title: Altered surfactant homeostasis and alveolar epithelial cell stress in amiodarone-induced lung fibrosis.
abstract: Amiodarone (AD) is a highly efficient antiarrhythmic drug with potentially serious side effects. Severe pulmonary toxicity is reported in patients receiving AD even at low doses and may cause interstitial pneumonia as well as lung fibrosis. Apoptosis of alveolar epithelial type II cells (AECII) has been suggested to play an important role in this disease. In the current study, we aimed to establish a murine model of AD-induced lung fibrosis and analyze surfactant homeostasis, lysosomal, and endoplasmic reticulum (ER) stress in this model. AD/vehicle was instilled intratracheally into C57BL/6 mice, which were sacrificed on days 7, 14, 21, and 28. Extent of lung fibrosis development was assessed by trichrome staining and hydroxyproline measurement. Cytotoxicity was assessed by lactate dehydrogenase assay. Phospholipids (PLs) were analyzed by mass spectrometry. Surfactant proteins (SP) and markers for apoptosis, lysosomal, and ER stress were studied by Western blotting and immunohistochemistry. AECII morphology was evaluated by electron microscopy. Extensive lung fibrosis and AECII hyperplasia were observed in AD-treated mice already at day 7. Surfactant PL and SP accumulated in AECII over time. In parallel, induction of apoptosis, lysosomal, and ER stress was encountered in AECII of mice lungs and in MLE12 cells treated with AD. In vitro, siRNA-mediated knockdown of cathepsin D did not alter the AD-induced apoptotic response. Our data suggest that mice exposed to intratracheal AD develop severe pulmonary fibrosis, exhibit extensive surfactant alterations and cellular stress, but AD-induced AECII apoptosis is not mediated primarily via cathepsin D.
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.
authors.forename: Poornima
authors.initials: P
authors.lastname: Mahavadi
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.
authors.forename: Ingrid
authors.initials: I
authors.lastname: Henneke
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.
authors.forename: Clemens
authors.initials: C
authors.lastname: Ruppert
authors.affiliation: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany REBIRTH Cluster of Excellence, Hannover, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Knudsen
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.
authors.forename: Shalini
authors.initials: S
authors.lastname: Venkatesan
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany.
authors.forename: Gerhard
authors.initials: G
authors.lastname: Liebisch
authors.affiliation: Centre for Inflammation and Tissue Repair, UCL Respiratory, University College London, London, UK Member of the European IPF Network.
authors.forename: Rachel C
authors.initials: RC
authors.lastname: Chambers
authors.affiliation: Institute of Functional and Applied Anatomy, Hannover Medical School, Hannover, Germany Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), Member of the German Center for Lung Research (DZL), Hannover, Germany REBIRTH Cluster of Excellence, Hannover, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Ochs
authors.affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University of Regensburg, Regensburg, Germany Member of the European IPF Network.
authors.forename: Gerd
authors.initials: G
authors.lastname: Schmitz
authors.affiliation: Member of the European IPF Network Department of Clinical and Molecular Biomedicine, University of Catania, Catania 95123, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Vancheri
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany Member of the European IPF Network.
authors.forename: Werner
authors.initials: W
authors.lastname: Seeger
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Korfei
authors.affiliation: Department of Internal Medicine, Justus-Liebig-University Giessen, Germany Universities of Giessen and Marburg Lung Center (UGMLC), Member of the German Center for Lung Research (DZL), Giessen, Germany Member of the European IPF Network Lung Clinic Waldhof-Elgershausen, Greifenstein, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Guenther
mesh_terms: D000638:Amiodarone / Q000633:toxicity*; D000818:Animals; D000889:Anti-Arrhythmia Agents / Q000633:toxicity*; D017209:Apoptosis / Q000187:drug effects; D002402:Cathepsin D / Q000235:genetics; D018929:Cell Culture Techniques; D002460:Cell Line; D004195:Disease Models, Animal; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D006706:Homeostasis / Q000187:drug effects*; D007322:Instillation, Drug; D008810:Mice, Inbred C57BL; D046529:Microscopy, Electron, Transmission; D011650:Pulmonary Alveoli / Q000187:drug effects* / Q000378:metabolism / Q000648:ultrastructure; D011658:Pulmonary Fibrosis / Q000139:chemically induced* / Q000378:metabolism / Q000473:pathology; D011663:Pulmonary Surfactants / Q000378:metabolism*
chemical_list: D000889:Anti-Arrhythmia Agents; D011663:Pulmonary Surfactants; D002402:Cathepsin D; D000638:Amiodarone
keywords: ER stress; alveolar epithelial cell apoptosis; amiodarone; lung fibrosis; lysosomal stress; surfactant