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Title: Investigating the role of CCN2 in a bleomycin model of Idiopathic Pulmonary Fibrosis
Author: Horwell, A. L.
ISNI:       0000 0004 7964 1520
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2018
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Idiopathic Pulmonary Fibrosis (IPF) is a chronic, progressive and ultimately lethal disease of the lung characterised by excessive synthesis and deposition of extracellular matrix (ECM). The 5-year survival rate for IPF is lower than that of breast, prostate and skin cancer, with the average mortality rate currently standing at 3 years post diagnosis. Incidence and mortality appear to be on the rise, and prevalence is expected to increase in an aging population. Recently several new drugs have been approved (Pirfenidone, Nintedanib and Pamrevlumab). However, the pathobiology of IPF still remains elusive and there is an ever more pressing need to elucidate the molecular processes behind this disease to identify potential therapeutic targets. It is widely believed that at the cellular level, the key mediators of fibrosis are the fibroblast cell and their profibrotic phenotype, the myofibroblast. In normal homeostasis the myofibroblast is activated to synthesise new ECM during wound healing. In IPF there is believed to be a dysregulation in this process leading to persistent activation of myofibroblasts after the healing response is complete. Transforming growth factor - β (TGF-β) is known to be one of the master regulators of wound healing has been shown to be capable of activating myofibroblasts. However, research has looked downstream in the TGF - β signalling pathway to another protein - connective tissue growth factor (CCN2). CCN2 is considered a key downstream mediator of the profibrotic effects of TGF- β and has been shown to induce a fibrotic response independently of TGF-β both in vitro and in animal models of IPF. A clinical trial of a monoclonal antibody to CCN2 (Pamrevlumab) has demonstrated the ability to reverse the fibrosis in various murine models of fibrosis and is showing promising efficacy in human trials. The mechanism of action of CCN2 in fibrosis is still yet to be fully understood. The aim of this thesis was to utilise the murine bleomycin model of IPF to further investigate the role of CCN2 in fibrosis. Two new double transgenic mouse lines were generated, containing a CCN2 floxed transcript and a tamoxifen-inducible cre recombinase (CreERT2). Two different CreERT2 were used to facilitate loss of function of CCN2 in fibroblast cells specifically (Col1α2-CreERT2), or in all cells (ROSA-CreERT2). A dosing regimen for initiating recombination in vivo was established, and the phenotype in the lungs characterised for both new mouse lines. A third double transgenic mouse line was generated to examine the expression pattern of the Col1α2-CreERT2 during adulthood and embryonic development using a dual fluorescent reporter construct (mT/mG). A protocol was established for the induction of fibrosis in these mice using bleomycin, and a novel imaging protocol using an ex vivo μCT scanner was developed to enable full imaging of unfixed lungs prior to genetic and histological analysis. In summary, the studies reported in this thesis presented novel findings on the role of CCN2 in the pathogenesis of fibrosis. Two novel transgenic mouse lines were established for the genetic manipulation of CCN2 in vivo. These were used in conjunction with the bleomycin model, which was established for the first time at the University of Liverpool. The results from these experiments unveiled unexpected results and raised further questions as to the role CCN2 plays in homeostatic regulation of ECM production by demonstrating a potent anti-fibrotic effect of CCN2. A third transgenic mouse line was used to characterise the expression profile of a fibroblast specific CreERT2 developed by the Bou-Gharios lab group and found that the fibroblast specificity was prevalent in a wider population of fibroblasts than described previously.
Supervisor: Bou-Gharios, George ; Spencer, Lisa ; Moots, robert Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral