| Background:Increased apoptosis in pulmonary microvascular endothelial cells and endothelial injury contribute to pathogenesis of COPD/emphysema. Endothelial repair is a process involved in directed migration and proliferation of local endothelial cells and circulating endothelial progenitor cells (EPCs). Smoking induces a markedly decrease in the level of circulating EPCs. The number of circulating EPCs is affected by the EPCs ability of mobilization from bone marrow to peripheral bood, so we hypothesize that smoking maybe inhibit c-Kit/s-SCF interaction of bone marrow EPCs in pathogenesis of COPD/emphysema, which is critical in recruitment of EPCs from bone marrow to peripheral bood.Objective:To observe changes of c-Kit(+)EPCs and s-SCF in the bone marrow in a CSE-induced emphysema mouse model and investigate mechanisms involved with the changes.Methods:1. Changes of c-Kit (+) endothelial progenitor cells and soluble stem cell factor in the bone marrow in a cigarette smoke extract-induced emphysema mouse model:twenty four mice were randomly divided into two groups:PBS-group (n=12), and CSE-group (n=12). Each mouse in the CSE-group was intraperitoneally injected with0.3ml CSE at day0,11, and22respectively. As a control, each mouse from the PBS-group received the intraperitoneal injection with the same volume of PBS. At day7, six mice from each group were sacrificed and harvested bone marrow samples. All the others were tested for lung function, sacrificed and harvested lung tissues and bone marrow samples at day28. Lung sections were stained with hematoxylin and eosin (HE), and morphology of lung tissues was observed by light microscopy. Mean linear intercept (MLI), destructive index (DI), and mean alveolar septum thickness (MAST) were measured. The protein expression of c-Kit in the lung tissue was detected by Western-blotting. EPCs/c-Kit(+) EPCs in bone marrow non-red blood cells were analyzed by flow cytometry, which were isolated from the harvested bone marrow cells by RBC lysis. The s-SCF levels in the supernatant of bone marrow flush fluid were measured in full-strength conditioned media using a colorimetric sandwich enzyme immunoassay.2. Mechanisms involved with the decrease in the level of s-SCF in the bone marrow in a CSE-induced emphysema mouse model: The activities of MMP-9were assayed by gelatin zymography. Concentrations of NO in the supernatant of bone marrow flush fluid were measured by a chemical method. MMP-9and TIMP-1levels in the supernatant of bone marrow flush fluid were measured in full-strength conditioned media using a colorimetric sandwich enzyme immunoassay. The protein expressions in the bone marrow, such as SCF, MMP-9, TIMP-1and NOS3, were detected by Western-blotting. SCF and MMP-9mRNA expressions in bone marrow cells were detected by real-time PCR.3. Effects of CSE on proliferation, survival and the frquency of c-Kit positive subset in cultured bone marrow-derived EPCs:Mononuclear cells were isolated from murine bone marrow by Ficoll density gradient centrifugation and cultured with endothelial growth medium-2(EGM-2). EPCs were identified by morphology, dual staining for Dil-acLDL and FITC-UEA-1, surface markers analysed by flow cytometry including CD34, CD133and Flk-1on the7th day of culture. The cultured EPCs were intervened with0.5%,1%,2%CSE respectively, then proliferation and apoptosis of EPCs was assayed after24hours. The proliferation capacity of EPCs was detected by MTT assay, and the apoptosis rate in EPCs was evaluated with Annexin V-FITC/PI Apoptosis Detection Kit. Effects of1%CSE on NO production, the ckit+subset frequency, and protein expressions of NOS3and c-Kit in cultured EPCs were investigated. The NO production was measured by the concentration of NO in the fluid supernatant of cultured EPCs. The ckit+subset in the cultured EPCs was analyzed by flow cytometry. The NOS3and c-Kit proteins were detected by Western-blotting.Results:1. Changes of EPCs and s-SCF in the bone marrow in a CSE-induced emphysema mouse model:At day28, we first measured the effect of CSE on mouse lung function. Cdyn was significantly lower (32.65vs.52.51μl/cmH20) and