4‐Methylumbelliferone inhibits tumour cell growth and the activation of stromal hyaluronan synthesis by melanoma cell‐derived factors

Background There is a close correlation between tumour progression and hyaluronan production, either by tumour cells or by stromal cells that are stimulated by tumour‐derived factors. Inhibition of tumour stimulation of fibroblast hyaluronan may suppress tumour growth and invasion. Objectives To examine the effect of the hyaluronan synthesis inhibitor 4‐methylumbelliferone (4‐MU) on the growth of and hyaluronan synthesis by fibroblasts and C8161 and MV3 melanoma cell lines, invasion, and inhibition of tumour cell‐derived factor activation of fibroblasts. Methods Effects of 4‐MU on growth and hyaluronan synthesis by fibroblasts and melanoma cells were examined in monolayer culture and fibroblast‐contracted collagen lattices, and their effects on the growth and invasion of tumour cells into collagen lattices were also studied. Results 4‐MU caused a dose‐dependent growth inhibition of fibroblast and melanoma cells with maximum inhibition at 0·5 mmol L^?1 4‐MU. At this dose, 4‐MU inhibited ³H‐glucosamine incorporation into fibroblast glycosaminoglycans by 52%, and hyaluronan synthesis by 64%. The relative inhibition was more pronounced when fibroblasts were stimulated with C8161 melanoma cell‐conditioned medium. 4‐MU reduced the level of hyaluronan in fibroblast‐contracted collagen lattices, and inhibited both the growth on and invasion into the lattices by melanoma cells. This growth inhibition appears to be predominantly independent of inhibition of hyaluronan synthesis. The effect on growth inhibition was reversible, and 4‐MU had no effect on apoptosis. Conclusions 4‐MU is a potent inhibitor of hyaluronan synthesis, induction of stromal hyaluronan accumulation by tumour cells, and fibroblast and melanoma cell proliferation, and results suggest that 4‐MU may have potential as a tumour cell anti‐invasive and antiproliferative agent.     

Evaluation of a near-senescent human dermal fibroblast cell line and effect of amelogenin

Background Fibroblast senescence may delay healing of chronic wounds. Objectives To characterize a chronic human dermal fibroblast cell line (CRL‐7815) with near‐senescent properties, cell proliferation and production of wound‐healing modulating cytokines, and biosynthesis and remodelling of collagen were compared with normal human dermal fibroblasts. Also, the response of CRL‐7815 fibroblasts to the extracellular matrix protein amelogenin that is beneficial in the treatment of stalled chronic wounds was studied. Methods Fibroblast proliferation was monitored by time‐resolved growth curves and factors secreted into the culture medium containing 10% fetal bovine serum were measured by enzyme‐linked immunosorbent assays. Fibroblast‐mediated reorganization was examined in three‐dimensional type I collagen matrices. Results Cell proliferation over 9 days was significantly (P < 0·01) slower for CRL‐7815 than for normal fibroblasts. Amelogenin at 1 mg mL^?1 increased (P < 0·01) CRL‐7815 proliferation to the level of the normal fibroblasts. The neutrophil chemoattractant interleukin (IL)‐8 was low while the constitutive production of monocyte chemoattractant protein (MCP)‐1 was highly elevated in medium from cultured CRL‐7815 fibroblasts. Amelogenin augmented IL‐8 but attenuated MCP‐1 secretion in CRL‐7815 fibroblasts. The elevated vascular endothelial growth factor production in CRL‐7815 fibroblasts was further increased with amelogenin while increased type I collagen synthesis by CRL‐7815 was reduced with 0·1 mg mL^?1 amelogenin. The dramatically impaired collagen matrix remodelling with CRL‐7815 fibroblasts (P < 0·001) was slightly improved with amelogenin (P = 0·0011). Conclusions The near‐senescent cell line CRL‐7815 shares functional anomalies with fibroblasts isolated from nonhealing chronic cutaneous wounds. Amelogenin has the capacity to switch chronic fibroblasts into an acute‐like phenotype.     

