| Endothelial progenitor cells (EPCs) are precursors that can differentiate to endothelial cells and promote the neovascularization of ischemic tissues. Previous studies on animals have shown that EPCs transplantation could improve the recovery of ischemia models in vivo and it may be a clinical therapy method to patients with ischemic diseases. The number of EPCs isolated from peripheral blood is reduced in patients with cardiovascular risk factors. Furthermore, the functions of EPCs are impaired in patients with aging, diabetes or hypercholesterolemia. The feasibility of using these cells for autografting in patients with ischemicdiseases are usually hampered by the rarity of the cells in the peripheral blood and the dysfunction of the cells although treatment of these patients with peripheral blood-derived cultured EPCs may be a potential therapeutic option. The umbilical cord blood may be an alternative source of EPCs for cell transplantation to promote neovascularization in patients with ischemic disease.Several populations of EPCs have been reported and isolated by different surface markers and showed different phenotype and characteristics as well as the origin. These differences should be further clarified for clinically cellular transplantation in the future. The hierarchy of EPCs from human umbilical cord blood was identified based on their clonogenic and proliferative potential but their function to promote the neovascularization has not been evaluated.It is a long journey for EPCs coming from bone marrow to ischemic tissue, including mobilization, migration, survival, proliferation, homing, transmigration across capillaries, migration in extracellular matrix and incorporation into new-formed capillaries. It is not yet known how EPCs transmigrate across capillaries although the knowledge about EPCs is increasing. diseases are usually hampered by the rarity of the cells in the peripheral blood and the dysfunction of the cells although treatment of these patients with peripheral blood-derived cultured EPCs may be a potential therapeutic option. The umbilical cord blood may be an alternative source of EPCs for cell transplantation to promote neovascularization in patients with ischemic disease.Several populations of EPCs have been reported and isolated by different surface markers and showed different phenotype and characteristics as well as the origin. These differences should be further clarified for clinically cellular transplantation in the future. The hierarchy of EPCs from human umbilical cord blood was identified based on their clonogenic and proliferative potential but their function to promote the neovascularization has not been evaluated.It is a long journey for EPCs coming from bone marrow to ischemic tissue, including mobilization, migration, survival, proliferation, homing, transmigration across capillaries, migration in extracellular matrix and incorporation into new-formed capillaries. It is not yet known how EPCs transmigrate across capillaries although the knowledge about EPCs is increasing.In the present study we used unfractioned MNCs isolated from human umbilical cord blood to culture EPCs and obtained two types of EPCs, HPP-EPCs and CACs, by using endothelial cell-conditioned medium and compared their phenotypes, growth characteristics and angiogenic potential. We further studied the role of adhesion molecular, CD11a and CD49d, in HPP-EPCs homing to ischemic tissue.Chapter 1 Culture of two types of endothelial progenitor cells from human umbilical cord bloodObjective: To establish the method of isolation and culture endothelial progenitor cells from human umbilical cord blood and find out the suitable culture system for EPCs.Methods: Mononuclear cell isolated by gradient density centrifuge from human umbilical cord blood were cultured in fibronectin-coated six-well tissue culture plates using cultures system supplemented with endothelial cell-conditioned medium. Two types of endothelial progenitor cells, circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs), were obtained and purified by mechanically picking up colonies. These cells were identified by two standards of EPCs, uptake of Ac-LDL and binding to lectin, and were analyzed by FACS for their phenotypes related to endothelial cells as well as for their origin.Results: We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs).CACs attached onto the fibronectin-coated plate at days 4-7 and appeared in spindle- or ellipse-shape, but not gathered into clusters. HPP-EPC colonies appeared after culture for 2-3 wk and had clear boundaries. HPP-EPCs appeared in "cobblestone-like" morphology with a higher volume ratio of nucleus/cytoplasm as compared with that of CACs. Two types of cells accorded with the standards of EPCs, uptake of DiI-Ac-LDL and binding to lectin, and expressed the markers of endothelial cell such as CD31, CD144 and vWF by immunocytochemistry. RT-PCR analysis disclosed the gene transcription of CD31,KDR,CD144 and ENOS in two types of cells. FACS analysis showed that in CACs the cells positive for CD31,CD144 and KDR were 90.6%±5.4%, 66.3%±18.2% and 31.7%±8.3%, respectively, and in HPP-EPCs 84.1%±9.5%,96.6%±1.4% and 97.2%±1.6%, respectively. There are differences in antigen expression between the two types of cells. CACs without expression of CD133, a marker of stem/progenitor cells, expressed CD14 (18.0%±6.4%), the marker of monocyte/macrophage, and