Peroxynitrite augments fibroblast-mediated tissue remodeling via myofibroblast differentiation

Ichikawa T, Sugiura H, Koarai A, Yanagisawa S, Kanda M, Hayata A, Furukawa K, Akamatsu K, Hirano T, Nakanishi M, Matsunaga K, Minakata Y, Ichinose M. Peroxynitrite augments fibroblastmediated tissue remodeling via myofibroblast differentiation. Am J Physiol Lung Cell Mol Physiol 295: L800-L808, 2008. First published September 12, 2008; doi:10.1152/ajplung.90264.2008.- Irreversible airflow limitation in asthma is associated with airway remodeling in which the differentiation of fibroblasts to myofibroblasts plays a pivotal role. In asthmatic airways, excessive production of reactive nitrogen species (RNS) has been observed. The aim of this study is to evaluate whether peroxynitrite, one of the RNS, can affect the differentiation of fibroblasts to myofibroblasts. Human fetal lung fibroblasts were treated with various concentrations of authentic peroxynitrite or a peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1), and the expressions of alpha-smooth muscle actin (alpha-SMA) and desmin, markers of myofibroblast differentiation, were evaluated. The releases of transforming growth factor-beta 1 (TGF-beta 1) and ECM proteins including fibronectin and collagen I were assessed. To clarify the mechanism in this differentiation, the effect of anti-TGF-beta antibody or NF-kappa B inhibitors on the alpha-SMA expression and ECM production was assessed. Peroxynitrite and SIN-1 significantly augmented the alpha-SMA expression compared with control in a concentration-dependent manner (P < 0.01 and P < 0.05, respectively). Peroxynitrite significantly increased desmin and TGF-beta 1 production (P < 0.01). Peroxynitrite enhanced the translocation of NF-kappa B into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappa B inhibitors, MG132 and caffeic acid phenethyl ester (CAPE), and anti-TGF-beta antibody. CAPE completely inhibited the peroxynitrite-augmented TGF-beta 1 release. The production of fibronectin and collagen I was significantly increased by peroxynitrite (P < 0.01) and inhibited by anti-TGF-beta antibody. These results suggest that RNS can affect the differentiation to myofibroblasts and excessive ECM production via a NF-kappa B-TGF-beta 1-dependent pathway.