ObjectiveTo explore optimal conditions of isolation, culture and labeled with superparamagnetic iron oxide (SPIO) in vitro of rat bone marrow endothelial progenitor cells, and lay the foundations for the further EPCs tracer study in vivo. MethodsThe EPCs derived from rat bone marrow were isolated and cultured by using density gradient centrifugation, which were labeled with different concentrations SPIO, Prussian blue staining was used to detect the cells labeling rate, MTT assay was used to detect the cells proliferation activity, and Trypan blue staining was used to detect the cells vitality. ResultsEPCs gradually growed in monolayer arrangement about 7 d after cultured. When the concentration of SPIO was 50μg/mL, the highest labeling rate of Prussian blue staining was 90%, the growth state of labeled EPCs were good, and could normal adherent growth and passage. At this time, the cell viability and proliferation activity were the highest through trypan blue staining and MTT assay. ConclusionsEPCs can be labeled with SPIO easily and efficiently when the concentration was 50μg/mL?without interference on the viability and proliferation activity, which lay the foundations for the further EPCs tracer study in vivo.
Objective To establish the three diamension-model and to observe the contribution of endothelial progenitor cell (EPC) in the angiogenesis and its biological features. MethodsEPC was obtained from the rats’ peripheral blood. Its cultivation and amplification in vitro were observed, and the function of the cultural EPC in vitro was detected. The three diamension-model was established and analyzed. ResultsEPC was obtained from the peripheral blood successfully. The proliferation of the EPC which induced with VEGF(experimental group) was better than that without VEGF （control group） at every different phase (P＜0.01). It was found that EPC grew into collagen-material from up and down in the three diamension-model, and its pullulation and infiltration into the collagen were seen on day 1 after cultivation. With the time flying, there were branch-like constructions which were vertical to the undersurface of collagen and interlaced to net each other. It showed that in experimental group the EPC grew fast, its infiltration and pullulation also were fast, the branch-like construction was thick. But in control group, the EPC grew slowly, infiltration and pullulation were slow, the branch-like construction was tiny and the depth of infiltration into collagen was superficial. The number of new vessels in experimental group was larger than that in the control group at every different phase (P＜0.01). ConclusionRat tail collagen can induce EPC involved in immigration, proliferation and pullulation in angiogenesis. The three-diamension model of EPC can be used to angiogenesis research. VEGF can mobilize and induce EPC to promote the angiogenesis.
Objective To measure the level of circulating endothelial progenitor cells ( EPCs) in peripheral blood of patients with acute exacerbation of chronic obstructive pulmonary disease ( AECOPD) , and to explore the relationship between EPCs and severity markers of the disease and cardiovascular adverse outcome predictors.Methods Forty patients with COPD were recruited, including 27 at acute exacerbation phase and 13 with stable COPD from December 2010 to December 2011. Sixteen healthy nonsmokers were included as controls. Circulating EPCs were isolated by Ficoll density-gradient centrifugation and purified by Magnetic Activated Cell Sorting system. High-sensitivity C-reactive protein ( hsCRP) was estimated by using a latex immunoturbidimetric assay kit, and matrix metalloproteinase-9 ( MMP-9) was measured by enzymelinked immunosorbent assay ( ELISA) . Arterial blood gas analysis and echocardiograph were performed in the AECOPD patients. The correlations between circulating EPCs, lung function, and cardiovascular markers were investigated. Results Circulating EPCs were significantly lower in AECOPD and stable COPD patients compared with the healthy controls [ ( 5.1 ±2.6) ×103 /mL and ( 6.0 ±3.2) ×103 /mL vs. ( 9.0 ±4.3) × 103 /mL, Plt;0. 05] . EPCs had a weak correlation with hsCRP ( P = 0. 033) , but not with MMP-9. In the AECOPD patients, EPC counts were significantly inversely correlated with PASP ( pulmonary artery systolic pressure) and NT-proBNP ( amino-terminal pro-brain natriuretic peptide) levels, and positively with left ventricular ejection fraction. No correlations were found between EPCs and lung function, blood gas, hospital stays or smoking index. Conclusions Circulating EPCs were significantly lower in AECOPD patients compared with healthy controls, in which systemic inflammation might be involved. Decreased EPCs were correlated with cardiac dysfunction in patients with AECOPD, which may account for the increased cardiovascular risk in this population.
