Antonio Serrano On Twitter: 8 Best Backup Software For Mac

Abstract In Duchenne muscular dystrophy (DMD), a persistently altered and reorganizing extracellular matrix (ECM) within inflamed muscle promotes damage and dysfunction. However, the molecular determinants of the ECM that mediate inflammatory changes and faulty tissue reorganization remain poorly defined. Here, we show that fibrin deposition is a conspicuous consequence of muscle-vascular damage in dystrophic muscles of DMD patients and mdx mice and that elimination of fibrin(ogen) attenuated dystrophy progression in mdx mice. These benefits appear to be tied to: (i) a decrease in leukocyte integrin α Mβ 2-mediated proinflammatory programs, thereby attenuating counterproductive inflammation and muscle degeneration; and (ii) a release of satellite cells from persistent inhibitory signals, thereby promoting regeneration. Remarkably, Fib-gamma(390-396A) (Fibγ 390-396A) mice expressing a mutant form of fibrinogen with normal clotting function, but lacking the α Mβ 2 binding motif, ameliorated dystrophic pathology. Delivery of a fibrinogen/α Mβ 2 blocking peptide was similarly beneficial. Conversely, intramuscular fibrinogen delivery sufficed to induce inflammation and degeneration in fibrinogen-null mice.

Thus, local fibrin(ogen) deposition drives dystrophic muscle inflammation and dysfunction, and disruption of fibrin(ogen)-α Mβ 2 interactions may provide a novel strategy for DMD treatment. INTRODUCTION Duchenne muscular dystrophy (DMD) is one of the most common X-linked lethal diseases, affecting 1 in 3500 newborn males. DMD results from mutations in the gene coding for the protein dystrophin, a cytoskeletal protein localized at the interface of the actin-based contractile apparatus and the sarcolemma.

In the absence of a functional dystrophin complex tethering the actin cytoskeleton inside the muscle cell to the extracellular matrix (ECM), forces generated by the muscle fiber contraction result in muscle fiber damage due to shearing of the sarcolemma (reviewed in ). The mdx mouse strain, which carries a naturally occurring nonsense mutation in exon 2 resulting in loss of dystrophin protein production, is the most widely used animal model for DMD (,). DMD patients and mdx mice exhibit progressive muscle degeneration, which is exacerbated by persistent inflammation via the production of free radicals and cytotoxic cytokines. Myofiber loss is initially compensated by proliferation and fusion of resident myogenic precursor cells (satellite cells) with pre-existing myofibers that thereby enlarge in size. Ultimately, however, after repetitive cycles of muscle degeneration and persistent inflammation, dystrophic myofibers become gradually replaced by fibrotic and fat tissue (; reviewed in,). Therapies based on restoration of dystrophin expression or the administration of dystrophin-positive stem cells are promising but still in the preclinical phase. Mounting evidence indicates a critical involvement of muscle extrinsic factors in DMD disease progression and the recovery of injured muscles.

The composition of the basal lamina ECM around the necrotic myofibers can influence the overall repair process. Indeed, immediately after injury, a provisional fibrin-rich matrix and/or fibrin-rich hematoma forms between damaged fibers that provide a scaffold for tissue reorganization/reparative processes resulting in newly formed connective tissue altering contractile function, and a supportive matrix controlling the activity of infiltrating inflammatory cells (especially macrophages). While infiltrating inflammatory cells undoubtedly play a generally positive role in normal repair (e.g.

By clearing myofiber debris), exuberant and persistent inflammatory cell action is likely to drive inopportune tissue reorganization in dystrophic muscle. These cells express several cytokines, growth factors and other soluble mediators e.g. Tumor necrosis factor alpha (TNFα), transforming growth factor beta, vascular endothelial growth factor that modulate the extent of myofiber degeneration as well as satellite cell-mediated regeneration. Thus, persistent and/or inappropriate ECM deposition around the myofiber is potentially pathogenic, and may promote inflammation in the damaged muscle tissue leading to inopportune tissue reorganization and loss of function. However, the specific matrix components and mechanisms that drive pathological inflammatory cell infiltration in the context of either muscular dystrophies or muscle damage remain largely unexplored. The local conversion of soluble fibrinogen to a provisional fibrin matrix plays a seminal role in controlling blood loss following vascular injury and is understood to support reparative tissue reorganization (,). However, in addition to being a classic acute-phase reactant, fibrin appears to be a potent regulator of the innate immune system by serving as a matrix-associated regulator of inflammatory cell function.

In macrophages, immobilized fibrin and fibrinogen (from here on we refer to both by the term ‘fibrin(ogen)’) induce activation of c-Jun N-terminal kinase and NF-κB via the α Mβ 2 (CD11b/CD18, Mac-1) integrin receptor, leading to the production of pro-inflammatory cytokines (; reviewed in ). Our previous studies showed that fibrin(ogen) and collagen matrices accumulated in diaphragms of aging mdx mice, while pharmacological fibrinogen depletion attenuated muscle fibrosis observed with age.

