The Role Of Proteinase With A Disintegrin And A Metalloproteinase Domain-9(ADAM9)in Airway Remodeling Of COPD

BackgroundChronic obstructive pulmonary disease(COPD)is the third leading cause of death in the world.In China,spirometry-defined Chronic obstructive pulmonary disease(COPD)is highly prevalent in the adult population,affecting 8.6%of the general population aged 20 years or older and 13.7%of people aged 40 years or older.Cigarette smoking,ambient air pollution and other noxious particles result in persistent airflow limitation and respiratory symptoms.Since the advent of global initiative for chronic obstructive lung disease(GOLD),people raised their knowledge for etiology,diagnosis,prevention,and treatment of COPD.But it still remained to be a big challenge to prevent and control COPD effectively.It is of great importance for physicians to investigate the mechanism of COPD pathogenesis,which also provided evidences for optimizing the treatment schemes of COPD patients.Small airway disease(SAD)and emphysema are two key features of COPD,and both contribute to the formation of airflow obstruction and heterogenous phenotypes.Multiple evidence had demonstrated that small airway remodeling is not only the initial step of COPD pathogenesis but also a key link between the two different phenotypes.In COPD,the small airways undergo marked remodeling,characterized by increased airway wall thickness,which stems from epithelial changes,mucus hypersecretion,increased infiltration of inflammatory cells,smooth muscle hyperplasia and fibrosis.Recent evidence has revealed that both the narrowing of small airway lumens and the loss of small airways could drive the formation of airflow obstruction,and the loss of small airways is related to increased severity of emphysema.Airway epithelium is the initial site that exposed to noxious gas or particles from the environment.Normal epithelium comprises of ciliated cells,secretory cells,intermediate cells,and basal cells.The chronic exposure to the cigarette smoke led to reprogram of the epithelial cells,such as basal and mucus-producing cell hyperplasia,loss of ciliated cells and squamous metaplasia.It appears that the disordered proliferation and differentiation of airway epithelial cell directly participated to the airway remodeling and the pathogenesis of COPD-related phenotype.The balance of proteinases and anti-proteinases is regulated precisely in healthy individuals,and the loss of balance is a key factor in the pathogenesis of COPD.Proteinases with a disintegrin and a metalloproteinase domain(ADAMs),which belong to the same metzincin superfamily as MMPs,show similar structural features but with several distinct domains,including a metalloproteinase domain,a disintegrin domain and a cysteine-enriched domain.These specific domains endow ADAMs with the biological functions of both matrix degradation and molecule shedding,allowing them to participate in cell adhesion,migration,proliferation,differentiation,and apoptosis.Multiple studies had showed that ADAM family members including ADAM8,ADAM 17,ADAM33 could participate in the pathogenesis of COPD.ADAM9,a member of the ADAM family,is normally expressed in lung epithelial cells,inflammatory cells,and smooth muscle cells.A murine study of ADAM9 knockout mice model showed alleviated airspace enlargement in response to cigarette smoking exposure,which indicated its involvement in the formation of emphysema.However,few studies had investigated the relationship between ADAM9 levels in airway epithelium and airway remodeling and imaging emphysema severity in COPD patients.In previous study,we confirmed the presence of ADAM9 in bronchial epithelia via immunohistochemistry,and its expression significantly increased in COPD patients.As a classic member of ADAM family,the clinical relevance of ADAM9 expression remained to be discussed.Moreover,how ADAM9 participate in airway remodeling during the pathogenesis of COPD is still unclear.We here tried to figure out these questions.Chapter ? The levels of airway epithelial ADAM9 in COPD and clinical significance Part ? Sputum ADAM9 levels in COPD patients and their clinical significance ObjectiveTo