Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal lung disease of unknown origin, characterized by alveolar epithelial cell damage, increased deposition of extracellular matrix (ECM) in the lung interstitium, enhanced fibroblast/myofibroblast proliferation and activation, which ultimately lead to distortion of normal lungarchitecture and loss of respiratory function. While the initial trigger of this disease is most likely an epithelial injury, the interstitial fibroblast/myofibroblast represents the key effector cell responsible for the increased ECM deposition characteristic of IPF. Fibroblasts secrete large amounts of fibrillar collagens, which are the key ECM proteinsthat are significantly increased in this disease. L-arginine is a precursor of many active compounds including: nitric oxide, asymmetrical dimethylarginine, and praline, an amino acid that is enriched in collagen. Thus, it was hypothesized that L-arginine metabolism is altered in pulmonary fibrosis, ultimately affecting collagen synthesis. In this study, the expression of key enzymes of the L-arginine pathway was characterized in bleomycin-induced pulmonary fibrosis in mice. Expression of arginase-1 and arginase-2 was significantly upregulated during bleomycin-induced lung fibrosis, which correlated with a decrease in lung L-arginine levels, as measured by high performance liquid chromatography. Furthermore, arginase-1 and arginase-2 mRNA and protein expression localized to fibroblasts, and their expression was increased in primaryfibroblasts isolated from bleomycin-treated mice, compared to controls, as assessed by semi-quantitative and quantitative RT-PCR, and immunoblotting. Moreover, TGF-beta 1, a key profibrotic mediator, induced arginase-1 mRNA expression in primary and NIH-3T3 fibroblasts. Finally, treatment of NIH-3T3 fibroblasts and primary human lung fibroblasts with the arginase inhibitor, NOHA, attenuated TGF-beta 1-stimulated collagen deposition, but not Smad signaling. Arginase-1 and arginase-2 mRNA expression, as well as their activity, however, was unchanged in total human lung homogenates from IPF patients compared to controls. These results demonstrated that arginase isoforms, key enzymes in nitric oxideand collagen metabolism, were expressed and functional in lung fibroblasts and upregulated in the early stages of bleomycin-induced pulmonary fibrosis. These changes were limited to the animal model of pulmonary fibrosis, as no changes in expression were observed in lungs from IPF patients. The TGF-beta 1-induced upregulation of arginase-1suggested an interplay between profibrotic agents and L-arginine metabolism during the course of experimental lung fibrosis, therapeutic manipulation of which may foster novel treatment options.
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