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Abstract (1) | Chronic Obstructive Pulmonary Disease | Epithelium

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  Abstract Background Chronic Obstructive Pulmonary Disease (COPD) is associated with bronchial epithelial changes, including squamous cell metaplasia andgoblet cell hyperplasia. These features are partially attributed to activation of the epidermal growth factor receptor (EGFR). Whereassmoking cessation reduces respiratory symptoms and lung function decline in COPD, inflammation persists. We determined epithelial proliferation and composition in bronchial biopsies from current and ex-smokers with COPD, and its relation to duration of smokingcessation. Methods 114 COPD patients were studied cross-sectionally: 99 males/15 females, age 62 ± 8 years, median 42 pack-years, no corticosteroids,current (n = 72) or ex-smokers (n = 42, median cessation duration 3.5 years), postbronchodilator FEV 1 63 ± 9% predicted. Squamous cellmetaplasia (%), goblet cell (PAS/Alcian Blue + ) area (%), proliferating (Ki-67 + ) cell numbers (/mm basement membrane), and EGFR expression (%) were measured in intact epithelium of bronchial biopsies. Results Ex-smokers with COPD had significantly less epithelial squamous cell metaplasia, proliferating cell numbers, and a trend towardsreduced goblet cell area than current smokers with COPD (p = 0.025, p = 0.001, p = 0.081, respectively), but no significant difference inEGFR expression. Epithelial features were not different between short-term quitters (<3.5 years) and current smokers. Long-term quitters (≥3.5 years) had less goblet cell area than both current smokers and short -term quitters (medians: 7.9% vs. 14.4%, p = 0.005; 7.9% vs.13.5%, p = 0.008; respectively), and less proliferating cell numbers than current smokers (2.8% vs. 18.6%, p < 0.001). Conclusion Ex-smokers with COPD had less bronchial epithelial remodelling than current smokers, which was only observed after long-termsmoking cessation (>3.5 years). Trial registration  NCT00158847 Background Chronic Obstructive Pulmonary Disease (COPD) is defined by progressive airflow limitation and airway inflammation[1], caused  predominantly by cigarette smoking. Additionally, the airway epithelium undergoes alterations, including squamous cell metaplasia,goblet and basal cell hyperplasia[2]. These findings are important for our understanding of the pathogenesis of COPD, since bronchial epithelial cells orchestrate an adequate maintenance of lung homeostasis by mucus production, ciliary beating, secretion of antimicrobial products and adequate immunological drive in response to noxious stimuli. Goblet cell hyperplasia is more pronounced in smokers withCOPD compared to those without, suggesting a role in the development of airflow limitation[3]. In addition, it contributes to mucus hypersecretion, which is associated with morbidity and mortality in COPD[4,5]. Squamous cell metaplasia impairs mucociliary clearance and contributes to the increased risk of squamous cell carcinoma as observed in COPD[6]. The mechanisms underlying epithelial alterations in COPD are incompletely understood. The epidermal growth factor receptor (EGFR)cascade has been shown to be involved in mucin production and goblet cell hyperplasia[7,8], repair of damaged epithelium[7,8], as well as development of squamous cell carcinoma[9]. A wide variety of stimuli can induce EGFR activation in vitro and in animals, includingcigarette smoke[7,8]. Additionally, epithelial EGFR expression is increased in bronchial biopsies from smokers with[10,11]and without[11,12]COPD compared to non-smokers. Previously, we have observed higher epithelial EGFR expression in ex-smokers with COPD compared to non-COPD, but not in current smokers, suggesting that current smoking may obscure differences in EGFR expression[13]. Therefore, EGFR activation may play a role in epithelial phenotypic alterations observed in COPD through active smoking.Smoking cessation improves respiratory symptoms and lung function decline in COPD, mostly within the first year after cessation[14,15], but interestingly bronchial airway inflammation persists or even worsens[16,17]. To our knowledge, there are no studies comparing bronchial epithelial features between current and ex-smokers with established COPD. Possibly, smoking cessation  contributes to restoration of epithelial characteristics in the large airways of COPD patients, which are directly and continuously exposedto the noxious substances present in cigarette smoke, thereby contributing to the clinical benefits observed after smoking cessation.Therefore, it needs to be addressed whether bronchial epithelial alterations and EGFR expression in large airways are reversible withsmoking cessation and related to the duration of smoking cessation in COPD.We hypothesised that bronchial epithelial cell proliferation