Herein we employ Myh11-CreERT2 ROSA floxed STOP eYFP Apoe−/− smooth muscle cell (SMC) lineage tracing mice to show that traditional methods for detecting SMCs based on immuno-staining fail to detect > 80% of SMC-derived cells within advanced atherosclerotic lesions. These unidentified SMC-derived cells exhibit phenotypes of other cell lineages including macrophages (Mϕs), and mesenchymal stem cells (MSCs). SMC-specific conditional knockout (KO) of Krüppel-like factor 4 (KLF4) resulted in reduced numbers of SMC-derived MSC-, and Mϕ-like cells, marked reductions in lesion size, and increases in multiple indices of plaque stability, including an increase in fibrous cap thickness. Results of in vivo KLF4 ChIP-Seq analyses, and studies in cultured SMC treated with cholesterol identified > 800 KLF4 target genes including many that regulate pro-inflammatory responses of SMC. Results indicate that the contribution of SMCs within atherosclerotic plaques has been greatly underestimated, and that KLF4-dependent transitions in SMC phenotype are critical in lesion pathogenesis.

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.

There are controversial claims that the embryonic stem cell (ESC) pluripotency factor OCT4 is activated in somatic cells, but there is no evidence it plays a functional role in these cells. Herein we demonstrate that smooth muscle cell (SMC)-specific conditional knockout of Oct4 within Apoe−/− mice resulted in increased lesion size and changes consistent with decreased plaque stability including a thinner fibrous cap, increased necrotic core, and increased intra-plaque hemorrhage. Results of SMC-lineage tracing studies showed that these changes were likely due to marked reductions in SMC number within lesions including impaired SMC migration and investment within the fibrous cap. Re-activation of Oct4 within SMCs was associated with hydroxymethylation of the Oct4 promoter and was HIF1α- and KLF4-dependent. Results provide the first direct evidence that OCT4 plays a functional role in somatic cells and highlight the importance of further investigation of possible OCT4 functions in normal and diseased somatic cells.

Despite decades of research, our understanding of the processes controlling late-stage atherosclerotic plaque stability remains poor. A prevailing hypothesis is that reducing inflammation may improve advanced plaque stability, as recently tested in the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS) trial, in which post-myocardial infarction subjects were treated with an IL-1β antibody. Here, we performed intervention studies in which smooth muscle cell (SMC) lineage-tracing Apoe mice with advanced atherosclerosis were treated with anti-IL-1β or IgG control antibodies. Surprisingly, we found that IL-1β antibody treatment between 18 and 26 weeks of Western diet feeding induced a marked reduction in SMC and collagen content, but increased macrophage numbers in the fibrous cap. Moreover, although IL-1β antibody treatment had no effect on lesion size, it completely inhibited beneficial outward remodeling. We also found that SMC-specific knockout of Il1r1 (encoding IL-1 receptor type 1) resulted in smaller lesions nearly devoid of SMCs and lacking a fibrous cap, whereas macrophage-selective loss of IL-1R1 had no effect on lesion size or composition. Taken together, these results show that IL-1β has multiple beneficial effects in late-stage murine atherosclerosis, including promotion of outward remodeling and formation and maintenance of an SMC- and collagen-rich fibrous cap.

Stable atherosclerotic plaques are characterized by a thick extracellular matrix (ECM)-rich fibrous cap populated by protective ACTA2 + myofibroblast (MF)-like cells, assumed to be almost exclusively derived from smooth muscle cells (SMC). Herein, we show that in murine and human lesions, 20 to 40% of ACTA2 + fibrous caps cells, respectively, are derived from non-SMC sources, including endothelial cells (EC) or macrophages that have undergone Endothelial-to-Mesenchymal (EndoMT) or Macrophage-to-Mesenchymal (MMT) transitions. In addition, we show that SMC-specific knockout of the platelet derived growth factor receptor beta (PDGFRB) in Apoe −/− mice fed a Western diet (WD) for 18 weeks resulted in brachiocephalic artery (BCA) lesions nearly devoid of SMC but with no changes in lesion size, remodeling, or indices of stability including percent of ACTA2 + fibrous cap cells. However, prolonged WD feeding of SMC-PDGFRB knockout mice resulted in reduced indices of stability, indicating that EndoMT and MMT-derived MFs cannot compensate indefinitely for loss of SMC-derived MFs. Using single cell and bulk RNA-seq analyses of the BCA region and in vitro models, we provide evidence that SMC to MF transitions (SMC-MFT) are induced by PDGF and TFGβ and dependent on aerobic glycolysis, while EndoMT is induced by IL1β and TGFβ. Together, we provide evidence that the ACTA2 + fibrous cap originates from a tapestry of cell types, which transition to an MF state through distinct signaling pathways that are either dependent on or associated with extensive metabolic reprogramming.

Rationale: Coronary artery disease (CAD) is a major cause of morbidity and mortality worldwide. Recent genome-wide association studies (GWAS) 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 twelve atherosclerosis-relevant phenotypes related to calcification, proliferation, and migration in VSMCs isolated from 151 multi-ethnic 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 GWAS for twelve atherosclerosis-relevant phenotypes and identified four genome-wide significant loci associated with at least one VSMC phenotype. We overlapped the previously identified CAD GWAS loci with our dataset 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 SNP rs67180937 was associated with lower VSMC MIA3 expression and lower proliferation. Lentivirus-mediated silencing of MIA3 in VSMCs resulted in lower proliferation, consistent with human genetics findings. Further, 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. Further, 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.

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 ) we have previously identified a sequence variant associated with age-related atherosclerosis, ) COL15A1 is a matrix organizer enhancing tissue structural integrity, and) small interfering RNA-mediated 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 knockout mice fed a Western diet for 18 wk failed to form advanced lesions. SMC-specific 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 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. 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 knockout markedly attenuated advanced lesion formation, likely through reducing SMC proliferation and impairing multiple proatherogenic inflammatory processes.

Background and aims:The atherosclerotic plaque microenvironment is highly complex, and selective agents that modulate plaque stability are not yet available. We sought to develop a scRNA-seq analysis workflow to investigate this environment and uncover potential therapeutic approaches. We designed a user-friendly, reproducible workflow that will be applicable to other disease-specific scRNA-seq datasets. Methods: Here we incorporated automated cell labeling, pseudotemporal ordering, ligand-receptor evaluation, and drug-gene interaction analysis into a ready-to-deploy workflow. We applied this pipeline to further investigate a previously published human coronary single-cell dataset by Wirka et al. Notably, we developed an interactive web application to enable further exploration and analysis of this and other cardiovascular single-cell datasets.Results: We revealed distinct derivations of fibroblast-like cells from smooth muscle cells (SMCs), and showed the key changes in gene expression along their de-differentiation path. We highlighted several key ligand-receptor interactions within the atherosclerotic environment through functional expression profiling and revealed several avenues for future pharmacological development for precision medicine. Further, our interactive web application, PlaqView (www.plaqview.com), allows lay scientists to explore this and other datasets and compare scRNA-seq tools without prior coding knowledge. Conclusions: This publicly available workflow and application will allow for more systematic and user-friendly analysis of scRNA datasets in other disease and developmental systems. Our analysis pipeline provides many hypothesis-generating tools to unravel the etiology of coronary artery disease. We also highlight potential