Aminoterminal extension peptides from type I procollagen normalize excessive collagen synthesis of scleroderma fibroblasts

Summary Fibroblasts derived from a skin biopsy of a patient with scleroderma in the sclerotic stage were shown to have a higher rate of DNA synthesis, and to synthesize more collagen than fibroblasts from a healthy control. The addition of procollagen peptides to the culture medium of scleroderma fibroblasts almost normalized the collagen synthesis. This observation indicates that the mechanism for the regulation of collagen synthesis by feed back inhibition of prollagen peptides is functioning in this disease. It is suggested that the level of biologically active procollagen peptides is lowered.

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Abbreviations BAPN -aminopropriontrile - CM carboxymethyl - DNA desoxyribonucleic acid - NCP non-collagenous proteins - MEM minimum essential medium     

Eosinophilic fasciitis. Increased collagen production and type I procollagen messenger RNA levels in fibroblasts cultured from involved skin

Two patients with eosinophilic fasciitis were studied to elucidate the activation of collagen production in this disorder. Histologic examination of biopsy specimens from the involved area of skin revealed the presence of inflammatory cell infiltrates and various degrees of collagen accumulation in the dermis, subcutis, fascia, and underlying muscle. Fibroblast cultures initiated from the involved skin exhibited 2.0- to 3.7-fold increased collagen production when compared with control fibroblast cultures established from the uninvolved skin of the same patients. Eosinophilic fasciitis fibroblasts also displayed 2.4- to 6.2-fold higher steady-state levels of type I procollagen messenger RNA than did the control cells, indicating pretranslational activation of type I procollagen gene expression. In addition, cellular fibronectin messenger RNA steady-state levels were elevated 1.9- to 3.3-fold in eosinophilic fasciitis fibroblasts. These results suggest that fibroblasts in the involved skin of patients with eosinophilic fasciitis exhibit an activated phenotype, similar to that of scleroderma fibroblasts, leading to accumulation of collagen in the skin and the underlying structures.     

Elevated prolidase activity in keloids: correlation with type I collagen turnover

BACKGROUND: Keloid pathogenesis involves an altered balance of extracellular matrix metabolism, mainly accumulation of type I collagen. This could be due to excessive synthesis or decreased degradation of matrix, or a combination of both processes. Prolidase, an imidodipeptide-cleaving cytosolic enzyme, plays an important role in the collagen catabolic process by recycling proline for collagen synthesis. Collagen accumulation in keloids is due to an imbalance in the steady state of collagen turnover. OBJECTIVES: To investigate prolidase activity and its role in the steady state of collagen turnover between normal skin and keloid tissue and their derived fibroblasts. METHODS: Ten sets of keloid and normal skin tissues and their derived fibroblasts were employed. Measurements were made of tissue prolidase activity, free proline level, and concentrations of the collagen synthesis product aminoterminal propeptide of type I procollagen (PINP) and the collagen degradative product carboxyterminal telopeptide of type I collagen (ICTP). Also, synthesis of collagens type I and III and matrix metalloproteinases 1 and 2 was investigated using Western blot analysis. RESULTS: Keloid tissues had a significant increase in prolidase activity, up to fourfold that in normal skin. The elevated prolidase activity was accompanied by an increase in tissue PINP and ICTP concentrations in keloid; in addition, the collagen turnover index (PINP/ICTP) was higher in keloids. CONCLUSIONS: The combination of elevated prolidase activity and associated higher collagen synthesis to degradation ratio in keloids suggests a possible metabolic process for the excessive accumulation of type I collagen in keloids.     

Ehlers-Danlos syndrome type I: a clinical and ultrastructural study of a family with reduced amounts of collagen type III

Ehlers-Danlos syndrome (EDS) type I was diagnosed in an 18-year-old girl on the basis of marked skin hyperextensibility with generalized loose-jointedness, pigmented paper-tissue scars, and a pronounced tendency to bruising. Her father and one of her sisters showed a similar phenotype. Her mother was normal. Light microscopy of skin biopsies showed large, irregular collagen fibres in the father and daughter, with normal findings in the mother. Electron microscopy of the skin sections revealed a variation in diameter and shape of the collagen fibrils as well as slight dilatation of the rough endoplasmic reticulum of fibroblasts in father and daughter, but normal findings in the mother. Cultured fibroblasts did not show these changes. Measurements of collagen synthesis by fibroblast cultures showed that type III collagen levels were reduced to 50% of normal in the father and daughter, and were normal in the mother. The alpha I (III) proteins had a normal molecular weight, determined by SDS-PAGE electrophoresis. The phenotypes and biochemical results in the family members tested were compatible with autosomal dominant transmission. To our knowledge, this is the first report of a type III collagen deficiency in Ehlers-Danlos syndrome type I. The findings in this family, especially the pronounced bruising tendency, illustrate the heterogeneity within type I EDS.     

