Exosomes secreted from bone marrow mesenchymal stem cells suppress cardiomyocyte hypertrophy through Hippo-YAP pathway in heart failure

Abstract Mesenchymal stem cells-derived exosomes (MSCs-exosomes) reportedly possess cardioprotective effects. This study investigated the therapeutic potential and mechanisms of MSCs-exosomes on heart failure (HF). H9c2 cells were used to establish a cardiomyocyte hypertrophy model by angiotensin II (Ang II) treatment. Isolated MSCs-exosomes were identified by transmission electron microscope and CD63 detection. Apoptosis rate was measured by terminal deoxynucleotidyl transferase (TdT) dUTP Nick-End Labeling (TUNEL) assay. Levels of inflammatory factors [interleukin (IL)-1β, IL-4, IL-6, and tumor necrosis factor (TNF)-α] and brain natriuretic peptide (BNP) were determined by ELISA. Expression of apoptosis-related proteins [Bax, B-cell lymphoma-2 (Bcl-2), and caspase 3] and Hippo-Yes-associated protein (YAP) pathway-related proteins [YAP, phosphor (p)-YAP, and tafazzin (TAZ)] was detected by western blotting. Cardiomyocyte hypertrophy of H9c2 cells induced by Ang II was ameliorated by MSCs-exosomes treatment. MSCs-exosomes downregulated Bax and caspase 3 levels and upregulated Bcl-2 level in Ang II-induced H9c2 cells. MSCs-exosomes also reduced the levels of BNP, IL-1β, IL-4, IL-6, and TNF-α in Ang II-induced H9c2 cells. Meanwhile, p-YAP was downregulated and TAZ was upregulated after MSCs-exosomes administration. In conclusion, MSCs-exosomes alleviate the apoptosis and inflammatory response of cardiomyocyte via deactivating Hippo-YAP pathway in HF.

摘要 已有研究证实，间充质干细胞来源外泌体（Mesenchymal stem cells-derived exosomes, MSCs-exosomes）具备心脏保护活性。本研究旨在探究MSCs-exosomes对心力衰竭（heart failure, HF）的治疗潜力及其潜在作用机制。
本研究通过血管紧张素II（angiotensin II, Ang II）处理H9c2细胞，构建心肌细胞肥大模型。采用透射电子显微镜与CD63分子标记检测，对分离获取的MSCs-exosomes进行身份鉴定。借助末端脱氧核苷酸转移酶dUTP缺口末端标记（terminal deoxynucleotidyl transferase TdT dUTP Nick-End Labeling, TUNEL）实验检测细胞凋亡率。通过酶联免疫吸附测定（enzyme-linked immunosorbent assay, ELISA）定量检测炎症因子[白细胞介素（interleukin, IL）-1β、IL-4、IL-6及肿瘤坏死因子（tumor necrosis factor, TNF）-α]与脑钠肽（brain natriuretic peptide, BNP）的表达水平。采用蛋白质印迹法（western blotting）检测凋亡相关蛋白[Bax、B细胞淋巴瘤-2（B-cell lymphoma-2, Bcl-2）及半胱氨酸天冬氨酸蛋白酶3（caspase 3）]与Hippo-Yes相关蛋白（Hippo-Yes-associated protein, YAP）通路相关蛋白[YAP、磷酸化YAP（phosphor p-YAP）及tafazzin (TAZ)]的表达水平。
实验结果表明，MSCs-exosomes处理可显著改善Ang II诱导的H9c2细胞心肌肥大表型。在Ang II诱导的H9c2细胞中，MSCs-exosomes可下调Bax与caspase 3的蛋白表达水平，并上调Bcl-2的蛋白表达水平。同时，MSCs-exosomes可降低Ang II诱导的H9c2细胞中BNP、IL-1β、IL-4、IL-6及TNF-α的分泌水平。此外，经MSCs-exosomes干预后，细胞内p-YAP的表达水平下调，而TAZ的蛋白表达水平上调。
综上，MSCs-exosomes可通过抑制Hippo-YAP信号通路，减轻心力衰竭过程中心肌细胞的凋亡与炎症反应，发挥心脏保护作用。