Simultaneous analysis of [3H]-thymidine uptake and α1(I) procollagen mRNA expression in systemic sclerosis skin fibroblasts – an in situ hybridization study

Abstract Heterogeneity of DNA synthesis and collagen synthesis has been reported in skin fibroblasts from systemic sclerosis (SSc) patients. The uptake of [³H]-thymidine and expression of α1(I) procollagen mRNA by cultured skin fibroblasts from four normal controls and four SSc patients was analyzed simultaneously. The grains overlying the cytoplasm representing α1(I) procollagen mRNA and overlying the nucleus representing [³H]-thymidine uptake were counted using computer-aided image analysis. The results were analyzed statistically. Procollagen mRNA expression by SSc fibroblasts was significantly greater than by control fibroblasts (P < 0.01). The distribution curve of [³H]-thymidine uptake showed two peaks representing low- and high-uptake cells. Significantly more SSc fibroblasts than control fibroblasts showed high [³H]-thymidine uptake (P < 0.05). The number of SSc fibroblasts expressing low amounts of α1(I) procollagen mRNA was significantly lower than the number of control fibroblasts (P < 0.05). [³H]-thymidine uptake by SSc fibroblasts expressing high amounts of α1(I) procollagen mRNA was significantly lower than by those expressing low amounts (P < 0.05). These results indicate that elevated DNA synthesis and elevated collagen mRNA synthesis in SSc skin fibroblasts are due to different clones with high DNA-synthesizing ability and high collagen-producing ability. Received: 5 October 1999 / Revised: 20 December 1999 / Accepted: 23 December 1999     

Melanoma cell-derived factors stimulate glycosaminoglycan synthesis by fibroblasts cultured as monolayers and within contracted collagen lattices

BACKGROUND: Various tumours exhibit glycosaminoglycan rich, and in particular hyaluronan rich matrices surrounding them that facilitate tumour growth and invasion. In many tumours, this matrix is predominantly synthesized by fibroblasts following stimulation by tumour cell-derived factors. OBJECTIVES: To determine what effect tumour cell-conditioned medium has upon fibroblast glycosaminoglycan synthesis when cells were cultured as monolayers and within contracted collagen lattices. METHODS: Serum-free conditioned medium from melanoma cell lines (C8161, MV3, A375 and Hs294T) was examined for its ability to stimulate the incorporation of 3H-glucosamine and 35SO4 into glycosaminoglycans synthesized by fibroblasts. RESULTS: Conditioned medium from all four melanoma cell lines exhibited potent glycosaminoglycan-stimulating activity. In monolayer culture, C8161-conditioned medium stimulated a 4.2-fold increase in fibroblast hyaluronan, and a 9.9-fold increase in sulphated glycosaminoglycan synthesis, while 35SO4 incorporation was increased only 2.1-fold. In collagen lattice cultures, C8161-conditioned medium stimulated a 4.9-fold increase in hyaluronan synthesis, a 5.4-fold increase in sulphated glycosaminoglycans, and a 1.3-fold increase in 35SO4 incorporation. CONCLUSIONS: Melanoma cells produce factors that are potent stimulators of fibroblast glycosaminoglycan synthesis, in both monolayer culture and within contracted collagen lattices. Synthesis of both hyaluronan and sulphated glycosaminoglycans with a reduced degree of polymer sulphation is stimulated. Such changes are likely to promote tumour cell proliferation and migration.     

Effects of retinoids on glycosaminoglycan synthesis by human skin fibroblasts grown as monolayers and within contracted collagen lattices

