The fibroblastic nature of dermatofibrosarcoma protuberans: Morphological investigations in vivo and in vitro

Summary Six cases of dermatofibrosarcoma protuberans were studied ultrastructurally. Biopsy material from all six cases as well as cell cultures derived from four cases were examined. In all cases, the tumor cells in vivo and in vitro were related to fibroblasts. The two cases with histiocytoid features and a small spiral pattern exhibited numerous lipid vacuoles but no histiocytic cell markers. In these two cases, cultured cells contained a higher number of intracytoplasmic vacuoles than the control fibroblasts. Two other cases exhibited some basement-membrane-like material surrounding tumor cells. All of the investigated cell strains obtained from the tumors showed synthesis of collagenous proteins similar to that found in fibroblasts. However, the level of total collagen was reduced, and type-III collagen was absent.The morphological variations in these cases of dermatofibrosarcoma protuberans appeared to be related to the histological pattern and may reflect the heterogeneity of normal fibroblasts.     

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.     

Alterations in scleroderma fibroblast surface glycoproteins associated with increased collagen synthesis

Fibroblasts were cultured from affected and unaffected skin sites of 6 patients with localized scleroderma. As a parameter of fibroblast activation, collagen synthesis and cellular pro alpha 1(I)collagen mRNA levels were measured. Cell surface glycoproteins were labelled with the periodate/borohydride method and fractionated electrophoretically. A distinct reduction in the relative amount of surface glycoproteins in the 120 kDa region was observed in two affected cell lines producing increased amounts of collagen and in one affected cell line with normal collagen production when compared to unaffected fibroblast lines. Other, non-systematic alterations in the surface glycoproteins of the affected cell lines were also detected. Compared to unaffected and healthy control fibroblasts no alterations in the surface protein profiles were seen in the other three affected cell lines. These cells did not show an increase in collagen production either. The results suggest that the activation of collagen synthesis found in scleroderma fibroblasts might be connected with alterations in the normal cell surface glycoprotein pattern.     

Necrobiosis lipoidica: ultrastructural and biochemical demonstration of a collagen defect

Ten patients with necrobiosis lipoidica lesions were studied. Five patients had diabetes mellitus. The age of the patients varied from 15 to 73 years and the duration of the skin lesions was from 2 to 20 years. Histologically, the lesions were characterized by degeneration of collagen and elastin. In some lesions elastin fibers could be seen in areas devoid of normal-looking collagen. Electron microscopy revealed loss of cross-striation of collagen fibrils and a marked variation in the diameter of individual collagen fibrils. The concentration of collagen, measured by assay of hydroxy-proline, a collagen-specific amino acid, was markedly decreased in the lesional skin, but the ratio of type I/III collagen was unchanged in the affected skin. Fibroblasts established from affected skin synthesized less collagen than cells derived from healthy-looking skin. The decreased collagen synthesis was due to a decreased amount of messenger RNA for type I procollagen, measured by hybridization with a specific human cDNA clone. The production of collagenase by these fibroblasts was not increased. Our results thus indicate that in necrobiosis lipoidica lesions, collagen fibrils are defective and the amount of collagen is reduced, probably due to decreased synthesis of collagen by affected fibroblasts.     

Factor XIII in scleroderma: in vitro studies

The administration of Factor XIII (FXIII) produces a beneficial effect on the skin lesions in about 50% of the treated patients with progressive systemic sclerosis (PSS). The effect of FXIII on various skin fibroblast functions (proliferation, attachment, biosynthetic activity and mechanical properties) was investigated in vitro using normal and PSS strains. In cell culture, most of the PSS fibroblast strains synthesized excessive amounts of collagen. Other cell functions such as adhesion to collagen I or III, to fibronectin, retraction of collagen lattices, proliferation in low serum concentration and degradation of newly synthesized collagen were not significantly different. The addition of FXIII (I U/ml) inhibited the synthesis of collagen by normal fibroblasts and reduced it in PSS fibroblasts to a level similar to that of normal fibroblasts. This effect was observed for cells cultured on plastic or in a collagen lattice. In the latter, an increased amount of collagen degradation was observed. No significant effect of FXIII on the other cell functions was noted. Excessive collagen production by PSS fibroblasts can be repressed by FXIII in vitro by at least two distinct mechanisms: a reduction of collagen synthesis and an increased degradation of the newly synthesized collagen.     

