Melbourne Veterinary School - Research Publications
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ItemTetrathiomolybdate Treatment Attenuates Bleomycin-Induced Angiogenesis and Lung Pathology in a Sheep Model of Pulmonary Fibrosis(FRONTIERS MEDIA SA, 2021-10-22)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.
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ItemEvaluation of microRNA expression in a sheep model for lung fibrosis(BMC, 2021-11-17)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.
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ItemThe efficacy and safety of pinocembrin in a sheep model of bleomycin-induced pulmonary fibrosis(PUBLIC LIBRARY SCIENCE, 2021-12-02)The primary flavonoid, pinocembrin, is thought to have a variety of medical uses which relate to its reported anti-oxidant, anti-inflammatory, anti-microbial and anti-cancer properties. Some studies have reported that this flavonoid has anti-fibrotic activities. In this study, we investigated whether pinocembrin would impede fibrosis, dampen inflammation and improve lung function in a large animal model of pulmonary fibrosis. Fibrosis was induced in two localized lung segments in each of the 10 sheep participating in the study. This was achieved via two infusions of bleomycin delivered bronchoscopically at a two-week interval. Another lung segment in the same sheep was left untreated, and was used as a healthy control. The animals were kept for a little over 5 weeks after the final infusion of bleomycin. Pinocembrin, isolated from Eucalyptus leaves, was administered to one of the two bleomycin damaged lung segments at a dose of 7 mg. This dose was given once-weekly over 4-weeks, starting one week after the final bleomycin infusion. Lung compliance (as a measure of stiffness) was significantly improved after four weekly administrations of pinocembrin to bleomycin-damaged lung segments. There were significantly lower numbers of neutrophils and inflammatory cells in the bronchoalveolar lavage of bleomycin-infused lung segments that were treated with pinocembrin. Compared to bleomycin damaged lung segments without drug treatment, pinocembrin administration was associated with significantly lower numbers of immuno-positive CD8+ and CD4+ T cells in the lung parenchyma. Histopathology scoring data showed that pinocembrin treatment was associated with significant improvement in inflammation and overall pathology scores. Hydroxy proline analysis showed that the administration of pinocembrin did not reduce the increased collagen content that was induced by bleomycin in this model. Analyses of Masson's Trichrome stained sections showed that pinocembrin treatment significantly reduced the connective tissue content in lung segments exposed to bleomycin when compared to bleomycin-infused lungs that did not receive pinocembrin. The striking anti-inflammatory and modest anti-fibrotic remodelling effects of pinocembrin administration were likely linked to the compound's ability to improve lung pathology and functional compliance in this animal model of pulmonary fibrosis.
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ItemIncreased Levels of ER Stress and Apoptosis in a Sheep Model for Pulmonary Fibrosis Are Alleviated by In Vivo Blockade of the KCa3.1 Ion Channel(HINDAWI LTD, 2021-03-20)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.
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ItemExamination of Novel Immunomodulatory Effects of L-Sulforaphane(MDPI, 2021-02-01)The dietary isothiocyanate L-sulforaphane (LSF), derived from cruciferous vegetables, is reported to have several beneficial biological properties, including anti-inflammatory and immunomodulatory effects. However, there is limited data on how LSF modulates these effects in human immune cells. The present study was designed to investigate the immunomodulatory effects of LSF (10 µM and 50 µM) on peripheral blood mononuclear cell (PBMC) populations and cytokine secretion in healthy adult volunteers (n = 14), in the presence or absence of bacterial (lipopolysaccharide) and viral (imiquimod) toll-like receptor (TLRs) stimulations. Here, we found that LSF reduced pro-inflammatory cytokines interleukin (IL)-6, IL-1β, and chemokines monocyte chemoattractant protein (MCP)-1 irrespective of TLR stimulations. This result was associated with LSF significantly reducing the proportion of natural killer (NK) cells and monocytes while increasing the proportions of dendritic cells (DCs), T cells and B cells. We found a novel effect of LSF in relation to reducing cluster of differentiation (CD) 14+ monocytes while simultaneously increasing monocyte-derived DCs (moDCs: lineage-Human Leukocyte Antigen-DR isotype (HLA-DR)+CD11blow-high CD11chigh). LSF was also shown to induce a 3.9-fold increase in the antioxidant response element (ARE) activity in a human monocyte cell line (THP-1). Our results provide important insights into the immunomodulatory effects of LSF, showing in human PBMCs an ability to drive differentiation of monocytes towards an immature monocyte-derived dendritic cell phenotype with potentially important biological functions. These findings provide insights into the potential role of LSF as a novel immunomodulatory drug candidate and supports the need for further preclinical and phase I clinical studies.
