Transforming growth factor-β1 and SMAD signalling pathway in the small airways of smokers and patients with COPD: potential role in driving fibrotic type-2 epithelial mesenchymal transition

BackgroundCOPD is a common disease characterized by respiratory airflow obstruction. TGF-β1 and SMAD pathway is believed to play a role in COPD pathogenesis by driving epithelial mesenchymal transition (EMT).MethodsWe investigated TGF-β1 signalling and pSmad2/3 and Smad7 activity in resected small airway tissue from patients with; normal lung function and a smoking history (NLFS), current smokers and ex-smokers with COPD GOLD stage 1 and 2 (COPD-CS and COPD-ES) and compared these with normal non-smoking controls (NC). Using immunohistochemistry, we measured activity for these markers in the epithelium, basal epithelium, and reticular basement membrane (RBM). Tissue was also stained for EMT markers E-cadherin, S100A4 and vimentin.ResultsThe Staining of pSMAD2/3 was significantly increased in the epithelium, and RBM of all COPD groups compared to NC (p <0.0005). There was a less significant increase in COPD-ES basal cell numbers compared to NC (p= 0.02). SMAD7 staining showed a similar pattern (p <0.0001). All COPD group levels of TGF-β1 in the epithelium, basal cells, and RBM cells were significantly lower than NC (p <0.0001). Ratio analysis showed a disproportionate increase in SMAD7 levels compared to pSMAD2/3 in NLFS, COPD-CS and COPD-ES. pSMAD negatively correlated with small airway calibre (FEF25–75%; p= 0.03 r= -0.36). EMT markers were active in the small airway epithelium of all the pathological groups compared to patients with COPD.ConclusionActivation of the SMAD pathway via pSMAD2/3 is triggered by smoking and active in patients with mild to moderate COPD. These changes correlated to decline in lung function. Activation of the SMADs in the small airways is independent of TGF-β1, suggesting factors other than TGF-β1 are driving these pathways. These factors may have implications for small airway pathology in smokers and COPD through the process of EMT, however more mechanistic work is needed to prove these correlations

Chronic Obstructive Pulmonary Disease and Lung Cancer: Underlying Pathophysiology and New Therapeutic Modalities

Chronic obstructive pulmonary disease (COPD) and lung cancer are major lung diseases affecting millions worldwide. Both diseases have links to cigarette smoking and exert a considerable societal burden. People suffering from COPD are at higher risk of developing lung cancer than those without, and are more susceptible to poor outcomes after diagnosis and treatment. Lung cancer and COPD are closely associated, possibly sharing common traits such as an underlying genetic predisposition, epithelial and endothelial cell plasticity, dysfunctional inflammatory mechanisms including the deposition of excessive extracellular matrix, angiogenesis, susceptibility to DNA damage and cellular mutagenesis. In fact, COPD could be the driving factor for lung cancer, providing a conducive environment that propagates its evolution. In the early stages of smoking, body defences provide a combative immune/oxidative response and DNA repair mechanisms are likely to subdue these changes to a certain extent; however, in patients with COPD with lung cancer the consequences could be devastating, potentially contributing to slower postoperative recovery after lung resection and increased resistance to radiotherapy and chemotherapy. Vital to the development of new-targeted therapies is an in-depth understanding of various molecular mechanisms that are associated with both pathologies. In this comprehensive review, we provide a detailed overview of possible underlying factors that link COPD and lung cancer, and current therapeutic advances from both human and preclinical animal models that can effectively mitigate this unholy relationship

Electronic Cigarette Aerosol Is Cytotoxic and Increases ACE2 Expression on Human Airway Epithelial Cells: Implications for SARS-CoV-2 (COVID-19)

