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.     

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.     

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.     

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.     

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 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.     

Effects of statin reloading before percutaneous coronary intervention on circulatory endothelial progenitor cells and inflammatory cytokines

AIM:To investigate the effects of atorvastatin reloading in pre-percutaneous coronary intervention (PCI) period on endothelial progenitor cell (EPC) count and inflammatory cytokine expression in the stable angina pectoris patients who had previously received long-term statin treatment. METHODS:The patients with stable angina pectoris that had received long-term statin therapy and planned to accept PCI were randomized into 3 groups: 80 mg atorvastatin 12 h and 40 mg 2 h before coronary angioplasty (80 mg reloading), pre-operatively with 40 mg/d atorvastatin for 7 d (40 mg reloading), and without atorvastatin reloading (no reloading). CD45-/CD133+/CD34+, CD45-/CD34+/KDR+ and CD45-/CD144+/KDR+ EPCs in 100 μL peripheral blood were determined by flow cytometry 1 h prior to PCI and 1 h, 6 h and 24 h after PCI. The serum concentrations of soluble intercellular adhesion molecule 1 (sICAM-1), C-reactive protein (CRP) and troponin I (TnI) were analyzed immediately prior to and 24 h after PCI. RESULTS: (1) In 80 mg reloading group, the numbers of circulating CD45-/CD133+/CD34+ and CD45-/CD34+/ KDR+ early differentiation stage EPCs 1 h and 6 h after coronary angioplasty was significantly elevated compared with those before PCI (P<0.05). (2) In control group, the serum concentrations of sICAM-1 and CRP 24 h after PCI were significantly elevated (P<0.05) compared with preoperative values. (3) The rise in serum TnI concentration from pre- to post-operation in 80 mg reloading group was lower than that in control group. CONCLUSION: The method of atorvastatin reload before PCI affects the number of EPCs in peri-operative period. High dose of atorvastatin application before PCI triggers early EPC circulation. The serum levels of post-operative inflammatory cytokine sICAM-1 as well as CRP are reduced by atorvastatin reloading before PCI.     

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.     

Effects of high-glucose on proliferation and apoptosis in endothelial progenitor cells of type 2 diabetes mellitus

AIM: This study aimed to observe the effects of high-glucose on proliferation and apoptosis of endothelial progenitor cells (EPCs) in type 2 diabetes mellitus patients,and tried to elucidate their possible role.METHODS: Various concentrations of glucose were added to the culture system of EPCs from 25 cases of type 2 diabetes mellitus patients (DM group) and 25 cases of healthy volunteers (control group).MTT assays were used to detect the proliferative rates.Annexin-V/PI stains were used to detect the apoptotic rates,and RT-PCR to detect the expression level of bcl-2 and bax.RESULTS: Proliferative activity of EPCs in both control group and DM group were attenuated when concentration of glucose was 33 mmol/L,while apoptotic rates increased.No significant change of proliferative rate and apoptotic rate of EPCs in DM group and control group in the presence of 5 mmol/L glucose was observed.The expression level of bax of EPCs in both DM group and control group increased while expression level of bcl-2 did not change much in the presence of 33 mmol/L glucose.CONCLUSION: High-glucose attenuates proliferative activity of EPCs and increases the apoptotic rate.Upregulation of bax may be its possible role.     

Effects of erythropoietin on expression of SDF-1 and its receptor in peripheral blood endothelial progenitor cells from rats with chronic renal failure

AIM:To investigate the effects of erythropoietin (EPO) on the expression of homing factors in peripheral blood endothelial progenitor cells (EPCs) from rats with chronic renal failure (CRF). METHODS:The CRF model was established by a two-stage 5/6 nephrectomy procedure in rats. Experimental rats were randomly divided into three groups: sham operation group, CRF model group and EPO treatment group. From the third week after the second stage of 5/6 nephrectomy procedure, rats in EPO treatment group received subcutaneous injection of human recombinant EPO at 50 U/kg every time and three times a week for 6 weeks, and then all the rats were sacrificed. EPCs were isolated from rat peripheral blood and primarily cultured. The mobilization, angiogenesis and functional activity of EPCs in vitro were detected. The mRNA and protein expression of EPO, EPO receptor (EPOR), stromal cell-derived factor 1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) in EPCs was also detected by the methods of real-time PCR and Western blotting. RESULTS:Compared with CRF model group, the expression of EPO and EPOR in EPCs in EPO treatment group was significantly up-regulated (P<0.05). Moreover, the expression of SDF-1 and its receptor CXCR4 in EPCs was also up-regulated by administration of EPO (P<0.05). CONCLUSION: EPO can mobilize EPCs from CRF rat peripheral blood, which may be associated with the increased expression of SDF-1 and its receptor CXCR4.     