Hexose sugars differentially alter collagen gene expression and synthesis in fibroblasts derived from granulation tissue,hypertrophic scar and keloid

Clinical observations have suggested that sugar and honey enhance granulation tissue formation and in vitro studies have shown that monosaccharide sugars stimulate mesenchymal and endothelial cells. In this study, the effects of glucose, fructose, galactose and mannose on type I and type III collagen gene expression and synthesis were studied in granulation tissue, hypertrophic scar and keloid fibroblast cultures. Glucose elevated both type I and type III collagen mRNAs in hypertrophic scar fibroblasts. Fructose increased type III collagen mRNA almost sevenfold in granulation tissue fibroblasts. Galactose caused an increase in type I and type III collagen mRNAs in granulation tissue fibroblasts and hypertrophic scar fibroblasts but, in contrast, mannose decreased type I and type III collagen levels in hypertrophic scar and keloid fibroblasts. Analysis of aminoterminal propeptides of type I and type III collagen (PINP and PIIINP) revealed that glucose decreased the amount of PINP in granulation tissue and keloid fibroblasts, whilst fructose decreased the amount in all the fibroblast cell lines studied. Galactose caused a decrease in the synthesis of type I collagen in all cell lines but a decrease was seen in type III collagen only in hypertrophic scar fibroblasts. Mannose decreased the amount of PINP in all cell lines but a decrease in the amount of PIIINP was seen only in granulation tissue fibroblasts. The effect of sugars on the ratio type I/type III collagen was negligible or decreasing with the exception of galactose, which increased the ratio in hypertrophic scar fibroblasts. The results suggest that glucose, fructose and galactose have no significant value in the stimulation of collagen synthesis in vitro. Mannose may have value in the prevention or treatment of abnormal scars.     

Lipoid proteinosis. A biochemical and ultrastructural investigation of two new cases

Lipoid proteinosis is a rare autosomal recessive disease characterized by cutaneous and visceral lesions, in which large amounts of amorphous material are constantly found in stroma. Morphological and biochemical studies indicate abnormal collagen production, but little attention has been paid to the lipid component of lesions. Microscopic and ultrastructural studies of skin, with special emphasis on fibroblasts, vessels, nerve endings and eccrine sweat glands, were conducted in two patients with lipoid proteinosis. Biochemical studies were undertaken in cultured fibroblasts. Evidence of lysosomal storage in epithelial cells of eccrine sweat glands and in dermal histiocytes, very similar to that found in some metabolic disorders, particularly Farber disease, was found in both cases. Our findings suggest that two alterations might coexist in lipoid proteinosis, one characterized by impaired normal collagen production and the other related to a metabolic defect which may lead to accumulation of ceramide or more complex lipids.     

Keloid-derived fibroblasts show increased secretion of factors involved in collagen turnover and depend on matrix metalloproteinase for migration