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ItemK(Ca)3.1 Channel-Blockade Attenuates Airway Pathophysiology in a Sheep Model of Chronic Asthma(PUBLIC LIBRARY SCIENCE, 2013-06-24)BACKGROUND: The Ca(2+)-activated K(+) channel K(Ca)3.1 is expressed in several structural and inflammatory airway cell types and is proposed to play an important role in the pathophysiology of asthma. The aim of the current study was to determine whether inhibition of K(Ca)3.1 modifies experimental asthma in sheep. METHODOLOGY AND PRINCIPAL FINDINGS: Atopic sheep were administered either 30 mg/kg Senicapoc (ICA-17073), a selective inhibitor of the K(Ca)3.1-channel, or vehicle alone (0.5% methylcellulose) twice daily (orally). Both groups received fortnightly aerosol challenges with house dust mite allergen for fourteen weeks. A separate sheep group received no allergen challenges or drug treatment. In the vehicle-control group, twelve weeks of allergen challenges resulted in a 60±19% increase in resting airway resistance, and this was completely attenuated by treatment with Senicapoc (0.25±12%; n = 10, P = 0.0147). The vehicle-control group had a peak-early phase increase in lung resistance of 82±21%, and this was reduced by 58% with Senicapoc treatment (24±14%; n = 10, P = 0.0288). Senicapoc-treated sheep also demonstrated reduced airway hyperresponsiveness, requiring a significantly higher dose of carbachol to increase resistance by 100% compared to allergen-challenged vehicle-control sheep (20±5 vs. 52±18 breath-units of carbachol; n = 10, P = 0.0340). Senicapoc also significantly reduced eosinophil numbers in bronchoalveolar lavage taken 48 hours post-allergen challenge, and reduced vascular remodelling. CONCLUSIONS: These findings suggest that K(Ca)3.1-activity contributes to allergen-induced airway responses, inflammation and vascular remodelling in a sheep model of asthma, and that inhibition of K(Ca)3.1 may be an effective strategy for blocking allergen-induced airway inflammation and hyperresponsiveness in humans.
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ItemIncreased Mast Cell Density and Airway Responses to Allergic and Non-Allergic Stimuli in a Sheep Model of Chronic Asthma(PUBLIC LIBRARY SCIENCE, 2012-05-14)BACKGROUND: Increased mast cell (MC) density and changes in their distribution in airway tissues is thought to contribute significantly to the pathophysiology of asthma. However, the time sequence for these changes and how they impact small airway function in asthma is not fully understood. The aim of the current study was to characterise temporal changes in airway MC density and correlate these changes with functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen. METHODOLOGY/PRINCIPAL FINDINGS: MC density was examined on lung tissue from four spatially separate lung segments of allergic sheep which received weekly challenges with HDM allergen for 0, 8, 16 or 24 weeks. Lung tissue was collected from each segment 7 days following the final challenge. The density of tryptase-positive and chymase-positive MCs (MC(T) and MC(TC) respectively) was assessed by morphometric analysis of airway sections immunohistochemically stained with antibodies against MC tryptase and chymase. MC(T) and MC(TC) density was increased in small bronchi following 24 weeks of HDM challenges compared with controls (P<0.05). The MC(TC)/MC(T) ratio was significantly increased in HDM challenged sheep compared to controls (P<0.05). MC(T) and MC(TC) density was inversely correlated with allergen-induced increases in peripheral airway resistance after 24 weeks of allergen exposure (P<0.05). MC(T) density was also negatively correlated with airway responsiveness after 24 challenges (P<0.01). CONCLUSIONS: MC(T) and MC(TC) density in the small airways correlates with better lung function in this sheep model of chronic asthma. Whether this finding indicates that under some conditions mast cells have protective activities in asthma, or that other explanations are to be considered requires further investigation.
