Tan Yaohong, Shao Hongwei, Eton Darwin, Yang Zhe, Alonso-Diaz Luis, Zhang Hongkun, Schulick Andrew, Livingstone Alan S, Yu Hong
Department of Surgery, Vascular Biology Institute, University of Miami, Miller School of Medicine, Miami, FL 33136, USA.
Cardiovasc Res. 2007 Mar 1;73(4):823-32. doi: 10.1016/j.cardiores.2006.12.015. Epub 2006 Dec 23.
Granulocyte colony-stimulating factor (G-CSF) mobilizes bone marrow mononuclear cells into the peripheral circulation. Stromal cell-derived factor-1 (SDF-1) enhances the homing of progenitor cells mobilized from the bone marrow and augments neovascularization in ischemic tissue. We hypothesize that SDF-1 will boost the pro-angiogenic effect of G-CSF.
NIH 3T3 cells retrovirally transduced with SDF-1alpha gene (NIH 3T3/SDF-1) were used to deliver SDF-1 in vitro and in vivo. Endothelial progenitor cells (EPCs) co-cultured with NIH 3T3/SDF-1 cells using cell culture inserts migrated faster and were less apoptotic compared to those not exposed to SDF-1. NIH 3T3/SDF-1 (10(6) cells) were injected into the ischemic muscles immediately after resection of the left femoral artery and vein of C57BL/6J mice. G-CSF (25 mug/kg/day) was injected intraperitioneally daily for 3 days after surgery. Blood perfusion was examined using a laser Doppler perfusion imaging system. The perfusion ratio of ischemic/non-ischemic limb increased to 0.57+/-0.03 and 0.50+/-0.06 with the treatment of either SDF-1 or G-CSF only, respectively, 3 weeks after surgery, which was significantly higher than the saline-injected control group (0.41+/-0.01, P<0.05). Combined treatment with both SDF-1 and G-CSF resulted in an even better perfusion ratio of 0.69+/-0.08 (P<0.05 versus the single treatment groups). Mice were sacrificed 21 days after surgery. Immunostaining and Western blot assay of the tissue lysates showed that the injected NIH 3T3/SDF-1 survived and expressed SDF-1. CD34(+) cells were detected with immunostaining, capillary density was assessed with alkaline phosphatase staining, and the apoptosis of muscle cells was viewed using an in situ cell death detection kit. More CD34(+) cells, increased capillary density, and less apoptotic muscle cells were found in both G-CSF and SDF-1 treated group (P<0.05 versus other groups).
Combination of G-CSF-mediated progenitor cell mobilization and SDF-1-mediated homing of EPCs promotes neovascularization in the ischemic limb and increases the recovery of blood perfusion.
粒细胞集落刺激因子（G-CSF）可促使骨髓单个核细胞进入外周循环。基质细胞衍生因子-1（SDF-1）可增强从骨髓动员的祖细胞的归巢能力，并增强缺血组织中的新生血管形成。我们推测SDF-1将增强G-CSF的促血管生成作用。
用携带SDF-1α基因的逆转录病毒转导的NIH 3T3细胞（NIH 3T3/SDF-1）用于在体外和体内递送SDF-1。与未接触SDF-1的内皮祖细胞（EPCs）相比，使用细胞培养插入物与NIH 3T3/SDF-1细胞共培养的EPCs迁移更快且凋亡更少。在切除C57BL/6J小鼠的左股动脉和静脉后，立即将NIH 3T3/SDF-1（10⁶个细胞）注射到缺血肌肉中。术后每天腹腔注射G-CSF（25μg/kg/天），共3天。使用激光多普勒灌注成像系统检查血液灌注情况。术后3周，仅用SDF-1或G-CSF治疗时，缺血/非缺血肢体的灌注率分别增加到0.57±0.03和0.50±0.06，显著高于注射生理盐水的对照组（0.41±0.01，P<0.05）。SDF-1和G-CSF联合治疗导致灌注率更高，为0.69±0.08（与单一治疗组相比，P<0.05）。术后21天处死小鼠。对组织裂解物进行免疫染色和蛋白质印迹分析表明，注射的NIH 3T3/SDF-1存活并表达SDF-1。用免疫染色检测CD34⁺细胞，用碱性磷酸酶染色评估毛细血管密度，并用原位细胞死亡检测试剂盒观察肌肉细胞的凋亡情况。在G-CSF和SDF-1治疗组中均发现更多的CD34⁺细胞、增加的毛细血管密度和更少的凋亡肌肉细胞（与其他组相比，P<0.05）。
G-CSF介导的祖细胞动员与SDF-1介导的EPCs归巢相结合，可促进缺血肢体的新生血管形成，并增加血液灌注的恢复。
