Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2

Transforming growth factor beta (TGF-beta) and connective tissue growth factor (CTGF) have been described to induce the production of extracellular matrix (ECM) proteins and have been reported to be increased in different fibrotic disorders. Skeletal muscle fibrosis is a common feature of Duchenne muscular dystrophy (DMD). The mdx mouse diaphragm is a good model for DMD since it reproduces the muscle degenerative and fibrotic changes. Fibronectin (FN) and proteoglycans (PG) are some of the ECM proteins upregulated in dystrophic conditions. In view of understanding the fibrotic process involved in DMD we have isolated fibroblasts from dystrophic mdx diaphragms. Here we report that regardless of the absence of degenerative myofibers, adult mdx diaphragm fibroblasts show increased levels of FN and condroitin/dermatan sulfate PGs synthesis. Fibroblasts isolated from non fibrotic tissue, such as 1 week old mice diaphragms or skin, do not present elevated FN levels. Furthermore, mdx fibroblast conditioned media is able to stimulate FN synthesis in control fibroblasts. Autocrine TGF-beta signaling was unaltered in mdx cells. When control fibroblasts are exposed to TGF-beta and CTGF, FN increases as expected. Paradoxically, in mdx cells it decreases in a concentration dependent manner and this decrease is not due to a downregulation of FN synthesis. According to this data we hypothesize that a pathological environment is able to reprogram fibroblasts into an activated phenotype which can be maintained through generations.     

Type IV collagen stimulates an increase in intracellular calcium. Potential role in tumor cell motility.

Type IV collagen (Coll IV), a component of the extracellular matrix, stimulates motility in the A2058 human melanoma cell line, a response that is inhibited by pertussis toxin (PT). Fibronectin (FN)-induced chemotaxis in this cell line is not affected by PT. To understand the mechanism of cellular signaling, single cell intracellular Ca2+ responses to Coll IV and FN were studied using Fura-2 and digital imaging fluorescence microscopy. Coll IV, at a dose that stimulates motility (100 micrograms/ml, 185 nM), induces a significant rise in cytosolic free Ca2+ concentration ([Ca2+]i) within 100 s. This response is not inhibited by PT. Treatment of the cells with FN 30 micrograms/ml (70 nM), a dose that stimulates near-maximal chemotaxis, does not increase [Ca2+]i appreciably. Removal of extracellular Ca2+ fails to inhibit the Coll IV-stimulated rise in Ca2+ in all cells. Depletion of extracellular Ca2+ and pretreatment of cells with Ca2+ channel blockers only partially inhibits Coll IV-induced motility. Depletion of intracellular Ca2+ inhibits both chemotaxis and the Coll IV-induced increase in intracellular Ca2+. Coll IV does not stimulate membrane phosphoinositide hydrolysis. We conclude that Coll IV treatment induces an inositol 1,4,5-trisphosphate-independent release of intracellular Ca2+ stores which appears to play a necessary role in the chemotactic response of A2058 cells but is not mediated by a PT-sensitive G-protein. This response is not seen in cells exposed to FN, suggesting different intracellular signaling mechanisms for stimulated motility between these two extracellular matrix molecules.     

Fibroblast remodeling of adsorbed collagen type IV is altered in contact with cancer cells

A series of co-culture experiments between fibroblasts and H-460 human lung carcinoma cells were performed to learn more about the fate of adsorbed type IV collagen (Coll IV). Fibroblasts were able to spatially rearrange Coll IV in a specific linear pattern, similar but not identical to the fibronectin (FN) fibrils. Coll IV partly co-aligns with fibroblast actin cytoskeleton and transiently co-localize with FN, as well as with beta1 and alpha2 integrin clusters, suggesting a cell-dependent process. We further found that this Coll IV reorganization is suppressed in contact with H460 cells. Zymography revealed strongly elevated MMP-2 activity in supernatants of co-cultures, but no activity when fibroblasts or cancer cells were cultured alone. Thus, we provide evidence that reorganization of substrate associated Coll IV is a useful morphological approach for in vitro studies on matrix remodeling activity during tumorigenesis.     

