Bader Andreas Matthaeus, Brodarac Andreja, Klose Kristin, Bieback Karen, Choi Yeong-Hoon, Kurtz Andreas, Stamm Christof
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Berlin, Germany.
Institute of Transfusion Medicine and Immunology, Mannheim, Germany.
Eur J Cardiothorac Surg. 2014 Jun;45(6):983-92. doi: 10.1093/ejcts/ezt576. Epub 2014 Feb 20.
Among the mechanisms by which somatic stem cells may improve left ventricular function in ischaemic heart disease are pro-survival stimuli mediated by secreted factors. This phenomenon is frequently referred to, but remains poorly understood. We therefore investigated the non-regenerative cardioprotective effects of cord blood mesenchymal stromal cells (CBMSCs) in vitro and sought to identify relevant intracellular signalling pathways.
Conditioned medium from CBMSCs and fibroblasts was prepared, and secreted factors were analysed by Luminex(®) immunobead assay. Murine cardiomyocyte-derived HL-1 cells were subjected to simulated ischaemia by glucose and serum deprivation and hypoxia in CBMSC-conditioned or cell-free control medium or in medium conditioned by foreskin fibroblasts. The proportions of vital, apoptotic and necrotic cells (poly-caspase activity, annexin V and ethidium homodimer-III staining) were quantified using a high-content imaging system. Metabolic activity and proliferation rate were determined via 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2-deoxyuridine assays. Phosphorylation of Akt, extracellular-signal-regulated kinase (ERK)1/2, signal transducer and activator of transcription 3 (STAT3) and glycogen synthase kinase 3β was determined by western blot, and experiments were repeated in the presence of specific small-molecule inhibitors (Wortmannin, UO126 and Stattic).
CBMSC medium reduced the proportion of dead HL-1 cardiomyocytes from 39 ± 3 to 28 ± 1% (P < 0.05) and the rate of late apoptotic cells to 68 ± 2% of that in control medium (P < 0.001). Metabolic activity was increased by 12 ± 1% compared with control (P < 0.05), while in fibroblast medium it was not (5 ± 2%, P = 1). This was associated with increased phosphorylation of Akt (2-fold, P < 0.05), ERK1/2 (3-fold, P < 0.01) and STAT3 (12-fold, P < 0.001). Combined blocking of the phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt and mitogen-activated protein kinase/ERK signalling abolished the protective CBMSC effect, while blocking the pathways individually had no effect. Inhibition of STAT3 phosphorylation drastically lowered HL-1 cell viability in control medium, but not in medium conditioned by CBMSCs.
The factors released by CBMSCs protect cardiomyocyte-like HL-1 cells from simulated ischaemia more than those released from fibroblasts. While CBMSC-triggered Akt and ERK1/2 activation provides protection in a compensatory manner, STAT3 is crucial for cardiomyocyte survival in ischaemia, but is not a key mediator of cytoprotective stem cell actions.
在缺血性心脏病中，体干细胞改善左心室功能的机制之一是由分泌因子介导的促生存刺激。这种现象常被提及，但仍了解甚少。因此，我们在体外研究了脐血间充质基质细胞（CBMSC）的非再生性心脏保护作用，并试图确定相关的细胞内信号通路。
制备来自CBMSC和成纤维细胞的条件培养基，通过Luminex®免疫磁珠分析法分析分泌因子。将小鼠心肌细胞来源的HL-1细胞在CBMSC条件培养基、无细胞对照培养基或包皮成纤维细胞条件培养基中，通过葡萄糖和血清剥夺以及缺氧进行模拟缺血处理。使用高内涵成像系统对活细胞、凋亡细胞和坏死细胞的比例（多聚半胱天冬酶活性、膜联蛋白V和乙锭同二聚体III染色）进行定量。通过3-(4,5-二甲基噻唑-2-基)-5-(3-羧甲氧基苯基)-2-(4-磺基苯基)-2H-四唑和5-溴-2'-脱氧尿苷测定法测定代谢活性和增殖率。通过蛋白质印迹法测定Akt、细胞外信号调节激酶（ERK）1/2、信号转导和转录激活因子3（STAT3）以及糖原合酶激酶3β的磷酸化水平，并在存在特异性小分子抑制剂（渥曼青霉素、UO126和Stattic）的情况下重复实验。
CBMSC培养基将死亡的HL-1心肌细胞比例从39±3%降至28±1%（P<0.05），晚期凋亡细胞率降至对照培养基中的68±2%（P<0.001）。与对照相比，代谢活性增加了12±1%（P<0.05），而在成纤维细胞培养基中则没有增加（5±2%，P=1）。这与Akt（2倍，P<0.05）、ERK1/2（3倍，P<0.01）和STAT3（12倍，P<0.001）的磷酸化增加有关。磷脂酰肌醇-4,5-二磷酸3-激酶/Akt和丝裂原活化蛋白激酶/ERK信号通路的联合阻断消除了CBMSC的保护作用，而单独阻断这些通路则没有效果。STAT3磷酸化的抑制显著降低了对照培养基中HL-1细胞的活力，但在CBMSC条件培养基中则没有。
CBMSC释放的因子比成纤维细胞释放的因子更能保护心肌样HL-1细胞免受模拟缺血的影响。虽然CBMSC触发的Akt和ERK1/2激活以补偿方式提供保护，但STAT3对缺血心肌细胞的存活至关重要，但不是细胞保护干细胞作用的关键介质。
