Vascular aging is highly associated with cardiovascular morbidity and mortality. Although the senescence of vascular smooth muscle cells (VSMCs) has been well-established as a major contributor to vascular aging, intracellular and exosomal micro-RNA (miRNA) signaling pathways in senescent VSMCs have not been fully elucidated. This study aimed to identify the differential expression of intracellular and exosomal miRNA in human VSMCs (hVSMCs) during replicative senescence (RS). To achieve this aim, intracellular and exosomal miRNAs were isolated from hVSMCs and subsequently subjected to whole-genome small RNA next-generation sequencing, bioinformatics analyses and qPCR validation. Three significant findings were obtained. First, senescent hVSMC-derived exosomes tended to cluster together during RS and the molecular weight of the exosomal protein tumor susceptibility gene 101 (TSG-101) increased relative to the intracellular TSG101, suggesting potential posttranslational modifications of exosomal TSG-101. Secondly, there was a significant decrease in both intracellular and exosomal hsa-miR-155-5p expression (n = 3, FDR < 0.05), potentially being a cell type-specific biomarker of hVSMCs during RS. Importantly, hsa-miR-155-5p was found to associate with cell cycle arrest and elevated oxidative stress. Lastly, miRNAs from the intracellular pool, i.e. hsa-miR-664a-3p, hsa-miR-664a-5p, hsa-miR-664b-3p, hsa-miR-4485-3p, hsa-miR-10527-5p and hsa-miR-12136,and that from the exosomal pool, i.e. hsa-miR-7704, were upregulated in hVSMCs during RS (n = 3, FDR < 0.05). Interestingly, these novel upregulated miRNAs were not functionally well-annotated in hVSMCs to date. In conclusion, hVSMC- specific miRNA expression profiles during RS potentially provide valuable insights into the signaling pathways leading to vascular aging.

中文翻译：

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复制衰老过程中人血管平滑肌细胞的细胞内和外泌体 microRNA 组分析

血管老化与心血管发病率和死亡率高度相关。尽管血管平滑肌细胞 (VSMCs) 的衰老已被证实是导致血管老化的主要因素，但衰老 VSMCs 中的细胞内和外泌体 micro-RNA (miRNA) 信号通路尚未完全阐明。本研究旨在确定复制衰老 (RS) 期间人类 VSMC (hVSMC) 中细胞内和外泌体 miRNA 的差异表达。为了实现这一目标，从 hVSMC 中分离出细胞内和外泌体 miRNA，然后进行全基因组小 RNA 下一代测序、生物信息学分析和 qPCR 验证。获得了三个重要发现。第一的，衰老的 hVSMC 衍生的外泌体在 RS 期间倾向于聚集在一起，并且外泌体蛋白肿瘤易感基因 101 (TSG-101) 的分子量相对于细胞内 TSG101 增加，表明外泌体 TSG-101 的潜在翻译后修饰。其次，细胞内和外泌体 hsa-miR-155-5p 表达均显着降低（n = 3，FDR < 0.05），这可能是 RS 期间 hVSMC 的细胞类型特异性生物标志物。重要的是，发现 hsa-miR-155-5p 与细胞周期停滞和氧化应激升高有关。最后，来自细胞内池的 miRNA，即 hsa-miR-664a-3p、hsa-miR-664a-5p、hsa-miR-664b-3p、hsa-miR-4485-3p、hsa-miR-10527-5p 和 hsa -miR-12136 和来自外泌体库的 hsa-miR-7704，在 RS 期间在 hVSMC 中上调（n = 3，FDR < 0.05）。有趣的是，迄今为止，这些新的上调 miRNA 在 hVSMC 中并未在功能上得到很好的注释。总之，RS 期间 hVSMC 特异性 miRNA 表达谱可能为导致血管老化的信号通路提供有价值的见解。