The Effect Of Urokinase Plasminogen Activator System Components On The Pathology Change Of COPD

BackgroundChronic Obstructive Pulmonary Disease (COPD) is one of the leading causes of morbidity and mortality worldwide, and its prevalence is increasing. While the cellular and molecular mechanisms that are involved in the pathogenesis of COPD have not yet been fully elucidated, there is still no effective therapy to cure the disease. The defining feature of COPD is a chronic, slowly progressive airflow limitation that results from the combination of pulmonary emphysema and small airways obstruction. The functional consequence of these abnormalities is airflow limitation. Bronchiolitis contributes to airflow limitation by narrowing and obliterating the airway lumen and by actively constricting the airway. Emphysema, by reducing the elastic recoil of the lung through parenchymal destruction and increase in alveolar size, as well as reducing the elastic load applied to the airways through destruction of alveolar attachments, also contributes to the airflow limitation characteristic of smokers. There are studies reported that the imbalance of extracllular metrix degradation and synethsis played important role in pathologensis of COPD. On the other hand, several studies report that increased programmed cell death mechanisms (i.e., apoptosis) can potentially contribute to epithelial cell loss, leading to the pathogenesis of emphysema. Thus, two kinds of balance could maintain the integrity of the lung.One is the balance of collagen synthetize and degradation and the other is apoptosis and proliferation. On previous study, we found that urokinase-type plasminogen activator (uPA) system components performed abnormal in both COPD patients and COPD animal model. We hypothesized that uPA system components may play important role on COPD pathology changes for their fibrinolysis activity and multiple other biological activities. ObjectiveTo explore the effect of uPA system components on pathology changes of COPD and the related mechanism.1. To observe the expression pattern of uPA system components on the lung tissues of COPD patients and to analysis the correlation between the level of uPA system components and the pathology changes of COPD.2. To explore the correlation between uPA system components and apoptosis index.3.To clarify the effect and mechanism of PAI-1on alveolar apoptosis,Methods1. Sample collectionSequential patients who underwent surgical resection for benign lung disease was enrolled. According to the Global Iniatitive for chonic obstructive pulmonary disease, all patients were devided to16COPD and20controls (10control smokers and10nonsmokers)2. Tests on human Lung tissue sections.(1) Immunohistochemistry was used to quantify uPA system components (uPA, uPAR, PAI-1and PAI-2) expression in peripheral lung tissues.(2) Collagen area pixel value with sirius red staining was used as the index of collagen deposition under small airway and low-attenuation area (LAA)%obtained from computed tomography as the index of emphysema.(3)TUNEL and Cleaved PARP immunohistochemistry were used to detecte the apoptosis in lung tissue of COPD patients and controls; and analyze the relationship between uPA system components expression and COPD apoptosis. 3. Experiments on cell line in vitro(1)For transfection treatment, A549cell line was transfected with PAI-1siRNA or nonsense siRNA using Lipofectamine2000according to the manual instructions.(2) PAI-1expression decresed after PAI-1siRNA transfection which further confirmed by quantitative real-time PCR, western blot and ELISA.(3)The survival of A549after CSC stimulate was detected by MTT.(4)Flow cytometry was used to detect the apoptosis of A549cells before and after transfection and CSC.(5) Chemiluminescence was used to detect the activity of caspase3/7, caspase8and caspase9.(6)Anti-Fas antibody (ZB4) was used before transfection to blocking Fas/FasL interaction and flow cytometry was used to detct the apoptosis as described above.Results1. Tests on human lung tissue sections(1)The level of uPA and uPAR were significantly elevated in COPD patients compared to control smokers and nonsmokers in small airway epitheliums, alveolar wall cells and in macrophages(P<0.05); the level of PAI-1significantly decresed in COPD patients compared to control smokers and nonsmokers in both small airway epitheliums and alveolar wall cells, but not in macrophages(P>0.05). As for PAI-2, the protein level elevated only in small airway epitheliums of COPD patients other than alveolar and macrohages.(2)uPA and PAI-2expression in small airway epitheliums were significantly related to collagen area pixel value (r=0.4187, p=0.0011;r=0.5653,P=0.0225),, while uPA positive alveolar closely related to LAA%respectively (r=0.5188, p=0.0359)(3)The apoptosis index (AI) was significantly elevated in COPD patients than control patients; PAI-1level on alveolar epithelium was negatively related to apoptosis index.(r=-0.5122, P＜0.05)2. Experiments on cell line in vitro(1)PAI-1expression decresed after PAI-1siRNA transfection which was confirmed by quantitative real-time PCR, western blot and ELISA.(2) The survival of A549was decreased after CSC and was related to the concentration and effect time of CSC.(3) The ratio of apoptosis cell elevated after PAI-1siRNA transfection and CSC input.(4) Caspase3/7and caspase8activity were elevated while caspase9had no change.(5) The ratio of apoptosis cell decreased when ZB4was used.Conclusion1. uPA system components may be a key on COPD pathology change.2. PAI-1could participant in COPD pathology change through affecting alveolar apoptosis.3.Low expression of PAI-1in COPD could enhance alveolar apoptosis through Fas-FasL pathway.