A new method to measure type I and III collagen synthesis in human skin in vivo: demonstration of decreased collagen synthesis after topical glucocorticoid treatment.

Collagen is synthesized as procollagen and large extra domains known as propeptides are cleaved off enzymatically. In the present study we have measured the carboxyterminal propeptide of type I collagen (PICP) and the aminoterminal propeptide of type III collagen (PIIINP) in blister fluids of human skin. High concentrations of PICP were found in the spontaneous blisters of patients with bullous pemphigoid, erysipelas, or erythema multiforme. Detectable amounts were also found in suction blisters induced on healthy skin. Because the concentrations in suction blisters were several times higher than in corresponding serum, most of PICP and PIIINP was derived from the underlying dermis. This method was used for assessing type I and type III collagen synthesis after topical glucocorticoid treatment. Clobetasol-17-propionate (CP) decreased the concentrations of PICP by 75% after 1 d of treatment, the maximum inhibition (92%) being found after 2 d treatment. PIIINP was also affected. Hydrocortisone and hydrocortisone-17-butyrate also decreased the concentrations of PICP and PIIINP, but less markedly than CP. Partial recovery was seen 3 d after stopping the treatment. Thus measurement of collagen type specific propeptides in suction blisters can be used as an estimate of collagen synthesis in vivo, avoiding both local anesthesia and skin biopsing. With radioimmunoassays for PICP and PIIINP a large number of samples can also be processed simultaneously.     

Constitutive synthesis of a 92-kDa keratinocyte-derived type IV collagenase is enhanced by type I collagen and decreased by type IV collagen matrices.

Human keratinocytes synthesize interstitial collagenase, a 72-kDa gelatinase, and a recently described 92-kDa gelatinase/type IV collagenase. We examined the synthesis of this novel enzyme by basal keratinocytes apposed to plastic, basement membrane collagen (type IV), and interstitial dermal collagen (type I). Samples of conditioned medium were electrophoresed on a 10% polyacrylamide, gelatin-ladened zymogram. Protein bands with gelatin-cleaving properties were identified by clarification of the gel and quantified by densitometry. A 92-kDa band had marked gelatinolytic activity and increased in culture over 72 h. The identification of this 92-kDa band as type IV collagenase was demonstrated by Western immunoblotting using monospecific antibody to the 92-kDa type IV collagenase. Keratinocytes apposed to type I collagen exhibited a threefold increase in the synthesis of the 92-kDa enzyme compared to cultures apposed to type IV collagen and a 1.5-times increase compared to plastic. The specificity of this enhancement was shown by constant levels of other proteins (e.g., the 72-kDa gelatinase). This study demonstrates that cell-matrix interactions modulate the synthesis of a recently described, keratinocyte-derived, 92-kDa gelatinase and that specific collagen types (I versus IV) have opposite effects upon the synthesis of this enzyme.     

Pseudoscleroderma associated with lung cancer: correlation of collagen type I and connective tissue growth factor gene expression

Pseudoscleroderma as a paraneoplastic syndrome is a rare disease. We report here a patient with lung cancer (undifferentiated squamous cell carcinoma), who developed acrosclerosis. Using in situ hybridization, marked expression of alpha1(I)-collagen and connective tissue growth factor (CTGF) mRNA was found in fibroblasts scattered throughout the dermis. However, transforming growth factor (TGF)-beta1 expression was not detected. The pattern of CTGF gene expression and collagen synthesis was similar to that in systemic scleroderma. The absence of TGF-beta1 mRNA could indicate that tumour-derived factors induce the expression of CTGF.     

