Machiko Nishihara-Fujihara1, Shunsuke Shoji2, Iori Maeda3, Terufumi Shimoda4, Sankei Nishima4, Kouji Okamoto3. 1. Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology,; Present address: Department of Dermatology, Kurume University School of Medicine, Fukuoka. Electronic address: fujihara_machiko@med.kurume-u.ac.jp. 2. Clinical Research Center, National Hospital Organization Tokyo National Hospital, Tokyo, Japan. 3. Graduate School of Life Science and Systems Engineering, Kyushu Institute of Technology. 4. Clinical Research Center, National Hospital Organization Fukuoka National Hospital.
Abstract
BACKGROUND: Airway remodeling is a repair process occurring after airway injury; its primary histopathological features are subepithelial fibrosis and smooth muscle thickening of the bronchi. These histopathological changes are considered to occur due to bronchial smooth muscle cells (bSMC) that secrete extracellular matrix (ECM) proteins, which work as chemoattractants and influence cell migration. Therefore, we examined the interaction between bSMCs and ECM proteins in vitro for understanding the remodeling process in the bronchi. METHODS: bSMCs were cultured to collect a bSMC-conditioned medium. Using the bSMC-conditioned medium thus obtained, we performed a cell migration assay, characterized beta integrin expression, and identified ECM proteins and matrix metalloproteinases by western blotting and gelatin zymography, respectively. RESULTS: The response of bSMC migration to bSMC-conditioned medium increased with time in culture, and fibronectin (FIB) was detected as a chemoattractant for bSMCs in bSMC-conditioned medium by western blot analysis and a cell migration assay using anti-FIB antibodies. The involvement of beta1 integrin in the migration of bSMCs toward FIB contained in bSMC-conditioned medium was demonstrated by inhibition of cell migration using anti-beta1 integrin antibodies. Expression of beta1 integrin on bSMCs was confirmed by using a beta-integrin-mediated cell adhesion array. In addition, metalloproteinases detected in bSMC-conditioned medium by gelatin zymography were suggested to be matrix metalloproteinase-1 and 2 by western blotting and amino acid sequencing. CONCLUSIONS: Our results suggest that FIB and matrix metalloproteinases secreted from bSMCs might play major roles in bSMC migration in the process of airway remodeling.
BACKGROUND: Airway remodeling is a repair process occurring after airway injury; its primary histopathological features are subepithelial fibrosis and smooth muscle thickening of the bronchi. These histopathological changes are considered to occur due to bronchial smooth muscle cells (bSMC) that secrete extracellular matrix (ECM) proteins, which work as chemoattractants and influence cell migration. Therefore, we examined the interaction between bSMCs and ECM proteins in vitro for understanding the remodeling process in the bronchi. METHODS:bSMCs were cultured to collect a bSMC-conditioned medium. Using the bSMC-conditioned medium thus obtained, we performed a cell migration assay, characterized beta integrin expression, and identified ECM proteins and matrix metalloproteinases by western blotting and gelatin zymography, respectively. RESULTS: The response of bSMC migration to bSMC-conditioned medium increased with time in culture, and fibronectin (FIB) was detected as a chemoattractant for bSMCs in bSMC-conditioned medium by western blot analysis and a cell migration assay using anti-FIB antibodies. The involvement of beta1 integrin in the migration of bSMCs toward FIB contained in bSMC-conditioned medium was demonstrated by inhibition of cell migration using anti-beta1 integrin antibodies. Expression of beta1 integrin on bSMCs was confirmed by using a beta-integrin-mediated cell adhesion array. In addition, metalloproteinases detected in bSMC-conditioned medium by gelatin zymography were suggested to be matrix metalloproteinase-1 and 2 by western blotting and amino acid sequencing. CONCLUSIONS: Our results suggest that FIB and matrix metalloproteinases secreted from bSMCs might play major roles in bSMC migration in the process of airway remodeling.
Authors: C Philippe; Y Boussadia; V Prulière-Escabasse; J F Papon; C Clérici; D Isabey; A Coste; E Escudier; M P d'Ortho Journal: Sleep Breath Date: 2014-07-04 Impact factor: 2.816