Masami Arakawa

Reduced collagenase gene expression in fibroblasts from hypertrophic scar tissue.

Summary 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 of α1 (I) and α1 (III) collagen, and collagenase in cultured fibroblasis from different portions of hypertrophic scars and normal dermis. the hypertrophic scar fibroblasts. increased levels of α1 (I) and α1 (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 fluorescent 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.

Reduced expression of mRNA for transforming growth factor β(TGFβ) and TGFβ receptors I and II and decreased TGFβ binding to the receptors in in vitro-aged fibroblasts

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 β (TGFβ) plays an important role in the regulation of type I collagen expression. The purpose of this study was to investigate the role of TGFβ in downregulation of type I collagen expression in in vitro-aged fibroblasts. We compared the expression of mRNA for α1(I) collagen, TGFβ ,TGFβ type I receptor and TGFβ type II receptor in early- and late-passage fibroblasts by Northern blot hybridizations. The mRNA levels of α1(I) collagen, TGFβ, and TGFβ 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 TGFβ receptor binding in early- and late-passage fibroblasts using receptor binding assays. The affinity of  125I-TGFβ 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 TGFβ and TGFβ receptors I and II and by decreased TGFβ receptor binding ability of the fibroblasts.

Analysis of collagen gene expression by cultured fibroblasts in morphoea

Collagen gene expression was studied in dermal fibroblasts derived from the inflammatory and sclerotic skin lesions of patients with localized or generalized morphoea. The levels of mRNA for type I collagen in early‐passage fibroblasts derived from inflammatory lesions were higher than those obtained from the uninvolved skin, whereas those fibroblasts obtained from sclerotic lesions were unaltered. No alteration in type I collagen mRNA levels was observed in late‐passage fibroblasts derived from the inflammatory lesions. The relative rate of collagen to total protein synthesis in earlypassage fibroblasts derived from inflammatory lesions was higher than that of fibroblasts from uninvolved skin, while no alteration or a slight decrease was observed in fibroblasts from the sclerotic lesions. The data suggest that the inflammatory reactions induce increased collagen synthesis by fibroblasts in the skin in scleroderma.

Effects of cyclosporin A on cell proliferation and collagen production by human skin fibroblasts

Cyclosporin A (CSA) is a potent immunosuppressive drug that has been used clinically for the treatment of organ rejection after transplantation as well as for patients with a wide variety of immune-mediated disorders. CSA has recently been reported to be effective in systemic sclerosis, which is a disease of the connective tissues leading to fibrosis of the skin and other involved organs. In this study, we investigated whether CSA affects the cell proliferation and collagen synthesis of human skin fibroblasts. CSA inhibited the DNA synthesis and cell growth of cultured fibroblasts at concentrations of 10(-8) M to 10(-5) M in a dose-dependent manner. The production of both collagen and non-collagenous protein at both the mRNA and protein levels was not affected by 10(-8) to 10(-6) M CSA, but was decreased in the presence of 10(-5) M CSA. These results suggest that CSA may inhibit the proliferation of fibroblasts, but not their synthesis of collagenous and non-collagenous proteins at therapeutic concentrations.

Effects of tumor necrosis factor-α on connective tissue metabolism in normal and scleroderma fibroblast cultures

Recent studies have demonstrated that tumor necrosis factor-α(TNF-α) selectively decreases production of collagens I and III, the major types of collagen in the dermis, and increases production of collagenase in cultured dermal fibroblasts. The effects of TNF-α on collagens I, III and VI, fibronectin and collagenase gene expression by fibroblasts derived from normal individuals and patients with systemic sclerosis (SSc) were studied. SSc is characterized by excessive accumulation of collagen in the skin and in certain organs. TNF-α inhibited collagen production and mRNA levels of collagens I and III and of fibronectin, and stimulated collagenase activity and collagenase mRNA levels in SSs fibroblasts. Levels of mRNA for α1(VI) and α3(VI) collagen and for Β-actin were unaltered in SSc fibroblasts incubated with TNF-α. Similar results were observed for mRNA levels in normal fibroblasts incubated with TNF-α. These results suggest that TNF-α could be expected to be beneficial in the treatment of SSc. In addition, our results indicated that collagen-VI expression is regulated independently from expression of collagens I and III, and expression of fibronectin and collagens I and III are regulated in parallel in fibroblasts treated with TNF-α.

Increased expression of type VI collagen genes in cutis laxa fibroblasts

Type VI collagen gene expression in cutis laxa was studied by measuring messenger RNA (mRNA) and protein production levels in four fibroblast strains from patients with congenital cutis laxa and comparing them with those in fibroblasts obtained from age-matched healthy subjects. Levels of type VI collagen mRNA were increased in all cutis laxa fibroblast strains and the levels of alpha 1 (VI) and alpha 3 (VI) chain mRNAs increased in parallel. Increases in type VI collagen mRNAs correlated well with production levels of the corresponding proteins, as determined by immunological assay. These results suggest that increased type VI collagen gene expression is one of the characteristics of cutis laxa dermal fibroblasts and that this abnormality may be related to the skin changes in cutis laxa.

Experimental immune complex arteritis under embolization with gelatin sponge powder: A new model for induction

Immune complexes may play an important role in causing necrotizing vasculitis of the skin. In our previous studies of active or reversed passive Arthuss reaction using horseradish peroxidase (HRP) as antigen, immune complexes were found to be deposited on venules and followed by neutrophil infiltrations [4 7]. Immune complexes did not, however, locate on arterial walls. Some conditions, such as emboli, may be necessary for antigen or immune complexes to stay in an artery long enough to bring about an immune reaction. In order to induce a new model of immune complex arteritis, gelatin sponge (Spongel, Yamanouchi) powder was saturated with an antigen solution and utilized as an embolizing agent. Three albino rabbits weighing 3 4 kg were sensitized by intracutaneous (IC) injection of 6 mg HRP (Type 6, Sigma) suspended in 0.3 ml Freunds incomplete adjuvant; the rabbits were then injected with 3 mg HRP after 2 and 4 weeks. At 5 weeks after sensitization, animals were checked by IC injection of 0.05 mg HRP, and positive erythematous reactions were confirmed. These sera showed only a single line against type-6 HRP solution in agar gel precipitate. Gelatin sponge was pulverized with a file as finely as possible. Sensitized rabbits auricular arteries were embolized by intraarterial injections of 10 mg gelatin sponge powder saturated with 10 mg HRP dissolved