Centre for Clinical Pharmacology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, EC1M 6BQ, UK.
Institute of Bioengineering, School of Engineering and Materials Science, Queen Mary University of London, London, EC1M 6BQ, UK.
Stem Cell Res Ther. 2020 Nov 3;11(1):465. doi: 10.1186/s13287-020-01989-w.
Inflammatory smooth muscle cells (iSMCs) generated from adventitial stem/progenitor cells (AdSPCs) have been recognised as a new player in cardiovascular disease, and microRNA-214-3p (miR-214-3p) has been implicated in mature vascular SMC functions and neointimal hyperplasia. Here, we attempted to elucidate the functional involvements of miR-214-3p in iSMC differentiation from AdSPCs and unravel the therapeutic potential of miR-214-3p signalling in AdSPCs for injury-induced neointimal hyperplasia.
The role of miR-214-3p in iSMC differentiation from AdSPCs was evaluated by multiple biochemistry assays. The target of miR-214-3p was identified through binding site mutation and reporter activity analysis. A murine model of injury-induced arterial remodelling and stem cell transplantation was conducted to study the therapeutic potential of miR-214-3p. RT-qPCR analysis was performed to examine the gene expression in healthy and diseased human arteries.
miR-214-3p prevented iSMC differentiation/generation from AdSPCs by restoring sonic hedgehog-glioma-associated oncogene 1 (Shh-GLI1) signalling. Suppressor of fused (Sufu) was identified as a functional target of miR-214-3p during iSMC generation from AdSPCs. Mechanistic studies revealed that miR-214-3p over-expression or Sufu inhibition can promote nuclear accumulation of GLI1 protein in AdSPCs, and the consensus sequence (GACCACCCA) for GLI1 binding within smooth muscle alpha-actin (SMαA) and serum response factor (SRF) gene promoters is required for their respective regulation by miR-214-3p and Sufu. Additionally, Sufu upregulates multiple inflammatory gene expression (IFNγ, IL-6, MCP-1 and S100A4) in iSMCs. In vivo, transfection of miR-214-3p into the injured vessels resulted in the decreased expression level of Sufu, reduced iSMC generation and inhibited neointimal hyperplasia. Importantly, perivascular transplantation of AdSPCs increased neointimal hyperplasia, whereas transplantation of AdSPCs over-expressing miR-214-3p prevented this. Finally, decreased expression of miR-214-3p but increased expression of Sufu was observed in diseased human arteries.
We present a previously unexplored role for miR-214-3p in iSMC differentiation and neointima iSMC hyperplasia and provide new insights into the therapeutic effects of miR-214-3p in vascular disease.
已发现来源于外膜干细胞/祖细胞（AdSPCs）的炎性平滑肌细胞（iSMCs）是心血管疾病的新发病机制，微小 RNA-214-3p（miR-214-3p）已被证实参与成熟血管平滑肌细胞的功能和新生内膜增生。本研究旨在探讨 miR-214-3p 在 AdSPCs 向 iSMC 分化中的功能作用，并阐明 miR-214-3p 信号对 AdSPCs 在损伤诱导的新生内膜增生中的治疗潜力。
通过多种生物化学测定评估 miR-214-3p 在 AdSPCs 向 iSMC 分化中的作用。通过结合位点突变和报告基因活性分析确定 miR-214-3p 的靶标。通过损伤诱导的动脉重塑和干细胞移植的小鼠模型研究 miR-214-3p 的治疗潜力。通过 RT-qPCR 分析检测健康和患病人类动脉中的基因表达。
miR-214-3p 通过恢复 Sonic hedgehog-Glioma-associated oncogene 1（Shh-GLI1）信号来阻止 AdSPCs 向 iSMC 分化/生成。在 AdSPCs 向 iSMC 生成过程中，鉴定出 Suppressor of fused（Sufu）为 miR-214-3p 的功能靶标。机制研究表明，miR-214-3p 过表达或 Sufu 抑制可促进 AdSPCs 中 GLI1 蛋白的核积累，SMαA 和血清反应因子（SRF）基因启动子中 GLI1 结合的共有序列（GACCACCCA）对于它们分别由 miR-214-3p 和 Sufu 调节是必需的。此外，Sufu 上调 iSMCs 中多种炎性基因表达（IFNγ、IL-6、MCP-1 和 S100A4）。在体内，miR-214-3p 转染到损伤血管中导致 Sufu 表达水平降低，iSMC 生成减少，并抑制新生内膜增生。重要的是，外膜周围移植 AdSPCs 会增加新生内膜增生，而移植过表达 miR-214-3p 的 AdSPCs 则可预防这种情况。最后，在患病的人类动脉中观察到 miR-214-3p 表达降低但 Sufu 表达增加。
本研究揭示了 miR-214-3p 在 iSMC 分化和新生内膜 iSMC 增生中的新作用，并为 miR-214-3p 在血管疾病中的治疗作用提供了新的见解。
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