investigate the sputum ADAM9 levels in COPD patients and analyze their correlation with clinical characteristicsMethods1.Patients:The sputum cohort consisted of healthy non-smokers,healthy smokers,and stable COPD patients.COPD was diagnosed based on a postbronchodilator FEV1/FVC ratio of less than 0.7 according to the guidelines of the Global Initiative for Chronic Obstructive Lung Disease(GOLD).2.Sputum collection and measurements:Sputum specimen were induced by hypertonic saline from subjects enrolled,following the safe measurement protocol as recommended.Slimy sputum was collected and disposed within 2 hours.Phlegm suppository was chosen and weighed.0.1%DTT solution was added to the phlegm with four times volume for splitting.After centrifugation,supernatants were collected for further measurements.Sputum ADAM9 levels and inflammatory cytokine IL-8 levels were detected via ELISA kits.3.Clinical characteristics(1)Pulmonary function parametersFEV1%of predicted,FEV1/FVC ratio,MMEF%of predicted,etc.(2)CT acquisition and quantitative analysisThe imaging data were quantitatively analyzed via 3D slicer software.Emphysema was defined as the percentage of voxels below-950 Hounsfield units(%LAA-950).Airway wall thickness was quantified using the following parameters:airway wall thickness(WT),measured along the middle third of each airway of the 3rd,4th and 5th bronchi from RB1,RB10,LB1 and LB 10;wall area percentage(WA%),quantified as WA%=(wall area/total bronchial area)×100 of the same bronchi;and Pi10,the square root of the wall area in a hypothetical airway with a 10 mm internal perimeter.4.Statistics analysisStudents't test and Mann-Whitney U test were used to compare group difference.Correlation was analyzed via Pearson's linear coefficients and Spearman's rank coefficients.Results1.Totally,21 Non-smoker controls,21 smokers and 42 stable COPD patients enrolled.2.Sputum ADAM9 levels was increased in COPD patients than non-smoker group and smoker group(P=0.036 and 0.039).No obvious difference was shown between smokers and non-smokers.3.Correlation analysis revealed that sputum ADAM9 levels was negatively correlated with FEV1%of predicted,FEV1/FVC ratio,MMEF%of predicted.4.There were no obvious correlation between sputum ADAM9 levels and%LAA-950,and WT,WA%,Pi-10 of the 3rd,4th,5th airway generations5.Sputum ADAM9 levels were positively correlated with sputum IL-8 levels(cc=0.558,P<0.001).Summary1.Sputum ADAM9 levels in COPD patients were increased than non-smoker group and smoker group.2.Sputum ADAM9 levels was negatively correlated with FEV1%of predicted,FEV1/FVC ratio,but not with%LAA-950 nor WA%.3.Sputum ADAM9 levels positively correlated with sputum IL-8 levelsPart II Airway epithelial ADAM9 expression in COPD patients and their clinical significanceObjectiveTo investigate the Airway epithelial ADAM9 levels in COPD patients and analyze their correlation with clinical characteristicsMethods1.Patients:Lung staining cohorts consisted of healthy non-smokers,healthy smokers,and patients with COPD.Lung tissue samples were collected at the surgery department from patients who underwent lobectomy or pneumonectomy for the reasons of peripheral lung mass or nodules.2.Lung tissue collection and measurements:Tissue sections with a 4-?m thickness were made from paraffin-embedded lung samples.The sections were then processed through dewaxing and antigen retrieval by citrate buffer(pH 6.0)for 15 minutes with the temperature in the range of 92?-98?.The sections were incubated with the primary antibody anti-ADAM9 IgG of a rabbit.A correlation analysis was performed by Pearson's linear correlation coefficients for continuous variables and Spearman's rank correlation coefficients for categorical variables.3.Clinical characteristics(1)Pulmonary function parametersFEV1%of predicted,FEV1/FVC ratio,MMEF%of predicted,etc.