The Change And Effect Of Endothelial Progenitor Cells In The Chronic Obstructive Pulmonary Disease

Objective:To observe the number and function of circulating endothelial progenitor cells(EPCs) in the patients with chronic obstructive pulmonary disease (COPD).Methods:The total study population included 20 COPD patients and 20 control subjects. Mononuclear cells were isolated from human peripheral blood by density gradient centrifugation, the number of EPCs (CD34+/CD133+/VEGFR-2+cells) was calculated by flow cytometer. Mononuclear cells were cultured in endothelial growth medium-2 (EGM-2), cells with following characteristic were identified as EPCs: cells with "slabstone"-like appearance, expression of von Willebrand factor(vWF) and endothelial nitric oxide synthase (eNOS), taking up DiI-acLDL and combining with FITC-UEA-I. The adherent activity was assessed by the number of EPCs adhering to the culture bottle, the proliferative activity was detected by MTT method, the concentration of NO in EPCs culture solution was determined indirectly by measuring the concentration of total nitrite and nitrate to reflect EPCs'secretory activity, protein expression of eNOS and phosphorated eNOS (P-eNOS) were evaluated by western blotting.Results:[1]. There were few EPCs in circulation. Circulating EPCs in COPD groop was lower than the control with the percentage of (0.54±0.16)% versus (1.15±0.57)% (P<0.05). [2]. EPCs from COPD patients'peripheral blood were increased after induction and culture.The percentage of EPCs was (3.18±0.80)% after 7 days'culture, and (9.41±2.25%) after 10 days'culture. [3]. The number of adhering EPCs in COPD groop was lower than the control[(18.7±4.8)/field vs (45.0±5.9)/field, P<0.05]. EPCs'proliferative activity in COPD groop was poorer than the control, A490nm in COPD groop was lower than the control[(0.135±0.038) vs (0.224±0.042), P<0.05]. The concentration of NO in the EPCs culture solution in COPD groop was lower than the control[(25.11±5.27)μmol/L vs (37.72±7.10)μmol/L, P<0.05]. [4]. ENOS and P-eNOS protein expression in EPCs in COPD groop were lower than the control[(112.06±10.00) vs (135.41±5.38), (88.89±4.98) vs (117.98±16.49), all P<0.05].Conclusion:[1]. Human peripheral blood-derived EPCs can increase successfully in vitro. [2]. The number and adherent, proliferative, secretory activity of circulating EPCs in COPD patients are decreased when compared with the control. [3]. ENOS protein expression is decreased in COPD patients.Objective:To observe the EPCs'location, the change of lung function, pulmonary alveoli and pulmonary blood vessels, airway inflammation, eNOS and P-eNOS protein expression in lung tissue in COPD model mice after EPCs'transplantation.Methods:Bone marrow-derived mononuclear cells (BMMNCs) were gained by density gradient centrifugation from male C57BL/6J mice cavitas medullaris washing solution, and then cultured in EGM-2. Cells with following characteristic were identified as EPCs:cells with "slabstone"-like appearance, expression of von Willebrand factor(vWF) and endothelial nitric oxide synthase (eNOS), taking up DiI-acLDL and combining with FITC-UEA-I. The COPD mice model was established by exposure the mice to the tobacco smoke (6 cigarettes for 15 minutes,4 times a day, for 90 days). There were six groups in this study:the control group:mice exposed to air, COPD group:mice exposed to smoke for 90 days, early-intervention group and late-intervention group:mice were transplanted with CM-DiI labeled EPCs through trachea after 1 month's and 3 months'smoke exposure respectively, early-pseudo-intervention group and late-pseudo-intervention group:in which mice were transplanted with PBS after 1 month's and 3 months'smoke exposure respectively. One month after the last smoke exposure, mice were anesthetized to measure lung function, the right lung were washed with PBS and BALF was collected, total cell number was countered and the differential cell number were calculated after hematoxylin and eosin(HE) staining. The concentration of NO in BALF was determined indirectly by measuring total nitrite and nitrate. After fixed with 4% paraformaldehyde through the tracheal cannula, the left lower lung was removed and stained with HE. The degree of emphysema was evaluated by mean alveolar septal thickness (MAST), mean linear intercept (MLI) and destructive index (DI). The pulmonary vascular characteristic was evaluated by external diameter of pulmonary arteries, thickness of vascular wall, lumen area and vessel area, the ratio of thickness of vascular wall to external diameter of vascular(WT%) and the ratio of lumen area to vessel area(VA%) by LOGENE-Ⅰimage analysis software.The protein expression of endothelial NOS (eNOS) and phosphorated eNOS (P-eNOS) in left upper lungs were evaluated by western blotting. Results:[1]. Mice BMMNCs could differentiate to endothelial cells and increased in the specific endothelial culture medium. [2]. EPCs could immigrate to the airway and pulmonary blood vessels after being