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Exploring the Role of Fibrocyte Derived Exosomes in the Pathogenesis of Lung Fibrosis
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A5796 - Exploring the Role of Fibrocyte Derived Exosomes in the Pathogenesis of Lung Fibrosis
Author Block: S. Chong1, P. Nadarajan2, L. Mthunzi3, M. O'Callaghan4, A. Fabre5, P. McLoughlin6, M. P. Keane2; 1Department of Respiratory, St. Vincent's University Hospital, School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland, 2Department of Respiratory, St. Vincent's University Hospital, School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland, 3School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland, 4Department of Respiratory, St. Vincent's University Hospital, School of Medicine, University College Dublin, Dublin 4, Ireland, 5Department of Histopathology, St. Vincent's University Hospital, School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland, 6School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
Background
Idiopathic pulmonary fibrosis (IPF) is the most common and severe of the idiopathic interstitial pneumonia (IIPs) characterised by inflammation and fibrosis..
Fibrocytes are cells that circulate in the peripheral blood and produce connective tissue proteins such as vimentin and collagens I and III, and have been shown to play a role in the pathogenesis of lung fibrosis
Exosomes are extracellular vesicles that are released when the cells are activated or undergo apoptosis under inflammatory conditions, and have been identified in other organs/processes such as renal fibrosis.
We investigated the role of exosomes from fibrocytes in lung fibrosis and their modulation by TGF beta, a potent pro-fibrotic factor.
Methods
We isolated human fibrocytes from blood buffy coat, and confirm their nature using Western Blot and immunofluorescence.
We then cultured the isolated fibrocytes for 72 hours with 10ng/ml of Transforming Growth Factor (TGF-β1) to assess for exosomes secretion.
Extracellular vesicles were extracted using ultracentrifugation. We validated the presence of fibrocytes-derived exosomes using Western Blot and Nanoparticle Tracking Analysis (Nanosight).
Subsequently, we assessed the potential role of exosomes in fibrogenesis by treating A549 cells and human lung fibroblasts with fibrocyte- derived exosomes.
Results
Human fibrocytes are spindle shaped cells and express collagen, CXCR4 and alpha smooth muscle actin. They secrete exosomes into their culture supernatant. This was validated by the expression of heat shock protein 70 (HSP70), CD 63 and the absence of calnexin.
The exosomes are of the size between 30-120nm by Nanosight anaylsis.
TGF-β1 increases the amount of exosomes secreted by fibrocytes.
Treatment of the A549 cells with fibrocyte-derived exosomes induces epithelial to mesenchymal transition (EMT).
Conclusion
Our results demonstrate that fibrocytes secrete exosomes which can induce EMT in A549 epithelial cells in vitro, a hallmark in the pathogenesis of IPF. Our findings suggest that manipulation of exosome secretion could offer potential therapeutic targets for IPF.
Author Block: S. Chong1, P. Nadarajan2, L. Mthunzi3, M. O'Callaghan4, A. Fabre5, P. McLoughlin6, M. P. Keane2; 1Department of Respiratory, St. Vincent's University Hospital, School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland, 2Department of Respiratory, St. Vincent's University Hospital, School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland, 3School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland, 4Department of Respiratory, St. Vincent's University Hospital, School of Medicine, University College Dublin, Dublin 4, Ireland, 5Department of Histopathology, St. Vincent's University Hospital, School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin 4, Ireland, 6School of Medicine and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland.
Background
Idiopathic pulmonary fibrosis (IPF) is the most common and severe of the idiopathic interstitial pneumonia (IIPs) characterised by inflammation and fibrosis..
Fibrocytes are cells that circulate in the peripheral blood and produce connective tissue proteins such as vimentin and collagens I and III, and have been shown to play a role in the pathogenesis of lung fibrosis
Exosomes are extracellular vesicles that are released when the cells are activated or undergo apoptosis under inflammatory conditions, and have been identified in other organs/processes such as renal fibrosis.
We investigated the role of exosomes from fibrocytes in lung fibrosis and their modulation by TGF beta, a potent pro-fibrotic factor.
Methods
We isolated human fibrocytes from blood buffy coat, and confirm their nature using Western Blot and immunofluorescence.
We then cultured the isolated fibrocytes for 72 hours with 10ng/ml of Transforming Growth Factor (TGF-β1) to assess for exosomes secretion.
Extracellular vesicles were extracted using ultracentrifugation. We validated the presence of fibrocytes-derived exosomes using Western Blot and Nanoparticle Tracking Analysis (Nanosight).
Subsequently, we assessed the potential role of exosomes in fibrogenesis by treating A549 cells and human lung fibroblasts with fibrocyte- derived exosomes.
Results
Human fibrocytes are spindle shaped cells and express collagen, CXCR4 and alpha smooth muscle actin. They secrete exosomes into their culture supernatant. This was validated by the expression of heat shock protein 70 (HSP70), CD 63 and the absence of calnexin.
The exosomes are of the size between 30-120nm by Nanosight anaylsis.
TGF-β1 increases the amount of exosomes secreted by fibrocytes.
Treatment of the A549 cells with fibrocyte-derived exosomes induces epithelial to mesenchymal transition (EMT).
Conclusion
Our results demonstrate that fibrocytes secrete exosomes which can induce EMT in A549 epithelial cells in vitro, a hallmark in the pathogenesis of IPF. Our findings suggest that manipulation of exosome secretion could offer potential therapeutic targets for IPF.
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