Junhui Zhu, Xiaojing He, Binquan Zhou, Xudong Xie, Junzhu Chen, Guosheng Fu
Department of Cardiology, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.
Cytotherapy. 2009;11(4):485-91. doi: 10.1080/14653240902887267.
Previous studies have shown that nicotine increases endothelial progenitor cell (EPC) numbers and functional activity. However, the mechanisms by which nicotine increases EPC numbers and activity remain to be determined. Recent studies have demonstrated that EPC numbers and activity are associated with EPC senescence, which involves telomerase activity. Therefore, we investigated whether nicotine might be able to prevent senescence of EPC through telomerase activation, leading to the potentiation of cellular function.
After prolonged in vitro cultivation, EPC were incubated with or without nicotine. The senescence of EPC was determined by acidic beta-galactosidase staining. The bromo-deoxyuridine incorporation assay and colony assay were employed to assess proliferative capacity and clonal expansion potential, respectively. To examine further the underlying mechanisms of these effects, we measured telomerase activity and the phosphorylation of Akt by Western blotting.
Nicotine dose-dependently prevented the onset of EPC senescence in culture. Moreover, nicotine increased the proliferation of EPC and colony-forming capacity. Nicotine significantly increased telomerase activity and phosphorylation of Akt, a downstream effector of phosphoinositide 3-kinase (PI3K). Moreover, pre-treatment with PI3K blockers, either wortmannin or LY294002, significantly attenuated the nicotine-induced telomerase activity. In addition, mecamylamine, a non-selective antagonist of nicotinic acetylcholine receptors (nAchR), abrogated the effects of nicotine on EPC.
The results of the present study indicate that nicotine delays the onset of EPC senescence, which might be related to activation of telomerase through the PI3K/Akt pathway. In addition, the effects of nicotine might be specifically mediated by nAchR activation.
既往研究表明，尼古丁可增加内皮祖细胞（EPC）数量及功能活性。然而，尼古丁增加EPC数量及活性的机制仍有待确定。近期研究显示，EPC数量及活性与EPC衰老相关，而这涉及端粒酶活性。因此，我们研究了尼古丁是否能够通过激活端粒酶来预防EPC衰老，从而增强细胞功能。
在体外长时间培养后，将EPC与尼古丁一起或不与尼古丁一起孵育。通过酸性β-半乳糖苷酶染色来确定EPC的衰老情况。分别采用溴脱氧尿苷掺入试验和集落试验来评估增殖能力和克隆扩增潜力。为了进一步研究这些效应的潜在机制，我们通过蛋白质免疫印迹法检测端粒酶活性和Akt的磷酸化水平。
尼古丁呈剂量依赖性地预防了培养的EPC衰老的发生。此外，尼古丁增加了EPC的增殖及集落形成能力。尼古丁显著增加了端粒酶活性以及磷酸肌醇3激酶（PI3K）下游效应分子Akt的磷酸化水平。此外，用渥曼青霉素或LY294002这两种PI3K抑制剂预处理可显著减弱尼古丁诱导的端粒酶活性。另外，烟碱型乙酰胆碱受体（nAchR）的非选择性拮抗剂美加明消除了尼古丁对EPC的作用。
本研究结果表明，尼古丁可延缓EPC衰老的发生，这可能与通过PI3K/Akt途径激活端粒酶有关。此外，尼古丁的作用可能是由nAchR激活特异性介导。
