title: Evaluation of permeability alteration and epithelial-mesenchymal transition induced by transforming growth factor-β1in A549, NCI-H441, and Calu-3 cells: Development of an in vitro model of respiratory epithelial cells in idiopathic pulmonary fibrosis.
abstract: BACKGROUND
  Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease, which is accompanied by changes in lung structure. With regard to treatment, aerosolized drugs administered intrapulmonarily are rapidly distributed into the plasma and do not remain in the lungs due to damage to the alveolar epithelium that occurs from pulmonary fibrosis. In this study, we sought to develop an in vitro model of respiratory epithelial cells in IPF for the evaluation of the intrapulmonary distribution of aerosolized drugs. We investigated transforming growth factor (TGF)-β1-induced epithelial-mesenchymal transition (EMT) and permeability alteration in A549, NCI-H441, and Calu-3 cell monolayers.
  
  
  METHODS
  After TGF-β1treatment of A549, NCI-H441, and Calu-3 cells, EMT markers including E-cadherin and vimentin and tight junction proteins including claudins-1, -3, and -5 were stained using immunofluorescence methods and detected using immunoblotting methods. Transport experiments were performed using TGF-β1-treated cell monolayers and fluorescein isothiocyanate dextrans (FD; 4.4, 10, and 70kDa). In addition, TGF-β1-induced apoptosis and necrosis were evaluated by flow cytometry using Annexin V and ethidium homodimer III, respectively.
  
  
  RESULTS
  In NCI-H441 cells, incomplete EMT, destruction of claudins-1 and -3, and enhancement of FD permeability were caused by TGF-β1treatment. In A549 cells, complete EMT occurred but was not adequate for transport experiments because of low transepithelial electrical resistance. Whereas in Calu-3 cells, no changes were observed. TGF-β1-induced apoptosis and necrosis were not observed in any of the cell lines.
  
  
  CONCLUSIONS
  Incomplete EMT and permeability enhancement were observed in the alveolar epithelium of IPF. Therefore, our results indicate that TGF-β1-treated NCI-H441 cell monolayers may serve as a useful in vitro model of respiratory epithelial cells for IPF.
authors.affiliation: Division of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8590, Japan. Electronic address: k-togami@hokuyakudai.ac.jp.
authors.forename: Kohei
authors.initials: K
authors.lastname: Togami
authors.affiliation: Division of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8590, Japan.
authors.forename: Kotaro
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Division of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8590, Japan.
authors.forename: Sumio
authors.initials: S
authors.lastname: Chono
authors.affiliation: Division of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8590, Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Tada
mesh_terms: D000072283:A549 Cells; D017209:Apoptosis / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D057167:Claudins / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000473:pathology*; D058750:Epithelial-Mesenchymal Transition / Q000187:drug effects*; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000473:pathology*; D009336:Necrosis / Q000473:pathology; D011650:Pulmonary Alveoli / Q000166:cytology / Q000187:drug effects; D020545:Respiratory Mucosa / Q000187:drug effects* / Q000473:pathology*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D057167:Claudins; D053773:Transforming Growth Factor beta1
keywords: Alveolar epithelial cells; Apoptosis; Claudins; Epithelial–mesenchymal transition; Idiopathic pulmonary fibrosis; Pulmonary permeability; Transforming growth factor-β(1)