title: C-type natriuretic peptide ameliorates pulmonary fibrosis by acting on lung fibroblasts in mice.
abstract: BACKGROUND
  Pulmonary fibrosis has high rates of mortality and morbidity; however, no effective pharmacological therapy has been established. C-type natriuretic peptide (CNP), a member of the natriuretic peptide family, selectively binds to the transmembrane guanylyl cyclase (GC)-B receptor and exerts anti-inflammatory and anti-fibrotic effects in various organs through vascular endothelial cells and fibroblasts that have a cell-surface GC-B receptor. Given the pathophysiological importance of fibroblast activation in pulmonary fibrosis, we hypothesized that the anti-fibrotic and anti-inflammatory effects of exogenous CNP against bleomycin (BLM)-induced pulmonary fibrosis were exerted in part by the effect of CNP on pulmonary fibroblasts.
  
  
  METHODS
  C57BL/6 mice were divided into two groups, CNP-treated (2.5 μg/kg/min) and vehicle, to evaluate BLM-induced (1 mg/kg) pulmonary fibrosis and inflammation. A periostin-CNP transgenic mouse model exhibiting CNP overexpression in fibroblasts was generated and examined for the anti-inflammatory and anti-fibrotic effects of CNP via fibroblasts in vivo. Additionally, we assessed CNP attenuation of TGF-β-induced differentiation into myofibroblasts by using immortalized human lung fibroblasts stably expressing GC-B receptors. Furthermore, to investigate whether CNP acts on human lung fibroblasts in a clinical setting, we obtained primary-cultured fibroblasts from surgically resected lungs of patients with lung cancer and analyzed levels of GC-B mRNA transcription.
  
  
  RESULTS
  CNP reduced mRNA levels of the profibrotic cytokines interleukin (IL)-1β and IL-6, as well as collagen deposition and the fibrotic area in lungs of mice with bleomycin-induced pulmonary fibrosis. Furthermore, similar CNP effects were observed in transgenic mice exhibiting fibroblast-specific CNP overexpression. In cultured-lung fibroblasts, CNP treatment attenuated TGF-β-induced phosphorylation of Smad2 and increased mRNA and protein expression of α-smooth muscle actin and SM22α, indicating that CNP suppresses fibroblast differentiation into myofibroblasts. Furthermore, human lung fibroblasts from patients with or without interstitial lung disease substantially expressed GC-B receptor mRNA.
  
  
  CONCLUSIONS
  These data suggest that CNP ameliorates bleomycin-induced pulmonary fibrosis by suppressing TGF-β signaling and myofibroblastic differentiation in lung fibroblasts. Therefore, we propose consideration of CNP for clinical application to pulmonary fibrosis treatment.
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. kimura@thoracic.med.osaka-u.ac.jp.|Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan. kimura@thoracic.med.osaka-u.ac.jp.
authors.forename: Toru
authors.initials: T
authors.lastname: Kimura
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. nojiri@ri.ncvc.go.jp.|Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan. nojiri@ri.ncvc.go.jp.
authors.forename: Takashi
authors.initials: T
authors.lastname: Nojiri
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. jhino@ri.ncvc.go.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Hino
authors.affiliation: Department of Regenerative Medicine and Tissue Engineering, National Cerebral and Cardiovascular Center, Suita-City, Osaka, Japan. hosoda.hiroshi.ri@ncvc.go.jp.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Hosoda
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. miurako@ncvc.go.jp.
authors.forename: Koichi
authors.initials: K
authors.lastname: Miura
authors.affiliation: Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan. yshintani@thoracic.med.osaka-u.ac.jp.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shintani
authors.affiliation: Department of General Thoracic Surgery, Kyoto Prefectural University of Medicine, Kyoto-City, Kyoto, Japan. mainoue@koto.kpu-m.ac.jp.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Inoue
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. zenitani@pedsurg.med.osaka-u.ac.jp.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Zenitani
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. takabatake-osk@umin.net.|Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan. takabatake-osk@umin.net.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takabatake
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. miyazato@ncvc.go.jp.
authors.forename: Mikiya
authors.initials: M
authors.lastname: Miyazato
authors.affiliation: Department of General Thoracic Surgery, Osaka University Graduate School of Medicine, Suita-City, Osaka, Japan. meinosin@thoracic.med.osaka-u.ac.jp.
authors.forename: Meinoshin
authors.initials: M
authors.lastname: Okumura
authors.affiliation: Department of Biochemistry, National Cerebral and Cardiovascular Center Research Institute, 5-7-1, Fujishirodai, Suita-city, Osaka, 565-8565, Japan. kangawa@ri.ncvc.go.jp.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kangawa
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000276:immunology*; D005347:Fibroblasts / Q000187:drug effects / Q000276:immunology* / Q000473:pathology*; D008168:Lung / Q000187:drug effects / Q000276:immunology / Q000473:pathology; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D020098:Natriuretic Peptide, C-Type / Q000008:administration & dosage* / Q000494:pharmacology; D011658:Pulmonary Fibrosis / Q000188:drug therapy* / Q000276:immunology* / Q000473:pathology; D016896:Treatment Outcome
chemical_list: D016207:Cytokines; D020098:Natriuretic Peptide, C-Type