Project description:Aims: Vascular smooth muscle cells (VSMCs) accumulate in atherosclerotic plaques and exhibit remarkable phenotypic plasticity, contributing to both plaque growth and stability. The plaque-stabilising fibrous cap is rich in VSMC-derived cells, yet the cellular transitions and regulatory mechanisms governing fibrous cap formation remain unclear. We aimed to delineate the VSMC phenotypic transitions associated with this critical process. Methods and Results: Mapping of lineage-traced VSMCs during plaque development revealed investment of VSMCs prior to fibrous cap formation. Using single-cell RNA-sequencing (scRNA-seq) profiles of lineage-traced VSMCs from atherosclerotic and acutely injured mouse arteries, we identified a disease-specific VSMC state co-expressing contractile genes with extracellular matrix (ECM) components (including fibrillar collagens and elastin) and NOTCH3, which are associated with fibrous cap formation. Computational trajectory analysis predicted that this proposed fibrous cap-related VSMC (fcVSMC) state arises from a previously described plastic, intermediate VSMC population expressing SCA1 and VCAM1. Clonal analysis further showed that NOTCH3+ fcVSMCs derive from intermediate VSMCs in both atherosclerosis and an acute vascular injury model, suggesting a conserved disease-relevant mechanism. The fcVSMCs were enriched in plaque fibrous caps compared to lesion cores, consistent with a role in fibrous cap formation. By combining scRNA-seq trajectory analysis and spatial transcriptomics of human atherosclerotic plaques, we identified protease-activated receptor-1 (PAR1) as a candidate regulator of fcVSMC generation. PAR1 was expressed by VSMCs in human plaque fibrous caps and, functionally, PAR1 activation by thrombin induced expression of contractile genes and ECM components associated with the fcVSMC state in cultured human VSMCs. Conclusions: Our findings identify a VSMC transition linked to fibrous cap formation in atherosclerosis and show this is modelled by vascular injury. We identify VSMC-expressed PAR1 as a potential therapeutic target for promoting plaque stability by driving the transition to the matrix-producing, fibrous cap-associated VSMC state.

Project description:Background and Aims: Atherosclerosis, a major contributor to cardiovascular and cerebrovascular diseases, remains a leading cause of global mortality and morbidity. This study focuses on the role of chemokine-like receptor 1 (CMKLR1) in VSMCs and its potential impact on atherosclerotic plaque formation and remodelling. Methods: Cmklr1-/-Apoe-/- mice, generated using CRISPR/Cas9 technology, were fed a high-fat western diet for 16 weeks to model atherosclerosis. Primary VSMCs were isolated from Cmklr1-/- and Cmklr1+/+ mice for in vitro experiments. Cell viability, senescence-associated β-galactosidase staining, and RNA interference were performed to assess the effect of CMKLR1 deficiency on VSMCs. Various assays, including flow cytometry, immunocytochemistry, and RNA-seq analysis, were employed to investigate the molecular mechanisms and downstream signals associated with CMKLR1 deficiency. Results: CMKLR1 deficiency was found to disrupt the cell cycle, leading to VSMC senescence both in vitro and in vivo. Cmklr1-/-Apoe-/- mice exhibited increased atherosclerotic plaque burden, emphasizing the role of CMKLR1 in plaque stability. Transcriptome analysis revealed significant changes in cellular components and biological processes, with a particular impact on the extracellular matrix and cellular response to stimulus. The TGFβ signalling pathway emerged as a potential downstream target of CMKLR1, affecting VSMC differentiation, vessel development, and extracellular matrix synthesis. The deficiency of CMKLR1 led to a reduction in TGFβ1 expression and downstream molecule SM22α, influencing plaque composition and stability. Conclusion: This study demonstrates that CMKLR1 deficiency promotes VSMC senescence and exacerbates atherosclerosis. The disrupted TGFβ signalling pathway, identified as a potential molecular mechanism, provides insights into the complex regulatory network associated with CMKLR1 in atherosclerotic plaque formation and remodelling. These findings highlight the importance of CMKLR1 in atherosclerosis and its potential as a therapeutic target for plaque stability.