transplanted to trachea. [3]. The COPD mice model could be established after 3 months'smoke exposure and it was confirmed by lung function and pathological change. [4]. Mean alveolar septal thickness(MAST) in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (8.09±1.02)μm,(4.32±0.62)μm,(6.29±1.01)μm,(5.48±0.65)μm,(4.51±0.61)μm,(4.44±0.63)μm, respectively. Mean linear intercept (MLI) in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (33.26±3.22)μm,(51.79±7.25)μm,(40.25±2.65)μm,(45.21±2.25)μm,(50.98±7.68)μm,(51.05±7.43)μm, respectively. Destructive index(DI) in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (12.85±3.22)%,(38.86±4.32)%,(20.37±3.43)%,(28.17±3.56)%,(37.47±4.76)%,(38.46±4.50)%, respectively.MAST in COPD group was thinner than the control(P<0.05), There were no statistical difference in MAST among COPD group,early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05). MAST in early-intervention group was thinner than the control, but thicker than COPD group and early-pseudo-intervention group (all P<0.05). MAST in late-intervention group was thinner than the control, but thicker than COPD group and late-pseudo-intervention group (all P<0.05). There was no statistical difference in MAST between early-intervention group and late-intervention group (P>0.05). MLI and DI in COPD group were higher than the control(all P<0.05). There were no statistical difference in MLI and DI among COPD group. early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).MLI and DI in early-intervention group were higher than the control, but lower than COPD group and early-pseudo-intervention group (all P<0.05). MLI and DI in late-intervention group were higher than the control, also higher than early-intervention group, but lower than COPD group and late-pseudo-intervention group(all P<0.05). [5]. WT% in the control. COPD group. early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (7.96±2.66)%,(26.66±2.57)%,(13.10±2.07)%,(17.67±1.44)%,(26.45±2.14)%,(26.33±2.26)%, respectively. VA% in the control. COPD group,early-intervention group,late-intervention group. early-pseudo-intervention group and late-pseudo-intervention group were (78.44±9.77)%,(54.88±4.80)%,(67.88±4.91)%,(63.66±7.89)%,(52.34±5.98),(53.46±5.04)%, respectively. WT% in COPD group was higher than the control(P<0.05). There were no statistical difference in WT% among COPD group. early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).WT% in early-intervention group was higher than the control, but lower than the COPD group and early-pseudo-intervention group(all P<0.05).WT% in late-intervention group was higher than the control, and also higher than early-intervention group, but lower than the COPD group and late-pseudo-intervention group(all P<0.05).VA% in COPD group was lower than the control(P<0.05). There were no statistical difference in VA% among COPD group. early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).VA% in early-intervention group was lower than the control, but higher than COPD group and early-pseudo- intervention group (all P<0.05). VA% in late-intervention group was lower than the control(P<0.05), there were no statistical difference in VA% among late-intervention group,COPD group and late-pseudo-intervention group(all P>0.05). [6]. Airway resistance(Raw) in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (0.0365±0.0196)Kpa/L/s,(0.3179±0.0969) Kpa/L/s,(0.1029±0.0460) Kpa/L/s,(0.1999±0.0113) Kpa/L/s,(0.2893±0.0624) Kpa/L/s,(0.3188±0.0889)Kpa/L/s, respectively. Lung compliance(CL) in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (0.3100±0.1067)L/Kpa,(0.1019±0.0004) L/Kpa,(0.2942±0.0228) L/Kpa,(0.2078±0.0823) L/Kpa,(0.1208±0.0186) L/Kpa, (0.1213±0.0216) L/Kpa,respectively.Peak expiratory flow(PEF) in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (8.38±1.08)L/S,(7.20±0.97) L/S,(7.95±0.95) L/S,(8.10±0.96) L/S,(7.45±1.05) L/S,(7.75±0.83) L/S,respectively. Raw in COPD group was higher than the control. There were no statistical difference in Raw among COPD group,early-pseudo-intervention group and late-pseudo-intervention group (all P>0.05).Raw in early-intervention group was lower than COPD group and early-pseudo-intervention group(all P<0.05), there was no statistical difference in Raw between early-intervention group and the control(P>0.05). Raw in late-intervention group was higher than the control,and also higher than early-intervention group, but lower than COPD group and late-pseudo-intervention group (all P<0.05). CL in COPD group was lower than the control(P<0.05). There were no statistical difference in CL among COPD group,early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).CL in early-intervention group was higher than COPD group and early-pseudo-intervention group (all P<0.05), there was no statistical difference in CL between early-intervention group and the control(P>0.05). CL in late-intervention group was lower than the control,and also lower than early-intervention group,but higher than COPD group and