ß-catenin, Twist and Snail: Transcriptional regulation of EMT in smokers and COPD, and relation to airflow obstruction.

COPD is characterised by poorly reversible airflow obstruction usually due to cigarette smoking. The transcription factor clusters of ß-catenin/Snail1/Twist has been implicated in the process of epithelial mesenchymal transition (EMT), an intermediate between smoking and airway fibrosis, and indeed lung cancer. We have investigated expression of these transcription factors and their "cellular localization" in bronchoscopic airway biopsies from patients with COPD, and in smoking and non-smoking controls. An immune-histochemical study compared cellular protein expression of ß-catenin, Snail1 and Twist, in these subject groups in 3 large airways compartment: epithelium (basal region), reticular basement membrane (Rbm) and underlying lamina propria (LP). ß-catenin and Snail1 expression was generally high in all subjects throughout the airway wall with marked cytoplasmic to nuclear shift in COPD (P < 0.01). Twist expression was generalised in the epithelium in normal but become more basal and nuclear with smoking (P < 0.05). In addition, ß-catenin and Snail1 expression, and to lesser extent of Twist, was related to airflow obstruction and to expression of a canonical EMT biomarker (S100A4). The ß-catenin-Snail1-Twist transcription factor cluster is up-regulated and nuclear translocated in smokers and COPD, and their expression is closely related to both EMT activity and airway obstruction.

Epithelial mesenchymal transition (EMT) and non-small cell lung cancer (NSCLC): a mutual association with airway disease.

NSCLC is a leading cause of morbidity and mortality worldwide. It includes adeno- and squamous cell carcinoma. In the background, COPD and smoking play a vital role in development of NSCLC. Local progression and metastasis of NSCLC has been associated with various mechanisms, but in particular by a process called epithelial mesenchymal transition (EMT), which is implicated in COPD pathogenesis. In this study, we have investigated whether expression of EGFR (activation marker) and S100A4, vimentin and N-cadherin (as EMT) is different both in central and leading edge of NSCLC and to what extent related to EMT activity of both small and large airways, stage and differentiation of NSCLC. We have investigated EMT biomarkers (S100A4, vimentin, and N-cadherin), an epithelial activation marker (EGFR) and a vascularity marker (Type-IV collagen) in surgically resected tissue from patients with NSCLC (adeno- and squamous cell carcinoma), and compared them with expression in the corresponding non-tumorous airways. EGFR, S100A4, vimentin, N-cadherin expression was higher in tumor cells located at the peripheral leading edge of NSCLC when compared with centrally located tumor cells of same subjects (P < 0.01). Type-IV collagen-expressing blood vessels were also more at the leading edge in comparison with central parts of NSCLC. EGFR and S100A4 expression was related to differentiation status (P < 0.05) and TNM stage (P < 0.05) of NSCLC. Moreover, EMT markers in the leading edge were significantly related to airway EMT activity, while peripheral edge vascularity of squamous cell carcinoma only was significantly related to large airway Rbm vascularity (P < 0.05). EGFR- and EMT-related protein expression was markedly high in the peripheral leading edge of NSCLCs and related to tumor characteristics associated with poor prognosis. The relationships between EMT-related tumor biomarker expression and those in the airway epithelium and Rbm provide a background for utility of airway changes in clinical settings.

The main rhinovirus respiratory tract adhesion site (ICAM-1) is upregulated in smokers and patients with chronic airflow limitation (CAL).

BACKGROUND: ICAM-1 is a major receptor for ~60% of human rhinoviruses, and non-typeable Haemophilus influenzae, two major pathogens in COPD. Increased cell-surface expression of ICAM-1 in response to tobacco smoke exposure has been suggested. We have investigated epithelial ICAM-1 expression in both the large and small airways, and lung parenchyma in smoking-related chronic airflow limitation (CAL) patients. METHODS: We evaluated epithelial ICAM-1 expression in resected lung tissue: 8 smokers with normal spirometry (NLFS); 29 CAL patients (10 small-airway disease; 9 COPD-smokers; 10 COPD ex-smokers); Controls (NC): 15 normal airway/lung tissues. Immunostaining with anti-ICAM-1 monoclonal antibody was quantified with computerized image analysis. The percent and type of cells expressing ICAM-1 in large and small airway epithelium and parenchyma were enumerated, plus percentage of epithelial goblet and submucosal glands positive for ICAM- 1. RESULTS: A major increase in ICAM-1 expression in epithelial cells was found in both large (p < 0.006) and small airways (p < 0.004) of CAL subjects compared to NC, with NLFS being intermediate. In the CAL group, both basal and luminal areas stained heavily for ICAM-1, so did goblet cells and sub-mucosal glands, however in either NC or NLFS subjects, only epithelial cell luminal surfaces stained. ICAM-1 expression on alveolar pneumocytes (mainly type II) was slightly increased in CAL and NLFS (p < 0.01). Pack-years of smoking correlated with ICAM-1 expression (r = 0.49; p < 0.03). CONCLUSION: Airway ICAM-1 expression is markedly upregulated in CAL group, which could be crucial in rhinoviral and NTHi infections. The parenchymal ICAM-1 is affected by smoking, with no further enhancement in CAL subjects.