and differentiation in patients with COPD is more pronounced in activesmokers than in those who stopped smoking, and that this difference is influenced by the duration of smoking cessation. Additionally, wequestioned whether the epithelial changes are associated with EGFR expression. We therefore investigated the extent of epithelial gobletcell hyperplasia, proliferation, squamous cell metaplasia, and EGFR expression in bronchial biopsies of current and ex-smokers withestablished COPD in a large cross-sectional study. Methods Subjects 114 patients with COPD, who participated in a two-centre trial (Groningen Leiden Universities and Corticosteroids in Obstructive LungDisease; GLUCOLD study), were included in this cross-sectional study. Patient characteristics and methods have been described indetail previously[17,18]. In brief, all patients had irreversible airflow limitation [postbronchodilator FEV 1 and FEV 1 /IVC <90% confidence interval  (CI) of predicted value] and chronic respiratory symptoms, they were all current or ex-smokers (quit = 1 year),with at least 10 pack-years of smoking. Patients did not use a course of steroids during the last three months, and did not havemaintenance treatment with inhaled or oral steroids during the last six months. They were allowed to use short-acting bronchodilators,and were in clinical stable condition. The medical ethics committees of each centre approved the study and all patients gave their writteninformed consent. Lung function Spirometry, reversibility to salbutamol, and diffusing capacity were measured according to previously described methods in order tocharacterise the patients[18]. Bronchoscopy Fiberoptic bronchoscopy was performed using a standardised protocol according to recent recommendations[19]as described  previously[17]. Smokers were requested to refrain from smoking on the day of the bronchoscopy. Patients received premedication (400 μg salbutamol p.i., 20 mg codeine p.o., 0.5 mg atropine s.c.) and local anaesthesia (lidocaine). The bronchoscopies were perf  ormed byexperienced pulmonary physicians using a fiberoptic bronchoscope (18×, outer diameter 6 mm, Pentax Optical Co., Japan) and pairs of cup forceps (Reda, Tuttlingen, Germany). Six macroscopically adequate bronchial biopsy specimens were taken from (sub) segmentalcarinae in the right or left lower lobe. Biopsy processing and staining Four paraffin- embedded biopsies were cut in 4 μm thick sections and haematoxylin/eosin staining was used for evaluation and selection of the two morphological best biopsies per patient for analysis (without crushing artefacts, large blood clots, or only epithelial scrapings).Sections were stained with Periodic acid-Schiff/Alcian blue (PAS/AB) and counterstained with Nuclear Fast Red to identify all secretorycells. Immunohistochemistry was performed as described previously for inflammatory cells[17], using specific antibodies against Ki-67 as a marker of proliferation (Dako M7240, dilution 1:100), and EGFR (Biogenex nr MU207-UC, dilution 1:50). Antigen retrieval wasobtained using citrate for Ki-67 and pepsin for EGFR. Analysis of bronchial biopsies Total biopsy images were prepared with a 3-chip colour camera and analysed by means of image analysis software (Zeiss Vision KS400system, Carl Zeiss, Göttingen, Germany) as follows. First, the length of the basement membrane was traced of all intact non-squamousmetaplastic epithelium (A), intact squamous metaplastic epithelium (B), and damaged epithelium (C) in PAS/AB stained sections, inorder to calculate the % intact epithelium [(A+B)/(A+B+C)] and the % metaplastic epithelium [B/(A+B)]. In addition, the presence of metaplastic epithelium was also scored as absent (0) or present (1). Intact epithelium (A+B) was defined as a layer of both basal andcolumnar cells without detachment from the basement membrane, including areas of goblet cell hyperplasia or squamousmetaplasia[20]. Consequently, damaged epithelium (C) was defined as all remaining epithelium, including denuded basement membrane. Squamous cell metaplasia was defined as pseudostratisfied multilayered epithelium consisting of polygonal cells covered byflattened layer of squamous cells and absence of ciliated cells[21]. Subsequently, the number of Ki-67 positively staining cells was counted in intact epithelium by a validated full automated procedure[22], and expressed as the number of Ki-67 + cells/mm basementmembrane. Densitometric analysis of PAS/AB and EGFR in intact epithelium (A+B) was also performed fully automated as  follows[22]. A linear combination of Red-, and Blue-filtered greyscale images was used, in order to derive a greyscale image (range 0  –  255) in which the purple staining (PAS/AB) and the brown-red staining (EGFR) highlighted above background. Results wereexpressed as the percentage of intact epithelium stained by PAS/AB and EGFR. In addition, EGFR staining