Constitutively activated dystrophic muscle fibroblasts show a paradoxical response to TGF-β and CTGF/CCN2

Transforming growth factor beta (TGF-β) and connective tissue growth factor (CTGF) have been described to induce the production of extracellular matrix (ECM) proteins and have been reported to be increased in different fibrotic disorders. Skeletal muscle fibrosis is a common feature of Duchenne muscular dystrophy (DMD). The mdx mouse diaphragm is a good model for DMD since it reproduces the muscle degenerative and fibrotic changes. Fibronectin (FN) and proteoglycans (PG) are some of the ECM proteins upregulated in dystrophic conditions. In view of understanding the fibrotic process involved in DMD we have isolated fibroblasts from dystrophic mdx diaphragms. Here we report that regardless of the absence of degenerative myofibers, adult mdx diaphragm fibroblasts show increased levels of FN and condroitin/dermatan sulfate PGs synthesis. Fibroblasts isolated from non fibrotic tissue, such as 1 week old mice diaphragms or skin, do not present elevated FN levels. Furthermore, mdx fibroblast conditioned media is able to stimulate FN synthesis in control fibroblasts. Autocrine TGF-β signaling was unaltered in mdx cells. When control fibroblasts are exposed to TGF-β and CTGF, FN increases as expected. Paradoxically, in mdx cells it decreases in a concentration dependent manner and this decrease is not due to a downregulation of FN synthesis. According to this data we hypothesize that a pathological environment is able to reprogram fibroblasts into an activated phenotype which can be maintained through generations.     

Immunofluorescent localization of extracellular matrix components during muscle morphogenesis. II. In chick embryos with hereditary muscular dysgenesis (cn/cn)

The immunofluorescent distribution of types I and III collagen, fibronectin, and laminin during muscle morphogenesis of the crooked neck dwarf mutant chick embryo differs from that of the normal chick. The drastic difference is related to the inability of the mutant embryo to maintain a harmonious muscle pattern. The first sign of the defect is the disaggregation of type I collagen fibers of the tendons and the disorganization of the intermuscular spaces. The organization of the connective tissue never proceeds beyond the appearance of an epimysial envelope, rich in types I and III collagen, which becomes disorganized shortly after. No perimysial envelopes displaying types I and III collagen fibers and fibronectin, nor endomysial sheaths develop. Only large spaces filled with types I and III collagen fibers subdivide groups of muscle cells irregularly. On the whole, type III collagen is less abundant than type I collagen. Fibronectin disappears from the periphery of the muscle cell. Laminin is more thickly deposited in the basal lamina around irregularly sized muscle cells than around the normal muscle cell. The results are discussed in terms of morphogenetic interactions between connective tissue cells and muscle cells, and in terms of fibrosis, which characterizes some muscle diseases.     

Characterization of muscle epimysium, perimysium and endomysium collagens

In the past it has been proven difficult to separate and characterize collagen from muscle because of its relative paucity in this tissue. The present report presents a comprehensive methodology, combining methods previously described by McCollester [(1962) Biochim. Biophys. Acta 57, 427-437] and Laurent, Cockerill, McAnulty & Hastings [(1981) Anal. Biochem. 113, 301-312], in which the three major tracts of muscle connective tissue, the epimysium, perimysium and endomysium, may be prepared and separated from the bulk of muscle protein. Connective tissue thus prepared may be washed with salt and treated with pepsin to liberate soluble native collagen, or can be washed with sodium dodecyl sulphate to produce a very clean insoluble collagenous product. This latter type of preparation may be used for quantification of the ratio of the major genetic forms of collagen or for measurement of reducible cross-link content to give reproducible results. It was shown that both the epimysium and perimysium contain type I collagen as the major component and type III collagen as a minor component; perimysium also contained traces of type V collagen. The endomysium, the sheaths of individual muscle fibres, was shown to contain both type I and type III collagen as major components. Type V collagen was also present in small amounts, and type IV collagen, the collagenous component of basement membranes, was purified from endomysial preparations. This is the first biochemical demonstration of the presence of type IV collagen in muscle endomysium. The preparation was shown to be very similar to other type IV collagens from other basement membranes on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and was indistinguishable from EHS sarcoma collagen and placenta type IV collagen in the electron microscope after rotary shadowing.     

Increase in decorin and biglycan in Duchenne Muscular Dystrophy: role of fibroblasts as cell source of these proteoglycans in the disease

Fibrosis is a common pathological feature observed in muscles of patients with Duchenne muscular dystrophy (DMD). Biglycan and decorin are small chondroitin/dermatan sulfate proteoglycans in the muscle extracellular matrix (ECM) that belong to the family of structurally related proteoglycans called small leucine-rich repeat proteins. Decorin is considered an anti-fibrotic agent, preventing the process by blocking TGF-beta activity. There is no information about their expression in DMD patients. We found an increased amount of both proteoglycans in the ECM of skeletal muscle biopsies obtained from DMD patients. Both biglycan and decorin were augmented in the perimysium of muscle tissue, but only decorin increased in the endomysium as seen by immunohistochemical analyses. Fibroblasts were isolated from explants obtained from muscle of DMD patients and the incorporation of radioactive sulfate showed an increased synthesis of both decorin and biglycan in cultured fibroblasts compared to controls. The size of decorin and biglycan synthesized by DMD and control fibroblasts seems to be similar in size and anion charge. These findings show that decorin and biglycan are increased in DMD skeletal muscle and suggest that fibroblasts would be, at least, one source for these proteoglycans likely playing a role in the muscle response to dystrophic cell damage.     