Effect of salidroside on activity of endothelial progenitor cells and phosphoinositide 3-kinase/Akt signaling pathway

AIM：To investigate whether salidroside has influence on the activities of endothelial progenitor cells (EPCs) and its mechanism. METHODS：Mononuclear cells from normal human peripheral blood were cultured in fibronectin coated flasks in endothelial progenitor medium. After 7 d, EPCs were characterized as adherent cells with acLDL-DiI uptaking and lectin binding by direct fluorescent staining. The proliferation and migration of EPCs were analyzed by MTT assay and Transwell chamber assay, respectively. The EPCs adhesion assay was performed by re-plating the cells on fibronectin-coated dishes, and then adherent cells were counted. NO and Akt protein were also detected. RESULTS：Salidroside promoted EPCs proliferative, migratory and adhesive capacities in a concentration dependent manner. Salidroside also increased NO secretion, and the level of phosphorylated Akt protein. However, the effects of salidroside on EPCs were inhibited by phosphoinositide 3-kinase inhibitor LY294002. CONCLUSION：Salidroside regulates the activity of EPCs by phosphoinositide 3-kinase/Akt signaling pathway.     

Bone marrow origin of endothelial progenitor cells responsible for postnatal vasculogenesis in physiological and pathological neovascularization

Endothelial progenitor cells (EPCs) exist in bone marrow, umbilical cord blood and peripheral blood of adult mammals, including humans. Furthermore, the discovery of EPCs has led to the notion of adult vasculogenesis, in which bone marrow (BM)-derived EPCs home to and incorporate into sites of new blood vessel formation, where they differentiate into endothelial cells, which is consistent with postnatal vasculogenesis. It has become apparent that circulating BM-derived EPCs are involved in promoting physiologic and pathologic neovascularization, such as wound healing and tumor growth. They are of great clinical importance in pro- or anti-angiogenic therapies.     

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.     

Overexpression of Jagged1 promotes aged rat-derived endothelial proge-nitor cells differentiating into mature endothelial cells

AIM: To investigate the effect of Jagged1 overexpression on endothelial cell-directional differentiation of aged rat-derived endothelial progenitor cells (EPC).METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 months old) or aged (19 to 26 months old) Sprague-Dawley rats and cultured in DMEM/F12 medium supplemented with 10% FBS. EPC were characterized as double positive for DiI-ac-LDL uptake and lectin binding. The experiments were divided into control group, PIRES2-EGFP transfection group, PIRES2-EGFP-Jagged1 transfection group and young rat-derived EPC group in which transfection was not performed. The GFP expression positive cell number was acquired by fluorescence microscopy and the transfection efficiency was calculated. Immunofluorescence, RT-PCR and Western blotting were used to detect the mRNA and protein expression. In vitro vasculogenesis kit was used to test the tube formation ability of EPC.RESULTS: EGFP-Jagged1 transfection induced a significant increase in the expression of Jagged1 in aged rat-derived EPC (P<0.01). Compared with the control, Jagged1 overexpression markedly enhanced the mRNA expression of von Willebrand factor (vWF) and kinase insert domain receptor (KDR) of vascular endothelial grouth factor vWF in aged rat-derived EPC (P<0.01) and improved the EPC-related tube formation (P<0.01). No significant difference between Jagged1 transfection and young rat-derived EPC groups in vWF and KDR mRNA expression and the ability of tube formation was found. CONCLUSION: In endothelial cell-conditioning medium, Jagged1 overexpression significantly promotes aged rat-derived EPC differentiation into mature endothelial cells.     

