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.

Project description:BACKGROUND: Neointimal hyperplasia is the major cause of significant vascular complications following arterial interventions. Despite the advancements in strategies such as drug-eluting stents (DES) to minimize neointimal hyperplasia, achieving consistently effective long-term outcomes remains a challenge. Protein-protein interactions (PPIs) mediated by PDZ domains are essential for numerous biological processes. However, little is known about the role of PDZ proteins in neointima formation. This study aims to explore the role of TAX1BP3, a singular PDZ protein, in phenotypic switching of VSMCs and its implication in neointimal hyperplasia. METHODS: Subcellular localization of TAX1BP3 was assessed in isolated VSMCs or arteries obtained from mice with neointima formation. TAX1BP3 mutants were constructed to study the role of SUMOylation on TAX1BP3 nucleocytoplasmic shuttling. 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 Co-immunoprecipitation (Co-IP) were performed to elucidate the underlying molecular mechanisms. AAV-mediated Tax1bp3 gene delivery and engineered AIENPs-TAX1BP3 were employed to investigate the potential translational relevance. RESULTS: TAX1BP3 exhibited dynamic nucleocytoplasmic shuttling during phenotypic switching of VSMCs. TAX1BP3 is SUMOylated at K116 and its SUMOylation is essential for maintaining the nuclear localization of TAX1BP3. Deficiency of TAX1BP3 facilitated the transition from a contractile to 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 VSMCs 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 significantly attenuated post-injury neointima formation and the progression of atherosclerosis. AIENPs-TAX1BP3 administration effectively reduced VSMCs phenotypic switching and neointimal hyperplasia. CONCLUSIONS: These results demonstrated SUMOylation of TAX1BP3 at K116 enables its nucleocytoplasmic shuttling during phenotypic switching of VSMCs. TAX1BP3 competitively interacts with YAP-TEAD complex in a non-canonical PDZ manner and exerted its protective role in vascular neointimal hyperplasia primarily through the regulation of cell proliferation.

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.

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Nucleocytoplasmic Shuttling of TAX1BP3: an Indispensable Player in Vascular Proliferative Diseases by Regulating YAP-TEAD Transcriptional Activity [RNA-seq]

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