Single cell analysis of smooth muscle cell phenotypic modulation in vivo during disease in mice and humans [mouse scRNA-seq]
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE131776 | GEO | 2019/07/29
REPOSITORIES: GEO
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Project description:In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human. We performed CITE-seq in mouse atherosclerotic lesions using antibodies directed against several macrophage surface markers. In the mice, we also performed SMC-specific knockout of TCF21, a causal CAD gene, to determine the effect of this gene on SMC phenotypic modulation. Finally, we performed ChIP-seq for the transcription factor TCF21 in a pooled DNA sample comprised of 52 different human coronary artery smooth muscle cell (HCASMC) lines to determine TCF21 target genes.
2019-07-29 | GSE131779 | GEO
Project description:In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human. We performed CITE-seq in mouse atherosclerotic lesions using antibodies directed against several macrophage surface markers. In the mice, we also performed SMC-specific knockout of TCF21, a causal CAD gene, to determine the effect of this gene on SMC phenotypic modulation. Finally, we performed ChIP-seq for the transcription factor TCF21 in a pooled DNA sample comprised of 52 different human coronary artery smooth muscle cell (HCASMC) lines to determine TCF21 target genes.
2019-07-29 | GSE131777 | GEO
Project description:In response to various stimuli, vascular smooth muscle cells (SMCs) can de-differentiate, proliferate and migrate in a process known as phenotypic modulation. However, the phenotype of modulated SMCs in vivo during atherosclerosis and the influence of this process on coronary artery disease (CAD) risk have not been clearly established. Using single cell RNA sequencing, we comprehensively characterized the transcriptomic phenotype of modulated SMCs in vivo in atherosclerotic lesions of both mouse and human. We performed CITE-seq in mouse atherosclerotic lesions using antibodies directed against several macrophage surface markers. In the mice, we also performed SMC-specific knockout of TCF21, a causal CAD gene, to determine the effect of this gene on SMC phenotypic modulation. Finally, we performed ChIP-seq for the transcription factor TCF21 in a pooled DNA sample comprised of 52 different human coronary artery smooth muscle cell (HCASMC) lines to determine TCF21 target genes.
2019-07-29 | GSE131778 | GEO
Project description: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.
2015-03-19 | GSE44461 | GEO
Project description:Aryl-hydrocarbon receptor protects against endochondral ossification of modulated smooth muscle cells in atherosclerosis Introduction: Smooth muscle cells (SMC) play a critical role in atherosclerosis. The Aryl hydrocarbon receptor (AHR) is an environment-sensing transcription factor that contributes to vascular development, and has been implicated in coronary artery disease (CAD) risk. We hypothesized that AHR can affect atherosclerosis by regulating phenotypic modulation of SMC. Methods: We combined RNA-Seq, ChIP-Seq, ATAC-Seq and in-vitro assays in human coronary artery SMC (HCASMC), with single-cell RNA-Seq (scRNA-Seq), histology, and RNAscope in an SMC-specific lineage-tracing Ahr knockout mouse model of atherosclerosis to better understand the role of AHR in vascular disease. Results: Genomic studies coupled with functional assays in cultured HCASMC revealed that AHR modulates HCASMC phenotype and suppresses ossification in these cells. Lineage tracing and activity tracing studies in the mouse aortic sinus showed that the Ahr pathway is active in modulated SMC in the atherosclerotic lesion cap. Furthermore, scRNA-Seq studies of the SMC-specific Ahr knockout mice showed a significant increase in the proportion of modulated SMC expressing chondrocyte markers such as Col2a1 and Alpl, which localized to the lesion neointima. These cells, which we term chondromyocytes (CMC), were also identified in the neointima of human coronary arteries. In histological analyses, these changes manifested as larger lesion size, increased lineage-traced SMC participation in the lesion, decreased lineage-traced SMC in the lesion cap, and increased alkaline phosphatase activity in lesions in the Ahr knockout compared to wild-type mice. We propose that AHR is likely protective based on these data and inference from human genetic analyses. Conclusion: Overall, we conclude that AHR promotes maintenance of lesion cap integrity and diminishes the disease related SMC-to-CMC transition in atherosclerotic tissues.