However, the precise mechanism(s) by which fibrin(ogen) influences disease progression remain obscure. In the present study, we show that fibrin(ogen), which is never detected outside of the vascular compartment in healthy muscle, is deposited in the muscle microenvironment immediately after injury, and upon disease onset in mdx mice. Using a combination of genetic and pharmacological approaches focused on fibrin(ogen), we directly tested the hypothesis that the severity of muscular dystrophy in mdx mice is dependent on fibrin(ogen) and that a mechanism supporting disease progression is coupled to the proinflammatory property of fibrin(ogen) linked to the α Mβ 2-binding motif. These studies establish for the first time that development of muscle pathology in mdx mice can be strongly attenuated by a simple fibrinogen mutation that has no influence on hemostasis and provide a proof-of-principle of the potential utility of interfering with the α Mβ 2-fibrin(ogen) interactions for treating DMD and other inflammatory myopathies. RESULTS Genetic or pharmacological depletion of fibrinogen attenuates degeneration, enhances regeneration and preserves function of dystrophic mouse muscle To investigate whether fibrin(ogen) may directly control dystrophic muscle degeneration, we first analyzed its presence in limb muscles before and after the disease onset in mdx mice. Prior to disease onset (14 days of age), fibrinogen was undetectable by immunostaining in gastrocnemius muscle of mdx mice (Fig. A).

In contrast, at the first signs of disease (21 days of age), and especially at the disease peak (30 days of age), fibrin(ogen) deposits were readily detectable in areas of muscle degeneration and inflammation (Fig. A), remaining elevated thereafter. As fibrin(ogen) deposition was not detected in muscles from healthy age-matched wild-type (WT) mice, these findings suggest that the accumulation of this provisional matrix protein specifically associates with dystrophinopathy in mdx mice. Fibrin(ogen) accumulates in mdx dystrophic muscle, and fibrin(ogen) deficiency results in reduced muscle degeneration and enhanced regeneration. ( A) Fibrin(ogen) deposition was analyzed by immunohistochemistry in gastrocnemius muscles of WT control mice and in mdx mice of 14, 30 and 90 days of age. Magnification bar: 50 µm.

( B) Immunostaining for fibrin(ogen) of gastrocnemius muscles of 90-day-old mice showing the absence of fibrin(ogen) deposition in Fib −/−mdx mice when compared with Fib +/+mdx mice. ( C) Left: reduced percentage of total muscle degeneration area in Fib −/−mdx versus Fib +/+mdx mice at 30 days of age, as determined by morphometric analysis on H&E-stained gastrocnemius muscle sections. Right: reduced muscle membrane damage in Fib −/−mdx versus Fib +/+mdx, as indicated by serum CK measurement. Data are mean ± SEM; n = 5 animals per group (. P. Fibrin(ogen) accumulates in mdx dystrophic muscle, and fibrin(ogen) deficiency results in reduced muscle degeneration and enhanced regeneration.

( A) Fibrin(ogen) deposition was analyzed by immunohistochemistry in gastrocnemius muscles of WT control mice and in mdx mice of 14, 30 and 90 days of age. Magnification bar: 50 µm.

( B) Immunostaining for fibrin(ogen) of gastrocnemius muscles of 90-day-old mice showing the absence of fibrin(ogen) deposition in Fib −/−mdx mice when compared with Fib +/+mdx mice. ( C) Left: reduced percentage of total muscle degeneration area in Fib −/−mdx versus Fib +/+mdx mice at 30 days of age, as determined by morphometric analysis on H&E-stained gastrocnemius muscle sections. Right: reduced muscle membrane damage in Fib −/−mdx versus Fib +/+mdx, as indicated by serum CK measurement. Data are mean ± SEM; n = 5 animals per group (. P. Pharmacological defibrination attenuates dystrophy progression in mdx muscle. Twelve-day-old mdx mice were subjected to a defibrination treatment by daily intraperitoneal injection with ancrod (or with vehicle–control saline solution), up to 2.5 months of age.

( A) Fibrin(ogen) staining of muscle sections of vehicle-treated and defibrinated mdx mice. ( B) Left: percentage of total muscle degeneration area. Right: muscle membrane damage indicated by serum CK levels. Data are mean ± SEM; n = 5 animals per group (. P. Pharmacological defibrination attenuates dystrophy progression in mdx muscle.