Platelet-activating factor receptor (PAFr) is upregulated in small airways and alveoli of smokers and COPD patients.

BACKGROUND AND OBJECTIVE: PAFr is a cell adhesion site for specific bacteria, notably non-typeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae. We previously published that PAFr expression is significantly upregulated in the large airways of smokers, especially in COPD. We have now investigated PAFr expression in the epithelium and Rbm of small airways and in the alveolar compartment in smokers and patients with both COPD and small airway disease. METHODS: We evaluated PAFr expression cross-sectionally in resected lung tissue from: eight smokers with normal lung function (NLFS); 10 with smoking-related small airway narrowing only; eight COPD smokers; 10 COPD ex-smokers, and compared these with nine control tissues. Anti-PAFr immunostaining was quantified using computer-aided image analysis. RESULTS: Significantly increased PAFr expression in small airway epithelium of all clinical groups was found compared with controls (P < 0.01). Moreover, epithelial PAFr expression was upregulated in COPD smokers compared with NLFS (P < 0.05), but not when compared with COPD ex-smokers or patients with only small airways disease. Smoking history (pack-year) correlated significantly with PAFr expression in the currently smoking individuals, especially in NLFS (r = 0.9; P < 0.002). An increase above normal in PAFr-expressing cells in the airway epithelial Rbm was only significant in COPD smokers (P < 0.007). An upregulation of PAFr-expressing cell in alveolar epithelium was uniformly found in all clinical groups compared with normal control (P < 0.01). CONCLUSION: Epithelial PAFr expression is upregulated in small airways and alveoli in smokers and COPD. Increased expression of PAFr could be crucial in facilitating acute and chronic respiratory infection with specific respiratory pathogens.

Epithelial mesenchymal transition in smokers: large versus small airways and relation to airflow obstruction.

BACKGROUND: Small airway fibrosis is the main contributor in airflow obstruction in chronic obstructive pulmonary disease. Epithelial mesenchymal transition (EMT) has been implicated in this process, and in large airways, is associated with angiogenesis, ie, Type-3, which is classically promalignant. OBJECTIVE: In this study we have investigated whether EMT biomarkers are expressed in small airways compared to large airways in subjects with chronic airflow limitation (CAL) and what type of EMT is present on the basis of vascularity. METHODS: We evaluated epithelial activation, reticular basement membrane fragmentation (core structural EMT marker) and EMT-related mesenchymal biomarkers in small and large airways from resected lung tissue from 18 lung cancer patients with CAL and 9 normal controls. Tissues were immunostained for epidermal growth factor receptor (EGFR; epithelial activation marker), vimentin (mesenchymal marker), and S100A4 (fibroblast epitope). Type-IV collagen was stained to demonstrate vessels. RESULTS: There was increased expression of EMT-related markers in CAL small airways compared to controls: EGFR (P<0.001), vimentin (P<0.001), S100A4 (P<0.001), and fragmentation (P<0.001), but this was less than that in large airways. Notably, there was no hypervascularity in small airway reticular basement membrane as in large airways. Epithelial activation and S100A4 expression were related to airflow obstruction. CONCLUSION: EMT is active in small airways, but less so than in large airways in CAL, and may be relevant to the key pathologies of chronic obstructive pulmonary disease, small airway fibrosis, and airway cancers.

Airway epithelial platelet-activating factor receptor expression is markedly upregulated in chronic obstructive pulmonary disease.