ObjectiveTo compare the biological features of early and late endothelial progenitor cells (EPCs) by isolating and culturing early and late EPCs from the human peripheral blood so as to find some unique properties of EPCs and to propose a suitable strategy for EPCs identification. MethodsMononuclear cells were isolated from the human peripheral blood using density gradient centrifugation. Then, the cells were inoculated in human fibronectin-coated culture flasks and cultured in endothelial cell basal medium 2. After 4-7 days and 2-3 weeks culture, early and late EPCs were obtained respectively. The morphology, proliferation potential, surface markers, cytokine secretion, angiogenic ability, and nitric oxide (NO) release were compared between 2 types of EPCs. Meanwhile, the human aortic endothelial cells (HAECs) were used as positive control. ResultsThe morphology of early and late EPCs was different:early EPCs formed a cell cluster with a spindle shape after 4-7 days of culture, and late EPCs showed a cobblestone appearance. Late EPCs were characterized by high proliferation potential and were able to form capillary tubes on Matrigel, but early EPCs did not have this feature. Both types EPCs could ingest acetylated low density lipoprotein and combine with ulex europaeus Ⅰ. Flow cytometry analysis showed that early EPCs did not express CD34 and CD133, but expressed the CD14 and CD45 of the hematopoietic stem cell markers;however, late EPCs expressed CD31 and CD34 of the endothelial cell markers, but did not express CD14, CD45, and CD133. By RT-PCR analysis, the expressions of vascular endothelial growth receptor 2 and vascular endothelial cadherin in early EPCs were significantly lower than those in the late EPCs and HAECs (P<0.05), but no significant difference was found in the expression of von Willebrand factor and endothelial nitric oxide synthase (eNOS) between 2 type EPCs (P>0.05). The concentrations of vascular endothelial growth factor, granulocyte colony-stimulating factor, and interleukin 8 were significantly higher in the supernatant of early EPCs than late EPCs (P<0.05). Western blot assay indicated eNOS expressed in both types EPCs, while the expression of eNOS in late EPCs was significantly higher than early EPCs at 5 weeks (P<0.05). Both cell types could produce similar amount of NO (P>0.05). ConclusionThe expression of eNOS and the production of NO could be used as common biological features to identify EPCs, and the strategy of a combination of multiple methods for EPCs identification is more feasible.
ObjectiveTo observe the effects of aquaporin 1 (AQP1) on the proliferation and migration of endothelial progenitor-endothelial progenitor cells (EPC).MethodsBone marrow cells of AQP1 wild-type (WT) (n=6) and knockout-type (KO) mice (n=6) were isolated and differentiated into EPC in vitro. Immunofluorescence was used to detect cell surface antigens to identify EPC. Live cell kinetic imaging and quantification technology, transwell migration assays, as well as scratch test were used to compare the function of EPC between AQP1 WT and KO mice.ResultsEPC culture showed that cells were initially suspended and gradually adhered to typical mesenchymal stem cells within 7 days. After cultured on special medium for endothelial cells they were adhered and differentiated, and fusiform or polygonal, paving stone-like EPC were observed around 14 days. When cultured by special medium of EPC, CD133 and CD31 were positively detected after 7 days, and CD34 and Flk-1 were positively detected after 14 days. Positive expression of AQP1 was only detected in EPC of AQP1 WT mice. Functional studies of EPC revealed there was no significant difference in the proliferation of EPC between AQP1 WT and KO group mice. Transwell assay showed that EPC migration ability of AQP1 KO mice was significantly weaker than that of WT mice. The scratch healing ability of EPC in AQP1 KO mice was significantly lower than that of WT mice.ConclusionsEPC initially shows the characteristics of stem cells and with the prolongation of culture time, EPC gradually shows the characteristics of endothelial cells. AQP1 affects the EPC migration rather than proliferation.
Objective To observe the effects of Galectin-3 on proliferation of vascular endothelial cells derived from peripheral blood endothelial progenitor cells. Methods The cultured peripheral blood endothelial progenitor cells in vitro were isolated and purified from human peripheral blood, and the cells were differentiated into vascular endothelial cells. Then the cells were cultivated with the galectin-3 of different concentrations, and to observe the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells. Results The abilities of proliferation of endothelial cells derived from peripheral blood endothelial progenitor cells of 0.1, 1.0, 2.5, 5.0, and 10.0 μg/ml groups were higher than that of 0 μg/ml group, there were not statistic significance of the differences between the 0.1，1.0, 2.5, and 0 μg/ml groups （P＞0.05）. But the abilities of proliferation of 5.0 and 10.0 μg/ml groups were obviously higher than that of 0, 0.1, 1.0, and 2.5 μg/ml groups （P＜0.05）, and the abilities of proliferation of 10.0 μg/ml group was also higher than that of 5.0 μg/ml group （P＜0.05）. Conclusion Galectin-3 can promote the proliferation of endothelial cells derived from peripheral blood endothelial progenitor cell.