intensity of positive areaswas expressed as the average greyvalue, after normalization of the distribution towards the background peak (white: greyvalue 255) andsubsequent inversion of the greyvalue distribution. Mean values of two biopsies analysed per patient were used for analysis. Statistical analysis Mean values and standard deviations (SD) were computed and presented, or median with interquartile range (IQR) in case of non-normaldistributed variables. Since most epithelial markers were still non-normal distributed after log-transformation, these data were analysedusing non-parametric tests. Differences between smokers and ex-smokers were explored using Chi-square tests or 2-tailed unpaired t-tests for patient characteristics, and Mann Whitney tests for epithelial features. To study the association with duration of smokingcessation, we compared smokers with ex- smokers who quit <3.5 years and those who quit ≥3.5 years ago, since this was the medianduration of smoking cessation, using Kruskal-Wallis tests. If these were significant, Mann Whitney tests were applied for further exploration of between-group differences. Univariate correlations were evaluated using Spearman rank correlation coefficient. Toanalyse correlations with years since smoking cessation, current smokers were included in the analysis as 0 years stopped. Multivariatelinear regression analysis was applied to adjust for significant differences in patient characteristics between the groups, such as age, pack-years, and FEV 1 /IVC. PAS/AB + area was measured in total intact epithelium, including squamous cell metaplasia, which bydefinition does not contain goblet cells. Therefore, linear regression analysis was also applied to adjust for % squamous cell metaplasiawhen analysing PAS/AB + differences between groups. SPSS 12.0 (SPSS Inc., Chicago, IL) software was used for statistical analysis. Results Patient characteristics Patient characteristics of the 114 patients and subgroups of smokers and ex-smokers have been described in detail previously[17,18]  (Table 1). Patients had moderate to severe COPD, based on a postbronchodilator FEV 1 of 63.0 (8.8)% predicted, and a median smokinghistory of 42 pack-years. Of the 114 COPD patients included in this study, 72 were current smokers and 42 were ex-smokers. Medianduration of smoking cessation in ex-smokers was 3.5 years (IQR: 1  –  10 years). Differences in patient characteristics between the groupsare presented in table 1.    Table 1.  Patient characteristics Epithelial features in smokers versus ex-smokers with COPD All 114 patients underwent bronchoscopy; from one patient (ex-smoker) none of the biopsies taken were adequate for analysis. Themedian analysed basement membrane length per biopsy was 5.03 (IQR: 3.64  –  6.14) mm, of which 1.12 (0.59  –  2.11) mm was intactepithelium. Characteristics of intact epithelium in the total group of patients were: 9.74 (3.54  –  34.0) Ki-67 + cells/mm BSM, 0 (0  –  19.7)%squamous cell metaplasia (37.3% of patients showed squamous cell metaplasia), 12.4 (4.69  –  18.9)% PAS/AB + area, and 10.4 (3.25  –  18.9)% EGFR  + area.Ex-smokers had significantly less Ki-67 + cell numbers, and % of patients with squamous cell metaplasia as well as % of epithelium withsquamous cell metaplasia (p = 0.001, p = 0.016, p = 0.025; respectively, Table 2)than current smokers. PAS/AB + area also tended to belower in ex-smokers, but this was not statistically significant (p = 0.081, Table 2). When adjusting for the presence of squamous metaplasia (which by definition does not contain goblet cells), the difference in PAS/AB + area became significant (p = 0.014). Whendifferences in sex, age, and FEV 1 /IVC were taken into account in multivariate linear and logistic regression analyses, all epithelialdifferences remained significant, except for PAS/AB + area. Epithelial EGFR  + areas and intensities of positive areas showed nodifferences between smokers and ex-smokers with COPD (Table 2).   Table 2.  Bronchial epithelial features of smokers and ex-smokers with COPD Duration of smoking cessation and epithelial features in COPD Ki-67 + cell numbers, the presence of squamous cell metaplasia, and the % PAS/AB + area, were different between current smokers, ex-smokers who quit <3.5 years ago, and ex- smokers who quit ≥3.5 years ago (Kruskal Wallis: p = 0.011, p < 0.001, p = 0.049; respectively, Table 2,Figur e1). Percentage squamous cell metaplasia and EGFR  + areas and intensities were not significantly different between these three groups (Table 2,Figure 1). There were no differences between current smokers and those who quit <3.5 years ago for any of the epithelial features. In contrast, those who quit ≥3.5 years ago had significantly less Ki -67 + cell numbers, presence of squamous cell metaplasia, and % PAS/AB + area than current smokers (p < 0.001, p = 0.029, p = 0.005, respectively; Table 2,Figure 1).  