Comparison of anticoagulant effects on vein grafts between human TFPI gene transfection and aspirin oral administration.

To develop a more efficient antithrombotic way after coronary artery bypass grafting (CABG), the anticoagulant effects were compared of human tissue factor pathway inhibitor (TFPI) gene transfection and aspirin oral administration (traditional method) on vein grafts. An eukaryotic expression plasmid pCMV-(Kozak) TFPI was prepared. Animal model of carotid artery bypass grafting was constructed. In operation, endothelial cells of vein grafts in TFPI group and empty plasmid control group were transfected with pCMV-(Kozak) TFPI and empty plasmid pCMV respectively, while no transfection was conducted in aspirin control group. After operation, aspirin (2 mg.kg(-1).(-1)) was administered (i.g.) in aspirin control group. Three days later, grafts (n=10) were harvested for RT-PCR, Western blotting and immunohistochemical analyses of exogenous gene expression and for pathological, scanning electron microscopic observation of thrombus. Thirty days later, the patency rates of remnant grafts (n=10) were recorded by vessel Doppler ultrasonography. Human TFPI gene products were detected in gene transferred vein grafts. Three days later, thrombi were found in 7 animals of aspirin control group and in 8 animals of empty plasmid control group, but in only 1 of TFPI group (P<0.01). Thirty days later, 5 grafts were occluded in empty plasmid control group, but none of grafts was occluded in the other groups (P<0.05). The endothelial surfaces of grafts in both of the control groups were covered with aggregated erythrocytes and platelets, and it were not seen in TFPI group. It was suggested that the anticoagulant effects on vein grafts of human TFPI gene transfection are better than those of aspirin.

Characteristics of ex vivo expansion of endothelial progenitor cells.

The characteristics for the ex vivo expansion of the endothelial progenitor cells (EPCs) were explored. CD34+ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded under the same conditions as those for total MNC, coincubation of CD34+ and CD34- from the same donor for EPCs. In addition, the effects of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis were examined. EPCs were determined and quantified by immunocytochemistry and flow cytometry. The results showed that both coculture of CD34+ and CD34- and total MNC led to a significant increase in the expansion of CD34+ cells as compared with CD34 enrichment (P < 0.05). There was a tendency toward decreased apoptosis in cultures when early passage was performed immediately after cord like structures appeared. VEGF had no significant effect on apoptosis (P > 0.05). These differentiated EPCs were positive for CD34+, von Willebrand factor (vWF), KDR, CD31 staining and phagocytized acetylated low-density lipoprotein (LDL). CD34+ cells accounted for (68.2 +/- 6.3)% of attaching (AT) cells at day 7 of culture. It was suggested the most efficient method to ex vivo expansion of EPCs was coculture of CD34+ and CD34- or total MNC. Early passage makes cell apoptosis rate decrease. VEGF had no significant effect on ex vivo expansion of EPCs.

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.