Studies Of Stromal Cell-derived Factor-1 Effects On Endothelial Progenitor Cell Transplantation For Therapeutic Neovascularization

PartⅠIsolation and differentiation of endothelial progenitor cells from human peripheral blood[Objective]: To elucidate a sample method of isolating and culture of endothelial progenitor cells (EPCs) from human peripheral blood mononuclear cells (MNCs), and observe the cell marker expression during proliferation and differentiation in vitro.[Methods]: Total MNCs were isolated from human peripheral blood by density gradient centrifugation, then cultured in M199 medium supplemented with vascular endothelial growth factor (VEGF, 20ng/ml), basic fibroblast growth factor (bFGF, 2ng/ml) and 20% fetal bovine serum (FBS). Attached spindle-shaped cells were detached at day 7, and then identified by endothelial-specific markers. The expression of CD34 and CD133 were determined by flow cytometry; KDR (VEGFR-2) was detected by reverse transcriptase-polymerase chain reaction (RT-PCR); the expression of CD31 was detected by immunocytochemistry andⅧfactor was detected by immunofluorescent staining; endothelial function was determined by cell absorption of DiI-acLDL and FITC-UEA-1．[Results] : Attached spindle-like cells appeared after 3 days culture and became more and larger at 7 days of culture. Cord-like structure was observed. The positive rates of CD34, CD133, CD31 andⅧfactor of ex vivo expanded EPC at 7 days of culture were 36．31%, 19．74%, 73．29% and 75．91%, respectively. RT-PCR demonstrated the presence of KDR mRNA. The differentiated cells had the capacity to incorporate DiI-acLDL and FITC-UEA-1 (>90%).[Conclusion] : EPCs have been shown to be present in circulation, and could be isolated and cultured from peripheral blood MNCs. PartⅡStromal cell-derived factor-1 effects on endothelial progenitor cell migration in vitro[Objective] : To investigate the effect of stromal cell-derived factor-1 (SDF-1) on ex vivo expanded EPCs migration, and the expression of CXCR4, the SDF-1 sole receptor, on EPCs.[Methods]: After 7 days culture of EPCs in vitro, the expression of CXCR4 was determined by immunocytochemistry and immunocytofluorescence. The migration function of EPC was determined using Transwell chamber. Four groups was determined according to SDF-1 concentration: control,10ng/ml 20ng.ml and 50ng/ml, respectively. Human EPCs cultured for 7 days were harvested, 5×10~4 cells were suspended in M199 supplemented with 5%BSA. After 6 hours of incubation at 37℃, the filter was removed and the cells on the filter were counted.[Results]: The expression of CXCR4 of 7 days culture of EPCs was 68．62% by immunocytochemistry, and immunocytofluorescence showed that 74．78% of day 7 EPCs express CXCR4．There was no significant difference between the two results (p >0．05) . In sum, the positive rate of CXCR4 was 71．7%. Ex vivo expanded EPCs demonstrated a potent dose-dependent migration activity toward SDF-1．The migration cells of SDF-1 different concentration of control, 10ng/ml, 20ng/ml and 50ng/ml were 3．5, 7．38, 24．88 and 28．0, respectively. The migration activity was significantly higher in group of 10ng/ml, 20ng/ml and 50ng/ml than control group (p<0．01) . The difference was determined in comparison of 10ng/ml vs 20ng/ml (p<0．01) , 20ng/ml vs 50ng/ml (p<0．05) , and 10ng/ml vs 50ng/ml (p<0．01) , respectively.[Conclusion]: The CXCR4 receptor is present on 7 days culture of EPC, and SDF-1 induces EPC migration in a dose-dependent manner. PartⅢStromal cell-derived factor-1 effects on endothelial progenitor cell transplantation for therapeutic neovascularization[Objective]: To explore the in vivo distribution of human EPC transplantation into nude mice model of unilateral ischemic hindlimb, and investigate if SDF-1 could augment vasculogenesis in vivo by inducing EPC recruitment in ischemic tissues. To elucidate whether endothelial nitric oxide synthase (eNOS) participate in the process of neovascularization.[Methods]: After establishment of unilateral hindlimb ischemia in athymic nude mice, 7 days ex vivo expanded EPCs with absorption of FITC-UEA-1 were injected intravenously. After the mice were put to death, the heart, liver, kidney, and the ischemic muscles of the animal were taken out and made into frozen section on day 3 and day 7．Then we observe the distribution of green fluorescence cells under fluorescent microscopy. 20 mice were randomly divided into four groups: group A (EPC intravenously±SDF-1 intramuscularly), Group B (EPC intravenously), group C (SDF-1 intramuscularly), and group D ( M199 intramuscularly). We observe the temperature and self-amputation rates in nude mice. 7 days after transplantation, tissue sections from the ischemic muscles were harvested. The ratio of the number of capillary and the number of skeletal muscle fiber was counted using NBT/BCIP staining methods. The expression of CD31, indicative of capillary density, and eNOS were detected in paraffin-embedded ischemic muscles by immunohistochemistry.[Results]: 3 days after EPC transplantation, green fluorescence cells could be seen in ischemic muscles, locating in the interstitial muscle tissue. 7 days after transplantation there were still a few fluorescence cells embedded in ischemic muscles. The labeled green fluorescence EPCs were not seen in the heart, the liver, and the kidney. Of the 20 nude mice, one died from heart failure by over infusion, the other died without reason. The rate of ischemic hindlimb reserving in group A, B, C and D was 80%, 75%, 20% and 0．Vessel numbers assessed by capillary/muscle fiber ratio in group A, B, C, and D was 1．01, 0．84, 0．57, and 0．46, respectively. Capillary/muscle fiber ratio of group A, B, C was greater than that of group D (p<0．01) . Group A was greater than group B and group B greater than group C (p<0．05) . The capillary density in group A, B, C, and D was 15．2,10．0,5．8, and 2．4, respectively. Group A and B were more than group D (p<0．01) , and group C was more than group D (p<0．05) . Group A was more than group B and group B was more than group C (p<0．05) . The expression of eNOS was detected in group A and B, and the positive rate in group A and B was 73．33% and 53．33%. There was no significance between the two groups, however.[Conclusion]: Ex vivo cultured EPC transplantation could be recruited to ischemic tissues in vivo, and contribute to neovascularization. Locally delivered SDF-1 augments this effect by inducing EPC recruitment to ischemic tissues. Reflections and ConclusionsWe draw the following the theoretical considerations based on our results:1．Isolation, culture, and expanded endothelial progenitor cell (EPC) ex vivo from the peripheral blood is an alternative method. This culture method would avoid the embarrassment of anesthesia and pain when isolated from the bone marrow, and immunological rejection and resource shortage when isolated from the cord blood, and would decrease the possibility risks of transplanting unselected mononuclear cells, such as excess inflammation and differentiation to undesired cell types;2．The CXCR4 receptor is present on 7 days culture of EPC, and SDF-1 induces EPC migration in a dose-dependent manner in vitro;3．Ex vivo cultured EPC transplantation could be recruited to i schemic tissues in vivo, and contribute to neovascularization. Locally delivered SDF-1 augments the effect of EPC neovascularization. This strategy might become a novel chemokine therapy for neovascularization of ischemia, especially important in situations when scarcer number and poorer function of EPC in patients combined with a variety of cardiovascular risks.The novelty of this study is threefold:1．The alternative of isolation, culture and expansion of EPC from the peripheral blood has been established;2．SDF-1 augments neovascularization by inducing EPC recruitment in ischemic tissues;3．The mechanism of neovascularization of EPC transplantation lies, partly, in the effects of eNOS.