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EHMT1/KMT1D drives neointima formation via epigenetic repression of the PPARγ–PDIA6 axis

ABSTRACT: Neointima formation underlies restenosis and other occlusive vascular diseases, yet the epigenetic regulators that drive this process remain poorly understood. We aimed to define the role of the histone methyltransferase EHMT1 in neointima formation and to delineate its downstream mechanisms. Here, we found that the EHMT1 expression and its histone marks H3K9me1/2 were markedly upregulated in proliferating VSMCs and in injured arteries exhibiting neointima formation. Deletion of EHMT1 in VSMCs suppressed phenotypic switching and significantly reduced neointimal hyperplasia in vivo. In vitro, EHMT1 knockdown inhibited, whereas EHMT1 overexpression promoted, VSMC proliferation, migration, and acquisition of a synthetic phenotype. Unbiased transcriptome sequencing revealed significant enrichment of peroxisome proliferator-activated receptor gamma (PPARγ) target genes following EHMT1 knockdown. Mechanistically, we demonstrated that EHMT1 directly repressed PPARγ expression via H3K9 dimethylation, and pharmacological activation of PPARγ with rosiglitazone reversed the proliferative, migratory, and synthetic effects induced by EHMT1 overexpression. Further analysis identified protein disulfide isomerase A6 (PDIA6) as a direct PPARγ target: PPARγ bound to the PDIA6 promoter (-567 to -385 bp) to suppress its expression. Overexpression of PDIA6 rescued the inhibitory effects of EHMT1 knockdown on VSMC proliferation and migration, whereas PDIA6 silencing abrogated the pro-proliferative and pro-migratory effects of EHMT1 overexpression. EHMT1 acts as a critical epigenetic regulator of neointima formation by controlling VSMC phenotypic switching through a novel EHMT1–PPARγ–PDIA6 axis. These findings provide mechanistic insight into epigenetic regulation of vascular remodeling and identify EHMT1–PPARγ–PDIA6 as a promising target for therapeutic intervention in restenosis.

ORGANISM(S): Homo sapiens

PROVIDER: GSE333141 | GEO | 2026/07/13

REPOSITORIES: GEO

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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:Pathological vascular remodeling—central to restenosis, atherosclerosis and vascu-lo-proliferative diseases—depends on phenotypic switching of vascular smooth muscle cells (VSMCs) from a quiescent, contractile state to a synthetic, proliferative program. Although the receptor tyrosine kinase c-KIT is implicated in proliferation, migration, and tissue repair, its role in VSMC plasticity has yet to be fully understood. Using c-Kit haploinsufficient mice subjected to right carotid artery ligation (CAL) and primary aortic VSMC cultures, we show that c-Kit is required for the contractile-to-synthetic transition. In vitro, c-Kit haploinsufficiency halved c-Kit expression, reduced BrdU incorporation, and blunted PDGF-BB–induced repression of contractile genes. c-Kit–deficient VSMCs ex-hibited a senescence program with increased p16INK4a/p21 expression and up-regulated senescence-associated secretory phenotype (SASP) mediators. RNA-seq of carotid arteries 7 days post-ligation revealed that wild-type arteries activated cell-cycle pathways and suppressed contractile signatures, whereas c-Kit–deficient carotid arteries failed to fully engage proliferative programs and instead maintained contractile gene expression. At 28 days post CAL in vivo, c-Kit haploinsufficiency produced markedly reduced neointima, fewer Ki67+ VSMCs, more p16INK4a+ cells, and impaired re-endothelialization. Because progenitor-to-VSMC differentiation contributes to remodeling, we tested adult cardiac stem/progenitor cells (CSCs) as a model system of adult progenitor differentiation. Wild-type CSCs efficiently generated induced VSMCs (iVSMCs) with appropriate smooth-muscle gene up-regulation; c-Kit–deficient rarely did so. Restoring c-Kit with a BAC transgene rescued both smooth-muscle differentiation and proliferative competence of c-Kit–deficient iVSMCs. Collectively, our data identify c-Kit as a gatekeeper of repar-ative VSMC plasticity. Adequate c-Kit enables progenitor-to-VSMC commitment and expansion of newly formed VSMCs while permitting injury-induced proliferation and matrix synthesis; reduced c-Kit locks cells in a hypercontractile, senescence-prone state and limits neointima formation. Modulating the c-Kit axis may therefore offer a strategy to fine-tune vascular repair while mitigating pathological remodeling.

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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EHMT1/KMT1D drives neointima formation via epigenetic repression of the PPARγ–PDIA6 axis

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