Raw was significantly higher (2.15vs.1.13cmH20/ml-sec) in the CSE-group (P<0.05). Following CSE injection and lung harvesting at day28, we observed normal staining patterns for the PBS-group, while we observed alveolar airspace enlargement, lung parenchyma destruction and bullae formation in the CSE-group. Additionally, the MLI and DI were significantly increased, and the MAST was significantly decreased in CSE-group (P<0.05). Using quantitative western blotting, we found significant decreases in the level of c-Kit (P<0.05). Further, mRNA expression was also significantly lower in lungs harvested from CSE-treated mice (P<0.05). While the number of non-red blood cells isolated from right tibia bone marrow was not different between the two groups (9.03×106±1.72×106[CSE-group] versus9.44×106±1.82×106[PBS-group], P>0.05), both EPCs and c-Kit(+) EPCs were markedly depleted in the CSE-induced emphysema model (P<0.05). Similar to EPCs levels, s-SCF concentrations in the supernatant of the bone marrow flush fluid were also decreased in the CSE-treated group (P<0.05).2. Mechanisms involved with the decrease in the level of s-SCF in the bone marrow in a CSE-induced emphysema mouse model: Gelatin zymography revealed a significant decrease in MMP-9activity in the bone marrow isolated from CSE-treated mice at day28(P<0.05). The concentrations of NO, MMP-9and TIMP-1were measured in the bone marrow flushing liquid. Significant increases in NO and MMP-9concentrations were found in CSE-group at-day7(P<0.05); there is no statistical difference between2groups in these concentrations at day28(P>0.05). Following CSE injection, the concentrations of TIMP-1were significantly elevated at day7or day28(P<0.05). The ratios of MMP-9/TIMP-1were not modified at day7(P>0.05) and markedly decreased at day28(P<0.05), which correlates with the above findings. Using quantitative western blot and real-time PCR analysis, we observed decreases in SCF protein and mRNA in the CSE-treated group at day7and day28(P<0.05). Although protein and mRNA expressions of MMP-9were increased in the CSE-treated group at day7(P<0.05), there was no statistically difference in MMP-9protein and mRNA levels between CSE-group and PBS-group at day28(P>0.05). TIMP-1, a known inhibitor of MMP-9activity, displayed increased protein levels in the CSE-treated group at day7and day28(P<0.05). Interestingly, the protein expression of NOS3was featured a trend of rising first and then falling in CSE-group in this study (P<0.05).3. Effects of CSE on proliferation, survival and the frquency of c-Kit positive subset in cultured bone marrow-derived EPCs: Cells on the7th day of culture showed fusiform, polygon, forming capillary structure or "cobblestone" morphology. The Dil-acLDL and FITC-UEA-1amphophilic cells’positive rate was (94.67±4.16)%. The concurrent positive expression rate of FITC-CD34> PE-CD133and APC-Flk-1was (95.07±1.73)%. When EPCs were exposed to increasing concentrations of CSE for24hours, the OD values were lower (P<0.01) and the apoptosis rates were higher (P<0.01) in all of CSE groups compared with control group. CSE induces EPCs apoptosis and impairs EPCs proliferation in a concentration-dependent manner. The concentrations of NO of1%CSE group were lower than that of control group (P<0.01). CSE did not alter c-Kit subset frequency in cultured EPCs (P>0.05). Compared with control group, the levels of NOS3and c-Kit were significantly decreased the CSE-treated group (P<0.05).Conclusions:1. C-Kit/s-SCF interactions of EPCs in the bone marrow were influenced in the CSE-induced emphysema model through depletion of c-Kit(+) EPCs and s-SCF.2. Inactivation of MMP-9and down-regulation of SCF expression contribute to depletion of s-SCF in the bone marrow in the CSE-induced emphysema mouse model.3. CSE induces cultured bone marrow derived EPCs apoptosis and impairs their proliferation in a concentration-dependent manner. NOS3/NO signals and c-Kit, involved with cell proliferation and survival, were down-regulated in cultured bone marrow derived EPCs exposure to CSE.28Figures,6Tables,140References... |
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