Twelve-day-old mdx mice were subjected to a defibrination treatment by daily intraperitoneal injection with ancrod (or with vehicle–control saline solution), up to 2.5 months of age. ( A) Fibrin(ogen) staining of muscle sections of vehicle-treated and defibrinated mdx mice. ( B) Left: percentage of total muscle degeneration area. Right: muscle membrane damage indicated by serum CK levels.

Data are mean ± SEM; n = 5 animals per group (. P. Intramuscular delivery of fibrinogen induces a tissue injury/repair-like response in fibrinogen-null mice. ( A) Fibrin(ogen) and α Mβ 2 co-immunostaining of muscle sections of non-injured WT mice and at 2, 4 and 12 h after CTX injury. ( B) Two-month-old Fib −/− mice were subjected to intramuscular delivery of saline (vehicle) or fibrinogen (9 mg/ml) into the right and left tibialis muscles, respectively. Left: representative pictures of saline and fibrinogen injected muscles stained for fibronectin and α Mβ 2. Right: quantification of the number of macrophages showed increased infiltration in fibrinogen- versus saline-treated muscles in Fib −/− mice at day 3 after injection.

Data are mean ± SEM; n = 4 animals per group (. P. Intramuscular delivery of fibrinogen induces a tissue injury/repair-like response in fibrinogen-null mice. ( A) Fibrin(ogen) and α Mβ 2 co-immunostaining of muscle sections of non-injured WT mice and at 2, 4 and 12 h after CTX injury.

• Play with a friend in two player mode (via built-in WiFi or your bluetooth controller). How satisfying is that? Can you download bubble struggle for mac. • Compete with players from around the world for the highest score.

( B) Two-month-old Fib −/− mice were subjected to intramuscular delivery of saline (vehicle) or fibrinogen (9 mg/ml) into the right and left tibialis muscles, respectively. Left: representative pictures of saline and fibrinogen injected muscles stained for fibronectin and α Mβ 2. Right: quantification of the number of macrophages showed increased infiltration in fibrinogen- versus saline-treated muscles in Fib −/− mice at day 3 after injection. Data are mean ± SEM; n = 4 animals per group (.

P. Interaction of fibrin(ogen) with α Mβ 2 integrin receptor on macrophages regulates inflammation, degeneration and regeneration in mdx muscle. ( A) Representative macrophage F4/80 immunostaining of muscle sections from Fib +/+mdx and Fibγ 390−396Amdx mice. ( B) Reduced muscle inflammatory macrophage infiltration analyzed by flow cytometry (.

P. Interaction of fibrin(ogen) with α Mβ 2 integrin receptor on macrophages regulates inflammation, degeneration and regeneration in mdx muscle. ( A) Representative macrophage F4/80 immunostaining of muscle sections from Fib +/+mdx and Fibγ 390−396Amdx mice. ( B) Reduced muscle inflammatory macrophage infiltration analyzed by flow cytometry (. P. Treatment with a fibrinogen-derived γ 377-395 peptide blocks inflammation and disease progression in mdx mice. ( A) Activation of macrophages by fibrin(ogen) is reduced by the fibrinogen-γ-derived 377–395 peptide.

Primary macrophages were treated with fibrinogen (Fg) in the absence or presence of the γ 377-395 peptide or scrambled peptide used as control. Additional experimental controls included treatment with an α Mβ 2 blocking antibody, or IgG control antibody, as indicated. Expression of inflammatory cytokines was analyzed by qRT–PCR. Results are fold-induction values with respect to untreated macrophages. Data are mean ± SEM; n = 3 experiments performed in triplicate (.

P. Treatment with a fibrinogen-derived γ 377-395 peptide blocks inflammation and disease progression in mdx mice. ( A) Activation of macrophages by fibrin(ogen) is reduced by the fibrinogen-γ-derived 377–395 peptide. Primary macrophages were treated with fibrinogen (Fg) in the absence or presence of the γ 377-395 peptide or scrambled peptide used as control. Additional experimental controls included treatment with an α Mβ 2 blocking antibody, or IgG control antibody, as indicated. Expression of inflammatory cytokines was analyzed by qRT–PCR. Results are fold-induction values with respect to untreated macrophages.

Data are mean ± SEM; n = 3 experiments performed in triplicate (. P. Paracrine effect of fibrinogen-activated macrophages on satellite cells.

Satellite cells obtained from mouse muscle were cultured in GM for 24 h with CM from macrophages treated or not with fibrinogen for 24 h (CM Fg), and incubated for 1 h with BrdU. When indicated, the γ 377-395 peptide or scramble peptide was added to fibrinogen-stimulated macrophages. Also, neutralizing antibodies against TNFα and IL-1β (or control IgG) were added to CM from fibrinogen-stimulated macrophages. ( A) Satellite cells subjected to the different macrophage-conditioned media were fixed and immunostained for BrdU, and positive cells were quantified. ( B) Satellite cells were cultured in GM and then shifted to differentiation medium, supplemented with different macrophage-conditioned media as above. Comparative qRT–PCR analysis of Myogenin.