The differences in PAS/AB and Ki-67 remained significant when adjusting for age and pack-year differences between both groups.Moreover, the % PAS/AB + area was also lower in long-term quitters than those who quit <3.5 years ago (p = 0.008), and tended to belower for Ki-67 + cell numbers (p = 0.050). When adjusting for differences in age, PAS/AB significance was lost (p = 0.061). Figure 1.   Epithelial phenotype in smokers (S) and ex-smokers with COPD . Individual values of: (A) %   PAS/AB + area of intact epithelium, (B) Ki-67 + cells (/mm basement membrane = BSM) in intact epithelium, (C) % squamous cellmetaplasia (SCM) of intact epithelium (note: in a large % of patients no SCM was observed), (D) % EGFR  + area of intact epithelium.Data are grouped by COPD smokers (S), COPD ex- smokers who quit < 3.5 years ago (<3.5 yr), and who quit ≥3.5 years ago (≥3.5 yr). Horizontal bars represent median values, * p < 0.05.There was a significant inverse relationship between the duration of smoking cessation (including current smokers as 0 years stopped)and Ki-67 + cell numbers (r  s = -0.354, p < 0.001), % squamous cell metaplasia (r  s = -0.212, p = 0.004), and % PAS/AB + area (r  s = -0.235; p= 0.013), but not with EGFR expression. Relation of epithelial features with smoking, symptoms and lung function The duration of smoking tended to be associated with the number of Ki-67 + cells (r  s = 0.180, p = 0.065), whereas the number of pack-years smoked was not associated with epithelial features. 46.5% of all patients reported symptoms of chronic bronchitis, and althoughex-smokers had significantly less symptoms of chronic bronchitis (Table 1)and % of PAS/AB + area, there was no relation between the presence of these symptoms and the % of PAS/AB + area (p = 0.78). Epithelial features were not associated with the degree of airflowlimitation. Relation between epithelial cell proliferation and differentiation Ki-67 + cell numbers and the % squamous cell metaplasia were positively associated with one another (r  s = 0.586, p < 0.001). Finally, Ki-67 + cell numbers were also positively associated with % EGFR  + area (r  s = 0.210, p = 0.031). Discussion In the present study, we demonstrated that long-term ex-smokers with COPD had less bronchial epithelial mucin stores, proliferatingcells, and squamous cell metaplasia than current smokers with COPD. Moreover, these epithelial differences in ex-smokers were onlysignificant after a long-term period of smoking cessation (>3.5 years). In contrast, epithelial EGFR expression was not different betweencurrent and ex-smokers with COPD. These results may indicate that smoking cessation reverses smoking-induced bronchial epithelialcell proliferation and differentiation in patients with COPD, and the magnitude of this effect increases with longer duration of smokingcessation. In addition, our results suggest that these smoking cessation-induced epithelial changes in COPD are not accompanied byreduced EGFR expression.Our observation of lower bronchial epithelial mucin stores, proliferating cells, and squamous cell metaplasia in large airways of ex-smokers as compared to current smokers with COPD, and the association with duration of cessation, is novel. These results are incontrast to other, smaller studies showing no differences in epithelial features in ex-smokers compared to smokers both with and withoutCOPD[21,23]. However, the finding that ex-smokers with chronic bronchitis (with or without airflow limitation), had less goblet cell metaplasia in small airways than current smokers[24]is in line with our findings in COPD patients. The effect of smoking cessation and duration of cessation on squamous cell metaplasia and proliferation has been examined previously in bronchial biopsies[25]. Although it was not mentioned whether these patients had respiratory symptoms and/or COPD, the latter study also reported less squamous cellmetaplasia and epithelial proliferation in ex-smokers. Our result of similar EGFR expression in ex-smokers compared to current smokerswith COPD, is also novel and in contrast with observations in smokers without COPD, where lower bronchial EGFR expression wasobserved in ex-smokers[12]. Taken together, it can now be inferred that smoking cessation results in decreased epithelial mucin stores,  proliferation, and squamous cell metaplasia, in large airways of patients with COPD, but that it does not affect EGFR expression.There are a few important considerations when interpreting our results. We included a large (n = 114) group of well-characterised patients with stable COPD, not inhaling steroids for at least six months or oral steroids for at least three months, and without a clinicaldiagnosis of asthma. First, it needs to be emphasised that this was a cross-sectional study, and it cannot be ruled out that our ex-smoking
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