Liu Zhenguo, Jiang Yuehua, Hao Hong, Gupta Kalpna, Xu Jian, Chu Ling, McFalls Edward, Zweier Jay, Verfaillie Catherine, Bache Robert J
Davis Heart & Lung Research Institute, The Ohio State University Medical Center, Columbus, OH 43210, USA.
Am J Physiol Heart Circ Physiol. 2007 Sep;293(3):H1760-5. doi: 10.1152/ajpheart.01408.2006. Epub 2007 Jun 1.
This study was designed to investigate the developmental expression of endothelial nitric oxide synthase (eNOS) during stem cell differentiation into endothelial cells and to examine the functional status of the newly differentiated endothelial cells. Mouse adult multipotent progenitor cells (MAPCs) were used as the source of stem cells and were induced to differentiate into endothelial cells with vascular endothelial growth factor (VEGF) in serum-free medium. Expression of eNOS in the cells during differentiation was evaluated with real-time PCR, nitric oxide synthase (NOS) activity, and Western blot analysis. It was found that eNOS, but no other NOS, was present in undifferentiated MAPCs. eNOS expression disappeared in the cells immediately after induction of differentiation. However, eNOS expression reoccurred at day 7 during differentiation. Increasing eNOS mRNA, protein content, and activity were observed in the cells at days 14 and 21 during differentiation. The differentiated endothelial cells formed dense capillary networks on growth factor-reduced Matrigel. VEGF-stimulated phosphorylation of extracellular signal-regulated kinase (ERK)-1 and ERK-2 occurred in these cells, which was inhibited by NOS inhibitor N(G)-nitro-L-arginine methyl ester. In conclusion, these data demonstrate that eNOS is present in MAPCs and is dynamically expressed during the differentiation of MAPCs into endothelial cells in vitro.
本研究旨在调查内皮型一氧化氮合酶（eNOS）在干细胞分化为内皮细胞过程中的发育表达情况，并检测新分化内皮细胞的功能状态。成年小鼠多能祖细胞（MAPCs）被用作干细胞来源，并在无血清培养基中用血管内皮生长因子（VEGF）诱导分化为内皮细胞。通过实时PCR、一氧化氮合酶（NOS）活性及蛋白质免疫印迹分析评估分化过程中细胞内eNOS的表达。结果发现，未分化的MAPCs中存在eNOS，但不存在其他NOS。诱导分化后，细胞内eNOS表达立即消失。然而，在分化第7天时eNOS表达再次出现。在分化第14天和21天时，细胞内eNOS mRNA、蛋白质含量及活性均增加。分化后的内皮细胞在生长因子减少的基质胶上形成致密的毛细血管网络。这些细胞中发生了VEGF刺激的细胞外信号调节激酶（ERK）-1和ERK-2磷酸化，而NOS抑制剂N（G）-硝基-L-精氨酸甲酯可抑制这一过程。总之，这些数据表明eNOS存在于MAPCs中，并且在体外MAPCs分化为内皮细胞的过程中动态表达。