BACKGROUND: A keloid is a specific skin lesion that expands beyond the boundaries of the original injury as it heals. Histologically, it is characterized by the excessive accumulation of collagen. However, the reasons for the expansion and the invasive nature of keloids remain unknown. OBJECTIVES: We evaluated collagen degradation and migration by cultured keloid fibroblasts based on the assumption that these variables were of functional relevance to the expanding and invasive nature of keloid lesions. METHODS: Collagen production was investigated by the detection of type 1 collagen (procollagen type 1C peptide: P1P). Matrix metalloproteinase (MMP)-1 (interstitial collagenase) and MMP-2 (gelatinase-A), were investigated as elements of the collagen degradation system. Enzyme immunoassays were performed to measure the production of P1P, MMP-1, MMP-2, and tissue inhibitor of metalloproteinase (TIMP)-1. To assess the production of MMP-2 its gelatinolytic activity was measured by zymography using gelatin-containing gels. The participation of transforming growth factor-beta1 (TGF-beta1) in the production and degradation of collagen was also investigated. Finally, the migratory activity of keloid fibroblasts was evaluated using a colony dispersion assay. RESULTS: The production of type 1 collagen, MMP-1, MMP-2, and TIMP-1 by keloid fibroblasts was 3-fold, 6-fold, 2.4-fold, and 2-fold greater than that of normal dermal fibroblasts, respectively. Production of P1P was increased when TGF-beta1 was added to cultures of keloid fibroblasts, while it was decreased when anti-TGF-beta1 antibody was added to the cultures. In contrast, the production of MMP-1 was decreased by the addition of TGF-beta1 to cultured keloid fibroblasts, while it was increased when anti-TGF-beta1 antibody was added to the cultures. The production of MMP-2 increased after treatment with TGF-beta1, but did not change significantly when anti-TGF-beta1 antibody was added to the cultures. Production of TIMP-1 did not change significantly when either TGF-beta1 or anti-TGF-beta1 antibody was added to the cultures. Keloid fibroblasts showed a 2.5-fold increase of migratory activity compared with normal dermal fibroblasts, while the migratory activity of these fibroblasts was reduced to the control level by treatment with a broad-spectrum MMP inhibitor (GM 6001). CONCLUSIONS: Cultured keloid fibroblasts showed increased production of collagen and MMPs, and TGF-beta1 played a role in this regulation of production. In addition, increased production of MMPs had a role in the high migratory activity of cultured keloid fibroblasts.     

Decorin and glycosaminoglycan synthesis in skin fibroblasts from patients with systemic sclerosis

We investigated the expression of decorin and the synthesis of sulphated glycosaminoglycans (GAGs) in cultured fibroblasts from patients with early-stage systemic sclerosis (SSc). Decorin mRNA levels were 1.8-fold higher in SSc fibroblasts than in control fibroblasts. SSc fibroblasts also produced 2.3-fold more decorin core protein and 2.2-fold more sulphated GAGs including dermatan sulphate and chondroitin sulphate. Newly synthesized GAGs, in the presence of p -nitrophenyl β-xylopyranoside, which elongates dermatan sulphate and chondroitin sulphate as an initiator, were increased tenfold and were mainly composed of dermatan sulphate and chondroitin sulphate. The rate of stimulation by the β-xyloside was similar in SSc and control fibroblasts. These results suggest that the increased amount of dermatan/chondroitin sulphate in SSc fibroblasts reflects an enhanced expression of decorin core protein. Type I collagen mRNA levels in SSc fibroblasts were also increased together with its synthesis. Therefore, our results indicate that an altered decorin and collagen production may affect the organization of collagen fibres and the fibrotic process observed in patients with SSc. Received: 24 September 1996     

Dermatofibrosarcoma protuberans: altered collagen metabolism in cell culture

Dermatofibrosarcoma protuberans is a low-grade malignant tumor that grows invasively but rarely forms metastases. Its origin is still controversial. We characterized the synthesis of collagen in detail in cells which were obtained from dermatofibrosarcoma protuberans tumors by enzymatic tissue disintegration. Similar to fibroblasts, all tumor cell strains produced considerable amounts of collagen. However, the rate was reduced compared to normal skin fibroblasts. Cells grown from the tumors synthesized type I collagen, but no type II could be detected. After serial passaging the cultures started to produce type III collagen, which is probably due to a slow overgrowth by normal fibroblasts.     

Collagen and lipid biosynthesis in a case of epitheliogenesis imperfecta in cattle

An analysis of the dermis and a study of collagen and lipid biosynthesis by fibroblast cultures were carried out in one case of epitheliogenesis imperfecta in a new born calf. This was found to be not only an epidermal disease with fissures and blisters within basal cells on the basement membrane but also a metabolic disease affecting the dermal fibroblasts. These fibroblasts showed a significant decrease in collagen biosynthesis with an increase in the percentage of type III collagen and also a decrease in the biosynthesis of lipids, especially glycerides and cholesterol.     

