Peking University People's Hospital, Peking University Institute of Hematology, Beijing Key Laboratory of Hematopoietic Stem Cell Transplantation, Peking University , Beijing, 100044, China.
Department of Cardiology, The General Hospital of the PLA Rocket Force, Beijing, 100088, China.
Stem Cell Res Ther. 2018 Oct 25;9(1):284. doi: 10.1186/s13287-018-1028-5.
Treatment with bone marrow mesenchymal stem cells (BM-MSCs) has been demonstrated to be an excellent cellular-based therapeutic strategy for treating myocardial infarction (MI). However, most of the patients suffering with MI are elderly. Hypoxic conditions can cause apoptosis of BM-MSCs, and this type of apoptosis is more prevalent in aged BM-MSCs. Decreased autophagy is one of the mechanisms underlying aging. The aim of this study is to uncover whether the increased hypoxic injury of aged BM-MSCs is due to autophagy and whether reducing autophagy diminishes the tolerance of hypoxia in aged BM-MSCs.
Young and aged BM-MSCs were isolated from male young and aged GFP/Fluc transgenic C57BL/6 mice respectively and then exposed to hypoxia and serum deprivation (H/SD) injury. The apoptosis level induced by H/SD was measured by terminal deoxynucleotidy transferase-mediated dUTP nick end-labeling (TUNEL) assay. Additionally, autophagy was analyzed via transfection with plasmids encoding green fluorescent protein-microtubule-associated protein lightchain3 (GFP-LC3), and autophagic vacuoles were visualized with transmission electron microscopy. Meanwhile, protein expression was measured by western blot analysis. Autophagic activity was manipulated by the administration of IGF-1 (insulin-like growth factor siRNA) and 3-methyladenine (3MA). Furthermore, young, aged, and the IGF-1 siRNA-transfected aged BM-MSCs were transplanted to myocardial infarcted adult C57BL/6 mice respectively. In vivo longitudinal in vivo bioluminescence imaging (BLI) of transplanted BM-MSCs was performed to monitor the survival of transplanted BM-MSCs in each groups.
Aged BM-MSCs exhibited a higher rate of apoptosis compared with young BM-MSCs under hypoxic conditions. Additionally, the level of autophagy was lower in aged BM-MSCs compared with young BM-MSCs under normoxic and hypoxic conditions. Meanwhile, hypoxia decreased the activity of the protein kinase B (Akt) and mammalian target of rapamycin (mTOR) signaling pathway in young and aged BM-MSCs, but aged BM-MSCs exhibited a relatively stronger Akt/mTOR activity compared with young BM-MSCs. In addition, IGF-1 knockdown significantly decreased the level of apoptosis in aged BM-MSCs under normoxic and hypoxic conditions. IGF-1 knockdown also decreased the activity of the Akt/mTOR signaling pathway and increased the level of autophagy in aged BM-MSCs under hypoxic condition. Furthermore, IGF-1 knockdown protected aged BM-MSCs from hypoxic injury by increasing the level of autophagy, thereby promoting the survival of aged BM-MSCs after myocardial infarction transplantation.
This study demonstrates that reducing autophagy decreases the hypoxia tolerance of aged BM-MSCs. Maintaining optimal levels of autophagy may serve as a new strategy in treating MI by BM-MSC transplantation in aged patients.
骨髓间充质干细胞（BM-MSCs）的治疗已被证明是治疗心肌梗死（MI）的一种极好的细胞治疗策略。然而，大多数患有 MI 的患者都是老年人。缺氧条件会导致 BM-MSCs 凋亡，而这种类型的凋亡在老年 BM-MSCs 中更为普遍。自噬减少是衰老的机制之一。本研究旨在揭示老年 BM-MSCs 中缺氧损伤的增加是否归因于自噬，以及减少自噬是否会降低老年 BM-MSCs 对缺氧的耐受性。
从雄性年轻和年老 GFP/Fluc 转基因 C57BL/6 小鼠中分别分离年轻和年老的 BM-MSCs，然后将其暴露于缺氧和血清剥夺（H/SD）损伤中。通过末端脱氧核苷酸转移酶介导的 dUTP 缺口末端标记（TUNEL）测定法测量 H/SD 诱导的细胞凋亡水平。此外，通过转染编码绿色荧光蛋白-微管相关蛋白轻链 3（GFP-LC3）的质粒分析自噬，并用透射电子显微镜观察自噬小体。同时，通过 Western blot 分析测量蛋白质表达。通过 IGF-1（胰岛素样生长因子 siRNA）和 3-甲基腺嘌呤（3MA）处理来操纵自噬活性。此外，分别将年轻、年老和 IGF-1 siRNA 转染的年老 BM-MSCs 移植到心肌梗死的成年 C57BL/6 小鼠中。通过体内纵向活体生物发光成像（BLI）监测各组移植 BM-MSCs 的存活情况。
与年轻 BM-MSCs 相比，缺氧条件下老年 BM-MSCs 的凋亡率更高。此外，与年轻 BM-MSCs 相比，在常氧和缺氧条件下，老年 BM-MSCs 的自噬水平较低。同时，缺氧降低了年轻和年老 BM-MSCs 中蛋白激酶 B（Akt）和哺乳动物雷帕霉素靶蛋白（mTOR）信号通路的活性，但与年轻 BM-MSCs 相比，老年 BM-MSCs 表现出相对更强的 Akt/mTOR 活性。此外，IGF-1 敲低显着降低了常氧和缺氧条件下老年 BM-MSCs 的细胞凋亡水平。IGF-1 敲低还降低了缺氧条件下老年 BM-MSCs 的 Akt/mTOR 信号通路活性并增加了自噬水平。此外，IGF-1 敲低通过增加自噬来保护老年 BM-MSCs 免受缺氧损伤，从而促进心肌梗死移植后老年 BM-MSCs 的存活。
本研究表明，减少自噬会降低老年 BM-MSCs 的耐缺氧性。维持最佳的自噬水平可能成为一种新策略，通过 BM-MSC 移植治疗老年患者的 MI。
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