Alterations of overused supraspinatus tendon: a possible role of glycosaminoglycans and HARP/pleiotrophin in early tendon pathology.

Supraspinatus tendon overuse injuries lead to significant pain and disability in athletes and workers. Despite the prevalence and high social cost of these injuries, the early pathological events are not well known. We analyzed the potential relation between glycosaminoglycan (GAG) composition and phenotypic cellular alteration using a rat model of rotator cuff overuse. Total sulfated GAGs increased after 4 weeks of overuse and remained elevated up to 16 weeks. GAG accumulation was preceded by up-regulation of decorin, versican, and aggrecan proteoglycans (PGs) mRNAs and proteins and biglycan PG mRNA after 2 weeks. At 2 weeks, collagen 1 transcript decreased whereas mRNAs for collagen 2, collagen 3, collagen 6, and the transcription factor Sox9 were increased. Protein levels of heparin affine regulatory peptide (HARP)/pleiotrophin, a cytokine known to regulate developmental chondrocyte formation, were enhanced especially at 4 weeks, without up-regulation of HARP/pleiotrophin mRNA. Further results suggest that the increased GAGs present in early lesions may sequester HARP/pleiotrophin, which could contribute to a loss of tenocyte's phenotype. All these modifications are characteristic of a shift towards the chondrocyte phenotype. Identification of these early changes in the extra-cellular matrix may help to prevent the progression of the pathology to more disabling, degenerative alterations.

Synthesis and biological activities of a library of glycosaminoglycans mimetic oligosaccharides.

Biologically active oligosaccharides related to glycosaminoglycans are accumulating increased attention because of their therapeutic potential and for their value in mechanistic studies. Heparan mimetics (HMs) are a family of dextran based polymer known to mimic the properties of glycosaminoglycans, and particularly those of heparan sulfates, as to interact with heparin binding proteins. HMs have shown to stimulate tissue repair in various animal models. Here, we use different methods to depolymerize HMs in order to produce a library of related oligosaccharides and study their biological activities. Since HMs were resistant to endoglycanases activities, depolymerization was achieved by chemical approaches. In vitro biological studies showed that HM oligosaccharides can differentially potentiate FGF-2 mitogenic and antithrombotic activities. In vivo, a selected oligosaccharide (H-dp12) showed to be able to regenerate tissue almost as well as the related polymeric product. The very low anticoagulant activity and high biological activity of low mass oligosaccharides give to these products a new therapeutic potential.

A synthetic glycosaminoglycan mimetic (RGTA) modifies natural glycosaminoglycan species during myogenesis.

Crucial events in myogenesis rely on the highly regulated spatiotemporal distribution of cell surface heparan sulfate proteoglycans to which are associated growth factors, thus creating a specific microenvironment around muscle cells. Most growth factors involved in control of myoblast growth and differentiation are stored in the extracellular matrix through interaction with specific sequences of glycosaminoglycan oligosaccharides, mainly heparan sulfate (HS). Different HS subspecies revealed by specific antibodies, have been shown to provide spatiotemporal regulation during muscle development. We have previously shown that glycosaminoglycan (GAG) mimetics called RGTA (ReGeneraTing Agent), stimulate muscle precursor cell growth and differentiation. These data suggest an important role of GAGs during myogenesis; however, little is yet known about the different species of GAGs synthesized during myogenesis and their metabolic regulation. We therefore quantified GAGs during myogenesis of C2.7 cells and show that the composition of GAG species was modified during myogenic differentiation. In particular, HS levels were increased during this process. In addition, the GAG mimetic RGTA, which stimulated both growth and differentiation of C2.7 cells, increased the total amount of GAG produced by these cells without significantly altering their rate of sulfation. RGTA treatment further enhanced HS levels and changed its sub-species composition. Although mRNA levels of the enzymes involved in HS biosynthesis were almost unchanged during myogenic differentiation, heparanase mRNA levels decreased. RGTA did not markedly alter these levels. Here we show that the effects of RGTA on myoblast growth and differentiation are in part mediated through an alteration of GAG species and provide an important insight into the role of these molecules in normal or pathologic myogenic processes.

Upregulation of HARP during in vitro myogenesis and rat soleus muscle regeneration.

Heparin affin regulatory peptide (HARP) is a heparin binding growth factor that belongs to a family of molecule whose biological function in myogenesis has been suspected without formal demonstration. In the present study, we investigated the expression and the distribution of HARP and its mRNA during soleus muscle regeneration using a crushed-induced regeneration model and also during differentiation of muscle satellite cells in primary cultures. We show that HARP mRNA and protein expression are increased during the regeneration process with a peak at day 5 after muscle crushing when new myotubes are formed. In situ hybridization and immunohistochemical studies showed that activated myoblasts expressed HARP at day two after crushing. Five days after muscle lesion, HARP is localised in newly formed myotubes as well as in prefused activated myoblasts. In regenerated myofibers, 15 days after crushing, expression of HARP was reduced. In vitro experiments using primary cultures of rat satellite cells indicated that HARP expression level increased during the differentiation process and peaked on fusion of myoblasts into myotubes. This is the first study demonstrating the presence of HARP in fusing myogenic cells suggests that this growth factor could play a function in myogenic differentiation.

Glycosaminoglycan mimetics (RGTA) modulate adult skeletal muscle satellite cell proliferation in vitro.

Muscle regeneration occurs through the activation of satellite cells, which are stimulated to proliferate and to fuse into myofibers that will reconstitute the damaged muscle. We have previously reported that a family of new compounds called "regenerating agents" (RGTAs), which are polymers engineered to mimic heparan sulfates, stimulate in vivo tissue repair. One of these agents, RG1192, a dextran derivative substituted by CarboxyMethyl, Benzylamide, and Sulfate (noted CMBS, RGTA type), was shown to improve greatly the regeneration of rat skeletal muscle after severe crushing, denervation, and acute ischemia. In vitro, these compounds mimic the protecting and stabilizing properties of heparin or heparan sulfates toward heparin-binding growth factors (HBGFs). We hypothesized that RGTA could act by increasing the bioavailability of some HBGF involved in myoblast growth and thus asked whether RGTA would alter the ability of satellite cells to proliferate. Its effect was tested on primary cultures of rat satellite cells. The RG1192 stimulated the proliferation of satellite cells in vitro in a dose-dependent manner. It appeared to be as efficient as natural glycosaminoglycans (GAGs; heparan sulfate, dermatan sulfate, or keratan sulfate) in stimulating satellite cell proliferation but was about 100 times more efficient than heparin. RG1192 stimulated satellite cell proliferation by increasing the potency of fibroblast growth factor 2 and scatter factor-hepatocyte growth factor. It also partially restored myoblast proliferation of satellite cells with chlorate-induced hyposulfation. Taken together, our results explain to some extent the improving effect of RGTA with a CMBS structure, such as the RG1192, on muscle regeneration in vivo by providing support for the hypothesis that RGTA may act by increasing the potency of some HBGFs during the proliferation phase of the regenerating muscle.