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In the fatal degenerative Duchenne muscular dystrophy (DMD), skeletal muscle is progressively replaced by fibrotic tissue. Here, we show that fibrinogen accumulates in dystrophic muscles of DMD patients and mdx mice. Genetic loss or pharmacological depletion of fibrinogen in these mice reduced fibrosis and dystrophy progression. Our results demonstrate that fibrinogen-Mac-1 receptor binding, through induction of IL-1␤, drives the synthesis of transforming growth factor-␤ (TGF␤) by mdx macrophages, which in turn induces collagen production in mdx fibroblasts. Fibrinogen-produced TGF␤ further amplifies collagen accumulation through activation of profibrotic alternatively activated macrophages. Fibrinogen, by engaging its ␣v␤3 receptor on fibroblasts, also directly promotes collagen synthesis. These data unveil a profibrotic role of fibrinogen deposition in muscle dystrophy.Supplemental material is available at http://www.genesdev.org.Received November 30, 2007; revised version accepted April 28, 2008. Duchenne muscular dystrophy (DMD) results from mutations in the gene coding for the protein dystrophin, which localizes at the inner face of the sarcolemma (Campbell 1995). Besides progressive muscle degeneration and inflammation, fibrotic transition of muscle tissue is critical in DMD as it progressively deteriorates locomotor capacity, posture maintenance, and the vital function of cardiac and respiratory muscles. Indeed, DMD individuals have a high degree of fibrosis increasing with age, which is reproduced in the diaphragm muscle of mdx mice (the mouse model of DMD) (Stedman et al. 1991). Importantly, the underlying mechanisms of fibrosis development within dystrophic muscle remain largely unknown.Fibrinogen is a soluble acute phase protein, which is released into the blood in response to stress. Apart from its key role in controlling blood loss following vascular injury, fibrinogen also extravasates at sites of inflammation or increased vascular permeability where it is immobilized and/or converted to fibrin (Rybarczyk et al. 2003) (from hereon we refer to both by the term "fibrin/ ogen"). We showed previously that mice with defective fibrinolysis exhibited impaired muscle regeneration after experimental injury (Suelves et al. 2002). In this study, we investigated the role of fibrin/ogen deposition in the development of fibrosis in dystrophic muscle. Results and DiscussionWe first analyzed fibrin/ogen deposition in muscles of DMD patients and its correlation with disease course. Compared with muscles of healthy individuals or of fibromyalgia patients, DMD muscles showed significant fibrin/ogen accumulation (Fig. 1A). Similarly, in mdx mice muscles, fibrin/ogen deposits were readily detectable after disease onset, while absent before disease onset (Fig. 1B,C). Thus, fibrin/ogen deposition is associated with muscle dystrophinopathy.Collagen deposition (fibrosis) was prominent in DMD muscles and particularly found in the same areas occupied by fibrin/ogen (Fig. 1D). To investigate the relationship between the e...

Mitochondria generate reactive oxygen species (ROS) as byproducts of molecular oxygen consumption in the electron transport chain. Most cellular oxygen is consumed in the cytochrome-c oxidase complex of the respiratory chain, which does not generate reactive species. The ubiquinone pool of complex III of respiration is the major site within the respiratory chain that generates superoxide anion as a result of a single electron transfer to molecular oxygen. Superoxide anion and hydrogen peroxide, derived from the former by superoxide dismutase, are precursor of hydroxyl radical through the participation of transition metals. Glutathione (GSH) in mitochondria is the only defense available to metabolize hydrogen peroxide. A small fraction of the total cellular GSH pool is sequestered in mitochondria by the action of a carrier that transports GSH from the cytosol to the mitochondrial matrix. Mitochondria are not only one of the main cellular sources of ROS, they also are a key target of ROS. Mitochondria are subcellular targets of cytokines, especially tumor necrosis factor (TNF); depletion of GSH in this organelle renders the cell more susceptible to oxidative stress originating in mitochondria. Ceramide generated during TNF signaling leads to increased production of ROS in mitochondria. Chronic ethanol-fed hepatocytes are selectively depleted of GSH in mitochondria due to a defective operation of the carrier responsible for transport of GSH from the cytosol into the mitochondrial matrix. Under these conditions, limitation of the mitochondrial GSH pool represents a critical contributory factor that sensitizes alcoholic hepatocytes to the prooxidant effects of cytokines and prooxidants generated by oxidative metabolism of ethanol. S-adenosyl-L-methionine prevents development of the ethanol-induced defect. The mitochondrial GSH carrier has been functionally expressed in Xenopus laevis oocytes microinjected with mRNA from rat liver. This critical carrier displays functional characteristics distinct from other plasma membrane GSH carriers, such as its ATP dependency, inhibitor specificity, and the size class of mRNA that encode the corresponding carrier, suggesting that the mitochondrial carrier of GSH is a gene product distinct from the plasma membrane transporters.