Tobacco smoking has emerged as a risk factor for increasing the susceptibility to infection from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via increased expression of angiotensin-converting enzyme-2 (ACE2) in the lung, linked to coronavirus disease 2019 (COVID-19) development. Given the modifiable nature of electronic cigarettes and the delivery of high concentrations of nicotine, we investigate whether electronic cigarette vaping has the potential to increase susceptibility to SARS-CoV-2 infection. We exposed BEAS-2B cells (bronchial epithelium transformed with Ad12-SV40 2B) and primary small airway epithelial cells (SAECs) to electronic cigarette aerosol condensates produced from propylene glycol/vegetable glycerin or commercially bought e-liquid (±added nicotine) and cigarette smoke extract to investigate if electronic cigarette exposure, like cigarette smoke, increases the expression of ACE2 in lung epithelial cells. In BEAS-2B cells, cytotoxicity (CCK-8), membrane integrity (LDH), and ACE2 protein expression (immunofluorescence) were measured for both 4- and 24 h treatments in BEAS-2B cells and 4 h in SAECs; ACE2 gene expression was measured using quantitative polymerase chain reaction (qPCR) for 4 h treatment in BEAS-2B cells. Nicotine-free condensates and higher concentrations of nicotine-containing condensates were cytotoxic to BEAS-2B cells. Higher LDH release and reduced membrane integrity were seen in BEAS-2B cells treated for 24 h with higher concentrations of nicotine-containing condensates. ACE2 protein expression was observably increased in all treatments compared to cell controls, particularly for 24 h exposures. ACE2 gene expression was significantly increased in cells exposed to the locally bought e-liquid condensate with high nicotine concentration and cigarette smoke extract compared with cell controls. Our study suggests that vaping alone and smoking alone can result in an increase in lung ACE2 expression. Vaping and smoking are avoidable risk factors for COVID-19, which, if avoided, could help reduce the number of COVID-19 cases and the severity of the disease. This is the first study to utilize electronic cigarette aerosol condensates, novel and developed in our laboratory, for investigating ACE2 expression in human airway epithelial cells

Endothelial-to-mesenchymal transition: a precursor to pulmonary arterial remodelling in patients with idiopathic pulmonary fibrosis

Background We have previously reported arterial remodelling in patients with idiopathic pulmonary fibrosis (IPF) and suggested that endothelial-to-mesenchymal transition (EndMT) might be central to these changes. This study aims to provide evidence for active EndMT in IPF patients. Methods Lung resections from 13 patients with IPF and 15 normal controls (NCs) were immunostained for EndMT biomarkers: vascular endothelial cadherin (VE-cadherin), neural cadherin (N-cadherin), S100A4 and vimentin. Pulmonary arteries were analysed for EndMT markers by using computer- and microscope-assisted image analysis software Image ProPlus7.0. All the analysis was done with observer blinded to subject and diagnosis. Results Increased expression of mesenchymal markers N-cadherin (p<0.0001), vimentin (p<0.0001) and S100A4 (p<0.05) was noted with downregulation of junctional endothelial VE-cadherin (p<0.01) in the intimal layer of the arteries from patients with IPF compared to NCs. Cadherin switch was observed in IPF patients, showing increase in endothelial N-cadherin and decrease in VE-cadherin (p<0.01). There was also VE-cadherin shift from junctions to cytoplasm (p<0.01), effecting endothelial cell integrity in patients with IPF. In IPF, individual mesenchymal markers vimentin and N-cadherin negatively correlated with diffusing capacity of the lungs for carbon monoxide (r′= −0.63, p=0.03 and r′= −0.66, p=0.01). Further, N-cadherin positively correlated with arterial thickness (r′=0.58, p=0.03). Conclusion This is the first study to demonstrate active EndMT in size-based classified pulmonary arteries from IPF patients and potential role in driving remodelling changes. The mesenchymal markers had a negative impact on the diffusing capacity of the lungs for carbon monoxide. This work also informs early origins of pulmonary hypertension in patients with IPF

Arterial remodelling in smokers and in patients with small airway disease and COPD: implications for lung physiology and early origins of pulmonary hypertension

Introduction Pulmonary vascular remodelling in chronic obstructive pulmonary disease (COPD) has detrimental consequences for lung physiology. The aim of our study was to provide a comprehensive size-based morphometric quantification of pulmonary arterial remodelling in smokers and in patients with small airway disease (SAD) or COPD. Method Movat's pentachrome staining was performed on lung resections for 46 subjects: 12 never-smoker normal controls (NC), six normal lung function smokers (NLFS), nine patients with SAD, nine patients with mild-to-moderate COPD who were current smokers (COPD-CS) and 10 patients with mild-to-moderate COPD who were ex-smokers (COPD-ES). Following a size-based classification of pulmonary arteries, image analysis software was used to measure their number, total wall thickness, individual layer thickness and elastin percentage. Results All pathological groups showed decreased numbers of pulmonary arteries compared with the NC group in all artery sizes. Arterial wall thickness was greater in NLFS and COPD-CS than in NC. Thickness in COPD-ES was decreased compared with COPD-CS. Intimal thickness was greater in all pathological groups in all arterial sizes than in the NC group. Medial thickness was also greater in small and medium arteries. Intimal thickness of larger arteries in COPD-CS correlated negatively to forced expiratory volume in 1 s/forced vital capacity (FVC) % and forced expiratory flow at 25–75% of FVC. Elastin deposition in small arteries was greatest in COPD-CS. Intimal elastin deposition had a more negative correlation with intimal thickness in NLFS and SAD than in COPD-CS. Conclusion Smoking, SAD and mild-to-moderate COPD are associated with pruning and a decrease in the number of pulmonary arteries, increased wall thickness and variable elastin deposition. These changes were associated with worse airway obstruction

