GM-CSF induces human vascular endothelial cells to form vessel-like structure and the role of VEGF

AIM: To investigate the influence of GM-CSF on human vascular endothelial cells induced to form new blood vessels and the role of VEGF. METHODS: HUVECs were cultured by Matrigel to set up a stable angiogenesis system with the stimulating factors. The rhGM-CSF concentration-dependent and time-dependent effects and the role of VEGF165 were detected. CD34 was measured by immunochemical staining and numbers of vessel formation was calculated under microscopic observation. RESULTS: After treatment with rhGM-CSF at various concentrations and at different time points, the numbers of vessel formation increased in a dose-dependent and time-dependent manner. In the presence of VEGF165, the numbers of vessel formation increased evidently. CONCLUSION: HUVECs were induced to develop tubular structure in vitro cultured with Matrigel. GM-CSF promotes human vascular endothelial cells to form vessel-like structure in vitro in a dose-dependent and time-dependent manner. VEGF also in vitro promotes human vascular endothelial cells to form new vessel-like structure.     

Comparative analysis of cultured endothelial progenitor cells in vitro from PBMCs and enriched CD133+ cells

AIM: To compare the methods of two currently employed isolation methods for endothelial progenitor cells (EPCs): from total peripheral blood mononuclear cells (PBMCs) and from enriched CD133+ cells, by defining the cell morphology, phenotype, reproductive activities and function in vitro, providing a reference for clinic application. METHODS: PBMCs from the healthy subjects were used for CD133+ sorting or not. The two groups of isolated cells were suspended in complete medium M199 for 7 d to 14 d. EPCs phenotype were characterized by FACS. The proliferation of differentiated EPCs was studied by MTT assay, and VEGF concentration was measured using an ELISA kit. Matrigel experiment and migration assay were imitated vascularization in vivo. RESULTS: PBMCs produced more colony-forming units (CFU) than CD133+ cells from the same volume of blood (P<0.01). From 7 d to 14 d, the two groups show decreased expression of hematopoietic stem cell markers and increased level of endothelial markers, but CD144+ cells in CD133+ group were lower than those in PBMCs groups (P<0.01). Cells in PBMCs group secreted more VEGF than that in CD133+ group on 7 d (P<0.01). Compared to CD133+ group, PBMCs group showed more potential of proliferation and vascularization in vitro. CONCLUSION: CD133+ sorted cells show a lower capacity of differentiation, secretion, proliferation and vascularization in vitro, which is unable to differentiate to mature endothelial cells, indicating that its not a preferential way to obtain EPCs for clinic therapy.     

The research development of endothelial progenitor cells

Endothelial progenitor cell (EPC) is a kind of directional cell that is able to differentiate to endothelial cell. The role of EPC is associated not only with vasculogenesis during embryonic development but also with physiological organ maintenance and angiogenesis during postnatal and adult period. There is a good clinical therapeutic prospective use for EPC in the treatment of ischemia diseases and inhibition of tumor angiogenesis.     

The ex vivo expansion characteristic of endothelial progenitor cells

AIM: To explore the ex vivo expansion characteristics of the endothelial progenitor cells (EPCs). METHODS: CD34⁺ cells were selected from umbilical cord blood mononuclear cells (MNC) by MiniMACS system, expanded at the same conditions as that for total MNC, coincubation of CD34⁺ and CD34^- from the same donation for EPCs. In addition, we tested the effect of vessel endothelial growth factor (VEGF) and passage on cell differentiation, expansion kinetics and apoptosis. EPCs were determined and quantified by immunocytochemistry and flow cytometry. RESULTS: Coculture of CD34⁺ and CD34^-,total MNC led to a significant increase in the expansion of CD34⁺ cells compared with CD34 enrichment (P＜0.05). There was a trend toward decreased apoptosis in cultures when early passage was performed once the linear cord like structures appeared. There was no significant effect on apoptosis between with VEGF and without VEGF group (P＞0.05). These differentiated EPCs were stained positive for CD34⁺, von Willebrand factor (vWF), KDR, CD31 and incorporate acetylated low-density lipoprotein (LDL). CD34⁺ and AC133⁺cells accounted for 68.2%±6.3% (n=6) and 57.2%±9.8% (n=6) of attaching (AT) cells at day 7 of culture, respectively. CONCLUSIONS: Coculture of CD34⁺ and CD34^- or culture of MNC enhances ex vivo expansion of EPCs. Early passage decreases apoptosis rate, VEGF has no significant effect on ex vivo expansion of EPCs.     

