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Ontogeny of Osteopontin Expression in a Non-BRICHOS Mutant Surfactant Protein C Mouse Model of Idiopathic Pulmonary Fibrosis
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A5758 - Ontogeny of Osteopontin Expression in a Non-BRICHOS Mutant Surfactant Protein C Mouse Model of Idiopathic Pulmonary Fibrosis
Author Block: S. Nureki1, Y. Tomer2, A. Venosa2, S. J. Russo2, J. Katzen2, S. Mulugeta2, J. Kadota1, M. F. Beers2; 1Department of Respiratory Medicine and Infectious Diseases, Oita University Faculty of Medicine, Yufu, Oita, Japan, 2Department of Medicine Division of Pulmonary and Critical Care Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States.
Rationale: A missense isoleucine to threonine substitution at position 73 (I73T) within the Non-BRICHOS linker domain is the most common SP-C gene [SFTPC] mutation linked to sporadic and familial forms of idiopathic pulmonary fibrosis (IPF). Osteopontin, a multifunctional cytokine, has been shown to possess both pro-inflammatory (including cell migration) and pro-fibrotic properties through effects on fibroblast migration and proliferation and has also been implicated as potential IPF biomarker. The goal of this study was to investigate osteopontin expression in a preclinical mouse model of spontaneous lung fibrosis.
Approach: A mouse line with tamoxifen inducible allelic expression of a mutant SP-CI73T sequence (IER-SP-CI73T) was generated by crossing a hypomorphic SP-CI73T founder line (wherein sftpcI73T knock-in alleles retained an intronic PGK-neomycin cassette) to a R26FlpOER deleter line for tamoxifen-mediated removal of PGK-neomycin cassette. Osteopontin mRNA and protein expression were assessed by quantitative RT-PCR (qRT-PCR), Western blotting, and immunohistochemistry in the lungs of IER-SP-CI73T and control mice.
Results: Intraperitoneal tamoxifen (iTAM) treatment of IER-SP-CI73T mice rapidly up-regulated sftpcI73T mRNA and proSP-CI73T protein (3-7 days) with subsequent rapid development of increased weight loss, enhanced mortality from progressive diffuse alveolar damage, and a substantial polycellular alveolitis (1-2 weeks). By 4-6 weeks post-iTAM, surviving IER-SP-CI73T mice exhibited a lung pathology containing fibroblastic foci and subpleural and septal collagen deposition. Western blotting detected significantly increased and sustained expression of osteopontin in bronchoalveolar lavage fluid beginning at 1 week post-iTAM treatment and continuing through the observed development of restrictive lung fibrosis in IER-SP-CI73T mice. qRT-PCR of whole lung mRNA from IER-SP-CI73T mice revealed increased levels of osteopontin beginning at 3-7 days post-iTAM. Immunohistochemistry localized osteopontin expression to alveolar type 2 (AT2) cells and macrophages in the lungs of IER-SP-CI73T mice. qRT-PCR of purified IER-SP-CI73T AT2 cells showed a 4-fold induction of osteopontin mRNA at 2 weeks iTAM treatment.
Conclusions: In a preclinical mouse model of spontaneous pulmonary fibrosis, osteopontin, the putative IPF biomarker in humans, is produced by both AT2 cells and other lung populations and emerges as a potential mediator of both acute IPF exacerbations and subsequent fibrotic remodeling.
Author Block: S. Nureki1, Y. Tomer2, A. Venosa2, S. J. Russo2, J. Katzen2, S. Mulugeta2, J. Kadota1, M. F. Beers2; 1Department of Respiratory Medicine and Infectious Diseases, Oita University Faculty of Medicine, Yufu, Oita, Japan, 2Department of Medicine Division of Pulmonary and Critical Care Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, United States.
Rationale: A missense isoleucine to threonine substitution at position 73 (I73T) within the Non-BRICHOS linker domain is the most common SP-C gene [SFTPC] mutation linked to sporadic and familial forms of idiopathic pulmonary fibrosis (IPF). Osteopontin, a multifunctional cytokine, has been shown to possess both pro-inflammatory (including cell migration) and pro-fibrotic properties through effects on fibroblast migration and proliferation and has also been implicated as potential IPF biomarker. The goal of this study was to investigate osteopontin expression in a preclinical mouse model of spontaneous lung fibrosis.
Approach: A mouse line with tamoxifen inducible allelic expression of a mutant SP-CI73T sequence (IER-SP-CI73T) was generated by crossing a hypomorphic SP-CI73T founder line (wherein sftpcI73T knock-in alleles retained an intronic PGK-neomycin cassette) to a R26FlpOER deleter line for tamoxifen-mediated removal of PGK-neomycin cassette. Osteopontin mRNA and protein expression were assessed by quantitative RT-PCR (qRT-PCR), Western blotting, and immunohistochemistry in the lungs of IER-SP-CI73T and control mice.
Results: Intraperitoneal tamoxifen (iTAM) treatment of IER-SP-CI73T mice rapidly up-regulated sftpcI73T mRNA and proSP-CI73T protein (3-7 days) with subsequent rapid development of increased weight loss, enhanced mortality from progressive diffuse alveolar damage, and a substantial polycellular alveolitis (1-2 weeks). By 4-6 weeks post-iTAM, surviving IER-SP-CI73T mice exhibited a lung pathology containing fibroblastic foci and subpleural and septal collagen deposition. Western blotting detected significantly increased and sustained expression of osteopontin in bronchoalveolar lavage fluid beginning at 1 week post-iTAM treatment and continuing through the observed development of restrictive lung fibrosis in IER-SP-CI73T mice. qRT-PCR of whole lung mRNA from IER-SP-CI73T mice revealed increased levels of osteopontin beginning at 3-7 days post-iTAM. Immunohistochemistry localized osteopontin expression to alveolar type 2 (AT2) cells and macrophages in the lungs of IER-SP-CI73T mice. qRT-PCR of purified IER-SP-CI73T AT2 cells showed a 4-fold induction of osteopontin mRNA at 2 weeks iTAM treatment.
Conclusions: In a preclinical mouse model of spontaneous pulmonary fibrosis, osteopontin, the putative IPF biomarker in humans, is produced by both AT2 cells and other lung populations and emerges as a potential mediator of both acute IPF exacerbations and subsequent fibrotic remodeling.
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