Zhang Xiaoyu, Zhou Yu, Ye Yanchen, Wu Ridong, Li Wen, Yao Chen, Wang Shenming
Division of Vascular Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China; National-Guangdong Joint Engineering Laboratory for Diagnosis and Treatment of Vascular Diseases, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China.
Laboratory of General Surgery, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, China.
Arch Biochem Biophys. 2022 Apr 15;719:109155. doi: 10.1016/j.abb.2022.109155. Epub 2022 Feb 24.
Restenosis is inevitable when patients undergo percutaneous transluminal angioplasty due to neointimal hyperplasia (NIH). Human umbilical cord mesenchymal stem cell-derived exosomes (hucMSC-Exos) have been studied in the field of cardiovascular diseases. However, the effects and mechanisms of hucMSC-Exos on NIH are unclear. We aimed to investigate whether MSC-Exos regulate vascular smooth muscle cell (VSMC) functions to inhibit NIH and explore the underlying mechanisms.
HucMSCs and mouse VSMCs were isolated and characterized by flow cytometry and immunofluorescence. HucMSC-Exos were identified by transmission electron microscopy, nanoparticle tracking analysis and western blots. Exosomes (Exos) were intravenously injected into mice with left common carotid artery ligation, and their effects on NIH were assessed by haematoxylin and eosin (H&E) and immunohistochemistry staining. The effects of hucMSC-Exos on VSMCs were evaluated by Cell Counting Kit-8, scratch wound, Transwell and Western blot assays. MicroRNA sequencing data in the Gene Expression Omnibus and mRNA sequencing results were used to identify potential molecules in hucMSC-Exos and target genes in VSMCs, respectively. We tested the regulatory effect of microRNAs in Exos and target genes in VSMCs using overexpression and knockdown experiments.
Primary hucMSCs, VSMCs and hucMSC-Exos were isolated and characterized. Administration of hucMSC-Exos suppressed NIH after artery ligation. H&E and immunohistochemistry results showed that hucMSC-Exos decreased the intima and media area and intima/media ratio, increased the contractile phenotype protein SM22a in the media layer and downregulated Serpine1 expression in the carotid artery. Exos were ingested by VSMCs, which inhibited migration and upregulated SM22a expression by suppressing Serpine1 expression in vitro. MiR-148a-3p was enriched in hucMSC-Exos and repressed Serpine1 by targeting its 3' untranslated region. Moreover, exosomal miR-148a-3p suppressed VSMC phenotypic switching and migration by targeting Serpine1.
We found that hucMSC-Exos inhibited NIH in a mouse carotid artery ligation model and that the inhibitory effects on VSMC phenotypic switching and migration were mediated by delivery of miR-148a-3p to VSMCs to target Serpine1.
由于新生内膜增生（NIH），患者接受经皮腔内血管成形术时再狭窄不可避免。人脐带间充质干细胞来源的外泌体（hucMSC-Exos）已在心血管疾病领域得到研究。然而，hucMSC-Exos对NIH的影响和机制尚不清楚。我们旨在研究间充质干细胞外泌体是否调节血管平滑肌细胞（VSMC）功能以抑制NIH，并探索潜在机制。
分离人脐带间充质干细胞和小鼠血管平滑肌细胞，并通过流式细胞术和免疫荧光进行鉴定。通过透射电子显微镜、纳米颗粒跟踪分析和蛋白质印迹法鉴定hucMSC-Exos。将外泌体（Exos）静脉注射到左颈总动脉结扎的小鼠体内，并通过苏木精和伊红（H&E）染色及免疫组织化学染色评估其对NIH的影响。通过细胞计数试剂盒-8、划痕伤口、Transwell和蛋白质印迹分析评估hucMSC-Exos对血管平滑肌细胞的影响。分别使用基因表达综合数据库中的微小RNA测序数据和mRNA测序结果来鉴定hucMSC-Exos中的潜在分子和血管平滑肌细胞中的靶基因。我们使用过表达和敲低实验测试了外泌体中微小RNA对血管平滑肌细胞中靶基因的调节作用。
分离并鉴定了原代人脐带间充质干细胞、血管平滑肌细胞和hucMSC-Exos。给予hucMSC-Exos可抑制动脉结扎后的NIH。H&E染色和免疫组织化学结果显示，hucMSC-Exos减小了内膜和中膜面积以及内膜/中膜比值，增加了中膜层收缩表型蛋白SM22a的表达，并下调了颈动脉中Serpine1的表达。血管平滑肌细胞摄取了外泌体，外泌体在体外通过抑制Serpine1表达抑制了血管平滑肌细胞的迁移并上调了SM22a的表达。MiR-148a-3p在hucMSC-Exos中富集，并通过靶向Serpine1的3'非翻译区抑制Serpine1。此外，外泌体miR-148a-3p通过靶向Serpine1抑制血管平滑肌细胞表型转换和迁移。
我们发现hucMSC-Exos在小鼠颈动脉结扎模型中抑制了NIH，并且对血管平滑肌细胞表型转换和迁移的抑制作用是通过将miR-148a-3p递送至血管平滑肌细胞以靶向Serpine1来介导的。
