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

人外周血单个核细胞（PB-MNCs）分化为内皮祖细胞（EPCs）参与出生后新生血管形成。虽然组织缺血可以动员骨髓中的EPCs，但缺氧对EPCs分化和血管生成功能的影响尚不清楚。我们研究了低氧条件是否会调节人PB-MNC衍生的EPCs的分化和功能。亚组的PB-MNCs在常氧或缺氧条件下均产生EPC样贴附（AT）细胞。然而，缺氧大大促进AT细胞从PB-MNCs的分化相比，常氧。AT细胞释放VEGF蛋白，并在其表面表达CD 31和KDR/VEGFR-2，内皮细胞谱系标志物，缺氧也增强。中和性抗VEGF mAb和KDR特异性受体酪氨酸激酶抑制剂SU 1498均以剂量依赖性方式抑制PB-MNC分化为EPC样AT细胞。AT细胞的迁移响应VEGF的检查由一个修改的Boyden室装置也增强了缺氧。最后，当细胞移植到免疫缺陷裸鼠的缺血后肢时，AT细胞的体外低氧条件处理显著增强了体内新血管形成的功效。总之，缺氧直接刺激人PB-MNC培养物中EPC样AT细胞的分化。此外，AT细胞在体内移植前的低氧预处理是一种有用的手段，以增强治疗性血管生成。

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.