The Role Of TNF-α In Smoking-induced Pulmonary Vascular Injury And The Intervention Mechanism Of Etanercept

Chapter 1.The Expression of TNF-a and Pulmonary Vascular Endothelial Cells Apoptosis in Smokers and COPD patientsObjective:To investigate the pathological mechanism of cigarette smoke induced pulmonary vessel injury through morphological observation of the pulmonary vascular, quantitative analysis of pulmonary arterial endothelial apoptosis and Tumor Necrosis Factor-a(TNF-a) express in the lungs tissues from smokers with or without Chronic Obstructive Pulmonary Disease(COPD).Methods:32 cases underwent pneumonectomy in cardiothoracic surgery in The First Affiliated Hospital of Nanjing Medical University during the were divided into 3 groups:Control group (Nonsmokers with normal lung function, n=8); Smokers group(Smokers with normal lung function, n=13); COPD group(Smokers with airflow obstruction, n=11). Before the operation, clinical data were record. The lung tissues obtained from the patients. The morphological changes of intra-acinar pulmonary arteries were observed by hematoxylin-eosin staining(HE), and collagen contents were investigated by Van Gieson staining(VG). The quantitative analysis of apoptosis of pulmonary vascular endothelial cells was undertaken by TdT-mediated dUTP nick end labeling (TUNEL). Immunohistochemistry was used to investigate the expression of TNF-a in the pulmonary vascular endothelial cells. The characteristics of pulmonary vessels and the correlations between smoke index, pulmonary vascular endothelial cells apoptosis and were analyzed.Results:1. Compared to the control group, the levels of pulmonary artery WT% were significantly increased in smokers group and COPD group. Collagen thickness of pulmonary artery walls were abnormally higher in pulmonary artery walls of smoke exposure groups, compared to the control group.2. The apoptotic rates of pulmonary vascular endothelia cells in COPD group and smoke group were higher than that in control group, significantly(P<0.05), but differences between COPD group and smoke group was not significantly. Compared to the control group, the expression rate of TNF-α in COPD group and smoke group were significantly increased, while no significant difference were find between COPD group and smoke group.3. The results of correlation analyses showed that the pulmonary vascular endothelial cells apoptosis rates were positive correlated with smoke index(r=0.701, P<0.01)in smokers with or without COPD, while expression of TNF-α was positively correlated with smoke index(r=0.417, P<0.05)in smokers with or without COPD.Conclusion:Smokers and smoking patients with COPD showed the similar characterizations in pulmonary arteries morphological changes. Both the apoptosis rate of pulmonary arterial endothelial cells and the expression of TNF-α were increased in smokers with or without COPD, which related to the smoke index.Chapter 2.Tumor necrosis factor-a Inhibitor Etanercept Confers Protection from Cigarette Smoke Extract-Induced Apoptosis in Human Pulmonary Artery Endothelia CellsObjective:Inflammation plays an important role in the pathogenesis of cigarette smoke-induced pulmonary vascular injury. This study aimed at the effects of early use of Etanercept, a recombinant human tumor necrosis factor receptor II:IgG Fc fusion protein(rhTNFR:Fc) on cigarette smoke etract(CSE) induced apoptosis in human pulmonary artery endothelia cells and the molecular mechanisms involved.Methods:Human pulmonary arterial endothelial cells(HPAECs) were cultured and tested viability and proliferation by MTT assay after stimulation of the different concentrate of cigarette smoke extract (CSE) and Etanercept intervention. According MTT assay results, HPAECs were divided into four groups:1.Control group; 2.Etanercept (ECT) group; 3.CSE group; 4.ECT+CSE group. The apoptosis rates was detected by flow cytometry with Annexin V/PI double staining, and Hochest 33358 staining was used to observed the apoptotic morphology. Western Blot method was employed to measure the Capase-3,-8,-9 protein expression.Results:1. The cell apoptosis rate of HPAECs was significantly increased in CSE group, compared to the control group. ECT+CSE group showed reduced HPAECs apoptosis rate, compared with the CSE group, while no significant different was found between the ECT group and CSE group.2. When HPAECs cells were treated with 5% CSE for 12 hours, actived Caspase-3 expression was significantly increased, compared with the control group. ECT+CSE group showed reduced actived Caspase-3 expression, compared with the CSE group.3. The protein level of active Caspase-8 was increased after CSE stimulation, compared with the control group, accompanying with the up-regulation of active Caspase-9. ECT+CSE group showed reduced both actived Caspase-8 and Caspase-9 expression, compared with the CSE group.Conclusion:1. Etanercept confers protection from cigarette smoke extract-induced apoptosis in HPAECs.2. Etanercept showed the protective effects in CSE-induced HPAECs apoptosis with the attenuation of the up-regulated activities of Caspase-3.3. Reduced activeities of Capase-8 and Capase-9 were involved in the protective effects of Etanercept in CSE-induced HPAECs apoptosis.Chapter 3.Etanercept attenuates short-term cigarette-smoke-exposure-induced pulmonary arterial remodeling in ratsObjective:To determine the effect of Etanercept on short-term smoke-induced pulmonary arteriole impairment and investigate its possible mechanism.Methods:24 Sprague-Dawley(SD) male rats were randomly divided into 3 group (6 in each):1.control group(CLT group); 2.cigarette smoke exposure group(SM group) were exposed to cigarette-smoke 6 days per week for 14 days; 3.cigarette smoke exposure+Etanercept group(SMET group) were administrated ECP for intervention, at the same condition as SM group. At the end of week 2, determine the mPAP by the right heart catheterization technique.Enzyme linked immunosorbent assay(ELISA) was used to assay the expression of TNF-a in lung tissue. Expression of MMP-2 and MMP-9 were examined by immunohistochemistry, and the activity of MMP-2 and MMP-9 were detected in gelatin zymography. NF-κB p65 protein expression were analysed by western blotting analysis.Results:1.After exposure of cigarette-smoke for two weeks, the mean pulmonary artery pressures (mPAP) and the percentage of vascular wall thickness/vascular external diameter(WT%) of rats significantly increased, compared to the control group. Elevation of mPAP and WT% were partially reduced by Etanercept intervention.2. Compared to the control group, significantly up-regulation of the expression and activities of MMP-2 and MMP-9 were found in cigarette smoke exposure group, while Etanercept intervention suppressed these changes induced by cigarette smoke significantly.3.The expression levels of TNF-a in lung homogenates were significantly higher in cigarette smoke exposure group than those in control group, while early use of Etanercept inhibit the rise of TNF-a in lung homogenates, compared to the cigarette smoke exposure groups.4. Furthermore, the expression levels of NF-κB p65 in nuclear were significantly higher than those in the control group. Etanercept treatment significantly attenuated cigarette-smoke-induced activation of nuclear factor NF-κB signal.Conclusion:1. Short-term cigarette smoke exposure induced elevation of mean pulmonary artery pressures and medial hypertrophy of pulmonary arterioles.2. Etanercept intervention partially reduced hemodynamic and morphological changes by Etanercept.3. Up-regulation of the expression and activities of MMP-2 and MMP-9, induced by cigarette-smoke, were also suppressed significantly by Etanercept.4. Etanercept treatment signifi cantly attenuated cigarette-smoke-induced TNF-a accumulation and activation of nuclear factor NF-κB signal.