atherogenic effects of Card9 deficiency in vivo. Transcriptomic analysis of human CARD9-deficient monocytes confirms the pathogenic signature identified in murine models. In summary, CARD9 is a key protective pathway in atherosclerosis, modulating macrophage CD36-dependent inflammatory responses, lipid uptake and autophagy.
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Aterosclerosis , Humanos , Animales , Ratones , Aterosclerosis/metabolismo , Autofagia/genética , Apolipoproteínas E/genética , Lípidos , Proteínas Adaptadoras de Señalización CARD/metabolismo , Ratones Noqueados , Ratones Endogámicos C57BLRESUMEN
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.
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Aterosclerosis , Enfermedad de la Arteria Coronaria , Placa Aterosclerótica , Femenino , Masculino , Humanos , Ratones , Animales , Placa Aterosclerótica/patología , Redes Reguladoras de Genes , Miofibroblastos/metabolismo , Enfermedad de la Arteria Coronaria/patología , Aterosclerosis/patología , Miocitos del Músculo Liso/metabolismoRESUMEN
Vascular pathologies show locational predisposition throughout the body; further insights into the transcriptomics basis of this vascular heterogeneity are needed. We analyzed transcriptomes from cultured endothelial cells and vascular smooth muscle cells from nine adult canine macrovessels: the aorta, coronary artery, vena cava, portal vein, femoral artery, femoral vein, saphenous vein, pulmonary vein, and pulmonary artery. We observed that organ-specific expression patterns persist in vitro, indicating that these genes are not regulated by blood flow or surrounding cell types but are likely fixed in the epigenetic memory. We further demonstrated the preserved location-specific expression of GATA4 protein in cultured cells and in the primary adult vessel. On a functional level, arterial and venous endothelial cells differed in vascular network morphology as the arterial networks maintained a higher complexity. Our findings prompt the rethinking of the extrapolation of results from single-origin endothelial cell systems.
Asunto(s)
Aorta , Células Endoteliales , Animales , Perros , Células Endoteliales/metabolismo , Vasos Coronarios , Venas Cavas , Vena Safena/metabolismo , Células CultivadasRESUMEN
Background: Leukocyte progenitors derived from clonal hematopoiesis of undetermined potential (CHIP) are associated with increased cardiovascular events. However, the prevalence and functional relevance of CHIP in coronary artery disease (CAD) are unclear, and cells affected by CHIP have not been detected in human atherosclerotic plaques. Methods: CHIP mutations in blood and tissues were identified by targeted deep-DNA-sequencing (DNAseq: coverage >3,000) and whole-genome-sequencing (WGS: coverage >35). CHIP-mutated leukocytes were visualized in human atherosclerotic plaques by mutaFISH™. Functional relevance of CHIP mutations was studied by RNAseq. Results: DNAseq of whole blood from 540 deceased CAD patients of the Munich cardIovaScular StudIes biObaNk (MISSION) identified 253 (46.9%) CHIP mutation carriers (mean age 78.3 years). DNAseq on myocardium, atherosclerotic coronary and carotid arteries detected identical CHIP mutations in 18 out of 25 mutation carriers in tissue DNA. MutaFISH™ visualized individual macrophages carrying DNMT3A CHIP mutations in human atherosclerotic plaques. Studying monocyte-derived macrophages from Stockholm-Tartu Atherosclerosis Reverse Networks Engineering Task (STARNET; n=941) by WGS revealed CHIP mutations in 14.2% (mean age 67.1 years). RNAseq of these macrophages revealed that expression patterns in CHIP mutation carriers differed substantially from those of non-carriers. Moreover, patterns were different depending on the underlying mutations, e.g. those carrying TET2 mutations predominantly displayed upregulated inflammatory signaling whereas ASXL1 mutations showed stronger effects on metabolic pathways. Conclusions: Deep-DNA-sequencing reveals a high prevalence of CHIP mutations in whole blood of CAD patients. CHIP-affected leukocytes invade plaques in human coronary arteries. RNAseq data obtained from macrophages of CHIP-affected patients suggest that pro-atherosclerotic signaling differs depending on the underlying mutations. Further studies are necessary to understand whether specific pathways affected by CHIP mutations may be targeted for personalized treatment.