Lu Haiyuan, Mei Hua, Wang Fan, Zhao Qian, Wang Siqi, Liu Lvjun, Cheng Lamei
Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, Hunan 410078, P.R. China.
National Center of Human Stem Cell Research and Engineering, Changsha, Hunan 410000, P.R. China.
Int J Mol Med. 2017 Jun;39(6):1492-1504. doi: 10.3892/ijmm.2017.2976. Epub 2017 May 5.
Human umbilical cord blood-derived endothelial progenitor cells (EPCs) have been proven to contribute to post-natal angiogenesis, and have been applied in various models of ischemia. However, to date, to the best of our knowledge, there is no available data on the angiogenic properties of EPCs during the process of in vitro expansion. In this study, we expanded EPCs to obtain cells at different passages, and analyzed their cellular properties and angiogenic ability. In the process of expansion, no changes were observed in cell cobblestone-like morphology, apoptotic rate and telomere length. However, the cell proliferative ability was significantly decreased. Additionally, the expression of CD144, CD90 and KDR was significantly downregulated in the later-passage cells. Vascular formation assay in vitro revealed that EPCs at passage 4 and 6 formed more integrated and organized capillary-like networks. In a murine model of hind limb ischemia, the transplantation of EPCs at passage 4 and 6 more effectively promoted perfusion recovery in the limbs on days 7 and 14, and promoted limb salvage and histological recovery. Furthermore, the phosphorylation levels of platelet‑derived growth factor receptor-β (PDGFR-β) were found to be significantly decreased with the in vitro expansion process, accompanied by the decreased activation of the PI3K/Akt signaling pathway. When PDGFR inhibitor was used to treat the EPCs, the differences in the angiogenic potential and migratory ability among the EPCs at different passages were no longer observed; no significant differences were also observed in the levels of phosphorylated PI3K/Akt between the EPCs at different passages following treatment with the inhibitor. On the whole, our findings indicate that the levels of phosphorylated PDGFR-β are decreased in EPCs with the in vitro expansion process, which impairs their angiogenic potential by inhibiting PI3K/Akt signaling. Our findings may aid in the more effective selection of EPCs of different passages for the clinical therapy of ischemic disease.
人脐带血来源的内皮祖细胞（EPCs）已被证明有助于出生后的血管生成，并已应用于各种缺血模型。然而，据我们所知，迄今为止尚无关于体外扩增过程中EPCs血管生成特性的可用数据。在本研究中，我们扩增EPCs以获得不同传代的细胞，并分析其细胞特性和血管生成能力。在扩增过程中，未观察到细胞鹅卵石样形态、凋亡率和端粒长度的变化。然而，细胞增殖能力显著下降。此外，后期传代细胞中CD144、CD90和KDR的表达明显下调。体外血管形成试验显示，第4代和第6代的EPCs形成了更完整、更有序的毛细血管样网络。在小鼠后肢缺血模型中，第4代和第6代EPCs的移植在第7天和第14天更有效地促进了肢体灌注恢复，并促进了肢体挽救和组织学恢复。此外，发现血小板衍生生长因子受体-β（PDGFR-β）的磷酸化水平随着体外扩增过程而显著降低，同时PI3K/Akt信号通路的激活也降低。当使用PDGFR抑制剂处理EPCs时，不同传代EPCs之间的血管生成潜能和迁移能力差异不再明显；抑制剂处理后不同传代EPCs之间磷酸化PI3K/Akt水平也未观察到显著差异。总体而言，我们的研究结果表明，随着体外扩增过程，EPCs中磷酸化PDGFR-β水平降低，通过抑制PI3K/Akt信号传导损害其血管生成潜能。我们的研究结果可能有助于更有效地选择不同传代的EPCs用于缺血性疾病的临床治疗。