(2)CT acquisition and quantitative analysis%LAA-950 was measured to evaluate emphysema severity.RB1,RB10,LB1,LB 10 were chosen as target airways,and WT,WA%,Pi-10 were measured of the 3rd,4th,5th airway generations.Airway counts were quantified via a visual inspection under the conditions of a window width of 1000 Hu and a level of-500 Hu.The right superior lobal bronchus(RB1)was identified as the initial site of airway counting and was designated as the 3rd airway generation.Slice-by-slice examination was performed to identify the bifurcation of the RB1.Here,airway counts of the 4th to 9th and 6th to 9th airways were summed to evaluate the loss of airway counts.4.Statistics analysisStudents't test and Mann-Whitney U test were used to compare group difference.Correlation was analyzed via Pearson's linear coefficients and Spearman's rank coefficients.Results1.Totally 98 subjects enrolled,including 28 Non-smoker controls,34 smokers and 36 stable COPD patients.2.In the tissue cohort,ADAM9 could be found in almost of all the subjects' airways.Immunostaining of surface AD AM9 in the airway was mainly localized to the apical side of epithelium,and could also be found in the basal side of the pseudostratified epithelium especially in the COPD patients.Significantly higher expressions of ADAM9 were observed in the airway epithelium of COPD patients than in smokers and nonsmokers,and the smokers furtherly showed higher positivity of ADAM9 than the non-smokers(P<0.05 for all).3.Correlation analysis revealed that epithelial ADAM9 expression was positively correlated with smoking pack-year,while negatively correlated with FEV1%of predicted,FEV1/FVC ratio,MMEF%of predicted.4.There was positively correlation between epithelial ADAM9 expression,%LAA-950,and WA%of 3rd,4th,5th airway generations,and Pi-10(P<0.05 for all).Furthermore,epithelial ADAM9 expression was negatively correlated with airway counts derived from the 4th to 9th bronchial generations(cc=-0.508,P<0.01).Summary1.Airway epithelial ADAM9 expression in COPD patients was increased than non-smoker group and smoker group,which positively correlated with cigarette exposure.2.Epithelial ADAM9 expression was negatively correlated with FEV1%of predicted,FEV1/FVC ratio.3.Epithelial ADAM9 expression was positively correlated with WA%of airways,but negatively correlated with airway counts of 4th to 9th bronchi.Conclusion I1.Airway epithelial ADAM9 levels was increased in COPD subjects than non-COPD controls.2.Airway epithelial ADAM9 Levels was closely correlated with airflow obstruction severity and CT measured airway remodeling.Chapter II The mechanisms of ADAM9 participate in airway remodeling of COPD Part I The role of ADAM9 on proliferation of HPBECsObjectiveTo investigate the effect of ADAM9 on proliferation of primary bronchial epithelial cells(HPBECs).Methods1.Patients:Cell brushing cohort consisted of healthy non-smokers.Brushing were conducted on the healthy sides of the patients.2.HPBECs Culture:HPBECs was collected during the bronchoscope examination.The brushing precipitants was collected after centrifugation.The cell precipitants were then resuspended and cultured in PneumaCultTM-Ex Medium.3.Immunofluorescence staining:Double staining indexes including cytokeratin 5+ADAM9,ADAM9+EGFR,cytokeratin 5+caspase 3.4.Western Blot:Protein quantitative measurements including ADAM9,cyclin D1,cleaved-caspase3,EGFR,etc.5.CCK-8 tests:After the application of gradient concentration of human recombinant ADAM9 protein,cell viability was detected with CCK-8 tests.6.Edu-594 cell proliferation tests:After the application of gradient concentration of human recombinant ADAM9 protein,cell proliferation was detected with Edu-594 cell proliferation detecting kits.7.Sphere formation experiments:HPBECs of logarithmic growth phases were collected.The cell precipitants were conducted through digestion,centrifugation,resuspension,cell counting,and then seeded into 96 well ultra-low attachment,U-bottom,cell plates.Cell density was 5000 cells for each well.gradient concentration of human recombinant ADAM9 protein and metalloproteinase inhibitors GM6001 were applied.After 72 hours,the diameters of the cell spheres were measured and compared.Results1.Totally 40 non-smoking subjects were included in cell brushing cohort.2.In the tissue cohort,ADAM9 could be found in almost of all the subjects' airways.Immunofluorescence staining showed that ADAM9 positive expressed cells were mainly located at the apical sides of the airway epithelium in