( C) eMHC mRNA expression. Results represent the mean of at least three experiments. Data are mean ± SEM (. P. Paracrine effect of fibrinogen-activated macrophages on satellite cells. Satellite cells obtained from mouse muscle were cultured in GM for 24 h with CM from macrophages treated or not with fibrinogen for 24 h (CM Fg), and incubated for 1 h with BrdU. When indicated, the γ 377-395 peptide or scramble peptide was added to fibrinogen-stimulated macrophages.

Also, neutralizing antibodies against TNFα and IL-1β (or control IgG) were added to CM from fibrinogen-stimulated macrophages. ( A) Satellite cells subjected to the different macrophage-conditioned media were fixed and immunostained for BrdU, and positive cells were quantified. ( B) Satellite cells were cultured in GM and then shifted to differentiation medium, supplemented with different macrophage-conditioned media as above. Comparative qRT–PCR analysis of Myogenin. ( C) eMHC mRNA expression. Results represent the mean of at least three experiments.

Data are mean ± SEM (. P. Fibrin(ogen) directly affects satellite cell functions. ( A) Fibrin(ogen) is deposited in the basal laminae of dystrophic muscle. Top: representative example of a double immunofluorescence staining showing an activated satellite cell in green (MyoD positive, see arrow) surrounded by fibrin(ogen) (red) in a 2.5-month-old mdx gastrocnemius muscle section. Nuclei were stained with 4'-6-diamidino-2-phenylindole. Magnification bars: 10 µm.

Bottom: immunohistochemistry showing fibrin(ogen) (red) and laminin (green) co-staining in a mdx muscle section. ( B) Fibrinogen alters satellite cell functions. Left: satellite cells were cultured in GM with and without increasing amounts of fibrinogen (500 and 1000 μg fibrinogen), and viable cells were counted after 72 h as an index of cell division. Data are mean ± SEM; n = 3 experiments performed in triplicate (. P.

Fibrin(ogen) directly affects satellite cell functions. ( A) Fibrin(ogen) is deposited in the basal laminae of dystrophic muscle. Top: representative example of a double immunofluorescence staining showing an activated satellite cell in green (MyoD positive, see arrow) surrounded by fibrin(ogen) (red) in a 2.5-month-old mdx gastrocnemius muscle section.

Nuclei were stained with 4'-6-diamidino-2-phenylindole. Magnification bars: 10 µm. Bottom: immunohistochemistry showing fibrin(ogen) (red) and laminin (green) co-staining in a mdx muscle section.

( B) Fibrinogen alters satellite cell functions. Left: satellite cells were cultured in GM with and without increasing amounts of fibrinogen (500 and 1000 μg fibrinogen), and viable cells were counted after 72 h as an index of cell division. Data are mean ± SEM; n = 3 experiments performed in triplicate (. P. Association of fibrin(ogen) deposits and macrophage infiltrates in dystrophic muscle of DMD patients. ( A) Representative examples of fibrin(ogen) accumulation and α Mβ 2 co-staining in sections of muscle biopsies from DMD patients compared with healthy individuals.

Scale bar: 25 µm. ( B) The proposed model for the deleterious action of persistently deposited fibrin(ogen) in the dystrophic muscle ECM, after extravasation. Exacerbated deposition of fibrin(ogen) promotes inflammation-mediated muscle degeneration and regeneration via α Mβ 2 integrin engagement on macrophages thus inducing expression of pro-inflammatory cytokines, which in turn may negatively regulate satellite cell functions. Fibrin(ogen) may also directly impact on satellite cells functions through α Vβ 3 integrin binding. Association of fibrin(ogen) deposits and macrophage infiltrates in dystrophic muscle of DMD patients.

( A) Representative examples of fibrin(ogen) accumulation and α Mβ 2 co-staining in sections of muscle biopsies from DMD patients compared with healthy individuals. Scale bar: 25 µm. ( B) The proposed model for the deleterious action of persistently deposited fibrin(ogen) in the dystrophic muscle ECM, after extravasation. Exacerbated deposition of fibrin(ogen) promotes inflammation-mediated muscle degeneration and regeneration via α Mβ 2 integrin engagement on macrophages thus inducing expression of pro-inflammatory cytokines, which in turn may negatively regulate satellite cell functions.

Fibrin(ogen) may also directly impact on satellite cells functions through α Vβ 3 integrin binding. DISCUSSION Despite intense research efforts, DMD is still an incurable and fatal degenerative muscle disorder that demands novel experimental and therapeutic advances.