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ItemNebulized perflubron and carbon dioxide rapidly dilate constricted airways in an ovine model of allergic asthma(BIOMED CENTRAL LTD, 2014-09-16)BACKGROUND: The low toxicity of perfluorocarbons (PFCs), their high affinity for respiratory gases and their compatibility with lung surfactant have made them useful candidates for treating respiratory diseases such as adult respiratory distress syndrome. We report results for treating acute allergic and non-allergic bronchoconstriction in sheep using S-1226 (a gas mixture containing carbon dioxide and small volumes of nebulized perflubron). The carbon dioxide, which is highly soluble in perflubron, was used to relax airway smooth muscle. METHODS: Sheep previously sensitized to house dust mite (HDM) were challenged with HDM aerosols to induce early asthmatic responses. At the maximal responses (characterised by an increase in lung resistance), the sheep were either not treated or treated with one of the following; nebulized S-1226 (perflubron + 12% CO2), nebulized perflubron + medical air, 12% CO2, salbutamol or medical air. Lung resistance was monitored for up to 20 minutes after cessation of treatment. RESULTS: Treatment with S-1226 for 2 minutes following HDM challenge resulted in a more rapid, more profound and more prolonged decline in lung resistance compared with the other treatment interventions. Video bronchoscopy showed an immediate and complete (within 5 seconds) re-opening of MCh-constricted airways following treatment with S-1226. CONCLUSIONS: S-1226 is a potent and rapid formulation for re-opening constricted airways. Its mechanism(s) of action are unknown. The formulation has potential as a rescue treatment for acute severe asthma.
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ItemStructural and functional correlations in a large animal model of bleomycin-induced pulmonary fibrosis(BMC, 2015-07-31)BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a severe and progressive respiratory disease with poor prognosis. Despite the positive outcomes from recent clinical trials, there is still no cure for this disease. Pre-clinical animal models are currently largely limited to small animals which have a number of shortcomings. We have previously shown that fibrosis is induced in isolated sheep lung segments 14 days after bleomycin treatment. This study aimed to determine whether bleomycin-induced fibrosis and associated functional changes persisted over a seven-week period. METHODS: Two separate lung segments in nine sheep received two challenges two weeks apart of either, 3U bleomycin (BLM), or saline (control). Lung function in these segments was assessed by a wedged-bronchoscope procedure after bleomycin treatment. Lung tissue, and an ex vivo CT analysis were used to assess for the persistence of inflammation, fibrosis and collagen content in this model. RESULTS: Fibrotic changes persisted up to seven weeks in bleomycin-treated isolated lung segments (Pathology scores: bleomycin12.27 ± 0.07 vs. saline 4.90 ± 1.18, n = 9, p = 0.0003). Localization of bleomycin-induced injury and increased tissue density was confirmed by CT analysis (mean densitometric CT value: bleomycin -698 ± 2.95 Hounsfield units vs. saline -898 ± 2.5 Hounsfield units, p = 0.02). Masson's trichrome staining revealed increased connective tissue in bleomycin segments, compared to controls (% blue staining/total field area: 8.5 ± 0.8 vs. 2.1 ± 0.2 %, n = 9, p < 0.0001). bleomycin-treated segments were significantly less compliant from baseline at 7 weeks post treatment compared to control-treated segments (2.05 ± 0.88 vs. 4.97 ± 0.79 mL/cmH20, n = 9, p = 0.002). There was also a direct negative correlation between pathology scores and segmental compliance. CONCLUSIONS: We show that there is a correlation between fibrosis and correspondingly poor lung function which persist for up to seven weeks after bleomycin treatment in this large animal model of pulmonary fibrosis.
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ItemThe Effects of Tumstatin on Vascularity, Airway Inflammation and Lung Function in an Experimental Sheep Model of Chronic Asthma(NATURE PUBLISHING GROUP, 2016-05-20)Tumstatin, a protein fragment of the alpha-3 chain of Collagen IV, is known to be significantly reduced in the airways of asthmatics. Further, there is evidence that suggests a link between the relatively low level of tumstatin and the induction of angiogenesis and inflammation in allergic airway disease. Here, we show that the intra-segmental administration of tumstatin can impede the development of vascular remodelling and allergic inflammatory responses that are induced in a segmental challenge model of experimental asthma in sheep. In particular, the administration of tumstatin to lung segments chronically exposed to house dust mite (HDM) resulted in a significant reduction of airway small blood vessels in the diameter range 10(+)-20 μm compared to controls. In tumstatin treated lung segments after HDM challenge, the number of eosinophils was significantly reduced in parenchymal and airway wall tissues, as well as in the bronchoalveolar lavage fluid. The expression of VEGF in airway smooth muscle was also significantly reduced in tumstatin-treated segments compared to control saline-treated segments. Allergic lung function responses were not attenuated by tumstatin administration in this model. The data are consistent with the concept that tumstatin can act to suppress vascular remodelling and inflammation in allergic airway disease.