Fibroblasts grown within contracted collagen lattices synthesize substantially less glycosaminoglycans than fibroblasts grown as monolayers on a plastic substrate. [3H]glucosamine incorporation into hyaluronate was reduced by 70%, and incorporation into sulphated glycosaminoglycans was reduced by 40%. However, incorporation into heparan sulphate and chondroitin sulphates was reduced by 14 and 49%, respectively, resulting in a substantial change in the proportions of the individual glycosaminoglycans. On the basis of [³H]glucosamine incorporation, hyaluronate constituted 80% of the total glycosaminoglycans synthesized in monolayer cultures, but only 67% in collagen lattice cultures. Incorporation of ³⁵SO₄ into chondroitin sulphates was reduced by 22%, whereas no change was observed in heparan sulphates following culture within collagen lattices. Exposure of the fibroblast cultures to retinotc acid (10^?6mol/l) and retinyl propionate (2 × 10^?6mol/l) resulted in a decrease in the incorporation of [³H]glucosamine into hyaluronate by up to 41 % in monolayer cultures, and 25% in collagen lattice cultures. The retinoids stimulated the incorporation of [3H]glucosamine into heparan sulphate by up to 72%, and chondroitin sulphates by up to 30%, whereas ³⁵SO₄ incorporation remained essentially unaltered. Only modest changes in the incorporation of both isotopes into tibroblasl sulphated glycosaminoglycans were observed following exposure to the retinoids in lattice cultures. Q-Sepharose ion-exchange chromatography at pH 2–0 revealed that there was no change in the degree of polymer sulphation of either chondroitin sulphate or heparan sulphate isolated from collagen lattice cultures compared with monolayer cultures. Retinoic acid (10^?6mol/l) treatment did, however, reduce the degree of polymer sulphation of heparan sulphates and chondroitin sulphates in both monolayer and lattice cultures. The molecular mass of the sulphated glycosaminoglycans was unaffected by culture conditions, but hyaluronate isolated from collagen lattice cultures had a higher molecular mass than that from monolayer cultures. Retinoic acid treatment had no effect on the molecular mass of the glycosaminoglycans isolated from either culture system.     

瘢痕疙瘩成纤维细胞胶原合成的实验研究

目的 探讨瘢痕疙瘩中胶原过度聚集的原因.方法 从正常皮肤与活跃增生瘢痕疙瘩标本分别培养真皮成纤维细胞,采用³H-脯氨酸掺入法分别检测单层培养及胶原凝胶三维培养体系中成纤维细胞的胶原合成量.用斑点杂交法检测单层培养细胞的人前.α1(Ⅰ)型胶原mRNA水平.结果 瘢痕疙瘩成纤维细胞Ⅰ型胶原mRNA水平升高,其胶原合成量也显著高于正常真皮成纤维细胞(P<0.01).结论 在增生活跃瘢痕疙瘩中,成纤维细胞胶原合成功能处于活化状态,胶原合成增加是导致胶原过度积聚的重要原因.     

Enhanced proliferation and collagen synthesis of human dermal fibroblasts in chronically photodamaged skin

Cutaneous aging can be divided into intrinsic aging and photoaging. We investigated the influence of aging and photoaging on the proliferation and collagen synthesis of human dermal fibroblasts cultured 3-dimensionally in a collagen gel. We examined 11 human dermal fibroblast cell lines cultured from 3 newborn skins (1 day old), and both exposed and unexposed skin from 4 elderly volunteers (60, 60, 73, 76 years old), respectively. Newborn fibroblasts actively proliferated within the attached collagen gels compared with the elderly cell lines. Within the attached collagen gels in the presence of 10% fetal calf serum (FCS), the fibroblasts from exposed skin proliferated rapidly compared with fibroblasts from unexposed skin from the same individuals. In collagen gel and monolayer cultures with 1% FCS, the percentage of collagen synthesized by photoaged and aged fibroblasts decreased significantly compared with that by newborn fibroblasts. When the fibroblasts were cultured three dimensionally in attached collagen gels in the presence of 1% FCS, the relative levels of collagen synthesis by cultured fibroblasts from photoaged skin were increased significantly compared with those of aged skin fibroblasts from the same individuals. These results suggest that fibroblasts of exposed skin may be more active than those of unexposed skin and that the three-dimensional culture of fibroblast can be used as a model to investigate the influence of aging and photoaging on cell functions.     

