Implantation of endothelial progenitor cells into laser-induced channels in rat ischemia hindlimb augments neovascularization.

The aim of this study was to investigate the effect of transmuscle laser revascularization (TMR) in combination with endothelial progenitor cell grafting on neovascularization of ischemic hindlimbs of nude rats. Mononuclear cells (MNCs) isolated from human umbilical cord blood (HUCB) by density gradient centrifugation were expanded in vitro. Spindle-shaped attaching (AT) cells and cord-like structures were developed from culture of MNCs. Acetylated low-density lipoprotein (Ac-LDL) incorporation by AT cells was performed. Phenotypic characterization was assessed by immunocytochemistry, and flow cytometry analysis. EPCs were labeled with 1, 1'-dioctadecyl-1 to 3,3, 3', 3'- tetramethyl-indocarbocyanine perchlorate (DiI) before being injected into the Nd:YAG laser channels or ischemic region. An acute ischemic limb model was created with the following four groups of nude rats by ligating the right external iliac artery: TMR + EPC group: rats with local transplantation of EPCs into laser channels; TMR group: those with transmuscular channels created without EPCs; EPC group: those with EPCs injected into the ischemic hindlimb; control group: an ischemic model without TMR or EPCs. All rats underwent femoral artery ultrasonic blood flow measurements of the ischemic and nonischemic limbs to obtain a flow ratio (femoral artery flow index [FAFI]: right femoral artery flow/left femoral artery flow) immediately after ligation of the artery (at baseline) and 28 days postoperation, and the ischemic limb muscle was sampled for histochemical and immunohistologic analysis. AT cells expressed AC 133 and endothelial cell (ECs) markers (KDR, CD34, CD31, and von Willebrand factor) and exhibited function similar to that of ECs as estimated by Ac-LDL incorporation. Flow cytometric analysis revealed that AT cells were positive for CD34 (62% +/- 7%) and AC133 (57.2% +/- 9.8%) at day 7 of culture. Twenty-eight days after the operation, the FAFI was significantly higher in the TMR+EPC group and EPC group than that in the control group. It was significantly higher in the TMR+EPC group, EPC group, and TMR group than that at their respective baselines. The FAFI in the control group was unchanged and no difference in FAFI was found between the TMR group and control group, and among the TMR+EPC, TMR, and EPC groups. TMR+EPC, TMR, and EPC treatment resulted in an increased number of capillaries in the treated regional area compared to the control group. Nd: YAG-laser transmuscle revascularization combined with the EPC grafting can significantly ameliorate perfusion and augment neovascularization in this ischemic hindlimb model of nude rats.