Department of Physiology and Pathophysiology, School of Basic Medical Sciences, Peking University; Key Laboratory of Molecular Cardiovascular Science, Ministry of Education, Beijing, P. R. China (Y.J., C.M., Z.M., J.H., W.L., X.M., S.Z., M.L., F.Y., J.F., Y.Z., Y.F., W.K.).
Beijing Institute of Biotechnology, Beijing, P. R. China (C.M.).
Circ Res. 2022 Oct 28;131(10):807-824. doi: 10.1161/CIRCRESAHA.122.321005. Epub 2022 Oct 6.
Phenotypic transition of vascular smooth muscle cells (VSMCs) accounts for the pathogenesis of a variety of vascular diseases during the early stage. Recent studies indicate the metabolic reprogramming may be involved in VSMC phenotypic transition. However, the definite molecules that link energy metabolism to distinct VSMC phenotype remain elusive.
A carotid artery injury model was used to study postinjury neointima formation as well as VSMC phenotypic transition in vivo. RNA-seq analysis, cell migration assay, collagen gel contraction assay, wire myography assay, immunoblotting, protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay were performed to clarify the phenotype and elucidate the molecular mechanisms.
We collected cell energy-regulating genes by using Gene Ontology annotation and applied RNA-Seq analysis of transforming growth factor-β or platelet-derived growth factor BB stimulated VSMCs. Six candidate genes were overlapped from energy metabolism-related genes and genes reciprocally upregulated by transforming growth factor-β and downregulated by platelet-derived growth factor BB. Among them, prohibitin 2 has been reported to regulate mitochondrial oxidative phosphorylation. Indeed, prohibitin 2-deficient VSMCs lost the contractile phenotype as evidenced by reduced contractile proteins. Consistently, mice were more susceptible to postinjury VSMC proliferation and neointima formation compared with mice. Further protein interactome analysis, co-immunoprecipitation, and mammalian 2-hybrid assay revealed that prohibitin 2, through its C-terminus, directly interacts with hnRNPA1, a key modulator of pyruvate kinase M1/2 (PKM) mRNA splicing that promotes PKM2 expression and glycolysis. Prohibitin 2 deficiency facilitated PKM1/2 mRNA splicing and reversion from PKM1 to PKM2, and enhanced glycolysis in VSMCs. Blocking prohibitin 2-hnRNPA1 interaction resulted in increased PKM2 expression, enhanced glycolysis, repressed contractile marker genes expression in VSMCs, as well as aggravated postinjury neointima formation in vivo.
Prohibitin 2 maintains VSMC contractile phenotype by interacting with hnRNPA1 to counteract hnRNPA1-mediated PKM alternative splicing and glucose metabolic reprogramming.
血管平滑肌细胞（VSMC）的表型转化是多种血管疾病早期发病机制的基础。最近的研究表明，代谢重编程可能与 VSMC 表型转化有关。然而，将能量代谢与不同的 VSMC 表型联系起来的确切分子仍然难以捉摸。
使用颈动脉损伤模型研究体内损伤后新生内膜的形成以及 VSMC 的表型转化。进行 RNA-seq 分析、细胞迁移试验、胶原凝胶收缩试验、线描肌描记术、免疫印迹、蛋白质相互作用分析、共免疫沉淀和哺乳动物 2 杂交试验，以阐明表型并阐明分子机制。
我们通过基因本体论注释收集了细胞能量调节基因，并对转化生长因子-β或血小板衍生生长因子 BB 刺激的 VSMC 进行了 RNA-Seq 分析。从能量代谢相关基因和转化生长因子-β上调而血小板衍生生长因子 BB 下调的基因中重叠了 6 个候选基因。其中，抑制素 2 已被报道可调节线粒体氧化磷酸化。事实上，抑制素 2 缺陷的 VSMC 失去了收缩表型，收缩蛋白减少。一致地，与 小鼠相比， 小鼠在损伤后 VSMC 增殖和新生内膜形成方面更易感性。进一步的蛋白质相互作用分析、共免疫沉淀和哺乳动物 2 杂交试验表明，抑制素 2 通过其 C 末端直接与 hnRNPA1 相互作用，hnRNPA1 是丙酮酸激酶 M1/2（PKM）mRNA 剪接的关键调节剂，促进 PKM2 表达和糖酵解。抑制素 2 缺陷促进了 PKM1/2 mRNA 剪接和从 PKM1 向 PKM2 的逆转，并增强了 VSMC 中的糖酵解。阻断抑制素 2-hnRNPA1 相互作用导致 PKM2 表达增加、糖酵解增强、VSMC 中收缩标志物基因表达受抑以及体内损伤后新生内膜形成加重。
抑制素 2 通过与 hnRNPA1 相互作用来维持 VSMC 的收缩表型，以拮抗 hnRNPA1 介导的 PKM 可变剪接和葡萄糖代谢重编程。
