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.     

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.     

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.     

Serum from rats at different ages modulates the functional activities of rat bone marrow-derived endothelial progenitor cells

AIM: To investigate the effects of serum from rats at different ages on the functional activities of rat bone marrow-derived endothelial progenitor cells (EPCs). METHODS: Mononuclear cells were obtained from bone marrow of young (1 to 2 month-old) and aged (19 to 26 month-old) Sprague-Dawley rats by Ficoll density gradient centrifugation and cultured with medium DMEM/F12 (containing 10% fetal bovine serum, endothelial cell growth supplement ECGs 100 mg/L, 1×10⁵ U/L of penicillin and streptomycin, respectively), 48 h later, the suspending cells were translocated to be cultured in new flasks coated with fibronectin, the secondary attached cells were used to perform the further experiments. EPCs were characterized as double positive for Dil-ac-LDL uptake and lectin binding. The cells were further identified by CD31 and vWF expression. Serum from young (1 to 2 month-old) and aged (19 to 26 month-old) rats was collected and used to culture EPCs. The experiments were divided into four groups: A= aged rat EPCs + aged rat serum; B= aged rat EPCs + young rat serum; C= young rat EPCs +aged rat serum and D= young rat EPCs + young rat serum. After cultured in DMEM/F12 supplemented with 10% serum from rats at different age (no fetal bovine serum addition in this medium), the average fluorescence intensities of EPCs stained with Dil-ac-LDL were tested by laser scanning co-focal microscopy. Migration and proliferation were assayed by modified Boyden chamber and MTT, respectively. Cell adhesion was performed by replacing EPCs onto cultured DAPI-labeled confluent smooth muscle cell monolayer and the adherent cells were counted. RESULTS: Young rat serum significantly improved the ability of aged rat EPCs for uptake of Dil-ac-LDL (P<0.01), increased the migration (P<0.01), adhesion (P<0.05) and proliferation activity (P<0.01) of aged rat EPCs, whereas aged rat serum obviously decreased the migration (P<0.05) and adhesion (P<0.05) activity of the young rat EPCs. CONCLUSION: Young rat serum significantly improves the activity of aged rat EPCs. On the contrary, aged rat serum partially inhibits the activity of young rat 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.     

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 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 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 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.     

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.     

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.     

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.     

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.     

Rosuvastatin inhibits up-regulation of inflammatory factors in endothelial outgrowth cells induced by C-reactive protein

AIM：To study the effects of rosuvastatin on C-reactive protein (CRP)-induced expression of inflammatory factors in endothelial outgrowth cells (EOCs). METHODS：Mononuclear cells (MNCs) were isolated from human umbilical cord blood by density gradient centrifugation. EOCs were treated with CRP at concentration of 5, 10, 25 and 50 mg/L and were exposed to 50 mg/L CRP for different time (0, 3, 6 and 12 h). In addition, EOCs were pre-incubated with rosuvastatin at different concentrations (10-8, 10-7 and 10-6mol/L) for 12 h and then stimulated with 50 mg/L CRP. The mRNA levels of IL-8, MCP-1, VCAM-1 and ICAM-1 in EOCs were measured by quantitative PCR. The protein levels of MCP-1 and VCAM-1 were detected by ELISA. RESULTS：CRP dose-dependently increased the mRNA levels of inflammatory factors (IL-8, MCP-1, VCAM-1 and ICAM-1) and protein levels of MCP-1 and VCAM-1. The mRNA levels of IL-8, VCAM-1, and ICAM-1 in EOCs reached to the peak at 6 h, while MCP-1 peaked at 3 h when treated with 50 mg/L CRP. The protein levels of MCP-1 and VCAM-1 increased in a time-dependent manner. The mRNA and protein levels of the inflammatory factors were significantly decreased by treatment with rosuvastatin at different concentrations. CONCLUSION：CRP is not just an inflammatory marker as CRP induces inflammation in EOCs. Rosuvastatin attenuates CRP-induced inflammatory response in EOCs, indicating a new target for the prevention of atherosclerosis.     

Effects of Danggui Buxue decoction on endothelial progenitor cells,serum VEGF and SDF-1 in atherosclerotic rabbits