The effects of interleukin-1 and prostaglandin E2 on accumulation of collagen and steady-state levels of proα1(I) collagen messenger RNA in experimental granulation tissue in rats

The effects of human interleukin 1β (IL-1β) and prostaglandin E ₂ (PGE ₂ ) on experimental granulation tissue in rats and on granulation tissue cells in culture were studied. IL-1β and PGE ₂ were injected into subcutaneously implanted sponges during the first 3 days after implantation. The rate of collagen synthesis in fibroblasts was measured as synthesis of protein-bound ³ H-hydroxyproline. The steady-state levels of proα1(I) and proα1(III) collagen chain mRNAs were estimated by Northern transfer analyses. By 7 days postoperatively IL-1β had decreased the hydroxyproline content of granulation tissue. PGE ₂ decreased nonsignificantly the amounts of hydroxyproline, but the steady-state levels of proα1(I) and proα1(III) collagen chain mRNAs were slightly elevated. In IL-1β-treated fibroblast cultures collagen production decreased by 15% and following PGE ₂ treatment by 34% compared with the controls. The latter effect could be abolished by indomethacin. Indomethacin alone stimulated collagen production by 40%. In vivo IL-1 decreases the formation of normal granulation tissue. This effect may be partly due to stimulation of secretion of PGE ₂ . Received: 13 September 1996     

Modulation of keratinocyte growth factor (KGF) mRNA expression in human dermal fibroblasts grown in monolayer or within a collagen matrix

Abstract In this study, we analysed the modulation of keratinocyte growth factor (KGF) mRNA expression in human dermal fibroblasts cultured either in monolayer or within a collagen matrix (dermal equivalent). In monolayer cultures, KGF expression by quiescent fibroblasts was stimulated by different growth substances such as serum, epidermal growth factor and basic fibroblast growth factor. Moreover, we demonstrated that the induction of this gene was mediated by at least 2 different signalling pathways involving protein kinase C (PKC) and cAMP. In dermal equivalents, we observed that the collagen matrix negatively modulated KGF mRNA expression. Indeed, among the growth substances used, only the serum slightly stimulated KGF expression. Nevertheless, as in monolayers, this induction involved at least PKC and cAMP signalling pathways. As the collagen matrix can modulate fibroblast growth, we also studied KGF expression in growing fibroblasts from either monolayer cultures or dermal equivalents. We then showed that this collagen matrix negatively influenced KGF expression independently of the proliferative state of fibroblasts. All these results underline the fact that KGF mRNA expression by human dermal fibroblasts is induced by different substances; however this expression can be modulated by fibroblast-matrix interactions.     

A collagen-based bi-layered composite dressing for accelerated wound healing

Aim of the studyThe aim of the study was to fabricate collagen-based composite dressings, evaluate the efficiency for wound healing and reveal the mechanism of promoting wound healing.Materials and methodsAn innovative bi-layered composite wound dressing was developed using two marine biomacromolecules (collagen and chitosan). Full-thickness skin defect model was performed to evaluate the wound healing activity in vivo. The levels of inflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin (IL-1, IL-6, IL-8) and growth factors like transforming growth factor beta (TGF-β), vascular epidermal growth factor (VEGF) and basic fibroblast growth factor (bFGF) were quantified by ELISA assays. The total amount of collagen was quantified by hydroxyproline content. The proliferation and viability of fibroblast cells cultured on collagen sponges were determined by CCK-8 assay.ResultsThe results of wound closure and histopathological analysis indicated that non-crosslinked collagen-based bi-layered composite dressing stimulated wound healing, accelerated re-epithelialization and accomplished wound healing within a time span of 28 days. The results of levels of inflammatory cytokines and growth factors showed that collagen-based composite dressings could reduce the inflammatory response and upregulate growth factors levels to accelerate the wound healing. The results of hydroxyproline content and CCK-8 assay indicated that collagen-based composite dressings could also promote collagen synthesis and fibroblasts viability and proliferation.ConclusionThe non-crosslinked collagen-based bi-layered composite dressing could be applied for an efficient and ideal wound dressing. Therefore, the findings provided the essential theoretical basis for the potential of collagen-based composite dressing applied in wound healing fields.     