Intravenous transfusion of endothelial progenitor cells reduces neointima formation

AIM: To investigate the feasibility of transplanting endothelial progenitor cells (EPCs) obtained from spleen in vascular endothelium repairmen after vascular injury. METHODS: EPCs were isolated by using a Ficoll density gradient centrifugation, and were cultured in plate. The endothelial characteristics of EPCs were identified by immunochemical staining and fluorescent labeling. Dil-Ac-LDL labeled spleen-derived EPCs were transplanted into the rats by intravenous injection directly after induction of arterial injury and again 24 hours later. Rats received FITC-labeled lectin intravenously before euthanasia. The distribution of fluorescent labeled EPCs was traced. The morphology of arterial intima and media was studied by optical microscopy and image analysing system. RESULTS: The adherent cells were considered EPCs that showed spindle shape and form blood-siland-like structures during development. The adherent cells had many endothelial characteristics. Fluorescent labeling showed that the intravenously injected EPCs specifically restricted to the vascular injury site, and lectin binding confirmed the endothelial phenotype. The ratio of neointimal/media area in EPCs transplantation group was obviously reduced than that in injury group and M199 group (0.82±0.09 vs 1.52±0.21, 1.48±0.19, P<0.01). The PCNA positive expression cells were evidently decreased compared with injury group and M199 group (19.25±3.96 vs 31.42±5.23, 29.37±3.16, P<0.05). CONCLUSION: EPCs incorporate into the process of injured carotid reendothelialization. EPCs transplantation induces an increase in the circulating EPCs, accelerates the process of endothelial repairmen and reduces neointima formation.     

Vascular endothelial growth factor on function of endothelial progenitor cells from peripheral blood

AIM: To investigate the effect of vascular endothelial growth factor(VEGF) on the biological function of peripheral endothelial progenitor cells (EPCs) and to find the suitable concentration to promote the growth of EPCs.METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation,and then the cells were plated on fibronectin-coated culture dishes.After culture for 4 days,attached cells were incubated with VEGF in a series of concentrations (0,10,20 and 50 μg/L) for 72 h,then attached cells were characterized with immunohistochemistry.EPC proliferation and migration activity were assayed with MTT assay and modified Boyden chamber assay,respectively.EPC adhesion assay was performed by replating MNCs on fibronectin-coated dishes,and then the adherent cells were counted.RESULTS: The EPCs from MNCs were successfully isolated and were differentiated to endothelial cells (ECs).Incubation of isolated human MNCs with VEGF increased the proliferative,migratory and adhesive capacities of EPCs,and this effect was most prominent when the concentration of VEGF was 20 μg/L after 72 hours.At the same concentration of VEGF,the functions of EPCs from patients with masculine coronary arteriography were lower than those of EPCs from patients with negative coronary arteriography.CONCLUSION: Functional activities of EPCs are decreased in patients with masculine coronary arteriography.The results suggest that the low concentration of VEGF may improve functional activities of EPCs.     

Preliminary study on induction of mouse embryonic stem cells into neural stem cells in vitro

AIM:To explore the feasibility of inducing mouse embryonic stem cells into neural stem cells in vitro. METHODS:Embryonic body induced by retinoic acid and retinal müller cells were selected in neural stem cell-defined medium for 7 days, and the morphological changes were observed. The selected cells were stained immunocytochemically with anti-nestin, anti-BrdU antibodies, and their ability of expansion and differentiation were analyzed. RESULTS:Large amounts of neurospheres were derived from embryonic body in the selected medium on the 7th day, which could be passaged and differentiated, stained positive with nestin and BrdU, and expressed nestin, glutaminase and Brn-3 genes. CONCLUSION:Neural stem cells could be derived from embryonic body induced by RA and müller cells in the selected medium, which would offer an alternative to treat neuropathy such as glaucoma and retinal degeneration in the future.     

The vascular endothelial progenitor cells and angiogenesis in ischemic cardiovascular diseases

The vascular endothelial progenitor cells are a population of functional endothelial precursors in circulating blood, which are derived from bone marrow or cord blood. CD34⁺, Flk-1⁺ and ACl33⁺ are their molecular markers. In this review, the functional characterization of vascular endothelial progenitor cells is introduced and the relationship between vascular endothelial progenitor cells and angiogenesis in is chemic cardiovascular diseases is discussed. These data may offer a foundation for the development of therapeutic angiogenesis for the prevention and treatment of ischemic cardiovascular diseases by transplantation of vascular endothelial progenitor cells.     

