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Title: The influence of the environment on cell-cell communication in the epithelial-mesenchymal trophic unit
Author: Bucchieri, Fabio
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2012
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Rationale: A key factor involved with the progressive decline in lung function in severe asthma is airway remodelling. This involves collagen deposition within the lamina reticularis, matrix deposition in the submucosa, smooth muscle hyperplasia, and microvascular and neuronal proliferation. Aims: The main aim of this thesis was to test the hypothesis that epithelial susceptibility to environmental injury and a prolonged tissue repair response result in activation of the epithelial mesenchymal trophic unit (EMTU) that in turn promotes airway wall remodelling. Methods: Investigation of epithelial mesenchymal signalling involved use of cells derived from normal or asthmatic volunteers and cultured in vitro. These were tested in simple monocultures and then a „tissue engineered‟ construct was developed to enable assessment of responses in a complex cell model that closely mimics the human bronchial mucosa. Results: Infection of asthmatic bronchial epithelial cells (AS PBEC) with RV-16 caused significantly less apoptosis when compared to infection of bronchial epithelial cells obtained from healthy controls (HC PBEC). As a consequence of this RV replicated more in AS PBEC; this defect may explain the higher susceptibility of the lower airways to RV infection in asthmatic subjects. Using primary bronchial fibroblast cultures, collagen type I and fibronectin significantly increased adhesion rates, while laminin reduced differentiation of fibroblasts into myofibroblasts. None of the ECM components that were evaluated affected fibroblast survival. Although asthmatic fibroblasts expressed more αSMA than normal fibroblasts, no significant difference in terms of fibroblast differentiation was found when different ECM components, of relevance to asthma pathogenesis, were studied. When bronchial fibroblasts were exposed to conditioned media obtained from PBEC infected with RV16, there was no effect on markers of remodelling but there was a marked amplification of the epithelial inflammatory response, mainly due to increased release of IL8, IL6, RANTES and IP-10, suggesting that during a virus-induced exacerbation the fibroblasts promote inflammation. To investigate susceptibility to oxidant stress, PBEC were treated with 20% cigarette smoke extract (CSE). AS PBECs were more susceptible to CSE with a significant increase of early apoptotic (EA) cells compared to HS PBEC. Reduced glutathione protected both HS and AS PBEC from CSE- induced cell death causing a significant increase in cell viability, with a concomitant decrease in apoptosis. Oxidative stress-induced apoptosis in PBECs did not follow the canonical caspase pathways, but rather depended on a more direct mitochondrial damage pathway. The ECM also appeared to play a role in oxidative stress-induced cell death, with collagen IV being most effective in reducing H2O2-induced apoptosis. Finally, we developed and characterised a novel 3D in vitro model of human bronchial mucosa that proved to be very similar to the normal in vivo counterpart with a well differentiated epithelial layer composed of ciliated and goblet cells producing mucus and supported by a functional basement membrane which separated the epithelium from a mesenchymal layer containing fibroblasts dispersed in a well organised ECM. This model allowed investigation of the long term effects of CSE exposure which showed that CSE caused many characteristic signs of remodelling such as thickening of the basement membrane, disarray of the ECM, loss of ciliated cells and hyperproduction of mucus. These effects occurred in the absence of inflammatory or immune cells. Conclusions: The finding that epithelial cells from asthmatic donors have increased susceptibility to viral and oxidative stress emphasises the importance of tissue susceptibility in asthma. These findings, together with the observations that there is substantial cross talk between epithelial cells and fibroblasts supports the concept that abnormal function of the EMTU is an important contributory factor for asthma pathogenesis.
Supervisor: Davies, Donna Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available