DHMEQ, a novel NF-kappaB inhibitor, suppresses growth and type I collagen accumulation in keloid fibroblasts

BACKGROUND: Keloid is a benign dermal tumor characterized by proliferation of dermal fibroblasts and overproduction of extracellular matrix (ECM). Nuclear factor kappaB (NF-kappaB) plays an important role in regulation of inflammation, immune response and cell proliferation. Activation of the NF-kappaB pathway is thought to be closely linked to abnormal cell proliferation and ECM production in keloid fibroblasts. OBJECTIVE: This study was set out to investigate the effects of a novel selective NF-kappaB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on keloid fibroblasts. METHODS: Primary normal and keloid dermal fibroblasts were used for this study. NF-kappaB activity was assessed by DNA-binding assay and immunohistochemistry. The effect of DHMEQ was evaluated by cell viability, cell growth and type I collagen accumulation. RESULTS: Basal NF-kappaB activity was constitutively elevated in keloid fibroblasts, indicating that this pathway is involved in keloid pathogenesis. DHMEQ markedly reduced cell proliferation and type I collagen accumulation in keloid fibroblasts. CONCLUSION: The inhibition of NF-kappaB by DHMEQ may be an attractive therapeutic approach for keloids.     

Analysis of the age-related composition of human skin collagen and collagens synthesized by fibroblast culture

Age-related differences in the composition and the post-translational modifications of human skin collagens were examined in the present study. The data were compared with results of collagen synthesis from in vivo-aged fibroblasts in culture. Skin extracts and newly synthesized collagen from fibroblast cultures derived from both old and young donor groups showed the same ratio of collagen III to collagen I. Furthermore, no difference was noted in the degree of prolyl and lysyl hydroxylation of collagen I and collagen III. Young and old fibroblasts synthesized a similar quantity of collagen in vitro. The data suggest that fibroblasts maintain a uniform level of collagen production, composition and modification independent of the age of the donor.     

Production of matrix metalloproteinase 2 in fibroblast reaction to mechanical stress in a collagen gel

The reaction of fibroblasts to mechanical forces generated by fibroblasts themselves in anchored collagen lattices was studied. Fibroblasts were cast in collagen gels. Free retracted gels (RG) were compared with stressed gels (SG) in 3-day and 14-day experiments. As previously described, SG showed an increase in protein, mainly collagen, biosynthesis. Matrix metalloproteinases (pro-MMP-2 and MMP-2) were studied by zymography. Certain cell membrane components, integrin alpha(2) and phosphatidylserine, were studied by flow cytometry with antibodies against the integrin alpha(2) subunit and with annexin V binding. Mechanical stress stimulated production of pro-MMP-2 both in the short-term (3-day) and the longer term (14-day) cultures. However, the pro-enzyme was not more activated and there was no difference in the amount of MMP-2 between RG and SG. There was only an increase with time under both conditions. The stressed fibroblasts reacted early with an increase in the integrin alpha(2) subunit, but the stimulated cells disappeared from the 14-day cultures. The number of cells measured in terms of the amount of DNA decreased between day 3 and day 14, mainly in the SG due to cytolysis. This cell stress was related to an alteration in the plasma membrane detected by the annexin V marker.     

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.     

Induction of matrix metalloproteinase‐1 by small interfering RNA targeting connective tissue growth factor in dermal fibroblasts from patients with systemic sclerosis