Gender differences in adult circulating endothelial progenitor cells (EPCs) and effect of estradiol on arousing of EPCs in vitro

AIM: To investigate the gender differences and influence of menstrual cycle on the number and activity of adult circulating endothelial progenitor cells (EPCs), and the effect of estradiol on EPCs. METHODS: Ten men and 10 women were enrolled in the study. Peripheral blood samples of the men were collected only once and peripheral blood samples of the women were collected at each menstrual cycle phase (menstrual, pre-ovulatory and mid-luteal phases). The number of CD34⁺/CD133⁺/kinase insert domain-containing receptor (KDR)⁺ EPCs was determined by flow cytometry analysis and the level of circulating estradiol was measured by radioimmunoassay. Mononuclear cells were isolated from the blood and cultured in vitro. After cultured for 7 days, the number and the adhesive capacity of EPCs were observed. The effect of estradiol on the EPCs were detected by transmembrane migration assay and proliferation assay. RESULTS: The number of circulating EPCs was significantly higher in women than that in men (P<0.01), and it was higher at the pre-ovulatory phase and the mid-luteal phase than that at the menstrual phase (P<0.05). After cultured in vitro, the activity of EPCs did not reveal gender difference. In the cells treated with estradiol at concentration of ≥1×10^-9 mol/L, the capacities of transmembrane migration and proliferation were significantly increased (P<0.05). CONCLUSION: There are the gender differences of adult circulating EPCs between men and women. The number and activity of adult circulating EPCs may be regulated by menstrual cycle. In addition, estrogen plays an important role in the arousing of EPCs.     

BYHWD promotes endothelial progenitor cells to repair damaged vascular endothelium

AIM: To investigate the role of Buyanghuanwu decoction (BYHWD) in promoting endothelial progenitor cells (EPCs)-induced recovery of damaged vascular endothelium. METHODS: The endothelial damaged rats were lavaged with BYHWD and injected with EPCs through vena caudalis. The repaired situation of damaged endothelium was observed. RESULTS: Compared with EPCs group and BYHWD group, the endothelial thickness was reduced, the levels of calcium, triglycerides and total cholesterol were decreased, but the high density lipoprotein levels were increased. In addition, the protein expression of vascular endothelial nitric oxide synthase and vascular stromal cell-drived factor-1 was sig-nificantly increased, but the expression of CXC chemokine receptor-4 was significantly reduced in BYHWD+EPC group. CONCLUSION: BYHWD promotes EPCs repairing damaged endothelium, the mechanism may be related to improve the internal environment and promotes the EPCs homing.     

Protective effects of salidroside on endothelial progenitor cells damaged by radiation

AIM: To explore the protective effects of salidroside on endothelial progenitor cells (EPCs) damaged by radiation and its mechanisms.METHODS: EPCs from normal peripheral blood were cultured in fibronectin-coated flasks with endothelial progenitor medium. The effects of salidroside on the viability, migration, adhesion and apoptosis of radiation-damaged EPCs were detected. The viability, apoptosis and migration of the cells were assayed by CCK-8 assay, flow cytometry and Transwell chamber experiment, respectively. The cell adhesion assay was performed by re-plating the cells on fibronectin-coated dishes, and then the adherent cells were counted. The expression of Akt protein in the cells was assessed by Western blotting. RESULTS: Salidroside improved the viability, and migratory and adhesive capacities of the EPCs, and decreased the apoptosis after radiation. Salidroside also increased the protein level of phosphorylated Akt. However, the effects of salidroside on radiation-damaged EPCs were inhibited by phosphatidylinositol 3-kinase inhibitor LY294002. CONCLUSION: Salidroside protects EPCs from radiation damages and its mechanism is associated with enhancing phosphatidylinositol 3-kinase/Akt signaling pathway.     

