Habgood, Anthony
(2018)
Refining in-vivo models of pulmonary fibrosis.
PhD thesis, University of Nottingham.
Abstract
Idiopathic pulmonary fibrosis (IPF) is a progressive disease with limited treatment options. IPF is characterized by excessive collagen deposition within the lung resulting in loss of lung function and a survival rate worse than most cancers. There is an urgent need to develop and better characterise animal models of this disease to further our understanding of its pathophysiology and to assist in the development of new pharmacological treatments. Researching respiratory diseases and the development of new therapies relies on the use of robust in vivo models.
The objective of this thesis was to better-characterise a range of animal models of pulmonary fibrosis. The principal objective was to evaluate a range of animal models of lung fibrosis to facilitate reproducibility of in vivo data across a range of models to increase confidence in the results of pre-clinical assessments. To try and minimise unnecessary animal usage, this thesis also investigated a number of techniques, such as non-invasive imaging, and ex vivo models such as the precision cut lung slice (PCLS) model to promote the principles of replacement, reduction and refinement of in vivo studies.
Firstly, I established and characterised the bleomycin model of acute lung injury and pulmonary fibrosis in both mice and rats. When this was achieved I used the bleomycin model to determine the effect of secretory leukocyte protease inhibitor (Slpi) gene deletion, and Tranilast treatment, on the development of bleomycin-induced lung injury and pulmonary fibrosis.
Using Slpi null mice, I set out to determine the role of SLPI in the development of pulmonary fibrosis. Slpi-/- mice have an increased ratio of active MMP-9 to pro-MMP-9 compared to wild-type animals demonstrating enhanced metalloproteinase activity within the lung. Wild-type mice treated with bleomycin have increased TGF-β activity, as measured by increased pSmad2 levels in nuclear extracts from macrophages taken from bronchoalveolar lavage fluid. Furthermore, wild-type mice have increased collagen type I, alpha 1, type III, alpha 1, type IV, alpha 1, mRNA expression and significantly more total lung collagen 28-days post bleomycin. However, TGF-β activity in Slpi-/- mice was not increased and collagen gene expression was not upregulated but they still had a significant increase in total lung collagen deposition 28-days post bleomycin. Overall these data suggested that a lack of Slpi expression does not significantly modify the development of lung fibrosis following bleomycin-induced lung injury.
In this thesis, I evaluate the efficacy of Tranilast at inhibiting fibrosis in a therapeutic bleomycin model. 36 rats were split into 6 treatment groups. Rats were dosed daily with compound from day 11 to day 21 post-bleomycin administration via the oropharyngeal or oral route. The study determined that bleomycin induced collagen deposition in all of the rats, with hydroxyproline levels as follows; saline / Tranilast oral 3.77 mg Vs bleomycin / Tranilast oral 6.73 mg **P<0.01, saline / Tranilast o.p. 2.39 mg Vs bleomycin / Tranilast o.p. 6.97 mg ****P<0.0001 but only a trend towards an increase in the lactose op control group saline / lactose o.p. bleomycin / lactose o.p. Tranilast treatment, regardless of route of administration, had no effect on collagen deposition in rats. pSmad2 levels should be determined to establish if Tranilast was affecting TGF-β activity. It is possible that Tranilast may have greater effect if used to inhibit mast cell degranulation therefore a treatment strategy investigating this may also be of interest in future studies.
A model of radiation-induced lung fibrosis was also established that showed mice administered thoracic irradiation developed substantial pulmonary fibrosis in a dose dependent manner. 17.5 Gy was determined as the optimum dose for future studies as it caused substantial fibrosis without high mortality and radiation associated side effects. The development of pulmonary fibrosis in irradiated animals was tracked with αvβ6 SPECT/CT imaging for the first time in this model in an effort to determine whether the duration of the model could be shortened. Specific binding of 111In-DTPA-A20FMDV2 to v6 integrins is increased in the lungs of 17.5 Gy irradiated animals, 8.68 %, as early as 17 weeks compared with ‘sham’ irradiated controls 3.76 % of initial 111In dose/mm3 of lung (P = 0.0517). This increase in v6 in the lung is detected in lungs that show some parenchymal damage in axial CT scans. Mice went on to establish dense areas of alveolitis and connective tissue deposition as well as pleural thickening at 26 weeks.
In this thesis, I also develop a precision cut lung slice model of pulmonary fibrosis where I induce fibrosis in a small number of mice using bleomycin then attain large numbers of slices, > 30 slice 150 µm thick, of fibrotic lung. Lung slices are cultured for 5 days and treated with various interventions. After culturing for 5 days, I was able to determine an increase in hydroxyproline in bleomycin treated mice compared with saline treated mice. Therefore, this model has the potential to be used to assess a compound’s ability to inhibit collagen deposition, this has the potential to greatly reduce the number of animals required when determining an EC50.
Finally, I investigated whether Elk1-/0 mice could be used as a potential model of age related fibrosis and demonstrated that Elk1-/0 mice have evidence of widespread, although mild, pulmonary fibrosis at 12 months of age when assessed via histology. Measurement of hydroxyproline established a significant increase in collagen deposition in Elk1-/0 mice compared with Elk1+/0 controls at 12 months of age (852.3, 758.4 µg/lung set, respectively, P = 0.0346)
This thesis has refined and further characterised a number of commonly used models of pulmonary fibrosis which were used in this thesis to further our understanding of the role of SLPI in the development of pulmonary fibrosis and also to assess the efficacy of novel drug treatments.
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