CD45 (31.4%±5.7%) of panleucocyte. HPP-EPCs expressed CD133 (64.2%±17.4%), but not CD14 and CD45. Furthermore, CACs did not express CD3, CD19 and CD15, the markers of lymphocyte T, B and granulocyte, respectively.Conclusions: We cultured mononuclear cells (MNCs) isolated from human umbilical cord blood using endothelial cell-conditioned medium and obtained two types of EPCs, referred as circulating angiogenic cells (CACs) and high proliferative potential endothelial progenitor cells (HPP-EPCs). Both types of the cells possess the characteristics of EPCs including expressing CD31, VE-cadherin, KDR and von Willebrand factor, uptake of Ac-LDL and binding to lectin. But contrary to CACs, which expressed CD14 but not CD133, HPP-EPCs expressed CD133 but not CD14. CACs may origin from monocytes/macrophages but not from lymphocyte T, B and granulocyte.Part 2 Differences between two types of EPCsObjective: To compare the biological characteristics and angiogenic potential between the two types of endothelial progenitor cells.Methods: FACS was performed to analyze the apoptosis in two types of EPCs induced by 48 h serum-free culture. In order to compare their growth characteristics, the growth curves were generated and the PDTs and CPDLs were calculated for the two types of cells. And the experiments of capillary-like structure formed on Matrigel were performed to evaluate the angiogenic potential in vitro. In ischemia models of nude mice, the effects to promote neovascularization of two types of cells were compared on three aspects: limb salvage, blood flow and capillary density. The incorporation of DiI-labelled EPCs into new-formed capillaries was confirmed under a confocal microscope.Results: The results of FACS showed that 1.2%±0.6％early-stage apoptosis cells and 0.1%±0.1% late-stage apoptosis cells were detected in HPP-EPCs after 48-h serum-free induction whereas 4.5%±1.4% early-stage apoptosis and 2.1%±0.8% late-stage apoptosis in CACs. As shown in growth curve, CACs could only reach 4-6 population doublings but HPP-EPCs could reach over 60 population doublings without any sign of senescence in vitro. The average CPDL of HPP-EPCs is 9.53-fold higher and the average PDT is shorter than those of CACs. The result of MTT assay showed optical density A490nm values (after 1:40 dilution) of HPP-EPCs (0.80±0.18) were about 4-fold higher than those of CACs (0.20±0.12) after 24-h culture with the same number (5×103) of seeded cells. The experiments of colony forming showed that no colony was formed in 960 individual cells from CACs, whereas more than 35 percent of 960 individual cells from HPP-EPCs colonies could survive and divide into 2-50 progeny cells, and about 45 percent of 960 individual cells formed colonies with more than 50 cells in a 2-wk culture period. And 5 tertiary colonies were formed in the total 96 individual cells from the secondary colonies. These data suggested that we have cultured different hierarchies of EPCs from human umbilical cord blood including CACs and HPP-EPCs, and the later contained HPP-ECFC and LPP-ECFC. The number of capillary-like structure in HPP-EPCs (28.6±15.8) was greater than that in CACs (4.7±3.4). The results in animal models showed that EPC transplantation raised the limb salvage rate (9 in 12 HPP-EPC-transplanted mice and 4 in 12 CAC-transplanted mice compared to 0 in 12 PBS-injected mice) and reduced the limb loss (including mild and severe loss of limb) rate (3 in 12 HPP-EPC-transplanted mice and 8 in 12 CAC-transplanted mice compared to 12 in 12 PBS-injected mice). And the results also disclosed that the improvements in relative ratio of radio counts of ischemic hind limb in the EPC-transplanted groups were greater than that in the control group (R-value increased from 0.153±0.038 to 0.713±0.081 in HPP-EPC- transplanted group, from 0.145±0.034 to 0.599±0.148 in CAC- transplanted group and from 0.128±0.034 to 0.444±0.120 in PBS- injected group, P＜0.01, n=12) and the degree of improvement in the HPP-EPC-transplanted group was higher than that in the CAC-transplanted group. The results revealed that the capillary density (capillaries/mm2) in both HPP-EPC- and CAC-transplanted groups increased as compared to the control group (370.3±55.4 in HPP-EPC-transplanted group, 318.8±48.4 in CAC- transplanted group and 246.9±59.6 in PBS-injected group, P＜0.001), and the increase degree of the capillary density in the HPP-EPC-transplanted group was higher than that in the CAC-transplant group, P＜0.01. Under the confocal microscope we observed that the DiI-labelled EPCs have incorporated into new-formed capillaries. Conclusions: HPP-EPCs showed stronger ability of proliferation, clonegenic potential and resistance to apoptosis in vitro as well as angiogenic potential in vitro and in vivo. EPCs could incorporate into new-formed capillaries.Part 3 Involvement of LFA-1 and VLA-4 in homing of EPCs to ischemic tissueObjective: EPCs transplantation can promote the neovasularization in ischemic tissue but the mechanism is still not fully understood. Previous studies have revealed that the trafficking of EPCs was induced by SDF-1 released from ischemic tissue, mediated directly by the effect of HIF. How is EPCs to interact with endothelial cell of capillaries in ischemic tissue and whether the integrinβ1 and integrinβ2 are involved in EPCs adhesion to and transmigration through endothelium. So in the present study we investigated the homing of EPCs to ischemic tissue.Material and methods: FACS was used to analyze the expression of integrinβ1 (VLA-4, CD49d) and integrinβ2 (LFA-1, CDlla) in HPP-EPCs and the expression of ICAM-1,2 and VCAM-1 in mouse bone marrow endothelial cells. The adhesion to and transmigration through endothelial cells of the HPP-EPCs blocked by functional grade neutralizing antibodies of VLA-4 and LFA-1 were studied in vitro. The proteins of ICAM-1, 2 and VCAM-1 of endothelial cells in ischemic muscles were detected by immunohistochemistry. The copies of mRNA of ICAM-1, 2 and VCAM-1 in muscles at 0, 6, 12, 24 and 48-h after ischemia were analyzed by Real time RT-PCR. The mouse models of ischemia were established and were transfused with HPP-EPCs that were labeled with CM-DiI and blocked by functional grade neutralizing antibodies of VLA-4 and LFA-1. The loss of limbs was observed and the length of necrosis limbs was measured. The blood flow of limb was analyzed by radionuclide examination. And the capillary density and the number of EPCs resided in ischemic muscles were analyzed by microscopy.Results: The results of FACS revealed that the cells positive for CD11a, CD49d and CXCR4 in HPP-EPCs were 85.7%±11.2%, 56.4%±13.8% and 87.3%±6.4%, respectively. In inactivated mouse endothelial cells the cells positive for ICAM-2 were 90.6%±4.3%, but for ICAM-land VCAM-1 were low, 3.1%±0.7% and 3.0%±0.8%, respectively. But the positive cells of ICAM-land VCAM-1 were raised greatly to 94.2%±4.6% and 93.8%±5.1% after activation of mBMECs by IL-1βand TNF-α. The expression of ICAM-1 and VCAM-1 were high in endothelium of muscles at 24-h after ischemia, but expressions of ICAM-2 were weak. The results of Real time RT-PCR revealed that the expression of ICAM-1 and VCAM-1 increased at 6-h after ischemia, reached the peak level during 12-h and 24-h and maintained at high level till 48-h ischemia. But the expression level of ICAM-2 was not of significant increase after ischemia. The results of adhesion in vitro revealed that the numbers of the adhered cells in the CD11a antibodies group (3175.7±567.2), in the CD49d antibody group (3237.44±555.6) and in the combinational antibody group (1125.3±205.1) were less than that in the isotype control antibody group (6562.1±717.4), each P＜0.01. Furthermore, the number of adhered cells in the combinational antibody group was less than the CD11a or the CD49d antibody group, each P＜0.05. The results of transmigration in vitro revealed that the numbers of migrated cells in the CD11a antibody group (1902.6±584.5), in the CD49d antibody group (1971.0±485.3) and in the combinational antibody group (801.8±309.4) were less than that in the isotype control antibody group (5255.3±849.7), each P＜0.01. Furthermore, the number of migrated cells in the combinational antibody group was less than the CD11a and the CD49d antibody group, each P＜0.05. There was no severe loss of limb in HPP-EPCs transplanted mouse model of ischemia. And there was no significant difference of limb salvage ratio among the CD11a antibody group (5 in 10), the CD49d antibody group (6 in 10), the combinational antibody group (5 in 10) and the isotype control antibody group (8 in 10). However, there were significant differences in length (cm) of necrosis limb among groups. The length of necrosis limb in the CD11a antibody group (0.73±0.24), in the CD49d antibody group (0.69±0.18) and the combinational antibody group (1.20±0.26) were less than that in the isotype control antibody group (0.34±0.21), each P＜0.01. Furthermore, the length of necrosis limb in the combinational antibody group was less than CD11a and CD49d antibody group, each P＜0.05. The results of radionuclide examination revealed that the improvement of blood flow in the CD11a antibody group (from 0.1280±0.0388 to 0.5820±0.0737), in the CD49d antibody group (from 0.1160±0.0369 to 0.6160±0.0721) and in the combinational antibody group (0.1250±0.0424) were less than that in the isotype control antibody group (0.1330±0.0392), each P＜0.01. Furthermore, the improvement of blood flow in the combinational antibody group was less than single CD11a or CD49d antibody group, each P＜0.05. The capillary density (capillaries/mm2) in the CD11a antibody group (316.2±56.5), in the CD49d antibody group (213.1±59.1) and in the combinational antibody group (801±309)were less than that in the isotype control antibody group (392.4±50.1), each P＜0.01. Furthermore, the capillary density in the combinational antibody group was less than the CD11a or the CD49d antibody group, each P＜0.05. The numbers of CM-DiI labeled HPP-EPCs resided in the ischemic tissue for the CD11a antibody group (56.0±12.7), in the CD49d antibody group (47.8±11.6) and in the combinational antibody group (23.9±10.9) were less than that for the isotype control antibody group (123.9±23.1 ), each P＜0.01. Furthermore, the number of migrated cells in the combinational antibody group was less than single antibody group of CD11a or CD49d, each P＜0.05.Conclusion: Both LFA-1 and VLA-4 are involved in HPP-EPCs homing to ischemic tissue and play an important role in EPCs adhesion to and transmigration through mature endothelial cells. This process may be mediated by the interaction between LFA-1 and VLA-4 of EPCs and their receptor, ICAM-land VCAM-1, expressed on surface of endothelial cells in the ischemic tissues.
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