Interleukin-1α enhances mast cell growth by a fibroblast-dependent mechanism

Abstract Mast cell hyperplasia is observed in various inflammatory skin diseases. Although the pathogenesis of these conditions remains largely uninvestigated, it has been speculated that lesional mediators provide a favorable microenvironment for mast cell growth. We investigated the effect of an inflammatory cytokine, IL-1α, on mast cell growth in a mast cell/fibroblast coculture system. When mouse bone marrow-derived cultured mast cells (BMMC) were cultured on a NIH/3T3 fibroblast monolayer, IL-1α stimulated mast cell proliferation. However, IL-1α did not stimulate ³H-thymidine incorporation in BMMC in the absence of fibroblasts. Separation of BMMC from fibroblasts by a permeable micropore membrane reduced the effect of IL-1α. When BMMC were prepared from W/W ^v mice, which lack a functional c-kit, or when NIH/3T3 fibroblasts were substituted with Sl/Sl ^d -derived fibroblasts, which lack membrane-bound stem cell factor (SCF), a lower, but significant, effect of IL-1α was observed. Flow cytometric analysis revealed no enhancement of SCF expression on fibroblasts following stimulation with IL-1α. Neutralizing antibodies against IL-3, IL-4, IL-10, and nerve growth factor (NGF) showed no inhibition. On the other hand, indomethacin inhibited the effect of IL-1α, and prostaglandin E₂ induced mast cell growth in the cocultures. These results indicate that IL-1α stimulates mast cell growth by a fibroblast-dependent mechanism, in which SCF/c-kit interaction may participate in a major way. The mast cell growth activity induced by this cytokine can, at least in part, be attributed to prostaglandins. Inflammatory cytokines may thus contribute to mast cell hyperplasia in skin diseases. Received: 6 September 1999 / Revised: 20 December 1999 / Accepted: 23 December 1999     

Inhibitory effects of bFGF,VEGF and minoxidil on collagen synthesis by cultured hair dermal papilla cells

Dermal papilla cells of rat vibrissa follicles cultivated in monolayers and in three-dimensional collagen gels show a different morphology in these culture systems. Dermal papilla cells cultured in lattices tend to express morphological features resembling those seen in vivo. Quantification of total collagen by incorporation of³H-proline in monolayer cultures and in collagen lattices show that the amount of collagen found in dermal papilla cells is higher than that secreted. Moreover, collagen synthesis measured in lattices is reduced to about 50% of that found in monolayer cultures. The influence of growth factors on collagen synthesis by hair dermal papilla cells was investigated. We studied the effects of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and minoxidil on collagen synthesis in monolayers and in lattices. VEGF, bFGF and minoxidil significantly decreased the total amount of collagen. In monolayer cultures, there was approximately a 30% inhibition of collagen production with 5ng/ml bFGF, 0.1 ng/ml VEGF and 100 ng/ml minoxidil. However, in the lattices this inhibition was reduced to about half. These results suggest that both culture substrate and growth factors influence collagen production by rat hair dermal papilla cells.     

Successful ultraviolet A1 phototherapy in the treatment of localized scleroderma: a retrospective and prospective study