2020-07-01 | GSE150768 | GEO
Project description:Recent meta-analyses of genome wide association studies (GWAS) have identified approximately 150 loci that are associated with coronary artery disease (CAD). To link the causal genes in these loci to functional transcriptional networks, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with human coronary artery smooth muscle cells (HCASMC) to investigate the cellular and molecular program downstream of one CAD associated transcription factor, TCF21. Analysis of the TCF21 downstream target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including growth factor binding (PDGF, VEGF), matrix interactions (“integrin binding,” “cell adhesion”), and smooth muscle contraction (“actin filament-based processes,” “actin cytoskeleton”). Motif searches of peak sequences confirmed the canonical E-box CAGCTG as the likely binding sequence for TCF21, but also identified bZip motifs that coordinate binding of AP1 family transcription factors. Follow-up ChIP-Seq studies verified enriched binding of bZip factors JUN and JUND to TCF21 target loci. Importantly, analysis of the representation of CAD genes among TCF21 target loci showed highly significant enrichment. Further, expression quantitative trait variation mapped to target genes of TCF21 were significantly enriched among variants with low P-values in the GWAS analyses, and single nucleotide polymorphisms within TCF21 peaks were shown to be in linkage disequilibrium with CAD-associated SNPs, suggesting a functional interaction between TCF21 binding and causal variants in other CAD disease loci. Thus, data and analyses presented here provide evidence for a transcriptional regulatory network that links TCF21 function in smooth muscle cells with a number of other CAD-associated genes, and suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology. We did ChIP-Seq on human coronary artery smooth muscle cells grown in SmGM-2 Smooth Muscle Growth Medium-2 including hEGF, insulin, hFGF-B and FBS, but without antibiotics (Lonza, #CC-3182. We did immunoprecipitations with two antibodies (Sigma HPA013189 and Abcam 49475). We conducted two biological replicates for each antibody, and also did an IgG control for these studies. For these experiments, sequencing was done on an Illumina GAII, single end reads. In separate set of experiments we performed ChIP-Seq for JUN (Ab sc-1694) and JUND (Ab sc-74) with the same HCASMC cells under the same culture conditions. For these studies we did one replicate for each antibody, and also included an IgG control. Sequencing for the JUN and JUND studies was paired ends, on an Illumina HiSeq machine.
2015-04-12 | E-GEOD-61369 | biostudies-arrayexpress
Project description:Genome-wide association studies (GWAS) for coronary artery disease (CAD, also termed coronary heart disease, CHD) have discovered and validated 48 loci genome-wide and recent 1000-Genomes based meta-analyses have discovered additional 8 loci with significant association, however, true follow-up studies on the mechanisms of association are still scarce. Here we analyze the relationship of two transcription factors, TCF21, one of the lead GWAS candidate genes for coronary artery disease and AHR, aryl hydrocarbon receptor, which previously was not implicated as fundamental for the atherosclerotic process. We show that both the predicted and in-vivo ChIP-Seq binding sites for TCF and AHR colocalize in the human genome with over 400 predicted sites and 119 ChIP-Seq sites directly overlapping. TCF and AHR-ARNT predicted binding sites longitudinal phasing was observed near the transcription start sites, outlining the position of +1 nucleosome. AHR-ARNT matrix was highly enriched in both TCF21 ChIP-Seq peaks and ATAC-Seq open chromatin regions in human coronary artery smooth muscle cells (HCASMC), implicating the role of AHR in this important cell type for vascular biology. Co-expression modules of TCF21 and AHR showed high degree of connectivity. Separation of rotationally phased and unphased predicted binding sites for TCF and AHR resolved the roles of direct and indirect TCF-AHR interactions, and implicated as highly-modulated various inflammatory, interleukin and cytokine related processes, while ChIP-Seq co-localization of TCF21 and AHR/ARNT emphasized the role of calcium related processes. We performed TCF21 overexpression analysis in human coronary artery smooth muscle cells (HCASMC) and obtained GO ontologies that recapitulate in vivo pathophysiology of the atherosclerotic vessel wall, and a set of chronic inflammatory ontologies was established with the colocalization of TCF21 and AHR ChIP-Seq sites as well as with the binomial testing of GWAS SNP overrepresentation. We experimentally confirmed that TCF21 binds to and regulates AHR gene expression in HCASMC, as well as to elements near AHR downstream genes, such as CYP1A1, using reporter assays. The functional relevance of AHR pathway in HCASMC is confirmed using AHR ligands TCDD and oxidized LDL. Finally, we show that AHR is elevated in atherosclerotic arteries using laser capture microdissection in mice in vivo and in human ex vivo. In conclusion, we extend GWAS results to functional assays and show that TCF21 and AHR functional connectivity provides a novel mechanism for diseased coronary vessel wall biology.
