Preparation of antibodies against the new collagen VI chains == The purified recombinant collagen VI fragments were used to immunize rabbits and guinea pigs

Preparation of antibodies against the new collagen VI chains == The purified recombinant collagen VI fragments were used to immunize rabbits and guinea pigs. mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed -smooth muscle actin. TGF-1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the Daidzein 6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the 6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the 6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the 5 chain was undetectable. Our results show a restricted and differential distribution of the novel 6 and 5 chains in skeletal ZPKP1 muscle when compared to the widely distributed, homologous 3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the 5 chain may have a specialized function in tissue areas subjected to tensile stress, the 6 chain appears implicated in ECM remodeling during muscle fibrosis. Keywords:Collagen VI, Skeletal muscle, Myotendinous junctions, Fibrosis, Duchenne muscular dystrophy == Highlights == We study collagen VI alpha 5 and alpha 6 chains expression Daidzein in human skeletal muscle. We show that alpha 5 chain is exclusively present at the myotendinous junctions. We show that alpha 6 chain is present in the ECM but not at the basement membranes. We show that alpha 6 chain increases in Duchenne muscular dystrophy muscle fibrosis. We hypothesize the possible formation of alpha1-2-6 or alpha1-2-5 chains trimers. == 1. Introduction == Collagen VI is an extracellular matrix Daidzein protein which forms a distinct microfibrillar network in most connective tissues. It was long considered to consist of three genetically distinct -chains (1, 2 and 3), secreted into the extracellular matrix where they form an extended microfilamentous network (Chu et al., 1988; Knupp and Squire, 2001). The collagen VI 1, 2 and 3 chains form heterotrimeric monomers that are assembled intracellularly to dimers and tetramers. After secretion, filaments are formed by end to end interactions of the pre-assembled tetramers, forming characteristic beaded filaments (Bruns, 1984) as visualized by the rotary shadowing electron microscopy technique (von der Mark et al., 1984; Sabatelli et al., 2001). Collagen VI has a critical role in maintaining skeletal muscle functional integrity; in fact, mutations in the COL6A1, COL6A2, and COL6A3 genes cause a group of inherited muscular dystrophies, namely Ullrich congenital muscular dystrophy (UCMD) (Camacho Vanegas et al., 2001), Bethlem myopathy (BM) Daidzein (Jbsis Daidzein et al., 1996; Lampe and Bushby, 2005; Gualandi et al., 2009), and Myosclerosis myopathy (Merlini et al., 2008). Col6a1/mice show a complete absence of collagen VI chains (Bonaldo et al., 1998; Gara et al., 2008) and display a myopathic phenotype associated with an impairment of the autophagy flux that determines the presence of dilated sarcoplasmic reticulum and abnormal mitochondria: these present a latent dysfunction, that causes an increased apoptosis of muscle fibers (Irwin et al., 2003; Grumati et al., 2010). Moreover, zebrafish models of the human collagen VI myopathies develop myopathy and display mitochondrial ultrastructural changes along with an increased rate of spontaneous apoptosis (Telfer et al., 2010). Collagen VI is also thought to be implicated in tissue remodelling and wound healing, as variations in collagen VI expression have been reported in many pathological conditions, like muscle and liver fibrosis (Freise et al., 2009). Fibrosis is the most conspicuous pathological change in dystrophic muscles; it is characterized by excessive accumulation of collagens and other ECM components, including collagen VI (Zanotti et al., 2007) and is regulated by mechanisms involving cellcell and cellmatrix interactions, as well as by factors secreted into the ECM. TGF-1 is one of the most potent regulators of tissue wound healing and fibrosis (Cutroneo, 2007). It is highly expressed in regenerating muscle after injury and in dystrophic muscles, including Duchenne muscular dystrophy patients and mdx mice (Zhou et al., 2006), carrying mutations in the dystrophin gene, and in MDC1A, caused by mutations in LAMA2 gene (Zanotti and Mora, 2006). Recently, three novel collagen VI chains, 4, 5 and 6 were identified (Fitzgerald et al., 2008; Gara et al., 2008). The new chains structurally resemble the collagen VI 3 chain, each of them consisting of seven VWA domains followed by a collagenous domain, two or three C-terminal VWA domains.