Ou Tiantong, Yang Wenlong, Li Wenjia, Lu Yijing, Dong Zheng, Zhu Hongming, Sun Xiaolei, Dong Zhen, Weng Xinyu, Chang Suchi, Li Hua, Li Yufan, Qiu Zhiwei, Hu Kai, Sun Aijun, Ge Junbo
Department of Cardiology, Zhongshan Hospital, Fudan University, Shanghai Institute of Cardiovascular Diseases, Shanghai, China.
Institute of Biomedical Sciences, Fudan University, Shanghai, China.
Clin Transl Med. 2020 Sep;10(5):e172. doi: 10.1002/ctm2.172.
Mesenchymal stem cells (MSCs) have therapeutic potential for multiple ischemic diseases. However, in vitro expansion of MSCs before clinical application leads to metabolic reprogramming from glycolysis to oxidative phosphorylation, drastically impairing their proliferative and therapeutic capacities. This study aimed to define the regulatory effects of Sirtuin 5 (SIRT5) on the proliferative and therapeutic functions of adipose-derived MSCs (ADMSCs) during in vitro expansion.
ADMSCs were isolated from wild-type (WT) and Sirt5-knockout (Sirt5 ) mice. Cell counting assay was used to investigate the proliferative capacities of the ADMSCs. Dihydroethidium and senescence-associated β-galactosidase stainings were used to measure intracellular ROS and senescence levels. Mass spectrometry was used to analyze protein succinylation. Oxygen consumption rates and extra cellular acidification rates were measured as indicators of mitochondrial respiration and glycolysis. Metabolic-related genes expression were verified by quantitative PCR and western blot. Hind limb ischemia mouse model was used to evaluate the therapeutic potentials of WT and Sirt5 ADSMCs.
SIRT5 protein levels were upregulated in ADMCs during in vitro expansion. Sirt5 ADMSCs exhibited a higher proliferation rate, delayed senescence, and reduced ROS accumulation. Furthermore, elevated protein succinylation levels were observed in Sirt5 ADMSCs, leading to the reduced activity of tricarboxylic acid cycle-related enzymes and attenuated mitochondrial respiration. Glucose uptake, glycolysis, and pentose phosphate pathway were elevated in Sirt5 ADMSCs. Inhibition of succinylation by glycine or re-expression of Sirt5 reversed the metabolic alterations in Sirt5 ADMSCs, thus abolishing their enhanced proliferative capacities. In the hind limb ischemia mouse model, SIRT5 ADMSCs transplantation enhanced blood flow recovery and angiogenesis compared with WT ADMSCs.
Our results indicate that SIRT5 deficiency during ADMSC culture expansion leads to reversed metabolic pattern, enhanced proliferative capacities, and improved therapeutic outcomes. These data suggest SIRT5 as a potential target to enhance the functional properties of MSCs for clinical application.
间充质干细胞（MSCs）对多种缺血性疾病具有治疗潜力。然而，在临床应用前对MSCs进行体外扩增会导致其代谢从糖酵解重编程为氧化磷酸化，从而严重损害其增殖和治疗能力。本研究旨在确定沉默调节蛋白5（SIRT5）在体外扩增过程中对脂肪来源的间充质干细胞（ADMSCs）增殖和治疗功能的调节作用。
从野生型（WT）和Sirt5基因敲除（Sirt5-/-）小鼠中分离ADMSCs。采用细胞计数法研究ADMSCs的增殖能力。使用二氢乙锭和衰老相关β-半乳糖苷酶染色来测量细胞内活性氧（ROS）水平和衰老程度。采用质谱分析蛋白质琥珀酰化修饰。测量氧消耗率和细胞外酸化率作为线粒体呼吸和糖酵解的指标。通过定量PCR和蛋白质印迹法验证代谢相关基因的表达。使用后肢缺血小鼠模型评估WT和Sirt5-/-ADSMCs的治疗潜力。
在体外扩增过程中，ADMCs中SIRT5蛋白水平上调。Sirt5-/-ADMSCs表现出更高的增殖率、延缓的衰老以及减少的ROS积累。此外，在Sirt5-/-ADMSCs中观察到蛋白质琥珀酰化水平升高，导致三羧酸循环相关酶的活性降低以及线粒体呼吸减弱。Sirt5-/-ADMSCs中的葡萄糖摄取、糖酵解和磷酸戊糖途径增强。用甘氨酸抑制琥珀酰化或重新表达Sirt5可逆转Sirt5-/-ADMSCs中的代谢改变，从而消除其增强的增殖能力。在小鼠后肢缺血模型中，与WT ADMSCs相比，SIRT5-/-ADMSCs移植增强了血流恢复和血管生成。
我们的结果表明，ADMSC培养扩增过程中SIRT5缺乏导致代谢模式逆转、增殖能力增强以及治疗效果改善。这些数据表明SIRT5作为增强MSCs功能特性以用于临床应用的潜在靶点。