2017-04-12 | GSE85565 | GEO
Project description:Objective – Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no FDA-approved anti-calcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results – We performed an unbiased quantitative proteomics and pathway network analysis that identified increased carnitine O-octanoyltransferase (CROT) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA restored mitochondrial proteome alterations and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in low-density lipoprotein receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density, or liver and plasma cholesterol and triglyceride concentrations. Conclusions – CROT is a novel inducer of vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an anti-fibrocalcific therapy.
2021-02-26 | PXD022575 | Pride
Project description: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 remain poorly understood. Single cell RNA sequencing (scRNAseq) data generated with SmartSeq2 (~8000 genes/cell) in nearly 19,000 single cells isolated during atherosclerosis progression in 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 (CAD) patients in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study.Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by three smooth-muscle cells (SMC), and three macrophage (MP) subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type pro-inflammatory/Trem2-high lipid-associated (MP) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of three arterial wall GRNs: GRN33 (MP), GRN39 (SMC) and GRN122(MP) with major contributions to CAD heritability and strong associations with clinical scores of coronary atherosclerosis severity (SYNTAX/Duke scores). The presence and pathophysiological relevance of GRN39 was verified in five independent RNAseq datasets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM, in cultured human vascular SMCs.By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a CAD framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced-stage, symptomatic atherosclerosis.
2024-03-05 | GSE260657 | GEO
Project description: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 remain poorly understood. Single cell RNA sequencing (scRNAseq) data generated with SmartSeq2 (~8000 genes/cell) in nearly 19,000 single cells isolated during atherosclerosis progression in 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 (CAD) patients in the Stockholm-Tartu Atherosclerosis Reverse Network Engineering Task (STARNET) study.Advanced stages of atherosclerosis progression and symptomatic carotid plaques were largely characterized by three smooth-muscle cells (SMC), and three macrophage (MP) subtype clusters with extracellular matrix organization/osteogenic (SMC), and M1-type pro-inflammatory/Trem2-high lipid-associated (MP) phenotypes. Integrative analysis of these 6 clusters with STARNET revealed significant enrichments of three arterial wall GRNs: GRN33 (MP), GRN39 (SMC) and GRN122(MP) with major contributions to CAD heritability and strong associations with clinical scores of coronary atherosclerosis severity (SYNTAX/Duke scores). The presence and pathophysiological relevance of GRN39 was verified in five independent RNAseq datasets obtained from the human coronary and aortic artery, and primary SMCs and by targeting its top-key drivers, FRZB and ALCAM, in cultured human vascular SMCs.By identifying and integrating the most gene-rich single-cell subclusters of atherosclerosis to date with a CAD framework of GRNs, GRN39 was identified and independently validated as being critical for the transformation of contractile SMCs into an osteogenic phenotype promoting advanced-stage, symptomatic atherosclerosis.
2024-03-05 | GSE260656 | GEO