Background Ultraviolet (UV) A1 phototherapy is an effective anti‐inflammatory treatment modality that influences fibroblast functions. Objectives To document the effects of UVA1 treatment in patients with localized scleroderma (LS) in a retrospective study (at least 6 months after UVA1 treatment) and in a prospective study before and immediately after medium‐dose UVA1 irradiation. Methods In total, 30 patients (retrospective study n = 17, prospective study n = 13) with LS receiving UVA1 phototherapy five times weekly (for 3–6 weeks) were investigated. Improvement was documented using standardized questionnaires and clinical evaluation (using modified Rodnan skin score, Cutometer and 7·5‐MHz ultrasound measurements). Levels of collagen I and collagen III metabolites were measured in serum and urine. Results In the retrospective study, medium‐dose UVA1 phototherapy had been performed 6 months–3 years earlier (cumulative dose 750–1400 J cm^?2; mean ± SD number of irradiations 19·3 ± 3·8). Fourteen of 17 patients (82%) reported an improvement in symptoms following UVA1 therapy. In the prospective study, skin elasticity increased in 77% of the patients following medium‐dose UVA1 phototherapy (cumulative dose 750–1250 J cm^?2; mean ± SD number of irradiations 20·8 ± 4·0). 7·5‐MHz ultrasound measurements showed a mean reduction of lesional skin thickness of 13% compared with skin thickness before UVA1 phototherapy. The ratio of deoxypyridinoline to creatinine was significantly elevated in about two‐thirds of the patients. Conclusions This open study showed a positive short‐ and long‐term efficacy of UVA1 phototherapy in patients with LS, with a reduction in sclerotic plaques, an increase in skin elasticity and a reduction of lesional skin thickness. UVA1 phototherapy had a significant effect on collagen metabolism. UVA1 phototherapy can be regarded as a safe treatment modality for patients with LS.     

Bleomycin increases steady-state levels of type I collagen, fibronectin and decorin mRNAs in human skin fibroblasts

Abstract Bleomycin is a drug capable of inducing tissue fibrosis. In this study, the effects of bleomycin on gene expression of extracellular matrix encoding ·1(I) collagen, fibronectin and decorin were determined in vitro in human dermal fibroblasts. Northern blot analysis showed that bleomycin upregulated ·1(I) collagen, fibronectin and decorin gene expression dose-dependently between 1 nM and 1 μM. Bleomycin at 100 nM upregulated α1(I) collagen, fibronectin and decorin mRNA expression with a peak at 6 h following stimulation in normal skin fibroblast monolayers. Bleomycin enhanced mRNA expression encoding these extracellular matrix proteins in both normal dermal and scleroderma fibroblasts. Concomitant stimulation with bleomycin and interferon-γ (1000 U/ml), a representative antifibrotic cytokine, decreased ·1(I) collagen mRNA expression. Bleomycin also mildly upregulated mRNA expression of transforming growth factor-‚ (TGF-‚) and connective tissue growth factor (CTGF) coordinately in normal skin fibroblasts. Our results indicate that bleomycin modulates gene expression of extracellular matrix proteins in dermal fibroblasts, and this effect may be mediated by TGF-‚ and CTGF. Received: 20 April 2000 / Revised: 19 June 2000 / Accepted: 4 September 2000     

Heparin induces fibroblast proliferation, cell-matrix interaction and epidermal growth inhibition

Abstract We have examined the effect of heparin on fibroblasts cultivated in monolayer or in a 3-dimensional culture system: the so-called collagen lattices. Thereafter, we have investigated the effect of heparin on the kinetics of epidermal growth on the collagen lattices. In monolayer culture, heparin stimulated the fibroblast growth with an optimal response at 0.01 mg/ml. The volume of treated fibroblasts was smaller than that of untreated controls. In the collagen lattices, heparin stimulated the fibroblast growth with an optimal respone at 0.1 mg/ml. The volume of treated fibroblasts was greater than that of untreated controls, the opposite to the result observed in monolayer culture. The beginning of the contraction of the collagen lattices was inhibited by heparin. Heparin inhibited epidermal growth on the immersion as well as on the emersion collagen lattices. These effects of heparin should be the consequences of heparin-induced modifications of cell-matrix interactions.     

Altered regulation of collagen metabolism in scleroderma fibroblasts grown within three-dimensional collagen gels