late-pseudo-intervention group (all P<0.05). There were no statistical difference in PEF among the groups(P>0.05). [7]. The concentration of NO in BALF in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (15.55±8.74)μmol/L,(52.65±12.16)μmol/L,(36.10±10.95)μmol/L,(43.76±13.19)μmol/L,(50.56±10.64)μmol/L,(53.37±12.83)μmol/L, respectively. The concentration of NO in BALF in COPD group was higher than the control(P<0.05). There were no statistical difference in the concentration of NO in BALF among COPD group,early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).The concentration of NO in BALF in early-intervention group was higher than the control, but lower than COPD group and early-pseudo-intervention group (all P<0.05). The concentration of NO in BALF in late-intervention group was higher than the control(P<0.05), there were no significant difference in the concentration of NO in BALF among late-intervention group,late-pseudo-intervention group and COPD group(P>0.05). [8]. The number of totel cells in BALF in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were (1.47±0.24) ×108/L,(5.85±0.67)×108/L,(3.61±0.38)×108/L,(5.47±0.71)×108/L,(5.67±0.75)×108/L,(5.87±0.72)×108/L,respectively.The number of macrophage in BALF in the control.COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were(1.34±0.14)×108/L,(4.45±0.63)×108/L,(2.86±0.34)×108/L,(4.11±0.69)×108/L,(4.32±0.73)×108/L,(4.54±0.65)×108/L,respectively.The number of neutrophil in BALF in the control.COPD group.early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were(0.77±0.09)×107/L,(7.76±0.92)×107/L,(5.01±0.78)×107/L,(7.58±0.96)×107/L,(7.62±0.89)×107/L,(7.87±0.81)×107/L, respectively.The percentage of macrophage in the control. COPD group.early-intervention group.late-intervention group.early-pseudo-intervention group and late-pseudo-intervention group were(86.55±8.90)％.(76.28±8.25)％.(78.81±7.96)％.(75.42±7.68)％.(76.36±8.03)％.(76.11±8.42)％,respectively.The percentage of neutrophil in the control.COPD group.early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were(8.68±0.97)％,(13.71±2.42)％,(12.31±1.98)％,(13.51±2.09)％,(13.01±2.13)％,(13.81±2.39)％,respectively.The number of totel cells and macrophage(M),neutrophil(N),the percentage of neutrophil(N%)in BALF in COPD group were all larger than those in the control(all P<0.05).There were no statistical difference in the number of totel cells and M,N,N% in BALF among COPD group,early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).The number of totel cells and M.N in BALF in early-intervention group were highr than those in the control, but lower than those in COPD group and early-pseudo-intervention group (all P<0.05), N% in early-intervention group was higher than the control(P<0.05), there were not statistical difference in N% among early-intervention group,early-pseudo-intervention group and COPD group(P>0.05). The number of totel cells and M,N in BALF in late-intervention group were highr than those in the control, also highr than those in early-intervention group(all P<0.05), there were no statistical difference in the number of totel cells,M,N in BALF among late-intervention group,late-pseudo-intervention group and COPD group(all P>0.05). [9]. OD value of eNOS and P-eNOS protein expression in lung tissue in the control,COPD group,early-intervention group,late-intervention group,early-pseudo-intervention group and late-pseudo-intervention group were [(253.89±18.69), (193.98±17.56)],[(142.53±10.27), (140.08±8.43)],[(162.66±13.61), (156.20±10.09)],[(168.28±13.65), (152.81±10.43)],[(150.43±10.71), (141.88±10.54)],[(148.57±11.16), (140.12±8.92)],respectively. ENOS and P-eNOS protein expression in lung tissue in COPD group were lower than those in the control(all P<0.05). There were no statistical difference in eNOS and P-eNOS protein expression in lung tissue among COPD group,early-pseudo-intervention group and late-pseudo-intervention group(all P>0.05).ENOS and P-eNOS protein expression in lung tissue in early-intervention group were lower than the control, but higher than COPD group and early-pseudo-intervention group(all P<0.05).ENOS and P-eNOS protein expression in lung tissue in late-intervention group were lower than the control, but higher than COPD group and late-pseudo-intervention group(all P<0.05).There were no significant difference in eNOS and P-eNOS protein expression in lung tissue between early-intervention group and late-intervention group(all P>0.05).Conclusion:[1]. Mice bone marrow-derived mononuclear cells (BMMNCs) can differentiate to endothelial cells and increase successfully in vitro. [2]. EPCs transplantation can alleviate lung impairment and pulmonary blood vessel hyperplasia in COPD model mice,early-transplantation is better than late-transplantation. [3]. EPCs transplantation can alleviate airway resistance and increase lung compliance in COPD model mice, early-transplantation is better than late-transplantation. [4]. Early-transplantation with EPCs can alleviate airway inflammation in COPD model mice.