Laboratory of Cardiovascular Regeneration, Division of Cardiology, Seoul St. Mary's Hospital, The Catholic University of Korea School of Medicine, Seoul, South Korea.
Int J Cardiol. 2013 Oct 25;169(1):73-82. doi: 10.1016/j.ijcard.2013.08.076. Epub 2013 Sep 9.
Although the rescue of cellular senescence during ex vivo expansion of human-derived cardiac progenitor cells (hCPC) is critical for the application of autologous stem cell therapy in cardiovascular disease, the underlying molecular pathways during replicative senescence in hCPC have not been fully defined. Thus, we examined whether the regulation of mitogen-activated protein kinases activation could facilitate the recovery of human c-kit-positive hCPCs (hCPC(c-kit+)) and whether senescence is reactive oxygen species (ROS)-dependent or -independent.
To investigate the molecular pathways of replicative cellular senescence, we first evaluated cellular senescence in ex vivo-expanded hCPC(c-kit+) by using senescence-associated β-galactosidase (SA-β-gal) activity with enlarged cytoplasm and observed increased expression of cell senescence-related pivotal molecules, including TP53, cleavage Mdm2 (cMdm2), and Mdm2. Unexpectedly, we found that the extracellular signal-regulated kinase (ERK) was markedly activated in aged hCPC(c-kit+), with reduced proliferative activity. SA-β-gal activity and cytoplasm size in senescent hCPC(c-kit+) were significantly reduced, with reduced TP53 and cMdm2 expression after treatment with a specific ERK inhibitor (U0126). We examined whether the signaling in ERK inhibitory rescue of hCPC(c-kit+) senescence is ROS-dependent. Interestingly, the increased ROS level was not changed after treatment with a specific ERK inhibitor. Similarly, the increased expression levels of endogenous antioxidant enzymes, e.g., peroxiredoxin (Prdx)-1 and 2, in senescent hCPC(c-kit+) were not changed after treatment with a specific ERK inhibitor.
From the above results, we conclude that the specific inhibition of ERK during cellular senescence might rescue bioactivities of senescent hCPC(c-kit+) in a ROS-independent manner.
尽管在体外扩增人心肌祖细胞（hCPC）过程中拯救细胞衰老对于心血管疾病中自体干细胞治疗的应用至关重要，但 hCPC 复制性衰老过程中的潜在分子途径尚未完全定义。因此，我们研究了是否调节有丝分裂原激活的蛋白激酶（MAPK）的激活可以促进人 c-kit 阳性 hCPC（hCPC(c-kit+))的恢复，以及衰老是否依赖或不依赖于活性氧（ROS）。
为了研究复制性细胞衰老的分子途径，我们首先通过使用衰老相关β-半乳糖苷酶（SA-β-gal）活性评估体外扩增的 hCPC(c-kit+)中的细胞衰老，观察到细胞衰老相关关键分子的表达增加，包括 TP53、裂解型 Mdm2（cMdm2）和 Mdm2。出乎意料的是，我们发现衰老的 hCPC(c-kit+)中的细胞外信号调节激酶（ERK）明显被激活，而增殖活性降低。SA-β-gal 活性和衰老 hCPC(c-kit+)的细胞质大小显著降低，用特异性 ERK 抑制剂（U0126）处理后，TP53 和 cMdm2 的表达减少。我们研究了 ERK 抑制对 hCPC(c-kit+)衰老的信号转导是否依赖于 ROS。有趣的是，用特异性 ERK 抑制剂处理后，ROS 水平的增加并没有改变。同样，衰老的 hCPC(c-kit+)中内源性抗氧化酶，如过氧化物还原酶（Prdx）-1 和 2 的表达水平增加，在用特异性 ERK 抑制剂处理后也没有改变。
从上述结果中，我们得出结论，在细胞衰老过程中特异性抑制 ERK 可能以 ROS 非依赖性方式拯救衰老 hCPC(c-kit+)的生物活性。
