Project description:IRF9 is ubiquitously expressed and mediates the effects of IFNs, previous study showed that IRF9 played an important role in immunity and cell fate decision. Our recent study revealed that IRF9 involved in cardiac hypertrophy, hepatic steatosis and insulin resistance. However, the function of IRF9 in VSMC and neointima formation was largely unknown. We found that IRF9 expression was significantly increased in the VSMCs of mouse carotid artery. More importantly, we generated SMC-specific IRF9 overexpression transgenic mice (IRF9 TG) and found that IRF9 TG significantly increased VSMC proliferation, migration and neointima formation compared with NTG mice in response to injury. To evaluate the underlying mechanism by which IRF9 promotes VSMC proliferation and migration after vascular injury, IRF9 TG and NTG mice were subjected to wire-injury and the carotid arteries were collected at 14 days post-injury. We combined 3-5 vessels for one sample, and 3 samples for each phenotype. Subsequently, a total of 400ng RNA was used following Affymetrix instruction and 10 ug of cRNA were hybridized for 16 hr at 45°. GeneChips were scanned using the Scanner 7G and the data was analyzed with Expression Console using Affymetrix default analysis settings and global scaling as normalization method. RMA analysis was employed to evaluate the gene expression. We used microarrays to detect the global gene expression in the carotid arteries of smooth muscle cell specific IRF9 transgenic mice(IRF9 TG) compared with non transgenic control mice (NTG) at 14 days post-injury and identified distinct classes of altered genes. non-transgenic controls mice (NTG) and smooth muscle specific IRF9 transgenic mice (IRF9 TG) were subjected to wire-injury and the carotid ateries were collected at 14 days post-injury. We combine 3-5 vessels in one tube and for a single Affymetrix microarray. Total RNA was extracted and a total of 400ng RNA was used following Affymetrix instruction. 3 biological samples for each genotype.

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: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: The phenotypic switching of vascular smooth muscle cells (VSMCs) plays a crucial role in vascular homeostasis. Protein-protein interactions (PPIs) mediated by PDZ domains is essential for cardiac functions. However, little is known about the role of PDZ proteins in regulating the phenotypic switching of VSMCs. In this study, we aim to explore the role of TAX1BP3, a singular PDZ protein, in maintaining the contractile phenotype of VSMCs. METHODS: Subcellular localization of TAX1BP3 was assessed in isolated VSMCs and arteries from mice or donors with in-stent restenosis and atherosclerosis. VSMC-specific Tax1bp3 knockout mice were generated to determine the relevant phenotypes in a carotid artery wire injury model. RNA sequencing, ATAC-sequencing, computational prediction of complex structures, and coCo-immunoprecipitation (Co-IP) were performed to elucidate the underlying molecular mechanisms. AAV-mediated Tax1bp3 gene delivery and recombinant TAX1BP3 were employed to investigate the potential translational relevance. RESULTS: TAX1BP3 exhibited dynamic nucleocytoplasmic shuttling during phenotypic switching of VSMCs. Deficiency of TAX1BP3 facilitated the transition from a contractile to a synthetic phenotype and aggravated neointima formation following vascular injury in mice. The integration of RNA sequencing and ATAC sequencing unveiled that TAX1BP3 primarily regulated the cell cycle progression and cell proliferation of VAMCs through YAP-TEAD transcription activity. The computational prediction of TAX1BP3/YAP1 complex structures and protein interaction related experiments revealed TAX1BP3 and TEAD1 compete for binding to YAP through its TEAD binding domain via a non-canonical PDZ manner. AAV-mediated Tax1bp3 gene delivery markedly attenuated post-injury neointima formation and the progression of atherosclerosis. Recombinant TAX1BP3 administration effectively reduced VSMCs proliferation and intimal hyperplasia following vascular injury in vitro and in vivo. CONCLUSIONS: Our results identified TAX1BP3 , a novel nucleocytoplasmic protein, a singular PDZ protein, that competitively interacts with YAP/-TEAD complex in a non-canonical PDZ manner and exerted its protective role in phenotypic switching and vascular intimal hyperplasia primarily through the regulation of cell proliferation.

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C-Kit is Essential for Vascular Smooth Muscle Cell Phenotypic Switch In Vitro And In Vivo After Injury

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