Time-dependent transcriptomic and phenotypic changes associated with repair and regeneration in the airway epithelium

Abstract

The airway epithelium demonstrates the ability to quickly repair following physical injury. The morphologic features of this dynamic repair process have been well characterised at the anatomic and cellular level using a number of animal model systems and these studies have provided a solid foundation upon which our understanding of normal repair is build. With the advent of molecular and bioinformatic tools and resources the opportunity exists to extend the value of these models in defining the molecular pathways and interactions that underlay the normal repair process. This thesis represents a realisation of this opportunity. A large animal model was developed in which selected areas of airway epithelium were subjected to bronchial brush biopsy as part of routine bronchoscopic examination procedures in anaesthetised sheep.

The process resulted in a physical perturbation of the normal pseudostratified structure of the sheep air-way epithelium at specific locations. By careful experimental design it was possible, within the same animals. to identify and sample from sites undergoing repair at different intervals subsequent in injury.

To supplement the histological evaluation of the repair process and align findings with established small animal models of airway epithelial repair proliferative cell labelling strategies were implemented in order to study the location and extent of cellular proliferation occurring during the repair process.

Molecular approaches towards defining the transcriptional response to physical injury comprised application of microarray technology using a commercially sources array platform. Such approach demanded preliminary effort directed towards optimising RNA integrity and yield from airway samples. Following preliminary studies directed towards optimising the model conditions patterns of airway epithelial repair following bronchial brush biopsy were studies in eight sheep at deigned time points (6 hours, 1,3, & 7 days) post-injury. Bronchial brush biopsy resulted in the acute removal of the epithelial cell layer and components of the underlying structures. repair processes were rapidly implemented through initial epithelial dedifferentiation, proliferation and migration at the wound margins and subsequent time-dependent changes in the proportion of subepithelial structures, including smooth muscle and blood vessels, as the epithelial surface moved towards repair. Transcriptional analysis revealed that over 13,000 probes showed evidence of differential expression at some point during the repair process (p<0.05), whilst of these, 1491 probes had in excess of a two-fold change in expression. array results were validated against conventional semi-quantitative RT-PCR for selected genes. Differentially expressed genes with previously characterised roles in epithelial migration, proliferation and differentiation were identified during the repair process. The relative emphasis of gene products with particular functional roles varied during the course of repair. Indeed gene ontology (GO) terms identified included those associated with the inflammatory response, cellular migration, extracellular matrix activities, differentiation, proliferation, cellular development, cell cycle activities, cellular adhesion, apoptosis and mitosis. In addition the Kyoto Encyclopedia of Genes and Gneomes (KEGG) databases were queried and such process indicated the involvement of cell communication, 053 and complement and coagulation cascade pathways throughout the repair process, initial (6h) Toll-like receptor and cytokine-cytoine receptor interaction pathways, and the progressive involvement of cell cycle, focal adhesion and extra-cellular matrix (ECM)-receptor, and cytokine interaction pathways as the epithelium repaired. The model of airway epithelial injury developed in this thesis generated features broadly consistent with those previously described in relation to various small animal model systems. Importantly, and in addition, this thesis defines the molecular features associated with repair in this model system and provides a useful resource with which to assess the comparative features of the airway transcriptional response to physical injury, It is through such comparison, using analogous methodology, that the fundamental pathways and interactions that underlay normal repair and regeneration can be identified and thereafter extended towards understanding the basis for variation associated with natural and experimental disease