Effect of activated human mast cells and mast cell-derived mediators on proliferation,type I collagen production and glycosaminoglycans synthesis by human dermal fibroblasts

Although an increased number of mast cells in fibrotic tissues such as scleroderma, keloid or healing wound has been highlighted, it is still unclear whether or not mast cells are fibrogenic. The aim of the present study is to determine whether functionally active human mast cells can provide human dermal fibroblasts directly with fibrogenic properties. In order to examine the effects of IgE-mediated mast cell activation on fibroblast proliferation and synthesis of type I collagen, we utilized an in vitro defined system in which cultured human mast cells were co-cultured with human dermal fibroblasts. We also employed a three-dimensional fibroblast culture system using supplementation of L-ascorbic acid as an assay system to investigate the effects of mast cell-derived mediators on synthesis of glycosaminoglycans by human fibroblast. Fibroblast proliferation was actively stimulated with IgE-activated mast cells. However, this stimulatory effect was canceled in co-cultures with a higher number of IgE-activated mast cells. In the presence of a higher number of activated mast cells, the fibroblast cell layer was destroyed, in contrast to an intact cell layer in the presence of same number of the mast cells without activation. Type I collagen synthesis was unchanged in fibroblasts co-cultured with mast cells. The total amount of main disaccharide units, particularly DELTADi-HA, was increased when fibroblasts were exposed to histamine. Thus, we conclude that other factors, in addition to mast cells, are important in the development of human tissue fibrosis or sclerosis.     

Collagen gene expression in keloids: analysis of collagen metabolism and type I, III, IV, and V procollagen mRNAs in keloid tissue and keloid fibroblast cultures

Regulation of collagen gene expression was studied in keloids and fibroblast cultures established from keloid biopsies from 9 patients. The collagen concentration in keloid tissue was not different from that in normal skin. The activities of 2 enzymes catalyzing intracellular collagen biosynthesis, prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT) were significantly elevated in the keloids, the mean increase in the former enzyme being 5-fold and in the latter 3-fold with respect to the controls. The mean procollagen production rate in the keloid fibroblasts was at the control level, with only 1 keloid cell line showing a procollagen synthesis rate higher than the mean value + 2 SD of the controls. The mean PH and GGT activities of the keloid fibroblasts were not elevated, but PH activity in 2 cell lines and GGT activity in 1 cell line were higher than the mean + 2 SD for the controls. Cellular type I, III, IV, and V procollagen mRNAs were measured by slot blot hybridization using specific human cDNA clones for the various collagen types. The amounts of type I, III, and V procollagen mRNAs corresponded to the ratios in which these collagen types are produced by fibroblasts. No synthesis of type IV procollagen mRNA by keloid fibroblasts was observed. The total amount of type I and III procollagen mRNAs correlated significantly (p less than 0.01) with the procollagen synthesis rate measured after radioactive labeling of the cells in the keloid and control fibroblasts, indicating that collagen production in these cells is mainly controlled by regulating the final steady state levels of collagen mRNA. The results suggest that fibroblasts isolated from keloids often synthesize normal amounts of collagen.     

Co-ordinate induction of collagen type I and biglycan expression in keloids

Proteoglycans are macromolecules displaying structural roles as well as regulatory functions in the maintenance of the extracellular matrix. Biglycan/PG-I and decorin/PG-II are two small proteoglycans that are structurally related but differ considerably in their localization in vivo and behaviour in vitro. Decorin and, to a minor extent, biglycan, can be located at the surface of type I collagen fibrils and have been shown to influence collagen fibrillogenesis. However, the physiological role of biglycan in the dermis is not known. Biopsies obtained from keloids were bisected and processed for total RNA extraction and immunohistochemistry. Northern blot analysis of total RNA obtained from keloids with high growth tendency in vivo showed a marked induction of biglycan and collagen α1(I) mRNA expression in comparison with total RNA obtained from normal skin or keloids with little growth tendency. In contrast, decorin mRNA expression remained largely unaltered. Studying these biopsies by immunohistochemistry, decorin expression in the dermis was unaltered comparing normal and keloid tissue, whereas a markedly increased staining for biglycan was observed in the keloid tissue, which was most pronounced in the nodular formations, and was a characteristie feature of keloids. The altered expression of biglycan in keloid tissue might be involved in the abnormal regulation of extracellular matrix deposition either through the binding of growth factors or by influencing the three-dimensional organization of collagen fibres or associated molecules.     