The present study explores a new mechanistic and therapeutic dimension by examining the molecular link between fibrin(ogen) deposition within persistently challenged muscle and inflammation-driven muscle damage, tissue reorganization and function loss. We provide direct evidence that fibrin(ogen), which is never present in normal muscle but rapidly accumulates in dystrophic muscle secondary to persistent tissue disruption and vascular leak, is a critical factor for inflammation-mediated DMD progression. Imposing either a genetic or pharmacological depletion of fibrinogen reduced macrophage numbers and activation in injured and dystrophic muscle and enhanced repair, while preserving locomotor capacity. However, more detailed studies revealed that muscle dystrophy pathology could also be ameliorated and muscle function preserved by interventions at the level of fibrinogen that do not impose any hemostatic alteration or hemorrhagic risk.

Specifically, the simple elimination of the α Mβ 2-binding motif on the fibrin(ogen) gamma chain that drives pro-inflammatory functions, but is irrelevant to fibrin polymer formation, sufficed to alleviate disease severity in mdx dystrophic muscle. Conversely, delivery of exogenous fibrinogen into intact muscle of fibrinogen-null mice caused a classical inflammatory tissue repair response. The relevance of these findings to human disease is underscored by the finding that fibrin(ogen) accumulation was positively associated with inflammation in degenerating muscles of DMD patients.

Hence, selectively targeting fibrin(ogen) inflammatory functions may represent a promising therapeutic strategy for DMD. The fact that fibrin(ogen) depletion affected several distinct aspects of muscle disease in mdx mice implies that recurrent fibrin deposition within the ECM may influence the progression of muscle disease through multiple mechanisms (Fig. B). Vascular damage early after disease onset in dystrophic muscle and secondary fibrin(ogen) accumulation in the ECM may be an early driver of the inflammatory changes known to exacerbate muscular dystrophy (,). Under this working hypothesis, intramuscular fibrin(ogen) deposits serve as temporospatial cue controlling local activation events in macrophages and other inflammatory cells that express α Mβ 2 (reviewed in ), and recurrent and/or exuberant fibrin(ogen) within diseased muscle tissue promotes counterproductive inflammatory tissue degeneration.

Best Free Backup Software

Our results demonstrate that upon fibrin(ogen) engagement, macrophages increase the expression of pro-inflammatory cytokines, including IL-1β, TNFα and IL-6 in vitro and in vivo, in an α Mβ 2-dependent manner. The presence of fibrin(ogen) appears to regulate the expression of cytokines known to promote muscle degeneration and impair regeneration, such as TNFα (,). Limiting these responses via experimentally imposed suppression of fibrin(ogen)-α Mβ 2 engagement by macrophages and other inflammatory cells may largely explain the reduced pathological changes observed in mdx muscle. Our data do not exclude other fibrinogen-mediated effects on inflammatory cells such as α Mβ 2-mediated inhibition of inflammatory cell apoptosis (,) and the increased release of free radicals (,).

Previous reports have shown that macrophage depletion or interference with pro-inflammatory cytokines transiently attenuated muscle dystrophy in mdx mice (,; reviewed in,). Our present findings further underscore the relevance of the α Mβ 2 integrin receptor in macrophage activation in DMD and directly establish for the first time that the engagement of α Mβ 2 with one of its ligands, fibrin(ogen), an early component of the muscle provisional matrix after injury, is a critical regulatory event in dystrophic muscle pathology. We show that mice bearing an endogenous fibrinogen γ chain gene with a specific mutation in the α Mβ 2-binding region (Fibγ 390-396A mice), which does not affect the clotting function of fibrinogen and does not limit the engagement of α Mβ 2 with other ligands (,), are significantly protected from acute muscle injury and dystrophic disease progression.

Furthermore, the effective amelioration of muscle dystrophy in mdx mice treated with the Fibγ 377-395 peptide demonstrates that inflammatory functions of fibrin(ogen) can, at least in principle, be pharmacologically suppressed, thus presenting an alternative treatment paradigm for impeding DMD disease progression. Impeding macrophage α Mβ 2/fibrin(ogen) binding could also enhance muscle regeneration, which is primarily mediated by activation of satellite cells, localized along the basal lamina. This effect might again be related to the inhibition of macrophage activation. Interestingly, we also found that fibrin(ogen) was deposited in the fibronectin + and laminin + ECM basal lamina of regenerating mdx muscle, where it physically entrapped satellite cells. Further, we demonstrated that fibrinogen regulates the proliferation and differentiation potential of satellite cells in culture through an α vβ 3-dependent mechanism. Thus, fibrin(ogen) depletion may promote regeneration at least in part by indirectly attenuating inflammation, and possibly by diminishing direct deleterious effects of persisting fibrinogen on satellite cell functions. Collectively, we propose that the development of exuberant and recurrent fibrin-rich matrices within challenged muscle tissue establishes a counterproductive inflammatory environment through α Mβ 2-mediated leukocyte activation events as well as a counterproductive environment for muscle tissue regeneration.

