Melbourne Veterinary School - Research Publications
Permanent URI for this collection
Search Results
1 - 5 of 5
-
ItemProtease-activated receptor-2 dependent and independent responses of bone cells to prostate cancer cell secretory products(WILEY, 2022-05)BACKGROUND: Metastatic prostate cancer lesions in the skeleton are frequently characterized by excessive formation of bone. Prostate cancer cells secrete factors, including serine proteases, that are capable of influencing the behavior of surrounding cells. Some of these proteases activate protease-activated receptor-2 (PAR2 ), which is expressed by osteoblasts (bone-forming cells) and precursors of osteoclasts (bone-resorbing cells). The aim of the current study was to investigate a possible role for PAR2 in regulating the behavior of bone cells exposed to metastatic prostate cancer cells. METHODS: The effect of medium conditioned by the PC3, DU145, and MDA-PCa-2b prostate cancer cell lines was investigated in assays of bone cell function using cells isolated from wildtype and PAR2 -null mice. Osteoclast differentiation was assessed by counting tartrate-resistant acid phosphatase-positive multinucleate cells in bone marrow cultured in osteoclastogenic medium. Osteoblasts were isolated from calvariae of neonatal mice, and BrdU incorporation was used to assess their proliferation. Assays of alkaline phosphatase activity and quantitative PCR analysis of osteoblastic gene expression were used to assess osteoblast differentiation. Responses of osteoblasts to medium conditioned by MDA-PCa-2b cells were analyzed by RNAseq. RESULTS: Conditioned medium (CM) from all three cell lines inhibited osteoclast differentiation independently of PAR2 . Media from PC3 and DU145 cells had no effect on assays of osteoblast function. Medium conditioned by MDA-PCa-2b cells stimulated BrdU incorporation in both wildtype and PAR2 -null osteoblasts but increased alkaline phosphatase activity and Runx2 and Col1a1 expression in wildtype but not PAR2 -null cells. Functional enrichment analysis of RNAseq data identified enrichment of multiple gene ontology terms associated with lysosomal function in both wildtype and PAR2 -null cells in response to MDA-PCa-2b-CM. Analysis of individual genes identified osteogenesis-associated genes that were either upregulated by MDA-PCa-2b-CM selectively in wildtype cells or downregulated selectively in PAR2 -null cells. CONCLUSIONS: Factors secreted by prostate cancer cells influence bone cell behavior through both PAR2 -dependent and -independent mechanisms. Both PAR2 -independent suppression of osteoclast differentiation and PAR2 -dependent stimulation of osteogenesis are likely to determine the nature of prostate cancer metastases in bone.
-
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.
-
ItemK(Ca)3.1 channel blockade attenuates microvascular remodelling in a large animal model of bleomycin-induced pulmonary fibrosis(NATURE PUBLISHING GROUP, 2019-12-27)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.
-
ItemThe vacuolar H+ ATPase V0 subunit d2 is associated with chondrocyte hypertrophy and supports chondrocyte differentiation.(Elsevier BV, 2017-12)Chondrocyte hypertrophy makes important contributions to bone development and growth. We have investigated a number of novel cartilage genes identified in a recent transcriptomic study to determine whether they are differentially expressed between different zones of equine foetal growth cartilage. Twelve genes (ATP6V0D2, BAK1, DDX5, GNB1, PIP4K2A, RAP1B, RPS7, SRSF3, SUB1, TMSB4, TPI1 and WSB2) were found to be more highly expressed in the zone of hypertrophic chondrocytes than in the reserve or proliferative zones, whereas FOXA3 and SERPINA1 were expressed at lower levels in the hypertrophic zone than in the reserve zone. ATP6V0D2, which encodes vacuolar H+ ATPase (V-ATPase) V0 subunit d2 (ATP6V0D2), was selected for further study. Immunohistochemical analysis of ATP6V0D2 in growth cartilage showed stronger staining in hypertrophic than in reserve zone or proliferative chondrocytes. Expression of ATP6V0D2 mRNA and protein was up-regulated in the mouse chondrocytic ATDC5 cell line by conditions inducing expression of hypertrophy-associated genes including Col10a1 and Mmp13 (differentiation medium). In ATDC5 cells cultured in control medium, knockdown of Atp6v0d2 or inhibition of V-ATPase activity using bafilomycin A1 caused a decrease in Col2a1 expression, and in cells cultured in differentiation medium the two treatments caused a decrease in nuclear area. Inhibition of V-ATPase, but not Atp6v0d2 knockdown, prevented the upregulation of Col10a1, Mmp13 and Vegf by differentiation medium, while Atp6v0d2 knockdown, but not inhibition of V-ATPase, caused an increase in the number of ATDC5 cells cultured in differentiation medium. These observations identify ATP6V0D2 as a novel chondrocyte hypertrophy-associated gene. The results are consistent with roles for V-ATPase, both ATP6V0D2-dependent and -independent, in supporting chondrocyte differentiation and hypertrophy.
-
ItemNormal inflammation and regeneration of muscle following injury require osteopontin from both muscle and non-muscle cells(BMC, 2019-02-26)BACKGROUND: Osteopontin is secreted by skeletal muscle myoblasts and macrophages, and its expression is upregulated in muscle following injury. Osteopontin is present in many different structural forms, which vary in their expression patterns and effects on cells. Using a whole muscle autograft model of muscle injury in mice, we have previously shown that inflammation and regeneration of muscle following injury are delayed by the absence of osteopontin. The current study was undertaken to determine whether muscle or non-muscle cells provide the source of osteopontin required for its role in muscle regeneration. METHODS: The extensor digitorum longus muscle of wild-type and osteopontin-null mice was removed and returned to its bed in the same animal (autograft) or placed in the corresponding location in an animal of the opposite genotype (allograft). Grafts were harvested at various times after surgery and analysed by histology, flow cytometry and quantitative polymerase chain reaction. Data were analysed using one- or two-way ANOVA or Kruskal-Wallis test. RESULTS: Immunohistochemistry confirmed that osteopontin was expressed by macrophages in osteopontin-null muscle allografts in wild-type hosts and by myoblasts in wild-type allografts in osteopontin-null hosts. The decrease in muscle fibre number observed in wild-type autografts following grafting and the subsequent appearance of regenerating fibres were delayed in both types of allografts to a similar extent as in osteopontin-null autografts. Infiltration of neutrophils, macrophages and M1 and M2 macrophage subtypes were also delayed by lack of osteopontin in the muscle and/or host. While the proportion of macrophages showing the M1 phenotype was not affected, the proportion showing the M2 phenotype was decreased by osteopontin deficiency. Expression of tumour necrosis factor-α and interleukin-4 was lower in osteopontin-null than in wild-type autografts, and there was no difference between the osteopontin-null graft types. CONCLUSIONS: Osteopontins from muscle and non-muscle sources are equally important in the acute response of muscle to injury, and cannot substitute for each other, suggesting that they play distinct roles in regulation of cell behaviour. Future studies of mechanisms of osteopontin's roles in acute muscle inflammation and regeneration will need to investigate responses to osteopontins derived from both myoblasts and macrophages.