Project description:Vascular smooth muscle cell (VSMC) dysregulation is a hallmark of vascular disease, including atherosclerosis. In particular, the majority of cells within atherosclerotic lesions are generated from pre-existing VSMCs and a clonal nature has been documented for VSMC-derived cells in multiple disease models. However, the mechanisms underlying the generation of oligoclonal lesions and the phenotype of proliferating VSMCs are unknown.Here we analyse clonal dynamics in multi-color lineage-traced animals over time after vessel injury to understand the cellular mechanisms underlying clonal VSMC expansion in disease.We demonstrate that VSMC proliferation is initiated in a small fraction of VSMCs that initially expand clonally in the medial layer and then migrate to form the oligoclonal neointima. Selective activation of VSMC proliferation also occurs in vitro, suggesting that this is a cell-autonomous feature. Mapping of VSMC trajectories using single-cell RNA-sequencing reveals a continuum of cellular states after injury and suggests that VSMC proliferation initiates in cells that have downregulated the contractile phenotype and show evidence of pronounced phenotypic switching. We show that proliferation is associated with induced expression of stem cell antigen 1 (SCA1) and the expression signature previously identified in SCA1+ VSMCs in healthy arteries. A remarkably increased proliferation of SCA1+ VSMCs, directly validated in functional assays, indicates that SCA1+ VSMCs act as "first responders" in vascular injury. Early atherosclerotic lesions also had clonal VSMC contribution and we show that the proliferation-associated injury response is conserved in plaque VSMCs, extending these findings to atherosclerosis. Finally, we identify VSMCs in healthy human arteries that correspond to the SCA1+ state in mouse VSMCs and show that genes identified as differentially expressed in this human VSMC subpopulation are enriched for genes showing genetic association with cardiovascular disease. We show that cell-intrinsic, selective VSMC activation drives clonal proliferation after injury and in atherosclerosis. Our study suggests that healthy mouse and human arteries contain VSMCs characterised by expression of disease-associated genes that are predisposed for proliferation. Targeting such "first responder" cells in patients undergoing vascular surgery could effectively prevent injury-associated VSMC activation and neoatherosclerosis.

Project description:BACKGROUND: Vascular smooth muscle cells (VSMC) are the most abundant cell type in the artery’s media layer and regulate vascular tone and lesion remodeling during atherogenesis. Like monocyte-derived macrophages, VSMCs accumulate excess lipids and contribute to the total intimal foam cell population in human coronary plaque and mouse aortic atheroma. While there are extensive studies characterizing the contribution of lipid metabolism in macrophage immunometabolic responses in atherosclerotic plaques, the role of VSMC lipid metabolism in regulating vascular function and lesion remodeling in vivo remains poorly understood. METHODS: We re-analyzed publicly available single-cell RNA sequencing (scRNA-seq) data from mouse atherosclerosis studies and found Liver X receptor (LXR) signaling in VSMCs was continuously activated during atherogenesis. We thus generated mice with an inducible SMC-specific knockout of LXR/LXR on a fate mapping background by crossbreeding LXRf/fLXRf/f mice with Myh11-CreERT2;mTmGf/f mice. Mice were administered with tamoxifen (1 mg/day) for 10 consecutive days and followed by a 2-week resting period to induce CreERT2 mediated recombination. To induce hypercholesterolemia and atherosclerosis, we injected Myh11-CreERT2;mT/mGf/f;LXRf/fLXRf/f mice and Myh11-CreERT2;mT/mGf/f mice with AAV8-PCSK9 and fed them with a Western diet (WD) for 16 weeks. Metabolic, immunocytochemistry and genomic analysis were conducted to assess lesion size and morphology and unravel the molecular mechanism by which LXR in VSMC contributes to lesion formation. RESULTS: Deficiency of LXR in SMCs under hypercholesterolemic condition influenced lesion remodeling by altering the fate of de-differentiated SMC and promoting the accumulation of VSMC-derived transitional cells. This phenotypic switching is accompanied with reduced index of plaque stability characterized by larger necrotic core area and reduced fibrous cap thickness. Moreover, SMC-specific LXR deficiency impaired vascular function and caused visceral myopathy characterized by bladder maladaptive remodeling and gut lipid malabsorption. CONCLUSIONS: We unveil an essential role of hypercholesterolemia-induced LXR signaling in atherosclerotic lesion remodeling, controlling the fate decision of VSMCs and regulating vascular and visceral SMCs functions.

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Vascular Smooth Muscle Cell-derived KIF13B Protects Against Atherosclerosis: Evidence from Humans and Mice

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