Consistent with this, our analysis on DMD patient samples suggests an association between fibrin(ogen) deposition and the presence of macrophages in degenerating dystrophic muscle. Interestingly, interference with the fibrin(ogen)/macrophage axis was recently shown to ameliorate neurodegeneration and halt demyelination in a mouse model of multiple sclerosis , to protect from collagen-induced arthritis and colitis-associated colon cancer in mice. Alzheimer disease has also been associated to fibrinogen deposition and inflammation (,). It is therefore possible that targeting fibrinogen–α Mβ 2 interactions could represent a quite broad strategy for inhibiting macrophage activation in neurodegenerative diseases coursing with inflammation. In this regard, our study provides a compelling proof-of-principle that the sole blockage of fibrinogen binding to α Mβ 2 on macrophages via therapeutic administration of the Fibγ 377-395 peptide improved muscle function in mdx mice. Although the activation of macrophages plays a central role in the pathogenesis of muscular dystrophies (reviewed in,), to the best of our knowledge agents that selectively inhibit macrophage inflammatory activation have not been developed and tried in this pathology.

A potential advantage of a fibrin(ogen)/macrophage interaction-based approach over current anti-inflammatory strategies is that interfering with α Mβ 2-fibrin(ogen) is likely to impose only a local block on the activation (and recruitment) of macrophages, i.e. Within the dystrophic muscle where fibrin(ogen) is deposited.

Since no therapies correcting the primary defect in DMD (i.e. Dystrophin replacement or rescue) are currently available, strategies selectively targeting fibrin(ogen)/inflammatory cell signaling, without affecting its pro-coagulant properties, may constitute an attractive alternative for DMD disease treatment.

MATERIALS AND METHODS Generation of double-mutant mice Fibrinogen (Fib) knock-out male mice were crossed with mdx female mice, Jackson Laboratories (USA). Male F1 mice were bred with mdx female mice, and their F2 heterozygous (Fib +/−) male and female offspring was intercrossed.

The resulting F3 generation showed the expected Mendelian distribution of Fib +/+, Fib +/− and Fib −/− genotypes, all of them in an mdx background. The Fib genotypes were confirmed by polymerase chain reaction (PCR) of ear biopsy genomic DNA, as previously described. The mdx genotype was confirmed by western blotting of muscle biopsies, using an anti-dystrophin antibody (Novocastra, UK, 1:200). Fibγ 390-396A knock-in mice were generated from double heterozygous matings to produce homozygous Fibγ 390-396A mice. Male Fibγ 390-396A mice were sequentially intercrossed with mdx female mice, as described above, to generate double-mutant mice. All animal experiments were approved by the Catalan Government of Animal Care Committee.

Morphometric analysis At selected times, muscles of vehicle and ancrod-treated mdx mice, Fib +/+, Fib −/−, Fibγ 390-396A, Fib +/+mdx, Fib −/−mdx and Fibγ 390-396Amdx mice were removed after cervical dislocation, frozen and stored at −80°C prior to analysis. Ten micrometer sections were collected from the mid-belly of muscles and stained with hematoxylin/eosin (H&E). The percentage of muscle degeneration was determined by morphometrical analysis of digital photomicrographs of H&E-stained cryosections.

Areas of necrosis and degeneration were identified by the presence of pale-stained myofibers, with irregular shape and often fragmented sarcoplasm. These areas contained few myonuclei and inflammatory cells were not conspicuous. Evans Blue Dye staining, a vital stain of myofiber permeability , showed that degenerating areas identified with H&E were composed of damaged fibers that had become permeable owing to muscular dystrophy ( ). The areas of degeneration on each individual sample were measured using the public domain ImageJ software on calibrated micrographs and were expressed as a percentage of the total muscle cross-sectional area (CSA). Myofiber CSA was determined as an indicator of muscle growth and regeneration.

All analyses and photography were performed on a Leica DC 500 microscope equipped with a video camera. All parameters relative to myofiber CSA were measured using the ImageJ program. Biochemical and functional assessment of muscle Serum CK was measured with the indirect CK colorimetric assay kit and standards (Thermo Electron, USA). Grip strength assay: Limb grip strength was measured as tension force using a computerized force transducer (Grip Strength Meter, Bioseb). Three trials of three measurements per trial were performed for each animal with a few minutes resting period between trials. The average tension force (grams) was calculated for each group of mice. Treadmill assay: the treadmill apparatus (Treadmill, Panlab) consisted of a motor-driven belt varying in terms of speed and slope.