Endothelial progenitor cell-conditioned medium improves lung structure in neonatal rats exposed to hyperoxia

AIM: To investigate the therapeutic effect of endothelial progenitor cell-conditioned medium (EPC-CM) on the lung structure of neonatal rat exposed to hyperoxia, and to explore the mechanisms.METHODS: Bone marrow-derived endothelial progenitor cells (EPCs) were collected from new born Sprague-Dawley (SD) rats and the EPCs were identified. The conditioned medium from the passage 3 EPCs was collected. Newborn SD rats (n=40) were randomly divided into 4 groups. The rats in room air group were exposed to the room air (21% O₂) for 21 d. The rats in hyperoxia group were exposed to hyperoxia (85% O₂) for 21 d. The rats in endothelial cell basal medium (EBM) group were exposed to hyperoxia for 21 d, and received 100 μL EBM on postnatal day 14 (P14) in a single intratracheal (IT) injection. The rats in EPC-CM group were exposed to hyperoxia for 21 d, and received 100 μL EPC-CM on P14 in a singlie IT injection. The rats were sacrified on the 21st day. The left lungs were excised, placed in 4% paraformaldehyde, serially dehydrated in ethanol and embedded by paraffin. Serial sectioning of the paraffin-embedded left lung tissues was prepared for 5 μm thickness, and stained with hematoxylin and eosin. The pulmonary radical alveolar count (RAC) and alveolar mean linear intercept (MLI) were then calculated. The microvascular density was determined by FVⅢ immunostaining. The mRNA expression of KGF, VEGF, SP-A and SP-C in the right lung tissues was detected by real-time fluorescence quantitative PCR. RESULTS: The cultured cells had typical EPC morphological characteristics, and had the abilities to bind to FITC-UEA-1 and uptake DiI-ac-LDL. The body weight of the rats on day 21, RAC, MLI and microvascular density were significantly lower in hyperoxia group and EBM group than those in room air group (P<0.05). The EPC-CM group had significantly higher RAC and microvascular density than those in hyperoxia group and EBM group (P<0.05), but the body weight and MLI had no significant difference. The mRNA expression levels of KGF, VEGF, SP-A and SP-C in hyperoxia group and EBM group were significantly lower than those in room air group (P<0.05). The mRNA expression levels of KGF, VEGF, SP-A and SP-C in EPC-CM group were significantly higher than those in hyperoxia group and EBM group (P<0.05). CONCLUSION: EPC-CM promotes the lung alveolarization and microvascular formation in neonatal rats exposed to hyperoxia. These benefits may be correlated with the increased KGF and VEGF mRNA expression.     

Effects of high glucose on proliferation,migration, adhesion and secretion of rat late endothelial progenitor cells

AIM: To investigate the effects of high glucose on the proliferation, adhesion, migration and secretion potentials of late endothelial progenitor cells (EPCs) from bone marrow. METHODS: Mononuclear cells were collected from rat bone marrow by density gradient centrifugation and cultured with M199 medium. The early EPCs were identified by DiI-ac-LDL and FITC-UEA-1 double staining. The late EPCs were identified by RT-PCR to detect the expression of von Willebrand factor (vWF) and VE-cadherin. Moreover, the cells were identified by FACS to detect the expression of CD133 and vascular endothelial growth factor receptor-2(VEGFR-2). The 3rd generation of EPCs was harvested and incubated with glucose in a series of concentrations (5, 10, 20 or 40 mmol/L). The cell proliferation, adhesion, migration and the secretion of chemokines such as monocyte chemoattractant protein-1(MCP-1) and interleukin-8 (IL-8) were assayed with MTT, adhesion test, modified Boyden chamber assay and ELISA, respectively. RESULTS: Compared with normal glucose (5 mmol/L)treatment, high glucose (10, 20, 40 mmol/L) dose-dependently degraded the proliferation and migration of late EPCs (P<0.05 or P<0.01). At the same time, treatment with glucose at the concentration of 40 mmol/L decreased the adhesion of EPCs (P<0.05) and increased the release of MCP-1 and IL-8 by late EPCs. CONCLUSION: High glucose inhibits proliferation, adhesion and migration of late EPCs, and enhances the secretion of inflammatory factors, indicating that the high glucose correlates with the vascular complications of patients with diabetes.     

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.     

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.