Basic research for the purpose of better outcome of therapeutic angiogenesis by cell tansplantation

人外周血单个核细胞(PB-MNC)的一个亚群可分化为内皮祖细胞(EPC)，参与出生后新生血管的形成。尽管组织缺血可以动员骨髓来源的内皮祖细胞，但低氧对其分化和血管生成功能的影响尚不清楚。我们检测了低氧条件是否会调节人外周血单核细胞来源的内皮祖细胞的分化和功能。PB-MNC亚群在常氧或低氧条件下均可形成EPC样贴壁(AT)细胞。然而，与常氧相比，低氧显著促进了PB-MNC向AT细胞的分化。AT细胞释放血管内皮生长因子蛋白，表面表达内皮细胞系标志CD31和KDR/VEGFR-2，缺氧后其表达增强。中和抗血管内皮生长因子单抗和KDR特异性受体酪氨酸激酶抑制剂SU1498均以剂量依赖的方式抑制PB-MNC向EPC样AT细胞的分化。用改良的Boyden小室装置检测，AT细胞对血管内皮生长因子的反应也在低氧条件下被增强。最后，当细胞被移植到免疫缺陷裸鼠的缺血后肢时，AT细胞的体外低氧预适应显著提高了体内新生血管的效率。结论：低氧直接刺激培养的人外周血单核细胞分化为EPC样AT细胞。此外，在体内移植前对AT细胞进行低氧预适应是促进治疗性血管生成的有效手段。

A subset of human peripheral blood mononuclear cells (PB-MNCs) differentiate into endothelial progenitor cells (EPCs) that participate in postnatal neovascularization. Although tissue ischemia can mobilize EPCs from bone marrow (BM), the effects of hypoxia on differentiation and angiogenic function of EPCs are little known. We examined whether hypoxic conditioning would modulate differentiation and function of human PB-MNC-derived EPCs. Subset of PB-MNCs gave rise to EPC-like attaching (AT) cells under either normoxic or hypoxic conditions. However, hypoxia much enhanced the differentiation of AT cells from PB-MNCs compared to normoxia. AT cells released VEGF protein and expressed CD31 and KDR/VEGFR-2, endothelial lineage markers, on their surface, which were also enhanced by hypoxia. Both a neutralizing anti-VEGF mAb and a KDR-specific receptor tyrosine kinase inhibitor, SU1498, suppressed PB-MNC differentiation into EPC-like AT cells in a dose-dependent manner. Migration of AT cells in response to VEGF as examined by a modified Boyden chamber apparatus was also enhanced by hypoxia. Finally, in vivo neovascularization efficacy was significantly enhanced by in vitro hypoxic conditioning of AT cells when cells were transplanted into the ischemic hindlimb of immunodeficient nude rats. In conclusion, hypoxia directly stimulated differentiation of EPC-like AT cells from human PB-MNC culture. Moreover, hypoxic preconditioning of AT cells prior to in vivo transplantation is a useful means to enhance therapeutic vasculogenesis.