At the end of the treadmill, an electrified grid was placed, on which footshocks (0.6 mA) were administered whenever the mice fell off the belt. The last week before sacrifice, after one daily acclimation session of 20 min at 6 m/min for 2 days, all mice were subjected to exhaustion treadmill tests at 5° inclination.

Each test was repeated twice at a temporal distance of 4 days and results averaged. The following scheme was adopted: 5 min at 5 m/min followed by incremental increase of speed of 1 m/min every minute until exhaustion. Exhaustion was defined as spending time on the shocker plate without attempting to re-engage the treadmill within 20 s.

The individual and the average times (min) and distances (meters) to exhaustion of running were measured. Induction of muscle regeneration Regeneration of skeletal muscle was induced by intramuscular injection of 300 µl of 10 −5 m CTX (Latoxan, France) in the gastrocnemius muscle group of the mice, usually of 8–12 weeks of age. This concentration and volume were chosen to ensure maximum degeneration of the myofibers.

The experiments were performed in right hindlimb muscles, and contralateral intact muscles were used as control. Morphological and biochemical examinations were performed at the indicated days after injury. Systemic defibrination Twelve-day-old mdx mice were daily injected intraperitoneally with ancrod (Sigma, 1–3 U ancrod/day) or with a saline solution for 60 days, and killed at 2.5 months of age. Muscles were dissected and frozen prior to analysis. Systemic delivery of Fibγ 377-395 peptide Fibrinogen γ 377-395 peptide (Y S M K E T T M K I I P F N R L S I G; Azco Pharmchem) or scramble peptide (K M M I S Y T F P I E R T G L I S N K; Azco Pharmchem) was resuspended in 0.9% NaCl at a concentration of 3 mg/ml, as described.

Peptides were delivered intraperitoneally every other day into 2-month-old mdx mice over the course of 3 weeks. Intramuscular fibrinogen delivery Fib −/− mice of 8–12 weeks of age were used for intramuscular injection of fibrinogen or saline. Fibrinogen (Calbiochem) was dissolved in endotoxin-free distilled water, diluted to 9 mg/ml with saline, aliquoted and kept at 37°C.

Fibrinogen (50 µl of 9 mg/ml) or saline was injected with a 10 µl Hamilton syringe attached to a 33-gauge needle into mouse tibialis anterior muscles. Samples were obtained at the indicated time points.

Analysis of muscle macrophages by FACS Hindlimb muscles were collected and weighed. Muscles were dissociated by incubation in Dulbecco's modified Eagle's medium (DMEM) containing pronase 0.05% (Calbiochem) at 37°C for 45 min twice, filtered and counted. Cells were separated using a Percoll gradient and stained with fluorescein isothiocyanate-conjugated anti-CD45, allophycocyanin-Cy7-conjugated F4/80 antibody (BD Pharmingen). Cells were sorted using a cell sorter fluorescence-activated cell-sorting (FACS) Aria II; BD. Total number of cells was normalized by milligram of wet tissue. Immunohistochemistry The following primary antibodies were used for immunohistochemistry: rat anti-F4/80 (Serotec, 1:200), goat anti-fibrin/ogen (Nordic, 1:100), rabbit anti-MyoD (Santa Cruz Biotechnology, 1:20), rabbit anti-fibronectin (Sigma, 1:40), rabbit anti-laminin (Sigma, 1:50); monoconal anti-human fibrin/ogen antibody (Accurate Chemical & Scientific Corporation, Westbury, NY, USA), clone NYBT2G1, 1:200, and rat anti-mouse α M/CD11b (ebioscience 1:100), eMHC (F1.652, neat hybridoma supernatant; Developmental Studies Hybridoma Bank). Depending on the antibody, immunohistochemistry was performed with the tyramide signal amplification Cyanine 3 system (PerkinElmer Life Sciences) or as previously described.

Control experiments without primary antibody demonstrated that signals observed were specific (data not shown). Cell culture and isolation of primary cells Isolation and culture of primary macrophages Bone-marrow-derived macrophages (BMDMs) were obtained from mdx mice and cultured as previously described (,).

In brief, bone-marrow cells were flushed from the femurs of mice. Cells were differentiated in DMEM supplemented with 20% heat-inactivated fetal calf serum (FCS) and 30% L929 supernatants containing macrophage-stimulating factor (MCSF) for 5–7 days. Cells were harvested and plated at a density of 2–4 × 10 5 per ml in RPMI supplemented with 5% fetal bovine serum. At 24–48 h after being replated, cells were stimulated for various times with lipopolysaccharides (10 ng/ml; Sigma). CM from WT BMDMs was obtained stimulating cell cultures for with fibrinogen (0.5 mg/ml; Sigma). When indicated, fibrinogen-stimulated macrophages were also treated with 200 μ m of γ 377-395 peptide a concentration shown to inhibit adhesion of α Mβ 2/Mac-1-overexpressing cells to immobilized fibrinogen and 200 μ m of scramble peptide. When indicated, CM was supplemented with blocking antibodies anti-IL-1β (30 ng/ml; R&D Systems) or anti-TNFα (50 ng/ml; BD Pharmingen) or unspecific IgG.