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The present study was undertaken to test whether endurance training in patients with COPD, along with enhancement of muscle bioenergetics, decreases muscle redox capacity as a result of recurrent episodes of cell hypoxia induced by high intensity exercise sessions. Seventeen patients with COPD (FEV(1), 38 +/- 4% pred; PaO2), 69 +/- 2.7 mm Hg; PaCO2, 42 +/- 1.7 mm Hg) and five age-matched control subjects (C) were studied pretraining and post-training. Reduced (GSH) and oxidized (GSSG) glutathione, lipid peroxidation, and gamma-glutamyl cysteine synthase heavy subunit chain mRNA expression (gammaGCS-HS mRNA) were measured in the vastus lateralis. Pretraining redox status at rest and after moderate (40% Wpeak) constant-work rate exercise were similar between groups. After training (DeltaWpeak, 27 +/- 7% and 37 +/- 18%, COPD and C, respectively) (p < 0.05 each), GSSG levels increased only in patients with COPD (from 0.7 +/- 0.08 to 1.0 +/- 0.15 nmol/ mg protein, p < 0.05) with maintenance of GSH levels, whereas GSH markedly increased in C (from 4.6 +/- 1.03 to 8.7 +/- 0.41 nmol/ mg protein, p < 0.01). Post-training gammaGCS-HS mRNA levels increased after submaximal exercise in patients with COPD. No evidence of lipid peroxidation was observed. We conclude that although endurance training increased muscle redox potential in healthy subjects, patients with COPD showed a reduced ability to adapt to endurance training reflected in lower capacity to synthesize GSH.

Post-training downregulation of muscle tumour necrosis factor (TNF)-a messenger ribonucleic acid (mRNA) expression and decrease in cellular TNF-a levels have been reported in the elderly. It is hypothesised that chronic obstructive pulmonary disease (COPD) patients may not show these adaptations due to their reduced ability to increase muscle antioxidant capacity with training.Eleven COPD patients (forced expiratory volume in one second 40¡4.4% of the predicted value) and six age-matched controls were studied. Pre-and post-training levels of TNF-a, soluble TNF receptors (sTNFRs: sTNFR55 and sTNFR75) and interleukin (IL)-6 in plasma at rest and during exercise and vastus lateralis TNF-a mRNA were examined.Moderate-intensity constant-work-rate exercise (11 min at 40% of pretraining peak work-rate) increased pretraining plasma TNF-a levels in COPD patients (from 17 ¡ 3.2 to 23¡2.7 pg?mL -1 ; pv0.005) but not in controls (from 19¡4.6 to 19¡3.2 pg?mL -1 ). No changes were observed in sTNFRs or IL-6 levels. After 8 weeks9 endurance training, moderate-intensity exercise increased plasma TNF-a levels similarly to pretraining (from 16¡3 to 21¡4 pg?mL -1 ; pv0.01). Pretraining muscle TNF-a mRNA expression was significantly higher in COPD patients than in controls (29.3¡13.9 versus 5.0¡1.5 TNF-a/18S ribonucleic acid, respectively), but no changes were observed after exercise or training.It is concluded that moderate-intensity exercise abnormally increases plasma tumour necrosis factor-a levels in chronic obstructive pulmonary disease patients without exercise-induced upregulation of the tumour necrosis factor-a gene in skeletal muscle. Eur Respir J 2003; 21: 789-794.

Extracellular proteolysis mediated by the uPA/PAI-1 system determines miR-21 expression in fibroblasts, which affects age-associated fibrogenesis and muscle deterioration in a muscular dystrophy model.