Modulation of collagen type synthesis in organ and cell cultures of fibroblasts

Fibroblast cultures are widely used to study abnormalities of collagen metabolism in both inborn and acquired diseases. However, there is reason to question the extent to which the experimental information obtained from in vitro culture systems in fact reflects the in vivo situation. In the present study we analyzed the proportions of collagens I and III synthesized by human and mouse skin fibroblasts maintained under various culture conditions. The amount of type III collagen extracted from skin specimens was lower than that which was newly synthesized in organ culture. Cells obtained by enzymatic disintegration of skin specimens synthesized more type III collagen than fibroblasts grown from explants. However, subcultivation of the enzymatically liberated cells resulted in a continuous decline of type III collagen production which eventually reached levels similar to those observed in explant cultures.     

Systemic glucocorticoids decrease the synthesis of type I and type III collagen in human skin in vivo, whereas isotretinoin treatment has little effect

The effects of systemic glucocorticoid and isotretinoin treatments on type I and type III collagen synthesis in intact skin were investigated by measuring the carboxyterminal and aminoterminal propeptides of type I procollagen, and the aminoterminal propeptide of type III procollagen, in suction blister fluid (SBF), in a study of 27 patients. All three parameters were significantly lower in the SBF of glucocorticoid-treated patients than in controls or patients undergoing treatment with isotretinoin, whereas the latter two groups did not differ significantly from each other. During glucocorticoid treatment, the concentrations of the procollagen propeptides were only about 20% of the corresponding control values, indicating that systemic therapy with prednisone at a dose of 0.48 mg/kg per day almost totally abolishes collagen synthesis in the skin. These results indicate that systemic glucocorticoid treatment suppresses the synthesis of both type I and type III collagen in the dermis, and suggest that many side-effects of these drugs, such as atrophy of the skin, are due to this inhibition. Systemic isotretinoin treatment did not stimulate skin collagen synthesis. Thus, its regenerative effect on connective tissue may be mediated by mechanisms other than direct stimulation of collagen synthesis.     

Activation of collagen gene expression in keloids: co-localization of type I and VI collagen and transforming growth factor-beta 1 mRNA

Untreated, clinically active keloids were examined as model system to study the spatial expression of extracellular matrix and transforming growth factor-beta 1 (TGF-beta 1) genes in fibrotic skin diseases. In situ hybridizations localized active expression of type I and VI collagen genes to the areas containing an abundance of fibroblasts and apparently representing the expanding border of the lesions. Within this zone, microvascular endothelial cells also expressed the type I collagen genes, as evaluated by simultaneous use of in situ hybridization for collagen gene expression and immunolocalization for factor VIII-related antigen, a marker for endothelial cell differentiation. Slot-blot hybridizations of RNA isolated from this zone suggested that the expression of type I and IV collagen genes was selectively enhanced, as compared to type III collagen gene expression. TGF-beta 1 protein and mRNA were also detected in areas active in type I and type VI collagen gene expression, indicating that TGF-beta 1 gene is transcribed and the corresponding protein is deposited in areas of elevated collagen gene expression, including microvascular endothelial cells. We conclude that the initial step in the development of fibrotic reaction in keloids involves the expression of the TGF-beta 1 gene by the neovascular endothelial cells, thus activating the adjacent fibroblasts to express markedly elevated levels of TGF-beta 1, as well as type I and VI collagen genes.     

Effects of UVA irradiation following treatment with 8-methoxypsoralen on type I and type III collagen synthesis in normal and scleroderma fibroblast cultures

Recent studies have demonstrated the efficacy of PUVA (psoralen plus ultraviolet A irradiation) therapy against sclerotic skin lesions in scleroderma, although the mechanisms underlying the improvement of the skin sclerosis by this therapy remain unknown. We investigated the effects of ultraviolet A (UVA) irradiation following the treatment with 8-methoxypsoralen on types I and III collagen synthesis and the gene expression of collagenase in cultured normal and scleroderma fibroblasts. The treatment reduced types I and III collagen synthesis and consequently, the types I and III collagen mRNA levels, in a UVA dose-dependent manner in both the normal and SSc fibroblasts, whereas the mRNA levels of collagenase remained almost unaltered. These results suggest that reduction of collagen synthesis by the fibroblasts may be one of the mechanisms underlying the efficacy of PUVA therapy against the sclerotic skin lesions in scleroderma.