Isolation and culture of muscle satellite cells Hindlimb muscles from mice were excised, separated from adipose and connective tissue, minced into coarse slurry and then digested for 1 h with 0.1% pronase (Sigma) in DMEM at 37°C with mild agitation. The digest was then mechanically dissociated by repeated trituration followed by filtration through a 100 mm vacuum filter (Millipore). The filtered digest was centrifuged through an isotonic Percoll gradient (60% overlaid with 20%).

Mononucleated cells in the Percoll interface were collected and resuspended in Ham's F10 medium supplemented with 20% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.001% Fungizone and 5 ng/ml bFGF growth medium (GM). Primary satellite cell cultures were maintained on a routinary basis on collagen-coated dishes in GM. The medium was changed daily and cultures were passaged 1:3 as they reached 60–70% confluence.

Experiments were performed by plating cells on Matrigel (BD Biosciences) Basement Membrane Matrix-coated dishes. To maintain the primary characteristics of the cells, all experiments were performed using cultures that had undergone between four and seven passages. All experiments were performed with independent cell isolates from at least three different animals for each genotype.

To induce muscle differentiation and fusion, GM was replaced by differentiation medium DM (DMEM supplemented with 2% horse serum, 2 m m l-glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.001% Fungizone) at satellite cell subconfluence. For detection of S-phase cells, satellite cells were cultured in GM, and cultures were pulsed with bromodeoxyuridine (BrdU, Sigma) for hours prior to fixation in 3.7% formaldehyde for 10 min and were immunostained using anti-BrdU antibody (Oxford Biotech) and a specific secondary biotinylated goat anti-rat antibody (Jackson Inmunoresearch Laboratories), followed by quantification. For fusion analysis, cells were immunostained with anti-eMHC antibody and nuclei within the eMHC-positive myofibers were quantified. When indicated, CM of cultured macrophages, control non-specific RGD peptide (GRGDNP; Biomol) or the cyclic RGD peptide (GpenGRGD; Bachem; 100 n m) or an alphaV neutralizing antibody (20 μg/ml; Chemicon) was added.

RNA isolation and quantitative reverse transcription–polymerase chain reaction RNA was analyzed by quantitative reverse transcription–polymerase chain reaction (RT–PCR). Total RNA was isolated from cells or muscle tissue using Tripure reagent (Roche Diagnostic Corporation). DNase digestion of 10 µg of RNA was performed using 2 U DNase (Turbo DNA-free™, Ambion). Complementary DNA was synthesized from 2 µg of total RNA using the First-Strand cDNA Synthesis kit (Amersham Biosciences).

PCRs were performed on a LightCycler ® 2.0 or in a LightCycler ® 480 System using a Light Cycler ® FastStart DNA Master PLUS SYBR Green I or Light Cycler ® 480 SYBR Green I Master (Roche Diagnostic Corporation), respectively, and specific primers shown in. Thermocycling conditions were as follow: initial step of 10 min at 95°C, then 45 cycles of 15 s denaturation at 94°C, 10 s annealing at 60°C and 15 s extension at 72°C. Reactions were run in triplicate, and automatically detected threshold cycle (Ct) values were compared between samples. Transcripts of the ribosomal protein L7 gene were used as endogenous normalization control. QPCRs primers: eMHC, 5′-AAAAGGCCATCACTGACGC-3′ and 5′-CAGCTCTCTGATCCGTGTCTC-3′ myogenin, 5′-GGTGTGTAAGAGGAAGTCTGTG-3′ and 5′-TAGGCGCTCAATGTACTGGAT-3′ TNFα, 5′-CGCTCTTCTGTCTACTGAACTT-3′ and 5′-GATGAGAGGGAGGCCATT-3′ IL-1β, 5′-CCAAAATACCTGTGGCCTTGG-3′ and 5′-GCTTGTGCTCTGCTTGTGAG-3′ IL-6, 5′-GAGGATACCACTCCCAACAGACC-3′ and 5′- AAGTGCATCATCGTTGTTCATACA-3′ L7, 5′-GAAGCTCATCTATGAGAAGGC-3′ and 5′-AAGACGAAGGAGCTGCAGAAC-3′. Statistical analysis Data are presented as mean ± SEM.

All quantitative data were analyzed with Prism software (GraphPad Software) for statistical analyses. The one-way analysis of variance and Dunnett's test were used for multiple comparisons. The Non-parametric Mann–Whitney test was used for single comparisons of unmatched groups.