Effects of Chinese yellow wine on homocysteine-induced dysfunction in rat endothelial progenitor cells

AIM: To investigate whether Chinese yellow wine has influences on homocysteine (Hcy)-induced dysfunction in rat endothelial progenitor cells (EPCs). METHODS: Rat bone marrow was extracted to harvest mononuclear cells (MNCs) by density gradient centrifugation. The MNCs were plated on fibronectin-coated culture dishes, and were induced into EPCs by EGM-2 complete medium supplemented with cell growth factor. The adherent cells were collected 7 d later for all studies. EPCs were characterized as adherent cells double positive for DiI-ac-LDL uptaking and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. The viability, migration, apoptosis and in vitro vasculogenic activity of the EPCs were determined by MTT assay, Transwell chamber assay, apoptosis kit and in vitro vasculogenesis kit, respectively. RESULTS: Compared with control group, the viability, migration and in vitro vasculogenic capacity of the EPCs in Hcy group were significantly decreased (P<0.01). Compared with Hcy group, yellow wine group and red wine group both significantly improved the viability, migration and in vitro vasculogenic capacity of Hcy-induced EPCs (P<0.01). Compared with control group, yellow wine group and red wine group both significantly improved the above-mentioned functions of EPCs (P<0.05). However, no significant difference of apoptosis in all groups was observed. CONCLUSION: Hcy may result in dysfuction of EPCs. Treatment with yellow wine improves Hcy-induced EPC functions.     

Effects of triptolide on changes of endothelial progenitor cells from peripheral blood

AIM: To investigate the effects of triptolide on endothelial progenitor cells from peripheral blood.METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by density gradient centrifugation.The cells were plated on fibronectin-coated culture dishes.After 7 d of culture,adherent cells were characterized by demonstrating the expression of CD34,CD31 and vWF with immunohistochemistry.Adherent cells were stimulated with triptolide (2.5,5.0,10.0,20.0 μg/L) or vehicle control for 24 h.Activities of EPCs in terms of proliferation and migration were determined by MTT assay and modified Boyden chamber assay,respectively.EPCs adhesion assay was performed by replating MNCs on fibronectin-coated dishes,and then the adherent cells were counted.RESULTS: The proliferative,migratory and adhesive capacities of EPCs decreased significantly after 24 h incubation with triptolide,maximum at 20 μg/L (compared to that in control group).In patients with coronary heart disease,the biological function of EPCs was lower than that in patients without coronary heart disease in low dosage of triptolide but almost the same in high dosage group.CONCLUSION: Triptolide may inhibit functional activities of EPCs,the reendothelization and neovascularization of vessel.     

Insulin protects endothelial progenitor cells against functional damage caused by high glucose

AIM: To investigate the impact of various levels of glucose on endothelial progenitor cells (EPCs) proliferation, senescence, and nitric oxide (NO) secretion,and the effect of insulin under high glucose conditions.METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation, cultured with medium 199, and identified to be EPCs at 7th day by flk-1 and AC133 double staining. EPCss were harvested and incubated with glucose (5, 10, 20, 40 mmol/L) or insulin (0.1, 1, 10, 100 nmol/L) under high glucose conditions for 24 h or 7 days. Proliferative capacity, senescence level and NO secretion (after 24 h of incubation) were subsequently determined.RESULTS: High glucose (40 mmol/L) markedly inhibited EPCs proliferation, accelerated EPCs senescence, and decreased NO production (all P<0.05). Compared with high glucose (40 mmol/L) group, insulin intervention promoted EPCs proliferation, inhibited EPCs senescence (prominent at 1 nmol/L, P<0.05), and enhanced NO secretion (prominent at 10 nmol/L, P<0.05).CONCLUSION: High glucose harms EPCs proliferation and function, while insulin alleviates this jeopardy, indicating the protective role of insulin for the cardiovascular system.     

Isolation of endothelial progenitor cells from cord blood with CD133 immunomagnetic sorting

AIM: To isolate, purify and differentiate endothelial progenitor cells from cord blood in vitro and to study their biological characteristics. METHODS: CD133+ cells were selected from fresh cord blood mononuclear cells (MNC) by magnetic activated cell-sorting system (MACS). EPC was studied by flow cytometry, immunocytochemistry and immunofluorescence staining. Isolated cells were cultured in IMDM medium supplemented with or without VEGF, bFGF, SCF. RESULTS: The percentage of CD133+ cells of cord blood MNC was (1.41±1.14)%, and purity was 75%-85% (FACS method). CD133+ cells were grown on fibronectin-coated chamber slides in the presence of VEGF, bFGF, SCF. Within 1-2 hours of culture cells became adherent. On day 7-10, the adherent cells displayed a typical “cobblestone” morphology. After 14 days of culture, the adherent cells revealed a heterogeneous cell population, comprising small-sized round cells, spindle-like cells and formed tube-like structure. Weibel-Palade bodies were shown on the transmission electron microscopy photomicrographs. Compared with the original, cell markers CD133 and CD34 decreased significantly (77.0%±3.3% to 1.6%±2.2% and 93.1%±4.7% to 37.4%±4.9%, P<0.05), while Flk-1 increased significantly (from 22.3%±3.3% to 94.3%±4.1%, P<0.05) after 14 days of culture with VEGF, bFGF, SCF. The vWF was strongly expressed (77.9%±3.3%) on the 14th day later. CONCLUSION: Vascular endothelial progenitor cells were isolated from cord blood with specific expression of CD133/CD34/Flk-1. With the stimulation of the growth factors, seven-ten days after culture EPCs could be turned to endothelial cells.     

