Implication of the satellite cell in dystrophic muscle fibrosis: a self-perpetuating mechanism of collagen overproduction.

Because of its mechanical function, skeletal muscle is heavily influenced by the composition of its extracellular matrix (ECM). Fibrosis generated by chronic damage, such as occurs in muscular dystrophies, is thus particularly disastrous in this tissue. Here, we examined the interrelationship between the muscle satellite cell and the production of collagen type I, a major component of fibrotic ECM, by using both C2C12, a satellite cell-derived cell line, and primary muscle satellite cells. In C2C12 cells, we found that expression of collagen type I mRNA decreases substantially during skeletal muscle differentiation. On a single-cell level, collagen type I and myogenin became mutually exclusive after 3 days in differentiation medium, whereas addition of collagen markedly suppressed differentiation of C2C12 cells. Primary cultures of satellite cells associated with isolated single fibers of the young (4 wk old) mdx dystrophic mouse and of C57BL/10ScSn wild-type controls expressed collagen type I and type III mRNA and protein. This pattern persisted in wild-type mice at all ages. But, curiously, in older (18-mo-old) mdx mice, although the myogenic cells continued to express type III collagen, type I expression became restricted to nonmyogenic cells. These cells typically constituted part of a cellular sheet surrounding the old mdx fibers. This combination of features strongly suggests that the progression to fibrosis in dystrophic muscle involves changes in the mechanisms controlling matrix production, which generates positive feedback that results in a reprogramming of myoblasts to a profibrotic function.

Bone marrow-derived cells do not contribute significantly to collagen I synthesis in a murine model of renal fibrosis.

Interstitial fibroblasts play a central role in kidney fibrosis. Their origin is debated, with recent data indicating a contribution of bone marrow (BM)-derived cells to the expanded population of interstitial cells after kidney damage in animals and humans. This study investigated whether these BM-derived cells would respond appropriately to a fibrotic drive by producing collagen. A transgenic mouse that expresses both luciferase and beta-galactosidase reporter molecules under the control of a 17-kb promoter and enhancer element of the gene encoding the alpha2 chain of the collagen I was used. Male transgenic BM was transplanted into female wild-type C57BL/6 mice (n=14), and unilateral ureteric obstruction was performed later to induce renal fibrosis. In the obstructed kidney of the BM-chimeric female mice, a mean of 8.6% of smooth muscle actin-positive interstitial cells were Y chromosome positive. Increased collagen I mRNA in the obstructed kidney was detected by in situ hybridization. No luciferase activity was detected by enzyme assays in tissue homogenates of BM recipients, and very few luciferase mRNA transcripts were seen, mainly in tubular cells. beta-Galactosidase activity was not a useful reporter molecule because it could not be distinguished from enhanced endogenous beta-galactosidase activity in the obstructed kidney. These results indicate that BM-derived interstitial cells do not make a significant contribution to collagen I synthesis in the context of renal injury.

Organ-specific collagen expression: implications for renal disease.

Chronic kidney disease is characterized by progressive accumulation of extracellular matrix and scarring, leading to the loss of kidney function. Excess deposition of the collagen family of proteins is the hallmark of kidney fibrosis. In this review, we survey the collagens that are associated with renal disease and we highlight the use of a transgenic approach to identify cis-acting sequences in the collagen type I promoter which are capable of directing collagen type I expression specifically in the kidney. Ultimately it may be possible to use this approach to halt the accumulation of collagen selectively in this organ.

Structurally different RGTAs modulate collagen-type expression by cultured aortic smooth muscle cells via different pathways involving fibroblast growth factor-2 or transforming growth factor-beta1.

We have engineered polymers called ReGeneraTing Agents (RGTAs), which mimic the protecting and potentiating properties of heparan sulfates toward heparin-binding growth factors (HBGF). RGTAs have been shown to optimize cell growth and regulate collagen production in vitro. Here, we studied relationships between RGTA structure and collagen-type expression in aortic smooth muscle cells by using two RGTAs, the carboxylmethylsulfate dextran RG-1503 and the carboxylmethylsulfate dextran with added benzylamide RG-1192. RG-1192 specifically induced a fivefold decrease in collagen III synthesis. This effect was abolished by FGF-2 neutralizing antibody. RG-1192 and FGF-2 acted synergistically to decrease collagen III. RG-1192 was more effective than heparin in this process. RG-1192 increased the pericellular localization of FGF-2 and protected FGF-2 from proteolysis. Surface plasmon resonance analysis indicated a Kd of 15.7 nM for the RG-1192/FGF-2 interaction (10.6 nM for the heparin/FGF-2 interaction). The structurally different RG-1503 (without benzylamide) did not interact with FGF-2 and worked synergistically with TGF-beta1 to specifically induce a twofold increase in collagen V. RGTAs with different structures exert different modulating effects on the collagen phenotype. Selection of appropriate RGTAs, which had been shown to enhance in vivo tissue repair, may provide a mean of correcting collagen abnormalities in vascular disorders and more generally in fibrotic diseases.

Col1a2 enhancer regulates collagen activity during development and in adult tissue repair.

An enhancer region in the type I collagen alpha 2 chain (pro-Col1a2) promoter has been previously identified approximately -17 kb away from the transcription start site. This upstream region termed the far-upstream-enhancer contains three DNAse I hypersensitive sites and has been shown to be conserved between mouse and human genes. In this study, we used transgenic mice harbouring the complete promotor sequence of the pro-Col1a2 gene up to -17 kb to examine the role of this enhancer in the expression and regulation of the collagen gene during development and in adult tissues pre and post injury. By careful histological mapping of the collagen type I endogenous gene distribution with that of the transgene driven by the mouse far upstream enhancer, we are able to show that in early days of collagen expression, E8.5-9.5, the endogenous gene preceded transgene expression. However, by E11.5 the overall pattern becomes synchronous with a few exceptions. In adult tissue, both endogenous and transgene expression are attenuated and both are reactivated in parallel in various organs by physical injury or fibrogenic cytokine injection. These findings suggest that the enhancer is central to the activation of the collagen type I and that mice harbouring this enhancer/reporter provide a useful model to follow collagen gene transcription activity and for investigating cellular activity in tissue fibrosis.

Heparin-like polymer improved healing of gastric and colic ulceration.

A family of chemically substituted biopolymers has been developed to protect and stabilize heparin binding growth factors and was shown to enhance tissue repair in various in vivo models. One of these compounds, a dextran derivative named RGTA11, was tested for its ability to treat acute gastritis and colic ulceration models induced by ethanol and acid. RGTA was not efficient in reducing nor in protecting against gastric acidic secretion compared to EGF. Ethanol gastritis measured by the alteration score of the injured mucosa was reduced by 56% with the oral administration of RGTA at doses of 100 microg/kg (p < 0.01). A similar effect was obtained by PGE2 at a similar dose. Alterations of the colic mucosa were reduced after 72 h by 75% after oral administration of RGTA11. RGTA presents both anti-inflammatory and tissue repair activities mediated by growth factor protection. These two properties would be beneficial for digestive ulcer treatment. The results presented here provide evidence for these effects.