Investigation of the molecular mechanism of progenitor cell-mediated therapeutic angiogenesis

使用自体干细胞/祖细胞进行治疗性血管生成是治疗严重缺血性疾病的一种新策略。最近的研究表明，脂肪组织可以提供脂肪来源的干细胞。因此，我们利用小鼠的后肢缺血模型研究了移植血管干细胞是否会增强血管生成。取C57BL/6J小鼠腹股沟脂肪垫，用标准培养液分离ASCs，FACS分析表明ASCs为SCA-1、CD31-CD34-Flk-1-c-kif-Lin。ASCs表达血管内皮生长因子(VEGF)和基质细胞衍生因子I(SDF-1)的mRNAs和蛋白。手术诱导大鼠后肢缺血，将体外扩增的ASCs(1.0x106个/只)、PBS或分化的成熟脂肪细胞(MAS)作为对照细胞注入缺血组织内。治疗3周时，ASC组激光多普勒血流指数(0.93±0.08比0.79±0.14，0.05)和毛细血管密度(1.51±0.08比1.13±0.07，0.01)均高于对照组。用流式细胞仪和培养法检测ASCs移植后循环内皮祖细胞数量的增加。ASC组大鼠缺血组织SDF-I mRNA丰度和血清SDF-1水平均高于对照组。最后，腹腔注射抗SDF-1中和抗体降低了ASCs植入的治疗效果。脂肪组织将是基于细胞的治疗性血管生成的有价值的来源。此外，SDF-1可能至少部分通过促进内皮祖细胞的动员，在ASCs介导的血管生成中发挥关键作用。

Therapeutic angiogenesis using antologous stem/progenitor cells represents a novel strategy for severely ischemic diseases. Recent reports indicated that adipose tissues could supply adipose-derived stem cells (ASCs). Accordingly, we examined whether implantation of ASCs would augment angiogenesis using a mouse model of hind limb ischemia. C57BL/6J mouse inguinal fat pad was obtained and ASCs were isolated using standard medium.Fluorescence-activated cell sorter (FACS) analysis revealed that ASCs were Sca-1^+CD31-CD34-flk-1-c-kif-Lin. ASCs expressed vascular endothelial growth factor (VEGF) and stromal cell-derived factor I (SDF-1) mRNAs and proteins. Hind limb ischemia was surgically induced and culture-expanded ASCs(1.0xl0^6cells/animal), PBS or differentiated mature adipocytes(MAs) as control cells were injected into the ischemic tissues. At 3 weeks, the ASC group had a greater laser Doppler blood flow index (0.93±0.08 vs. 0.79±0.14 p<0.05) and a higher capillary density (1.51±0.08 vs. 1.13±0.07 p<0.01) compared to the control group. Implantation ofASCs increased circulating EPCs measured by FACS and culture assay. SDF-I mRNA abundance at ischemic tissues and serum SDF-1 levels were greater in the ASC group than control group. Finally, intraperitoneal injection of an anti-SDF-1 neutralizing antibody reduced therapeutic efficacies of ASCs implantation.Adipose tissue would be a valuable source for cell-based therapeutic angiogenesis. Moreover, SDF-1 may play a pivotal role in the ASCs-mediated angiogenesis at least in part by facilitating mobilization of EPCs.