AIM: To investigate the effects of Danggui Buxue decoction (DBD) on serum vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1 (SDF-1), as well as the activity of circulating endothelial progenitor cells (EPCs) in atherosclerotic rabbits. METHODS: The model of atherosclerosis was established using immune injury and fatty diet for 4 weeks in New Zealand rabbits (n=25). All modeled rabbits were randomized into 5 groups with 5 animals in each group. The rabbits in atherosclerosis group were intragastrically administered with distilled water. The rabbits in simvastatin group were treated with simvastatin at the dose of 1.7 mg/kg. The rabbits in DBD high-dose, middle-dose and low-dose treatment groups were given DBD at the doses of 6 g/kg, 3 g/kg and 1.5 g/kg, respectively. All drugs were given once a day for 2 weeks. After treatment, the levels of serum VEGF and SDF-1 were measured. The mononuclear cells isolated from the rabbit peripheral blood were cultured for 7 days in vitro, and then attached cells were cultured with both DiI-ac-LDL and FITC-UEA-1 for identification. The proliferation was detected by MTT method. The cell migration was observed using Transwell chambers. The adhesion determination and in vitro angiogenesis assay were also performed. RESULTS: Compared with atherosclerosis group, the levels of serum VEGF and SDF-1 were elevated (P<0.05), the proliferation, migration, adhesion and angiogenesis of EPCs were all improved in DBD high-dose, middle-dose treatment groups and simvastatin group (P<0.05). CONCLUSION: DBD elevates the levels of serum VEGF and SDF-1 to improve the activity of EPCs in the process of atherosclerosis.     

Protective effect of salidroside on rats with hypoxic pulmonary hypertension by suppressing oxidative stress

AIM: To study whether salidroside plays a protective role in hypoxia-induced pulmonary hypertension by suppressing oxidative stress. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia (N) group, hypoxia for 4 weeks (H₄) group, low-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 16 mg/kg, H₄S16) group and high-dose salidroside (hypoxia for 4 weeks and treatment with salidroside at 32 mg/kg, H₄S32) group. The mean pulmonary arterial pressure (mPAP), the weight ratio of right ventricle/(left ventricle+septum)[RV/(LV+S)] and vessel wall area/vessel total area (WA/TA) were evaluated. The levels of malondialdehyde (MDA) in the serum and lung tissues were detected by colorimetric method. The levels of 8-iso-prostaglandin F_2α (8-iso-PGF_2α) in the serum and lung tissues were measured by ELISA. The activity of superoxide dismutase (SOD) in the serum was analyzed by hydroxylamine method. The expression of NAPDH oxidase 4 (NOX4) and SOD1 in the lung tissues was determined by Western blot. RESULTS: Compared with N group, the levels of mPAP, RV/(LV+S) and WA/TA in H₄ group were significantly increased, which were apparently attenuated by salidroside injection in a dose-dependent manner. Meanwhile, salidroside administration apparently decreased the levels of MDA and 8-iso-PGF_2α in the serum and lung tissues, as well as the expression of NOX4 in the lung tissues. Besides, compared with N group, the activity of SOD in the serum and the expression of SOD1 in the lung tissues in H₄ group were significantly decreased, while administration of salidroside increased the activity of SOD in the serum and the expression of SOD1 in the lung tissues in a dose-dependent manner. CONCLUSION: Salidroside protects the pulmonary vessels from remodeling and attenuates hypoxia-induced pulmonary hypertension by inhibiting oxidative stress.     

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.     

Protective effect of procyanidin single active ingredient B2 on human endothelial progenitor cells under high glucose stimulation

AIM: To study the protective effect of procyanidin single active ingredient B2(PC-B2) on human endothelial progenitor cells(EPCs) stimulated with high glucose. METHODS: The human EPCs were isolated from peripheral blood of healthy people and identified. The EPCs were divided into control group(PBS treatment), hypertonic control group(25 mmol/L mannitol treatment), high glucose(30 mmol/L) group, and different concentrations(2, 10 and 50 mg/L) of PC-B2+30 mmol/L glucose groups. The viability of EPCs was detected by CCK-8 assay. The levels of LDH, MDA, SOD and GSH in the EPCs were detected. The changes of NO, ET-1, ICAM-1 and VCAM-1 in the EPCs cultured medium were measured by ELISA. The cell apoptotic rate and reactive oxygen species(ROS) in the EPCs were analyzed by flow cytometry. The expression of VEGF and VEGFR-2 in the EPCs were determined by Western blot. RESULTS: Compared with control group, the viability of human EPCs was decreased significantly in 30 mmol/L glucose group(P<0.05). The LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were induced significantly(P<0.05), but SOD and GSH activity and NO production were decreased significantly(P<0.05). The ROS and cell apoptotic rate were increased significantly(P<0.05). The expression of VEGF and VEGFR-2 in the EPCs were decreased(P<0.05). When human EPCs were treated with different concentrations of PC-B2 and 30 mmol/L glucose, the viability was obviously rebounded(P<0.05), the LDH leakage, MDA content and the releases of ET-1, ICAM-1 and VCAM-1 were decreased gradually(P<0.05), the SOD, GSH activity and NO production were increased significantly(P<0.05), the ROS and cell apoptotic rate were decreased significantly(P<0.05), and the expression of VEGF and VEGFR-2 in the EPCs was increased gradually(P<0.05).CONCLUSION: PC-B2 enhances the viability of human EPCs under high glucose condition, reduces high glucose-induced oxidative damage, restores the EPCs normal function, and reduces the releases of inflammatory cytokines and apoptosis, thus playing a protective effect on human EPCs through inducing the expression of VEGF and VEGFR-2.