Effects of homocysteine on number and activity of endothelial progenitor cells from peripheral blood

AIM: To investigate whether homocysteine (Hcy) has influences on endothelial progenitor cell (EPCs) number and activity from peripheral blood. METHODS: Total mononuclear cells (MNCs) were plated on fibronectin-coated culture dishes and cultured for 7 days, and then attached cells were stimulated with Hcy or vehicle control for 6 h, 12 h, 24 h and 48 h. The adhesion, proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed, respectively. RESULTS: Incubation of isolated human MNCs with Hcy dose and time-dependently decreased the number of EPCs with maximum at 200 μmol/L for 24 hours (35.7±6.7 vs 62.5±10.6， P＜0.01). In addition, Hcy impaired EPC proliferative (0.531±0.061 vs 0.328±0.055， P＜0.05), migratory (26.3±6.4 vs 6.4±3.7， P＜0.01), adhesive (33.1±8.1 vs 17.4±7.5， P＜0.01) and vasculogenesis capacity (25.4±9.1 vs 10.4±4.7， P＜0.01) in a dose and time-dependent manner. CONCLUSION: It is suggested that Hcy may result in the reduction of EPCs and decrease EPC functional activity.     

Effects and possible mechanism of recombinant human erythropoietin on proliferation of endothelial progenitor cells

AIM: To observe the effects of recombinant human erythropoietin on proliferation of endothelial progenitor cells (EPCs) from healthy volunteers and patients with renal failure,and tried to elucidate the possible mechanism.METHODS: Various concentrations of rhEPO were added to the culture system of EPCs from 15 cases of patients with renal failure (RF group) and 15 cases of healthy volunteers (control group).MTT assays were used to detect proliferative rates.Annexin-V/PI stains were used to measure the apoptotic rates.Western blotting was used to determine the expression of Akt protein kinase.RESULTS: Numbers and proliferative ability of EPCs from control group and RF group were improved in dose-dependent manner when concentrations of rhEPO were 100 U/L,600 U/L and 1 200 U/L.However,compared to the control group,numbers and function of EPCs from RF group were remarkably decreased.The apoptosis rate of EPCs was decreased and the activity of Akt protein kinase was improved in the presence of 1 200 U/L rhEPO.Wortmannin was able to block the effects.CONCLUSION: rhEPO improves the number and function of EPCs from both healthy volunteer and patients with renal failure.PI3K/Akt might play an important role in it.     

Barrier function of human retinal microvascular endothelial cells cultured in vitro

AIM: To investigate the blood-retinal barrier (BRB) function of human retinal microvascular cells (hRMECs) cultured in vitro.METHODS: Primary hRMECs were cultured and identified. The fourth generation was inoculated in a micro porous filter with the filter membrane of Polyester (PET). Cells on the surface of filter membrane was observed by phase microscope at different time points, and occludin staining was used to show the tight junction (TJ) indirectly. Transendothelial electrical resistance (TER) was detected at 2, 3, 4 and 5 weeks, respectively and the permeability was measured with horseradish peroxidase (HRP) in both normal condition and stimulated with 5 μg/L VEGF 15 min, 30 min, 45 min, 60 min at 2 weeks monolayer. RESULTS: Primary hRMECs were cultured successfully and purity was up to 95.8%. Cells formed a monolayer l week after seeding. 2, 3 and 4 weeks after inoculation, the density of hRMECs stayed unchanged. Occludin staining showed TJ indirectly 2 weeks after inoculation. Distribution of occludin showed the TJ maturing with time delaying. TER reached (120.62±3.97) Ω/cm2 at 2 weeks and kept stable within 4 weeks (P>0.05). VEGF at the concentration of 5 μg/L increased HRP penetration significantly compared to that in normal culture condition (P<0.05).CONCLUSION: The filter of PET membrane may be a useful method to set up the model monolayer of hRMECs and investigate the barrier function of blood retina barrier.