Microvascular remodelling in a sheep model of bleomycin-induced pulmonary fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF) is a lethal, progressive, and chronic lung disease with unknown cause and dismal prognosis. The median survival time of IPF patients is 2-4 years after diagnosis and the treatment options available for IPF are very limited. The pathogenesis of IPF is complex and not fully understood. The current paradigms focus on alveolar epithelial cell injury as a key initiating event followed by dysregulated wound healing process resulting in fibrosis and distortion of the lung’s architecture. During this process, injuries to the lung are not only associated with fibrosis of the lung parenchyma, but also with changes to the pulmonary vasculature, leading to an aberrant vascular remodelling. However, the precise relationship between vascular remodelling, and the development of fibrosis, in the lung parenchyma is not well understood. One of the reasons for this is that the underlying mechanisms that operate in these disease processes are largely unexplored.

Initially, using tissues from previously published studies, I confirmed the presence of microvascular remodelling in small airways as well as in the parenchyma of bleomycin-infused fibrotic lung segments. Blood vessel density, particularly those of blood vessels >30um in diameter, was significantly lower in small airways. The small airways that were exposed to bleomycin injury had increased levels of connective tissues in the airway walls which led to functional changes of increased resistance in these lung segments. In contrast to the lower blood vessel density levels found in bleomycin exposed airways, there was increased neovascularisation in the parenchyma of fibrotic lung segments injured by bleomycin. The blood vessel density in the parenchyma correlated with the level of fibrosis in bleomycin infused lung segments.

After confirming the microvascular remodelling in response to bleomycin, the effects of different anti-fibrotic and anti-angiogenic treatments on this process were investigated. I found that these microvascular remodelling changes were significantly attenuated by treatment with the KCa3.1 channel inhibitor, senicapoc. The microvascular remodelling was associated with increases in vascular endothelial growth factor (VEGF) expression and endothelial cell proliferation which were significantly reduced with the senicapoc treatment. These parameters were not significantly suppressed with pirfenidone treatment. Senicapoc treatment attenuated microvascular remodelling through inhibition of capillary endothelial cell proliferation and VEGF expression.

I further assessed whether blocking of angiogenesis, using a known antiangiogenic agent tetrathiomolybdate, can attenuate bleomycin-induced fibrosis in the sheep model. In this experiment, I used the segmental approach, where two bronchoscopic boluses of bleomycin, administered two weeks apart, were infused into lower lung segments to induce fibrosis. Saline was infused into the contralateral lung segment to act as an internal healthy-control lung segment. Tetrathiomolybdate treatment, via intravenous injection, was started one week after the final bleomycin infusion and continued twice weekly for six weeks. The sheep were then culled, and a full histopathological and other analyses were performed on the extracted lung tissues. The functional parameter of compliance was measured via a wedged bronchoscope procedure prior to and seven weeks following final bleomycin infusion. Bronchoalveolar lavage (BAL) fluid was also assessed sequentially during the study periods. I found that copper lowering by tetrathiomolybdate chelation, results in the attenuation of angiogenesis and fibrosis in lung segments injured by bleomycin. Moreover, tetrathiomolybdate treatment attenuated the histopathological changes, and improved the poorer lung function that were associated with the development of bleomycin-induced pulmonary fibrosis. Tetrathiomolybdate also prevented accumulation of inflammatory cells in BAL fluid two weeks after bleomycin injury.

Overall, the results presented in this thesis indicate the presence of microvascular remodelling in bleomycin-induced pulmonary fibrosis. Senicapoc and tetrathiomolybdate treatments attenuated microvascular remodelling as well as pulmonary fibrosis. This is in contrast with the application of the FDA-approved drug pirfenidone, which only attenuated fibrosis, without any effects on the vasculature, in this model. It will be interesting to see whether intervention strategies derived from this thesis can be applied clinically to treat IPF.