Objective. The goal of this study was to quantitatively analyze the distribution of collagen synthesis in normal and systemic sclerosis (SSc) fibroblast populations in order to determine the extent of activation in SSc populations.Methods. We used quantitative in situ hybridization to assess the population distribution of type I collagen synthesis. Fibroblast cultures were derived from both clinically involved and uninvolved skin regions of patients with SSc, and from healthy adults, and assessed for levels of cyl(1) procollagen messenger RNA (mRNA).Results. Dermal fibroblasts from both patients with SSc and normal adults were heterogeneous for distribution of d ( I ) procollagen mRNA when assessed by in situ hybridization, with a wide range of grains per cell. In contrast, clones of neonatal fibroblasts showed a relatively homogeneous distribution of grain counts. Involved SSc skin fibroblasts had a larger proportion of cells in the high collagen-producing mRNA subpopulation (mean f SEM 28.4 f 6.85%), compared with normal fibroblasts (1.75 f 1.44%) and uninvolved fibroblasts (9.6 f 6.73%). Conversely, within the low collagen- Submitted for publication August 9, 1995; acceptcd in revised form March 4, 1996. producing mRNA subpopulation, involved fibroblasts had a smaller proportion of cells (mean f SEM 14.0 f 5.63%) than did uninvolved fibroblasts (37.8 2 13.690/0), while normal fibroblasts had a majority of the cells in this subpopulation (53.5 f 8.68%).Conclusion. These results suggest that only a specific subset of fibroblasts are activated in SSc, as evidenced by an increased proportion of cells with high levels of al(1) procollagen mRNA. Differences between the SSc and normal fibroblast populations appeared to be quantitative rather than qualitative. This may be a result of either clonal selection or selective activation in the pathogenesis of SSc.
The major histopathological feature of hypertrophic scar lesions is fibrosis, characterized by excessive accumulation of collagen. The purpose of this study was to determine if there is not only increased expression of collagen but also decreased expression of collagenase in hypertrophic scar fibroblasts. We compared the expression of mRNA for alpha 1 (I) and alpha 1 (III) collagen, and collagenase in cultured fibroblasts from different portions of hypertrophic scars and normal dermis. In hypertrophic scar fibroblasts, increased levels of alpha 1 (I) and alpha 1 (III) collagen mRNAs were observed in fibroblasts from the edge and outside of scar tissue, while normal levels were noted in fibroblasts from the centre of this tissue. In contrast, decreased levels of collagenase mRNA were found in the hypertrophic scar fibroblasts. The reductions were: centre (25% of the control) greater than the edge (43% of the control) greater than the outside (84% of the control). The changes in the collagenase mRNA levels of the hypertrophic scar fibroblasts correlated well with decreased collagenolytic activity as determined by the degradation rate of fluorescein isothiocyanate-labelled type I collagen in fibroblast culture supernatant. These results suggest that decreased expression of collagenase in hypertrophic scar fibroblasts may be one possible cause for the excessive accumulation of collagen in the skin lesions of hypertrophic scars.
Previous studies have demonstrated that the expression of type I collagen, the most abundant protein in the dermis, is reduced in in vitro-aging fibroblast cultures, but the mechanism controlling the reduction of type I collagen expression is not understood. Recent studies, however, have demonstrated that transforming growth factor beta (TGF beta) plays an important role in the regulation of type I collagen expression. The purpose of this study was to investigate the role of TGF beta in downregulation of type I collagen expression in in vitro-aged fibroblasts. We compared the expression of mRNA for alpha 1 (I) collagen, TGF beta, TGF beta type I receptor and TGF beta type II receptor in early and late-passage fibroblasts by Northern blot hybridizations. The mRNA levels of alpha 1(I) collagen, TGF beta, and TGF beta receptors I and II in late-passage fibroblasts were reduced to 62%, 62%, 59% and 59%, respectively, of those in early-passage fibroblasts. We also compared TGF beta receptor binding in early- and late-passage fibroblasts using receptor binding assays. The affinity of 125I-TGF beta in late-passage fibroblasts was lower than that in early-passage fibroblasts. These results suggest that the reduction of type I collagen expression in in vitro-aged fibroblasts is regulated by reduced expression of TGF beta and TGF beta receptors I and II and by decreased TGF beta receptor binding ability of the fibroblasts.
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