In systemic scleroderma (SSc) excessive deposition of collagen leads to fibrosis of various tissues including the skin. Previous studies have demonstrated that scleroderma fibroblasts in explant monolayer cultures are heterogeneous with respect to their levels of collagen synthesis. The critical role played by the extracellular matrix (ECM) in the modulation of fibroblast metabolism prompted us to study the regulation of collagen synthesis in scleroderma fibroblasts grown within three-dimensional collagen gels, a culture system representing more physiological conditions than monolayer cultures. Normal fibroblasts grown in this system dramatically reduce their collagen synthesis as compared to monolayer cultures. Quantification of total protein and collagen synthesis showed that scleroderma fibroblasts did not demonstrate the down regulation of collagen synthesis as observed in control fibroblasts, resulting in a much higher collagen synthesis in scleroderma fibroblasts compared to controls. However, also in this system scleroderma fibroblasts were heterogeneous in their response to the collagenous lattice. Ten strains were investigated, of which 3 were indistinguishable from controls, while 7 maintained higher levels of collagen production. In addition, our data showed that the changes in collagen synthesis on the protein level were accompanied by respective up- or downregulation on the mRNA level. These results indicate that an altered response to the surrounding ECM is an important factor in the disturbed regulation of connective tissue synthesis in scleroderma fibroblasts observed in vivo.     

Phenytoin modulates connective tissue metabolism and cell proliferation in human skin fibroblast cultures

Phenytoin has been proposed for the treatment of certain dermatologic conditions involving connective tissue abnormalities. To understand the biochemical basis of connective tissue changes, we incubated human skin fibroblasts in culture with varying concentrations of phenytoin. The results indicated that fibroblast proliferation, detected by tritiated thymidine incorporation into cells, was slightly stimulated when short incubation periods and low concentrations of phenytoin were employed. However, with longer incubation times and higher phenytoin concentrations, a significant reduction in fibroblast proliferation was observed. Further studies demonstrated that incubation of cells with phenytoin did not affect the production of procollagen, measured as synthesis of radioactive hydroxyproline in the cultures. However, assay of prolyl hydroxylase, an enzyme participating in the post-translational synthesis of hydroxyproline during collagen biosynthesis, was significantly reduced in the fibroblast cultures. The activity of collagenase, an enzyme participating in degradation of collagen, was markedly decreased in cultures treated with phenytoin. Thus, phenytoin may modulate collagen metabolism primarily by affecting the degradation of collagen. The results support previous suggestions that phenytoin may be useful for treatment of patients with increased levels of collagenase, such as in recessive dystrophic epidermolysis bullosa.     

Clinical and genetic features of 20 Japanese patients with vascular‐type Ehlers–Danlos syndrome

Background Vascular‐type Ehlers–Danlos syndrome (vEDS) is a severe autosomal dominant inherited disorder resulting from mutations within the α1 type III collagen gene (COL3A1). The majority of published mutations are base changes leading to the substitution of single glycine residues within the triple‐helical domain of type III collagen. Although clinical characteristics and mutations in the COL3A1 gene have been analysed for some patients from Europe and America, similar analyses have not yet been performed for Japanese patients with vEDS. Objectives To analyse the genetic and phenotypic findings in Japanese patients with vEDS. Methods We analysed the clinical features of 20 unrelated individuals with vEDS. To quantify type III collagen production, the fibroblasts were cultured with ³H‐proline, and the radiolabelled collagenous proteins were analysed using sodium dodecyl sulphate–polyacrylamide gel electrophoresis and fluorography. Mutations in COL3A1 were detected by sequence analysis of cDNA from patients’ fibroblasts and subsequently by a genomic DNA sequence analysis. Results Thin and translucent skin with extensive bruising and hypermobility of the small joints were observed in about 90% of the patients, whereas the prevalence of serious clinical findings such as rupture/dissection/aneurysm of the arteries (30%) or rupture of the gastrointestinal tract (25%) was relatively low. Sequence analyses of the COL3A1 gene demonstrated heterozygous point mutations leading to glycine substitution in only nine patients (45%), while heterozygous splice‐site mutations at the junction of the triple‐helical exons were observed in the remaining 11 patients (55%). The average type III collagen production level in the cultured dermal fibroblasts was 14·6% of the normal value. The types of complication were not associated with specific mutations in COL3A1. Conclusion The analysis in the present series revealed a low frequency of patients presenting with serious clinical findings such as arterial rupture/arterial dissection/aneurysm and perforation or rupture of the gastrointestinal tract, and revealed a higher prevalence of splice‐site mutations at the junction of the triple‐helical exons than of glycine substitution mutations in COL3A1.