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: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: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:Atherosclerosis represents an age-related disease, which remains one of the leading cause of deaths in developed countries. It begins with a local deposition of lipid in the innermost part of the artery – intima. Thereafter a number of immune cells are attracted and infiltrate into the artery wall, where, together with endothelia and smooth muscle cells, form an atherosclerotic plaque (Libby, 2008). Thus, atherosclerosis is considered as an inflammatory disease, characterized by intense immunological activity (Libby P, 2012). One of the main risk factor for atherosclerosis is aging. It was demonstrated that there is an accumulation of senescent cells within atherosclerotic plaque (..). Accordingly, vascular smooth muscle cells (VSMCs) derived from the lesion demonstrate a phenotype characteristic for senescent cells: diminished proliferative potential, shorter telomeres, increased number of DNA damage and upregulation of SA-β-gal activity (Gorenne, 2006; Matthes, 2006, Mahomoudi, 2008). Accumulation of senescent VSMCs within the area of plaque development promotes its instability due to lack of proliferation and impaired production of extracellular matrix, both leading to weakening of fibrous cap (Gorenne, 2006). This has been further confirmed by the observation, that elimination of senescent cells in the atherosclerosis-prone low-density lipoprotein receptor-deficient (Ldlr-/-) mice slowed down the disease progression (27789842). One of the most important feature of senescent cells, that has the biggest impact on tissue microenvironment, is their ability to secrete a number of cytokines, chemokines, matrix metalloproteinases and growth factors collectively known as the Senescence Associated Secretory Phenotype (SASP) (20078217, 28165648). Apart from soluble factors, senescent cells secret also an increased number of extracellular vesicles (EVs), research of which has only recently begun (32461143). EVs are small membranous vesicles that can be categorized based on their size and origin, into: exosomes, microvesicles and apoptotic bodies. EVs play crucial role as carriers of proteins, different types of RNA and DNA, lipids and metabolites that, take part in intercellular communication (23420871). They were shown to participate in many physiological but also pathological conditions. Studies performed over the last decade proved that EVs affect atherosclerosis progression at different stages by several distinct mechanisms (as reviewed by Knokhot, 2020, 33261815). The increased concentrations of EVs have been found in plasma of patients suffering from cardiovascular diseases as well as in atherosclerotic plaque itself (Yin, 2015, 26142082; Rautou, 2011, 21852557). However, the role of EVs derived from senescent cells in atherosclerosis, particularly in the context of the plaque development, remains unknown. The functioning of immune cells within atherosclerotic plaque is modulated by local milieu. Thus, senescent cells present in the lesion can influence plaque development by non-cell autonomous mechanism. Indeed, recently it was demonstrated that SASP factors secreted by senescent VSMCs promote recruitment of monocytes, induce expression of adhesion receptors on endothelial cells and activate adjacent normal VSMCs, promoting inflammation in the plaque (Gardner, 2015). However, the role of EVs in this context has not been studied. Thus, we have undertaken research aimed at investigating the role of senescent cell derived EVs on immune cells activity. To this end, we isolated and characterized the EVs secreted by human VSMCs undergoing senescence. We performed unbiased quantitative proteomic analysis of both soluble and insoluble SASP components. Moreover, we analyzed the influence of EVs derived from senescent and non-senescent cells on T lymphocytes and monocytes. Altogether our studies revealed that EVs produced by senescent VSMCs strengthen inflammatory response of the immune cells, what would have a tremendous significance in atherosclerotic plaque development.

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.

Project description:Rationale: Neointima formation is a common pathological feature of atherosclerosis and restenosis after angioplasty and involves the proliferation and migration of vascular smooth muscle cells (VSMCs). N6-methyladenosine (m6A), the most prevalent mRNA internal modification and being proposed to be primarily produced by RNA methyltransferase METTL3, plays a vital role in post-transcriptional regulations. Nevertheless, the role of RNA m6A modification in VSMCs and neointima formation remains disputable and undetermined. Objective: To determine the role of METTL3 and its produced RNA modification m6A in VSMCs and neointima formation after vascular injury. Methods and Results: We examined the expression of m6A writers and erasers in the carotid artery collected from human carotid endarterectomy (CEA) as well as in that of mice and unanimously found that METTL3 expression is increased significantly after vascular injury. Then, VSMC-confined METLL3 knockout mice (Myh11CreERT2 METTL3flox/flox) were generated, and carotid artery injury was induced. METTL3 knockout markedly attenuated artery neointima formation induced by wire injury. Moreover, we discovered that METTL3 deficiency repressed both ex vivo and in vivo proliferation of VSMCs. Through a joint analysis of the data from bulk RNA and m6A sequencing, serum- and glucocorticoid-inducible kinase 1 (SGK1) was identified due to its well-documented role in promoting VSMC proliferation and migration. Mechanistically, METTL3-mediated SGK1 mRNA methylation (A146 and A210) was proposed to facilitate SGK1 transcription by recruiting the m6A reader YTHDC1, as shown by well-designed experiments involving methylation site mapping, m6A RNA immunoprecipitation (m6A-RIP), chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) and reporter gene analysis. As anticipated, VSMC proliferation and intimal hyperplasia that had already been mitigated by METTL3 ablation were both restored by SGK1 overexpression. Conclusions: Our findings suggest that METTL3 promotes SGK1 expression via mRNA methylation-mediated facilitation of its own transcription, thus predisposing VSMCs to a proliferative state and contributing to neointima formation after vascular injury, underscoring the essential role METTL3 plays in vascular remodeling.

Project description:Atherosclerosis is a chronic inflammatory disease of the blood vessels, characterized by atherosclerotic lesions. Early vascular injury initiates excessive inflammatory and immune responses, communicated by blood leukocytes and cells from the vasculature. Key factors contributing to early stages of atherosclerosis and plaque development include the pro-inflammatory cytokine IFNγ, which is vital for both innate and adaptive immunity and is also expressed at high levels in atherosclerotic lesions. Recent discoveries provide novel evidence to suggest that IFNγ-activated STAT1 orchestrates a platform of cell-type common and specific inflammatory responses in Vascular Smooth Muscle Cells (VSMC) and macrophages (MΦ) that are instrumental in the onset and progression of atherosclerotic plaque formation. Therefore, we compared genome-wide transcriptional responses of mouse VSMCs and bone marrow-derived macrophages (BMDMs) in time-dependent IFNγ response using RNA-seq. We identified genes upregulated both in a common and cell-type-specific manner. Next, comparative analysis with RNAseq data from aortic plaques from HFD-treated ApoEKO mice identified 225 differentially expressed genes with a BMDM-specific character. These included many known IFNγ target genes containing putative GAS and ISRE sites in their promoters. Likewise, using a scRNAseq data set obtained from human coronary atherosclerotic plaques, identified 98 pro-inflammatory and pro-atherogenic genes that were characterized as BMDM-specific and exclusively expressed in foamy, inflammatory, monocytes and tissue-resident BMDM subtypes. Finally, we identified a 25 BMDM-specific gene subset commonly expressed in mouse and human plaques, over-representing STAT1-binding sites and predominantly associated with Leukocyte Chemotaxis and Migration. These results provide strong evidence for a BMDM-specific role of STAT1 in coronary artery plaque development and identify a BMDM-specific STAT1-dependent pro-inflammatory gene signature that could serve to monitor and diagnose plaque progression in human atherosclerosis.

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Premature cell senescence promotes vascular smooth muscle cell phenotypic modulation in atherosclerosis and after injury

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