Carlin, Helen Victoria
Characterising epithelial to mesenchymal transition and pro-fibrotic processes in chronic obstructive pulmonary disease
, PhD thesis, University of Salford.
Chronic Obstructive Pulmonary Disease (COPD) is an irreversible disease reducing lung function via processes of inflammation, emphysema and fibrosis. Patient treatment options are limited including bronchodilators and inhaled corticosteroids that are poorly effective. Linked with a significant history of cigarette smoking, pathology is characterised by recruitment of inflammatory cells to the lung (alveolar macrophages, neutrophils and lymphocytes: TH1 and CD8); dysregulated neutrophils and macrophages release tissue lysing proteases causing enlargement of airspaces (emphysema). Most severe pathology includes dysregulated epithelial repair and overgrowth of lung fibroblasts (fibrosis). The source of fibroblasts is not clear. Fibrosis in renal, liver and heart disease is partly mediated by epithelial-mesenchymal transition (EMT) differentiation whereby epithelia acquire fibrotic biomarkers such as desmin, S100A4 and α-smooth muscle actin. In lung fibrosis this has not been characterised, further the molecular mechanisms in the lung are poorly understood. Identifying these processes is key to developing improved future therapeutic interventions.
To characterise human COPD lungs for expression of EMT biomarkers compared to control subjects to determine the processes of lung fibrosis in COPD. Further, to identify the signalling mechanisms within the EMT tissue to identify the regulatory processes that may be intervened in future therapies. Finally, to assess primary human fibroblast responses to cigarette smoke extract (CSE) and stretch forces to establish a new model of emphysema and lung fibrosis for future research.
Patients were recruited from the North West Lung Centre at the University Hospital of South Manchester who were undergoing lobectomy surgery as part of their normal clinical care. Lung resections were prepared for histochemical analysis by formalin fixing and wax embedding. 5ul microtome slices were immunohistochemically stained for typical mesenchymal (EMT) and epithelia biomarkers. Statistical comparisons were made between control groups: non-smokers (NS), current smokers (CS) ex-smokers (EXS), and COPD patients: COPD current smokers (CCS) and COPD ex-smokers (CEX). Identified regions of EMT tissue were laser capture microscopy dissected, RNA isolated, and target genes assessed by Q-RTPCR. Tissue culture experiments were performed by expanding fibroblasts from 1mm lung sections in RPMI culture media that were seeded in wells and exposed to stretch forces and doses of cigarette smoke extract (1 cigarette bubbled into 25ml RPMI media =100%) over time. IL-6, IL-8 and TGFβ1 responses were measured by ELISA and statistical comparisons made.
EMT marker S100A4 was significantly (P<0.05) raised in COPD patients. The other EMT biomarkers (desmin, αSM actin and collagen) also showed numerical increases over NS but were not significant. S100A4, desmin and collagen expression were all significantly raised in current smokers and ex-smokers compared to never smokers regardless of airway obstruction. S100A4+ve regions were selected for EMT identification for immune-lazer capture microscopy. SMAD3, SNAI1, TGFβ and IL6 were all numerically raised in EMT/S100A4+ve regions however the small subset sample numbers did not offer enough statistical power for the study. Primary fibroblast cultures released cytokines IL6, IL8 and TGFβ dose dependently to CSE: % (1, 5, 10, 20, 50), particularly at later time points (24 and 48hours) assessed by % change to 0%CSE. Absolute levels (pg/mL) were much higher at 24 and 48 hours compared to 1, 4 and 6 hours, which was particularly notable in COPD patients for all 3 cytokines. MTT assay showed viability was good 100-80% when cells were cultured and a small (~5% decrease) dose response to CSE for all cytokines. Approximately 5% viability is lost over the time course 0-48 hours; which is more notable (an extra 5%) in COPD patients.
This study showed EMT processes are associated with S100A4 expression and particularly with smoking pack year history rather than airway obstruction. We therefore demonstrated EMT processes are involved in smoking related pulmonary fibrosis. Further, S100A4 EMT regions are linked with TGFβ1 signalling components SMAD3 and SNAI1 and downstream outputs TGFβ1 and IL-6; this study needs expanding with more patients to determine if this molecular mechanism is EMT related in COPD. CSE directly induced pro-inflammatory mediator release and raised fibroblast responses seen in COPD patients and current smokers imply a pre-activated state contributing to their lung pathology. Stretch forces did not increase cytokine release beyond responses to CSE alone. Good starting cell viability indicates this is a suitable lung fibrosis model. CSE doses induced raised cell death responses in COPD patients over time; implicating their enhanced susceptibility to apoptosis and supporting contemporary literature. This in vitro primary fibroblast model will be useful for testing effectiveness of novel anti-inflammatory and anti-fibrotic drugs in future.
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