Kohei Togami1, Kotaro Yamaguchi2, Sumio Chono2, Hitoshi Tada2. 1. 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. 2. Division of Pharmaceutics, Hokkaido Pharmaceutical University School of Pharmacy, 7-Jo 15-4-1 Maeda, Teine, Sapporo, Hokkaido 006-8590, Japan.
Abstract
INTRODUCTION: 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-β1 treatment 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-β1 treatment. 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. DISCUSSION: 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.
INTRODUCTION: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-β1 treatment 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-β1 treatment. 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. DISCUSSION: 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: Thijs Pasman; Danielle Baptista; Sander van Riet; Roman K Truckenmüller; Pieter S Hiemstra; Robbert J Rottier; Naomi M Hamelmann; Jos M J Paulusse; Dimitrios Stamatialis; André A Poot Journal: Membranes (Basel) Date: 2021-03-11
Authors: Carolin Schilpp; Robin Lochbaum; Peter Braubach; Danny Jonigk; Manfred Frick; Paul Dietl; Oliver H Wittekindt Journal: Pflugers Arch Date: 2021-01-02 Impact factor: 3.657