BACKGROUND: We recently published that platelet-activating factor receptor (PAFr) is upregulated on the epithelium of the proximal airways of current smokers and also in bronchial epithelial cells exposed to cigarette smoke extract. These treated cells also showed upregulation of Streptococcus pneumoniae adhesion. Bacterial wall phosphorylcholine specifically binds to PAFr expressed on airway epithelium, thus facilitating adherence and tissue invasion, which may be relevant to chronic obstructive pulmonary disease (COPD). Moreover, the use of inhaled corticosteroids (ICS) in COPD patients is associated with an increased risk of invasive respiratory pneumococcal infections. OBJECTIVE: In this study, we have investigated whether PAFr expression is especially upregulated in airway epithelium in COPD patients and whether this expression may be modulated by ICS therapy. METHODS: We cross-sectionally evaluated PAFr expression in bronchial biopsies from 15 COPD patients who were current smokers (COPD-smokers) and 12 COPD-ex-smokers, and we compared these to biopsies from 16 smokers with normal lung function. We assessed immunostaining with anti-PAFr monoclonal antibody. We also used material from a previous double-blinded randomized placebo-controlled 6-month ICS intervention study in COPD patients to explore the effect of ICS on PAFr expression. We employed computer-aided image analysis to quantify the percentage of epithelium stained for PAFr. RESULTS: Markedly enhanced expression of PAFr was found in both COPD-smokers (P<0.005) and COPD-ex-smokers (P<0.002) compared to smokers with normal lung function. There was little evidence that PAFr expression was affected by ICS therapy over 6 months. CONCLUSION: Epithelial PAFr expression is upregulated in smokers, especially in those with COPD, and is not obviously affected by ICS therapy.

Changes in airway histone deacetylase2 in smokers and COPD with inhaled corticosteroids: a randomized controlled trial.

The expression of HDAC2 is reported as reduced in chronic obstructive pulmonary disease (COPD). We assessed HDAC2 expression within the airways of smokers and subjects with COPD and effects of inhaled corticosteroids (ICS), using immuno-histology to contrast with previous molecular methodology. Endobronchial biopsies (ebb) from current smokers with COPD (COPD-CS; n = 15), ex-smokers with COPD (COPD-ES; n = 17), smokers with normal lung function (NS; n = 16) and normal controls (NC; n = 9) were immunostained for HDAC2. A double-blinded, randomized, placebo-controlled 6 months intervention study assessed effects of ICS on HDAC2 in 34 COPD subjects. There was no difference in epithelial HDAC2 staining in all groups. There was a significant reduction in total cell numbers in the lamina propria (LP) in COPD-CS and NS (p<0.05). LP cellularity correlated inversely with smoking history in COPD-CS (R = -0.8, p<0.003). HDAC2 expression increased markedly in NS (p<0.001); in contrast COPD-CS was associated with suppressed signal (p<0.03), while normal in COPD-ES. ICS did not affect HDAC2 cell staining. Our findings suggest that airway HDAC2 expression is increased in the LP by smoking itself, but is reduced in COPD. Ex-smokers have normalised HDAC2 cell expression, but ICS had no effect. The paper emphasise the pit-falls of relying on molecular data alone to define airway changes. NAME OF REGISTRY: The Australian New Zealand Clinical Trials Registry (ANZCTR). REGISTRY NUMBER: ACTRN12612001111864.

Distinctive characteristics of bronchial reticular basement membrane and vessel remodelling in chronic obstructive pulmonary disease (COPD) and in asthma: they are not the same disease.

AIMS: This study compared reticular basement membrane (Rbm) and vascular remodelling within the bronchial mucosa of subjects with chronic obstructive pulmonary disease (COPD) with those from patients with asthma, to test the 'Dutch hypothesis' of whether these are essentially the same or different pathological conditions. METHODS AND RESULTS: Bronchoscopic biopsies were stained with anti-collagen IV antibody; 18 current smoking COPD, 10 symptomatic asthmatics and 13 healthy non-smoking controls were studied. The Rbm in COPD was fragmented, non-homogeneous, variable in thickness and hypervascular, whereas in asthma the Rbm was compact and homogeneous with no evidence of increased vascularity compared to controls. Length of Rbm splitting presented as percentage of Rbm length was used to measure fragmentation; it was greater in COPD than in controls and asthmatics [median (range) 20.7% (0.4-68.5) versus 5.3% (0.0-21.7) versus 1.5% (0.0-15.1), P < 0.001]. The number of Rbm vessels/mm Rbm [median (range) 10.1 (1.6-23.0) versus 4.5 (0.0-26.4) versus 4.4 (0.4-8.1), P < 0.01] and area of Rbm vessels, µm(2) /mm Rbm [median (range) 953 (115-2456) versus 462 (0-3263) versus 426 (32-2216), P < 0.05] was also increased in COPD compared to normal subjects and asthmatics. CONCLUSIONS: The characteristics of Rbm remodelling are quite different in asthma and COPD.