non-smokers.In the airway epithelium of smokers and COPD patients,ADAM9 positive expressed cells were significantly increased.Meanwhile,the ratio of ADAM9 positive cells in cytokeratin 5 positive cells were also increased in COPD patients obviously.3.The expression ofA DAM9 in HPBECs was increased after the application of gradient concentration of cigarette smoking extracts(CSE),4%,6%and 8%.4.After the application of gradient concentration of human recombinant ADAM9 protein,cell viability was detected with CCK-8 tests shown no obvious changes.5.After the application of gradient concentration of human recombinant ADAM9 protein(10ng/ml,50ng/ml,100ng/ml,),the diameters of HPBECs spheres were reduced.6.100ng/ml rhADAM9 inhibited the proliferation activity of HPBECs by Edu-594 tests.7.The application of rhADAM9 downregulated the expression of cyclin D1 protein,and upregulated the expression of cleaved-caspase 3 protein.8.The EGFR levels on cell surface of HPBECs was reduced after the application of rhADAM9.Summary1.RhADAM9 could inhibit the sphere formation capacity and proliferation activity of HPBECs;downregulate the expression of cyclin D1 and upregulate the level of cleaved-caspase 3.2.A possible mechanism for rhADAM9 suppress HPBECs proliferation was mediated by reducing the level of EGFR on cell surface.Part II The role of AD AM9 on differentiation of HPBECsObjectiveTo investigate the effect of ADAM9 on differentiation of HPBECs and function of fibroblast.Methods1.ALI differentiation model:HBPECs were cultured in the upper chamber of transwell cell plates which then were exposed to the air,while the cultural medium whether including stimulation(CSE or rhADAM9)or not were added to the lower chamber.The air-liquid models were built to investigate the effect of ADAM9 to the differentiation of HPBECs.2.Differentiation markers including involucrin,MUC5AC,MUC5B,EMT markers were tested at the end of air-liquid cultures as well as monolay er culture of HPBECs.3.Co-cultural models were built using HPBECs seeded in the upper chambers and fibroblast cell in the lower chambers.The role of rhADAM9 on the function of fibroblast cell were studied which including synthesis of collagen-III and elastin.4.Other methods were same as above PARTs.Results1.Human recombinant ADAM9 protein increased the squamous metaplasia markers involucrin expression in HPBECs and ALI differentiation models.Furthermore,combination application of rhADAM9 and GM6001 decreased the expression of involucrin than rhADAM9 interfering group.2.RhADAM9 inhibited the MUC5AC expression of HBPECs.After the application of rhADAM9 of lng/ml,10ng/ml,50ng/ml,the expression of MUC5AC significantly decreased.3.The application of rhADAM9 shown no obvious effects on expression of EMT markers such as vimentin,E-cadherin.4.In ALI differentiation models,rhADAM9 reduced the expression of EGFR on differentiated epithelium.10ng/ml rhADAM9 group shown declined EGFR levels than control group and CSE group5.After the application of rhADAM9 to HPBECs grown in the upper chambers,the synthesis of collagen ? in fibroblast grown in the lower chambers increased,while the expression of elastin was reduced accompanied by the increasing concentration of rhADAM9.Summary1.RhADAM9 could increase the expression of involucrin,promote the formation of squamous metaplasia,while MUC5AC were decreased.2.RhADAM9 could performed on the HPBECs-HFL co-culture model,and lead to the increased synthesis of collagen ? and dereased level of elastin.Conclusion ?1.RhADAM9 suppressed the proliferation of HPBECs,and the downregulation of cell surface EGER could be one of the possible mechanisms.2.RhADAM9 promoted the HBPECs differentiating towards squamous metaplasia,and affected the fiber synthesis capacity of fibroblast.Entire conclusions1.Airway epithelial ADAM9 expression were increased in patients with COPD,and the expression were correlated with airflow obstruction and CT measured airway remodeling.2.ADAM9 suppressed the proliferation of HPBECs and led to the shedding of EGFR.3.ADAM9 promoted the squamous metaplasia of HPBECs,and could led to the decreased MUC5AC expression as well as increased collagen ? synthesis.