Objective To determine the transfection efficiency of recombinant adenovirus to endothelial progenitor cells(EPCs) and provide the base of lung cancer therapy by transfecting human herpes simplex virusthymidine kinase（HSV-TK） gene to EPCs. Methods Admove recombinant adenovirus 5F35(AD5F35) which transfected with βgalactosidase(AD5F35LacZ) to the 24 well plate cultivated with EPCs and transfect the EPCs. Stain the EPCs with LacZ kit and calculate the transfection efficiency. Results The blue stain cells were cells transfected successfully with AD5F35LacZ under the optical microscope. The transfection efficiencies of adenovirus to EPCs were different under the premise of the different multiplicity of infection(MOI). In a certain range, the transfection efficiencies rise with the MOI rise. When MOI was 400,the proportion of blue stain cell is the highest, which was 98.38%±1.25%. Conclusion Recombinant adenovirus can transfect EPCs successfully. The transfection efficiencies rise with the MOI rise. When the MOI is 400,the transfection efficiency is the highest.
Objective To study the short and medium term effect of myocardial contractile force by implantation of endothelial progenitor cells （EPCs） in the myocardial infarction model. Methods Hundred and twenty SD rats were equally and randomly divided into experimental group and control group （60 rats in each group）. Acute myocardial infarction model was created by ligation of LAD. Autologous EPCs were purified from peripheral blood then implanted into the acute myocardial infarct site via topical injection. IMDM were used in control group. Specimens and muscle strip were harvested at 3, 6 weeks, 6, 8 and 12 months after EPCs implantation for contractile force study and to detect the expression of vascular endothelial growth factor（VEGF）, basic fibroblast growth factor （bFGF） and Ⅷ factor by immunohistology and video image digital analysis system. Results The expression of VEGF, bFGF and the microvessel counts in experimental group were much higher than those of control group（P〈 0.01） at 3, 6 weeks and 6 months after transplantation. The contractile force in experimental group was better than that in control group（P〈0.01） at the same time. But from 8 months after implantation, the contractile force and so on were not up in the experimental group. Conclusion EPCs, after being implanted into infarct myocardium, shows the ability of improvement of the contractile performance in infarcted myocardium by means of angiogenesis and vasculogenesis and the medium term results are persistent.
ObjectiveTo review the research progress of neural regulation mechanism of vasculogenesis. MethodsThe relevant literature on neural regulation mechanism of vasculogenesis was extensively reviewed. ResultsNeural regulation of vasculogenesis depends on synergistic effect among various cells of neurovascular unit, and co-participation of multiple cytokines, and it is closely related to a variety of repair mechanism, such as nerve regeneration and synaptic plasticity, but the specific mechanism need to be further investigated. ConclusionThe research of the neural regulation mechanism of vasculogenesis will contribute to further understanding repair mechanism of nerves and vessels injuries.
ObjectiveTo review the current progresses in purification strategies, biological characters, and functions of endothelial progenitor cells (EPCs) derived extracellular vesicles (EVs) (EPC-EVs). MethodsRecent relevant publications on the EPC-EVs were extensively reviewed, analyzed, and summarized. ResultsEPC-EVs are usually isolated by differential centrifugation and exhibit a homogenous pattern of spheroid particles with a diameter ranging from 60 to 160 nm under transmission electron microscopy. EPC-EVs are positive for cell-surface markers of EPCs (CD31, CD34, and CD133), and negative for markers of platelets (P-selectin and CD42b) and monocytes (CD14). Recent studies have shown the effectiveness of EPC-EVs in ischemic injuries, anti-Thy1 glomerulonephritis, and cardiomyocyte hypertrophy, and also shown their predictive role in cardio-cerebral-vascular diseases. ConclusionAn alluring prospect exists on the EPC-EVs-related research. Further studies are required to decipher the composition of EPC-EVs and their precise role in pathophysiological processes, and to investigate the molecular mechanisms for their targeting and function.