The distribution of connective tissue content in swimming muscle of cod (Gadus morbua)

The amount and distribution of connective tissue in the swimming muscle of cod (Gadus morhua) have been determined. By biochemical analysis, the collagen was found to make up 1,5 % of the total protein content. Measured by light microscopical morphometric analysis, the areal fraction of the connective tissue elements was found to be 3,0 % of the total muscle area. The areal fraction of myocommatal connective tissue mentioned above was found to be 2.3 %. The thickness of the endomysial sheath was calculated by morphometry based on electron microscopy, and was found to be 0.30 and 0.16 μ for red and white fibres, respectively. The areal fraction of the endomysial sheaths was 2.3 % in red muscle and 0.5% in white muscle. The endomysial sheaths make up 25% of the total connective tissue in the swimming muscles. These sheaths influence the binding properties of fish muscle products.     

Liver connective tissue cells isolated from human schistosomal fibrosis or alcoholic cirrhosis represent a modified phenotype of smooth muscle cells

Hepatic connective tissue cells associated with schistosomal fibrosis and alcoholic cirrhosis were studied in vitro. Primary cell lines were isolated from all biopsies: they were identified as specific homogeneous cell populations, named liver connective tissue cells (LCTC). They were recognized as analogous to smooth muscle cells, different from true fibroblasts by morphological and physiological criteria. The proliferative capacity of LCTC is directly proportional to the degree of fibrosis in hepatic tissues. LCTC are able to secrete type I, III and IV collagen, fibronectin, laminin and amyloid P component. Their relationship with specific pathology of intrahepatic vascular tree in schistosomiasis is hypothesized.     

A Sensitive,Reproducible and Objective Immunofluorescence Analysis Method of Dystrophin in Individual Fibers in Samples from Patients with Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is characterized by the absence or reduced levels of dystrophin expression on the inner surface of the sarcolemmal membrane of muscle fibers. Clinical development of therapeutic approaches aiming to increase dystrophin levels requires sensitive and reproducible measurement of differences in dystrophin expression in muscle biopsies of treated patients with DMD. This, however, poses a technical challenge due to intra- and inter-donor variance in the occurrence of revertant fibers and low trace dystrophin expression throughout the biopsies. We have developed an immunofluorescence and semi-automated image analysis method that measures the sarcolemmal dystrophin intensity per individual fiber for the entire fiber population in a muscle biopsy. Cross-sections of muscle co-stained for dystrophin and spectrin have been imaged by confocal microscopy, and image analysis was performed using Definiens software. Dystrophin intensity has been measured in the sarcolemmal mask of spectrin for each individual muscle fiber and multiple membrane intensity parameters (mean, maximum, quantiles per fiber) were calculated. A histogram can depict the distribution of dystrophin intensities for the fiber population in the biopsy. This method was tested by measuring dystrophin in DMD, Becker muscular dystrophy, and healthy muscle samples. Analysis of duplicate or quadruplicate sections of DMD biopsies on the same or multiple days, by different operators, or using different antibodies, was shown to be objective and reproducible (inter-assay precision, CV 2–17% and intra-assay precision, CV 2–10%). Moreover, the method was sufficiently sensitive to detect consistently small differences in dystrophin between two biopsies from a patient with DMD before and after treatment with an investigational compound.     

Collagen, cross-linking, and advanced glycation end products in aging human skeletal muscle.

We examined intramuscular endomysial collagen, cross-linking, and advanced glycation end products, as well as the general and contractile protein concentration of 20 young (25 +/- 3 yr) and 22 old (78 +/- 6 yr, range: 70-93 yr) sedentary men and women to better understand the underlying basis of changes in skeletal muscle mass and function that occur with aging. The old individuals had an impaired ability (increased time) (P < 0.05) to climb stairs (80%), rise from a chair (56%), and walk (44%), as well as lower (P < 0.05) quadriceps muscle volume (-29%), muscle strength (-35%), muscle power (-48%), and strength (-17%) and power (-33%) normalized to muscle size. Vastus lateralis muscle biopsies revealed that intramuscular endomysial collagen (young: 9.6 +/- 1.1, old: 10.2 +/- 1.2 microg/mg muscle wet wt) and collagen cross-linking (hydroxylysylpyridinoline) (young: 395 +/- 65, old: 351 +/- 45 mmol hydroxylysylpyridinoline/mol collagen) were unchanged (P > 0.05) with aging. The advanced glycation end product, pentosidine, was increased (P < 0.05) by approximately 200% (young: 5.2 +/- 1.3, old: 15.9 +/- 4.5 mmol pentosidine/mol collagen) with aging. While myofibrillar protein concentration was lower (-5%, P < 0.05), the concentration of the main contractile proteins myosin and actin were unchanged (P > 0.05) with aging. These data suggest that the synthesis and degradation of proteins responsible for the generation (myosin and actin) and transfer (collagen and pyridinoline cross-links) of muscle force are tightly regulated in aging muscle. Glycation-related cross-linking of intramuscular connective tissue may contribute to altered muscle force transmission and muscle function with healthy aging.     

