Arginine metabolism in experimental and idiopathic pulmonary fibrosis

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 lung architecture 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 proteins that 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 primary fibroblasts 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 oxide and 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-1 suggested 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.Die Idiopathische Lungenfibrose (Idiopathic pulmonary fibrosis – IPF) ist eine progrediente und fatale Lungenerkrankung unbekannten Ursprungs. Sie ist gekennzeichnet durch eine Schädigung der alveolären Epithelzellen, vermehrter Ablagerung von extrazellulärer Matrix im Interstitium der Lunge, verstärkter Proliferation und Aktivierung von Fibroblasten bzw. Myofibroblasten, was schließlich zu einer Zerstörung des normalen Gewebes führt, sowie zum Verlust der Lungenfunktion. Während die Krankheit initial höchstwahrscheinlich durch eine Verletzung es Lungenepithels ausgelöst wird, gelten die Fibroblasten bzw. die Myofibroblasten als Schlüsselzellen, da sie für die charakteristische Ablagerung extrazellulärer Matrix während des Krankheitsverlaufes der IPF verantwortlich sind. Fibroblasten produzieren große Mengen an fibrillärem Kollagen, welches die Hauptkomponenten der Extrazellulären Matrix darstellen, die bei der Lungenfibrose signifikant erhöht vorkommen. L-Arginin ist eine wichtige biologische Vorstufe vieler aktiver Verbindungen, wie zum Beispiel Stickoxid, asymmetrisches Dimethylarginin, und Prolin – einer Aminosäure, die besonders häufig in Kollagen vorkommt. Daher stellten wir die Hypothese auf, dass der L-Arginin-Metabolismus in der Lungenfibrose verändert ist, was schließlich die Kollagensynthese in Fibroblasten beeinflusst. In dieser Studie wurde die Expression der Schlüsselenzyme des L-Arginin-Stoffwechselwegs während der Bleomycin-induzierten Lungenfibrose in Mäusen untersucht. Arginase-1 und Arginase-2 waren während der Bleomycin-induzierten Lungenfibrose signifikant hochreguliert, was mit einer gleichzeitigen Verringerung der L-Arginin-Level in der Lunge, gmessen mit high performance liquid chromatography (HPLC), einherging. Weiterhin war die Arginase-1 und Arginase-2 in den Fibroblasten exprimiert und war in primären Fibroblasten, isoliert aus Bleomycin-behandelten Mäusen im Vergleich zu unbehandelten Kontrollen, erhöht. Interessanterweise induzierte TGF-beta 1, ein profibrotischer Schlüsselmediator, die Arginase-1 mRNA-Expression in primären Fibroblasten und in NIH3T3-Zellen. Schließlich verringerte eine Behandlung von NIH3T3-Fibroblasten sowie humaner Lungenfibroblasten mit dem Arginase-Inhibitor NOHA, die TGF-beta 1-induzierte Kollagenablagerung, jedoch nicht die Signaltransduktion durch Smads. Die mRNA-Expression von Arginase-1 und Arginase-2, sowie deren Aktivitäten zeigten jedoch in Lungenhomogenaten von Fibrosepatienten im Vergleich zu gesunden Kontrolllungen keine Veränderung. Diese Ergebnisse zeigen, dass Isoformen der Arginase, die Schlüsselenzyme im Stickoxid- und im Kollagenstoffwechsel sind, in Lungenfibroblasten exprimiert werden und funktional von Bedeutung sind. Dies konnte im Tiermodell gezeigt werden, während keine Veränderungen der Expression in Lungen von Fibrosepatienten festgestellt wurden. Die TGF-beta 1-induzierte Hochregulierung von Arginase-1 deutet auf eine Wechselwirkung zwischen profibrotischen Molekülen und dem L-Arginin-Metabolismus während des Verlaufs der experimentellen Lungenfibrose hin, welche ein Ansatzpunkt für eine Entwicklung neuer medizinischer Behandlungsmethoden darstellen könnte

Analysis of methylarginine metabolism in the cardiovascular system identifies the lung as a major source of ADMA

Protein arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). Three forms of methylarginine have been identified in eukaryotes: monomethylarginine (l-NMMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA), all characterized by methylation of one or both guanidine nitrogen atoms of arginine. l-NMMA and ADMA, but not SDMA, are competitive inhibitors of all nitric oxide synthase isoforms. SDMA is eliminated almost entirely by renal excretion, whereas l-NMMA and ADMA are further metabolized by dimethylarginine dimethylaminohydrolase (DDAH). To explore the interplay between methylarginine synthesis and degradation in vivo, we determined PRMT expression and DDAH activity in mouse lung, heart, liver, and kidney homogenates. In addition, we employed HPLC-based quantification of protein-incorporated and free methylarginine, combined with immunoblotting for the assessment of tissue-specific patterns of arginine methylation. The salient findings of the present investigation can be summarized as follows: 1) pulmonary expression of type I PRMTs was correlated with enhanced protein arginine methylation; 2) pulmonary ADMA degradation was undertaken by DDAH1; 3) bronchoalveolar lavage fluid and serum exhibited almost identical ADMA/SDMA ratios, and 4) kidney and liver provide complementary routes for clearance and metabolic conversion of circulating ADMA. Together, these observations suggest that methylarginine metabolism by the pulmonary system significantly contributes to circulating ADMA and SDMA levels

Wnt-inducible protein (WISP-1) is a key regulator of alveolar epithelial cell hyperplasia in pulmonary fibrosis

Fibrotic lung disease is characterized by distorted lung architecture and severe loss of respiratory function secondary to alveolar epithelial cell (AEC) hyperplasia, enhanced extracellular matrix (ECM) deposition and fibroblast proliferation. Repetitive epithelial injuries with impaired alveolar wound healing and altered AEC gene expression represent a trigger mechanism for development of fibrosis. To reveal gene regulatory networks in lung fibrosis, we compared gene expression profiles of freshly isolated AEC obtained from mice 14 days after saline or bleomycin (BM) instillation using whole genome microarray analysis. Several genes of the Wnt signaling pathway, in particular WISP-1, a member of the CCN family, were highly regulated. WISP-1 protein expression was demonstrated in proliferating AEC in BM-treated lungs by immunofluorescence. When analyzing all six CCN family members, WISP-1 was upregulated the most 14 days after BM challenge, as analyzed by qRT-PCR. To elucidate WISP-1 function, cultured primary mouse AEC were stimulated with WISP-1 and demonstrated a 230% increase in proliferation, analyzed by 3H-thymidine incorporation. This was mediated through enhanced phosphorylation, but not expression of protein kinase B (PKB/Akt), as detected by immunoblot. Finally, increased expression of WISP-1 was detected in lung homogenates and isolated AEC from IPF patients, using qRT-PCR. Immunohistochemical analysis of WISP-1 and Ki67 verified the existence of hyperplastic and proliferative AEC expressing WISP-1 in vivo. Our study thus identifies WISP-1 as a novel regulator of AEC injury and repair, and suggests that WISP-1 is a key mediator in pulmonary fibrosis