Airway inflammatory changes in the lungs of patients with asthma-COPD overlap (ACO): a bronchoscopy endobronchial biopsy study

Abstract Background Although asthma and chronic obstructive pulmonary disease (COPD) are two distinct chronic airway inflammatory diseases, they often co-exist in a patient and the condition is referred to as asthma-COPD overlap (ACO). Lack of evidence regarding the inflammatory cells in ACO airways has led to their poor prognosis and treatment. The objective of this endobronchial biopsy (EBB) study was to enumerate inflammatory cellular changes in the airway wall of ACO compared with asthma, COPD current smokers (CS) and ex-smokers (ES), normal lung function smokers (NLFS), and non-smoker controls (HC). Methods EBB tissues from 74 patients were immunohistochemically stained for macrophages, mast cells, eosinophils, neutrophils, CD8+ T-cells and CD4+ T-cells. The microscopic images of stained tissues were evaluated in the epithelium, reticular basement membrane (RBM) cells/mm RBM length, and lamina propria (LP) cells/mm2 up to a depth of 120 µM using the image analysis software Image-Pro Plus 7.0. The observer was blinded to the images and disease diagnosis. Statistical analysis was performed using GraphPad Prism v9. Results The tissue macrophages in ACO were substantially higher in the epithelium and RBM than in HC (P < 0.001 for both), COPD-ES (P < 0.001 for both), and -CS (P < 0.05 and < 0.0001, respectively). The ACO LP macrophages were significantly higher in number than COPD-CS (P < 0.05). The mast cell numbers in ACO were lower than in NLFS (P < 0.05) in the epithelium, lower than COPD (P < 0.05) and NLFS (P < 0.001) in RBM; and lower than  HC (P < 0.05) in LP. We noted lower eosinophils in ACO LP than HC (P < 0.05) and the lowest neutrophils in both ACO and asthma. Furthermore, CD8+ T-cell numbers increased in the ACO RBM than HC (P < 0.05), COPD-ES (P < 0.05), and NLFS (P < 0.01); however, they were similar in number in epithelium and LP across groups. CD4+ T-cells remained lower in number across all regions and groups. Conclusion These results suggest that the ACO airway tissue inflammatory cellular profile differed from the contributing diseases of asthma and COPD with a predominance of macrophages

Angiotensin-Converting Enzyme 2 (ACE2), Transmembrane Peptidase Serine 2 (TMPRSS2), and Furin Expression Increases in the Lungs of Patients with Idiopathic Pulmonary Fibrosis (IPF) and Lymphangioleiomyomatosis (LAM): Implications for SARS-CoV-2 (COVID-19) Infections

We previously reported higher ACE2 levels in smokers and patients with COPD. The current study investigates if patients with interstitial lung diseases (ILDs) such as IPF and LAM have elevated ACE2, TMPRSS2, and Furin levels, increasing their risk for SARS-CoV-2 infection and development of COVID-19. Surgically resected lung tissue from IPF, LAM patients, and healthy controls (HC) was immunostained for ACE2, TMPRSS2, and Furin. Percentage ACE2, TMPRSS2, and Furin expression was measured in small airway epithelium (SAE) and alveolar areas using computer-assisted Image-Pro Plus 7.0 software. IPF and LAM tissue was also immunostained for myofibroblast marker α-smooth muscle actin (α-SMA) and growth factor transforming growth factor beta1 (TGF-β1). Compared to HC, ACE2, TMPRSS2 and Furin expression were significantly upregulated in the SAE of IPF (p < 0.01) and LAM (p < 0.001) patients, and in the alveolar areas of IPF (p < 0.001) and LAM (p < 0.01). There was a significant positive correlation between smoking history and ACE2 expression in the IPF cohort for SAE (r = 0.812, p < 0.05) and alveolar areas (r = 0.941, p < 0.01). This, to our knowledge, is the first study to compare ACE2, TMPRSS2, and Furin expression in patients with IPF and LAM compared to HC. Descriptive images show that α-SMA and TGF-β1 increase in the IPF and LAM tissue. Our data suggests that patients with ILDs are at a higher risk of developing severe COVID-19 infection and post-COVID-19 interstitial pulmonary fibrosis. Growth factors secreted by the myofibroblasts, and surrounding tissue could further affect COVID-19 adhesion proteins/cofactors and post-COVID-19 interstitial pulmonary fibrosis. Smoking seems to be the major driving factor in patients with IPF.Medicine, Faculty ofNon UBCAnesthesiology, Pharmacology and Therapeutics, Department ofReviewedResearcherOthe