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.     

Connective tissue metabolism in muscular dystrophy. Amino acid composition of native types I, III, IV and V collagen isolated from the gastrocnemius muscle of embryonic chickens with genetic muscular dystrophy

The amino acid composition data on types I, III, IV and V collagen isolated from embryonic dystrophic skeletal muscle strongly indicate that alterations in collagen synthesis occur in intramuscular connective tissue of developing muscles in embryonic dystrophic chickens. The changes observed in the amino acid composition of dystrophic collagen were: (a) a selective removal of polar amino acids and substitution with non-polar amino acids; (b) significant decreases in basic (lysine, hydroxylysine and arginine) and hydroxylated (4-hydroxyproline and hydroxylysine) amino acids; and (c) significant increases in the amounts of glycine, proline and alanine. The amino acid substitutions suggest a genetic alteration in the collagen synthesizing process and a change in its structure. The variations in amino acid composition of collagen from dystrophic chickens could give rise to a decrease in both inter- and intramolecular cross-linking, thus decreasing the stability and functionality of newly formed collagen fibrils. The differences associated with the dystrophic collagen reported in this study are probably due to the differences in primary structure in terms of amino acid sequence rather than post-translational modifications. The structural differences noted would also lead to an alteration of the role collagen plays in regulating the differentiation of developing muscles. The changes in amino acid structure strongly suggest that the 'collagen' formed by dystrophic chickens should be considered a collagen-like protein or 'collagenoid'.     

Nebulin and titin expression in Duchenne muscular dystrophy appears normal

Monoclonal antibodies which recognize different epitopes on either titin or nebulin show normal staining patterns on frozen sections of three muscle biopsies of Duchenne muscular dystrophy (DMD). Gel electrophoresis and immunoblotting performed on two of these muscle biopsies show the normal pattern of titin and nebulin polypeptides. Since the donor of one of these biopsies has a large deletion of the 5'-region of the DMD gene, our results argue against the recent proposal that nebulin is the gene mutated in DMD.     

Melanocytes—A novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal–epidermal junction. In addition the full‐length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323–1331, 2013. © 2012 Wiley Periodicals, Inc.     

Eosinophil Adhesion and Maturation is Modulated by Laminin

Eosinophils (Eo) participate in the inflammatory response to parasites, allergins, toxins, and epitopes recognized by autoimmune antibodies. Nonetheless, little attention has heretofore been paid to the interactions of Eo with extracellular matrix (ECM) proteins during their migration through the subendothelial basement membrane and into the surrounding tissue. Therefore, we have studied the adhesion of Eo to specific ECM proteins and the effect of this adhesion on Eo viability and maturation. Control Eo (from normal donors) adhere no better to substrates coated with laminin (LM), fibronectin (FN), cytotactin (CT), or collagen types I or IV (Col IV) than they do to human serum albumin coated substrates. In contrast, Eo activated in vitro with IL-5 or in vivo in patients with eosinophilia bind well to LM, FN and Col IV. LM is by far the most avid ligand among these molecules. For example, 43% of input cells bind to a substrate bearing 200 fmol/cm² of LM; a similar level of adhesion to FN requires 30 times as much adsorbed protein. Antibody inhibition experiments suggest that the αβ1 integrin heterodimer is the predominant LM receptor on these cells. Flow cytometry showed similar levels of these subunits on control and activated Eo, suggesting that Eo adhesion to LM is not regulated simply by cell surface integrin concentration. The effects of ECM proteins on Eo behavior were also examined. A LM-coated substrate (with no added cytokine) was found to be almost as effective as IL-5 in maintaining Eo viability while an equally adhesive FN-coated substrate had much less effect. Normally, even in the presence of 10% serum, no Eo survive a 5-day incubation in vitro unless IL-3, IL-5, or GM-CSF is added to the medium. Conditions that inhibit adhesion to LM (anti-integrin antibodies in the medium or CT on the substrate) and certain anti-cy-tokine antibodies inhibited the promotion of Eo viability by LM. During incubation on LM, Eo become hypodense, as they do in the presence of IL-5, indicating that they have become activated. These observations suggest that the interactions of Eo and ECM proteins may be important both for their potential to direct Eo migration and for their ability to regulate Eo viability, cytokine production, and maturation.     

A micro-anatomical model of the distribution of myocardial endomysial collagen

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.