Ichikawa Tomohiro, Sugiura Hisatoshi, Koarai Akira, Yanagisawa Satoru, Kanda Masae, Hayata Atsushi, Furukawa Kanako, Akamatsu Keiichiro, Hirano Tsunahiko, Nakanishi Masanori, Matsunaga Kazuto, Minakata Yoshiaki, Ichinose Masakazu
Third Department of Internal Medicine, Wakayama Medical University, School of Medicine, 811-1 Kimiidera, Wakayama, Japan.
Am J Physiol Lung Cell Mol Physiol. 2008 Nov;295(5):L800-8. doi: 10.1152/ajplung.90264.2008. Epub 2008 Sep 12.
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-kappaB 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-kappaB into the nucleus confirmed by immunocytostaining and immunoblotting. Peroxynitrite-augmented alpha-SMA expression was blocked by NF-kappaB 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-kappaB-TGF-beta(1)-dependent pathway.
哮喘中的不可逆气流受限与气道重塑相关，其中成纤维细胞向肌成纤维细胞的分化起关键作用。在哮喘气道中，已观察到活性氮物质（RNS）的过度产生。本研究的目的是评估RNS之一的过氧亚硝酸盐是否会影响成纤维细胞向肌成纤维细胞的分化。用不同浓度的纯过氧亚硝酸盐或过氧亚硝酸盐供体盐酸3-吗啉代 sydnonimine（SIN-1）处理人胎儿肺成纤维细胞，并评估肌成纤维细胞分化标志物α-平滑肌肌动蛋白（α-SMA）和结蛋白的表达。评估转化生长因子-β1（TGF-β1）和包括纤连蛋白和I型胶原在内的细胞外基质蛋白的释放。为阐明这种分化的机制，评估抗TGF-β抗体或NF-κB抑制剂对α-SMA表达和细胞外基质产生的影响。与对照组相比，过氧亚硝酸盐和SIN-1以浓度依赖性方式显著增强α-SMA表达（分别为P < 0.01和P < 0.05）。过氧亚硝酸盐显著增加结蛋白和TGF-β1的产生（P < 0.01）。免疫细胞化学和免疫印迹证实过氧亚硝酸盐增强了NF-κB向细胞核的转位。过氧亚硝酸盐增强的α-SMA表达被NF-κB抑制剂MG132和咖啡酸苯乙酯（CAPE）以及抗TGF-β抗体阻断。CAPE完全抑制过氧亚硝酸盐增强的TGF-β1释放。过氧亚硝酸盐显著增加纤连蛋白和I型胶原的产生（P < 0.01），并被抗TGF-β抗体抑制。这些结果表明，RNS可通过NF-κB-TGF-β1依赖性途径影响向肌成纤维细胞的分化和细胞外基质的过度产生。
