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Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by excessive deposition of extracellular matrix in the interstitial lung parenchyma, often manifested by dyspnea and progressive loss of lung function. The role of inflammation in the pathogenesis of IPF is not well understood. This study evaluated the histopathological and inflammatory components of bleomycin-induced pulmonary fibrosis in mouse and sheep models, in terms of their ability to translate to the human IPF. Merino sheep ( n  = 8) were bronchoscopically administered with two bleomycin infusions, two weeks apart, into a caudal lung segment, with a saline (control) administered into a caudal segment in the opposite lung. Balb/c mice were twice intranasally instilled, one week apart, with either bleomycin ( n  = 7); or saline (control, n  = 7). Lung samples were taken for the histopathological assessment 28 days in sheep and 21 days in mice after the first bleomycin administration. We observed tertiary lymphoid aggregates, in the fibrotic lung parenchyma of sheep, but not in mouse lung tissues exposed to bleomycin. B-cell and T-cell infiltration significantly increased in sheep lung tissues compared to mouse lung tissues due to bleomycin injury. Statistical analysis showed that the fibrotic score, fibrotic fraction, and tissue fraction significantly increased in sheep lung tissues compared to murine lung tissues. The presence of tertiary lymphoid aggregates in the lung parenchyma and increased infiltration of T-cells and B-cells, in the sheep model, may be useful for the future study of the underlying inflammatory disease mechanisms in the lung parenchyma of IPF patients., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2023 Udari Eshani Perera et al.)

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibroproliferative disorder that has one of the poorest prognoses amongst interstitial lung diseases. Recently, the finding of aberrant expression levels of miRNAs in IPF patients has drawn significant attention to the involvement of these molecules in the pathogenesis of this disease. Clarification of the differential expression of miRNAs in health and disease may identify novel therapeutic strategies that can be employed in the future to combat IPF. This study evaluates the miRNA expression profiles in a sheep model for lung fibrosis and compares them to the miRNA profiles of both IPF patients and the mouse bleomycin model for pulmonary fibrosis. Pathway enrichment analyses were performed on differentially expressed miRNAs to illustrate which biological mechanisms were associated with lung fibrosis., Results: We discovered 49 differentially expressed miRNAs in the sheep fibrosis model, in which 32 miRNAs were significantly down regulated, while 17 miRNAs were significantly upregulated due to bleomycin-induced lung injury. Moreover, the miRNA families miR-29, miR-26, miR-30, let-7, miR-21, miR-19, miR-17 and miR-199 were aberrantly expressed in both sheep and mouse models, with similar differential miRNAs expression observed in IPF cases. Importantly, 18 miRNAs were aberrantly expressed in both the sheep model and IPF patients, but not in mice., Conclusion: Together with pathway enrichment analyses, these results show that the sheep model can potentially be used to characterize previously unrecognized biological pathways associated with lung fibrosis., (© 2021. The Author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Derseh, Perera, Dewage, Stent, Koumoundouros, Organ, Pagel and Snibson.)

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease, characterized by progressive damage to the lung tissues. Apoptosis and endoplasmic reticulum stress (ER stress) in type II alveolar epithelial cells (AECs) and lung macrophages have been linked with the development of IPF. Therefore, apoptosis- and ER stress-targeted therapies have drawn attention as potential avenues for treatment of IPF. The calcium-activated potassium ion channel KCa3.1 has been proposed as a potential therapeutic target for fibrotic diseases including IPF. While KCa3.1 is expressed in AECs and macrophages, its influence on ER stress and apoptosis during the disease process is unclear. We utilized a novel sheep model of pulmonary fibrosis to demonstrate that apoptosis and ER stress occur in type II AECs and macrophages in sheep with bleomycin-induced lung fibrosis. Apoptosis in type II AEC and macrophages was identified using the TUNEL method of tagging fragmented nuclear DNA, while ER stress was characterized by increased expression of GRP-78 ER chaperone proteins. We demonstrated that apoptosis and ER stress in type II AECs and macrophages increased significantly 2 weeks after the final bleomycin infusion and remained high for up to 7 weeks post-bleomycin injury. Senicapoc treatment significantly reduced the rates of ER stress in type II AECs and macrophages that were resident in bleomycin-infused lung segments. There were also significant reductions in the rates of apoptosis of type II AECs and macrophages in the lung segments of senicapoc-treated sheep. In vivo blockade of the KCa3.1 ion channel alleviates the ER stress and apoptosis in type II AECs and macrophages, and this effect potentially contributes to the anti-fibrotic effects of senicapoc., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Udari E. Perera et al.)

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.

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 K Ca 3.1 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.

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic lung disease with unknown cause. While the drugs nintedanib and pirfenidone have been approved for the treatment of IPF, they only slow disease progression and can induce several side-effects, suggesting that there is still an unmet need to develop new efficacious drugs, and interventions strategies, to combat this disease. We have recently developed a sheep model of pulmonary fibrosis for the preclinical testing of novel anti-fibrotic drugs. The aim of this study was to assess the effects of pirfenidone to ascertain its suitability as a benchmark for comparing other novel therapeutics in this sheep model. To initiate localized fibrosis, sheep were given two infusions of bleomycin (0.6 U/ml per infusion), a fortnight apart, to a specific lung segment. The contralateral lung segment in each sheep was infused with saline to act as an internal control. Two weeks after the final bleomycin infusion, either pirfenidone or methylcellulose (vehicle control) were administered orally to sheep twice daily for 5 weeks. Results showed that sheep treated with pirfenidone had improved lung function, ameliorated fibrotic pathology, lower numbers of active myofibroblasts, and reduced extra cellular matrix deposition when compared with the relevant measurements obtained from control sheep treated with vehicle. This study showed that pirfenidone can attenuate bleomycin-induced pulmonary fibrosis in sheep, and can therefore be used as a positive control to assess other novel therapeutics for IPF in this model.