Tetrathiomolybdate Treatment Attenuates Bleomycin-Induced Angiogenesis and Lung Pathology in a Sheep Model of Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic lung disease characterized by excessive extracellular matrix (ECM) deposition in the parenchyma of the lung. Accompanying the fibrotic remodeling, dysregulated angiogenesis has been observed and implicated in the development and progression of pulmonary fibrosis. Copper is known to be required for key processes involved in fibrosis and angiogenesis. We therefore hypothesized that lowering bioavailable serum copper with tetrathiomolybdate could be of therapeutic value for treating pulmonary fibrosis. This study aimed to investigate the effect of tetrathiomolybdate on angiogenesis and fibrosis induced in sheep lung segments infused with bleomycin. Twenty sheep received two fortnightly infusions of either bleomycin (3U), or saline (control) into two spatially separate lung segments. A week after the final bleomycin/saline infusions, sheep were randomly assigned into two groups (n = 10 per group) and received twice-weekly intravenous administrations of either 50 mg tetrathiomolybdate, or sterile saline (vehicle control), for 6 weeks. Vascular density, expressed as the percentage of capillary area to the total area of parenchyma, was determined in lung tissue sections immuno-stained with antibodies against CD34 and collagen type IV. The degree of fibrosis was assessed by histopathology scoring of H&E stained sections and collagen content using Masson's trichrome staining. Lung compliance was measured via a wedged bronchoscope procedure prior to and 7 weeks following final bleomycin infusion. In this large animal model, we show that copper lowering by tetrathiomolybdate chelation attenuates both bleomycin-induced angiogenesis and pulmonary fibrosis. Moreover, tetrathiomolybdate treatment downregulates vascular endothelial growth factor (VEGF) expression, and improved lung function in bleomycin-induced pulmonary fibrosis. Tetrathiomolybdate also suppressed the accumulation of inflammatory cells in bronchoalveolar lavage fluid 2 weeks after bleomycin injury. The molecular mechanism(s) underpinning copper modulation of fibrotic pathways is an important area for future investigation, and it represents a potential therapeutic target for pulmonary fibrosis.

Small airway remodeling in a sheep model of bleomycin-induced pulmonary fibrosis.

BACKGROUND: Although IPF is described traditionally as a disease affecting lung parenchyma, there is renewed interest in the alterations in the structure and function of the small airways in both IPF patients, and animal models of pulmonary fibrosis. Small airway remodeling may contribute to the pathophysiology of pulmonary fibrosis. Given the dearth of knowledge of small airway changes in pulmonary fibrosis, this study aims to assess the structural remodeling, as well as functional changes associated with bleomycin-injured small airways in a sheep model of pulmonary fibrosis. MATERIALS AND METHODS: Two separate lung segments in ten sheep received two challenges of either 3 IU bleomycin, or saline (control), two weeks apart. The animals were euthanized seven weeks after the final bleomycin injury. Airflow resistance in the infused segments was measured with a wedged-bronchoscope procedure. This parameter was measured at baseline before bleomycin/saline-infusion, and at 2-, 4-, and 7-weeks after the final bleomycin-infusion. Inflammation and fibrosis in the airways were assessed by semi-quantitative morphological parameters. The density of blood vessels in the small airway walls was assessed in lung tissue sections immuno-stained with antibodies against collagen type IV. RESULTS: There were a number of changes in the distal airways of bleomycin-infused lung segments. Bleomycin exposure significantly elevated airway resistance in these lung segments when compared to saline-infused control lung segments. In the peribronchial and peribronchiolar regions of the small airways, there were significantly increased levels of inflammation, fibrosis, airway wall area, and collagen deposition in bleomycin-infused airways when compared to saline-infused airways. Bronchial blood vessel density was not significantly different between bleomycin-and saline-infused lung segments. CONCLUSIONS: In summary, our results indicate that the distal airways are involved in the pathology induced by bleomycin in this sheep model. This suggests that the sheep model may be useful for studying small airway remodeling in pulmonary fibrosis.

KCa3.1 channel blockade attenuates microvascular remodelling in a large animal model of bleomycin-induced pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with limited therapeutic options and poor prognosis. IPF has been associated with aberrant vascular remodelling, however the role of vascular remodelling in pulmonary fibrosis is poorly understood. Here, we used a novel segmental challenge model of bleomycin-induced pulmonary fibrosis in sheep to evaluate the remodelling of the pulmonary vasculature, and to investigate the changes to this remodelling after the administration of the KCa3.1 channel inhibitor, senicapoc, compared to the FDA-approved drug pirfenidone. We demonstrate that in vehicle-treated sheep, bleomycin-infused lung segments had significantly higher blood vessel density when compared to saline-infused control segments in the same sheep. These microvascular density changes were significantly attenuated by senicapoc treatment. The increases in vascular endothelial growth factor (VEGF) expression and endothelial cell proliferation in bleomycin-infused lung segments were significantly reduced in sheep treated with the senicapoc, when compared to vehicle-treated controls. These parameters were not significantly suppressed with pirfenidone treatment. Senicapoc treatment attenuated vascular remodelling through inhibition of capillary endothelial cell proliferation and VEGF expression. These findings suggest a potential new mode of action for the novel drug senicapoc which may contribute to its efficacy in combatting pulmonary fibrosis.