Please cite this paper as: Induction of matrix metalloproteinase‐1 by small interfering RNA targeting connective tissue growth factor in dermal fibroblasts from patients with systemic sclerosis. Experimental Dermatology 2010; 19 : e111–e116. Abstract: We aimed to evaluate the effect of small interfering RNA (siRNA) targeting CTGF on extracellular matrices (ECMs) metabolism in normal and SSc fibroblasts. Normal and SSc fibroblasts were transfected with CTGF‐specific siRNAs to silence CTGF synthesis. After silencing CTGF, production of type I collagen and matrix metalloproteinase (MMP)‐1 by fibroblasts stimulated with TGF‐β was examined. Then quantitative analyses of protein production or mRNA expression of type I collagen, MMP‐1,‐2,‐9 and tissue inhibitor of metalloproteinase (TIMP)‐1 with TGF‐β stimulation were carried out. Furthermore, after silencing CTGF, proliferations of normal and SSc fibroblasts were investigated. CTGF‐specific siRNA significantly reduced CTGF production. The production of type I collagen was significantly reduced by CTGF silencing in normal fibroblasts. The CTGF silencing significantly increased the production of MMP‐1 and decreased the production of TIMP‐1 in SSc fibroblasts. The mRNA expression of MMP‐1 was increased in CTGF‐silenced SSc fibroblasts, but not in normal fibroblasts. There were no significant changes in the production or mRNA expression of other ECM‐related genes in normal and SSc fibroblasts. Fibroblast proliferations were suppressed by CTGF silencing in normal and SSc fibroblasts. Our data showed that MMP‐1 was increased by CTGF‐specific siRNA transfection only in SSc fibroblasts. RNAi targeting CTGF could be a novel therapeutic strategy for SSc.     

Investigation of irradiation by different nonablative lasers on primary cultured skin fibroblasts

Background. A variety of lasers with different wavelengths and biological effects are widely used for nonablative skin rejuvenation, but the underlying mechanisms have not been fully investigated. Aim. To investigate the effects of irradiation by different nonablative lasers on collagen synthesis and the antioxidant status of cultured fibroblasts to identify a possible mechanism for laser photorejuvenation. Methods. Cultured skin fibroblasts were irradiated with three different lasers: 532 nm potassium–titanyl phosphate (KTP), 1064 nm Q‐switched neodymium:yttrium–aluminium–garnet (Nd:Yag) and 1064 nm long‐pulse Nd:YAG, and production of collagen and changes in lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH‐Px) were assayed. Results. Irradiation by all three lasers led to a marked increase in collagen production. Two major antioxidant enzymes, SOD and GSH, were significantly increased, whereas MDA was markedly reduced after laser irradiation. No change in LDH activity was found between nonirradiated and irradiated fibroblasts. Conclusion. This study indicates that the increased collagen synthesis by fibroblasts after laser treatment may be partly due to improved antioxidant capacity, which reduces oxidative stress and thus stimulates new collagen production.     

增生性瘢痕发生和演变过程中成纤维细胞生物学功能变化及其意义

目的 探索增生性瘢痕在发生和演变过程中,成纤维细胞生物学功能变化的规律及其意义.方法 选取人不同时期增生性瘢痕组织和正常皮肤组织,进行HE染色观察.另外,分离和培养不同时期瘢痕和正常皮肤中成纤维细胞,RT-PCR分别检测成纤维细胞在转移生长因子（TGF-β1）、血管内皮细胞生长因子（VEGF）、和Ⅰ、Ⅲ胶原mRNA表达水平的变化.结果 HE染色可见正常皮肤细胞和微血管数目较少,早期瘢痕增多,炎症细胞浸润明显.增生期瘢痕成纤维细胞和微血管增多.随病情进展,微血管呈缩窄倾向.消退期瘢痕成纤维细胞减少,微血管狭窄或闭塞.成熟期瘢痕见微血管、成纤维细胞数目进一步减少,微血管管腔小,大部分开放.RT-PCR检测结果发现,正常皮肤来源的成纤维细胞TGF-β1、VEGF和Ⅰ、Ⅲ胶原mRNA有较低表达,早期瘢痕成纤维细胞TGF-β1、VEGF和Ⅰ、Ⅲ胶原mRNA表达水平开始升高,在增生期表达到达高峰,消退期瘢痕的表达开始下降,到成熟期表达最低.结论 增生性瘢痕发生和演变过程中,成纤维细胞功能存在动态改变,这种动态改变与瘢痕的发生和消退成熟的病理变化相关.