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     

Increased expression of GAB1 promotes inflammation and fibrosis in systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease mainly characterized by persistent inflammation and fibrosis. The receptor tyrosine kinase (RTK) signal pathway plays an important role in the process of SSc, and Grb2‐associated binding protein (GAB) is crucial in activating RTK signalling. A previous study found elevated levels of GAB1 in bleomycin (BLM)‐induced fibrotic lungs, but the effects of GAB1 in SSc remain unclear. Our aim was to investigate whether GAB1 was dysregulated and its potential role in SSc. Compared with healthy donors, we found GAB1 expression was 1.6‐fold higher in peripheral blood mononuclear cells (PBMC), 2.5‐fold higher in CD4 + T cells, and 2‐fold higher in skin from of SSc patients (P < .01). At the same time, the levels of type one collagen (COLI) were also significantly increased (1.8‐fold higher) in SSc skin. Additionally, BLM‐induced SSc mice showed mRNA levels of Gab1 2‐fold higher than saline‐treated controls, and Gab1 expression correlated positively with collagen content. A further in vitro study showed silencing of GAB1 suppressed inflammatory gene expression in TNF‐α induced fibroblasts. Additionally, GAB1 deficiency prominently inhibited cell proliferation and reduced COLI protein levels in TGF‐β induced fibroblasts. Taken together, these data suggest that GAB1 has a relatively high expression rate in SSc, and knockdown of GAB1 may attenuate SSc by stimulating inflammatory and fibrotic processes.     

Modulation of endothelial dysfunction and apoptosis: UVA1-mediated skin improvement in systemic sclerosis

UVA1-mediated effects regarding vascular dysregulation as a primary pathogenetic factor of systemic sclerosis skin lesions have so far not been investigated. Pre- and posttherapy skin biopsies of four patients were evaluated immunohistochemically for angiostatic, angiogenic and angioapoptotic features. Immunohistochemistry revealed a partial pretherapy loss of endothelial CD31 and CD34 expression accompanied by a posttherapy increase of CD34⁺ cells. Simultaneously, VEGF and M30 CytoDEATH immunolabeling demonstrated UVA1-induced neovascularization and decreased endothelial apoptosis. Our results suggest that UVA1 irradiation exerts its positive effects by a modulation of endothelial regulation/transformation beside the proposed induction of T cell apoptosis and collagenases.     

Epidemiological study of patients with systemic sclerosis in Tokyo

Summary We conducted an epidemiological study of systemic sclerosis in the city of Tokyo using the records of patients who had been registered to receive free medical service for intractable diseases. A total of 636 patients were registered as having systemic sclerosis in 1987, and we sent questionnaires to the doctor of each patient. The contents of the questionnaires included the patient's name, sex, age, occupation, major symptoms, therapy and laboratory findings. We received 357 completed replies, and were able to analyse them. Our study estimated that at 1 January 1988 the prevalence rate in Japan was between 2.1 and 5.3 per 100000. The male/female ratio was 14:1. The ages of the patients when surveyed ranged from 17 to 82 years, with a mean age of 51 years, peaking with the most numerous group being 50–59 years. The characteristic signs of systemic sclerosis were as follows: proximal scleroderma, 75%; sclerodactyly, 91%; pitting scars, 49%; short sublingual frenulum, 49%; pulmonary fibrosis, 45%; diffuse pigmentation, 45%; and phalangeal contracture, 35%. Raynaud's phenomenon was present in 93% of patients, and was the initial symptom in 59% of cases. With respect to specific antinuclear antibodies, anticentromere antibody was present in 19% and antitopoisomerase I antibody was present in 27%.     

A potential contribution of altered cathepsin L expression to the development of dermal fibrosis and vasculopathy in systemic sclerosis

Cathepsin L (CTSL) is a lysosomal proteolytic enzyme involved in inflammation and vascular and extracellular matrix remodelling, which are the three cardinal pathological events associated with systemic sclerosis (SSc). To elucidate the potential role of CTSL in the development of SSc, we here investigated CTSL expression in the lesional skin of patients with SSc and SSc animal models and the clinical correlation of serum CTSL levels. CTSL expression was elevated in dermal small vessels of SSc patients compared with those of healthy controls. Consistently, CTSL mRNA levels were increased in SSc lesional skin samples, but not in cultivated SSc dermal fibroblasts, compared with corresponding control samples from healthy individuals. Serum CTSL levels were significantly higher in SSc patients than in healthy controls and inversely correlated with skin score. Furthermore, the elevation of serum CTSL levels was linked to SSc vasculopathy. Supporting these results, Ctsl mRNA levels were decreased in the skin of bleomycin‐treated mice, an SSc animal model recapitulating its fibrotic aspect, and CTSL expression was enhanced in dermal small vessels of endothelial cell‐specific Fli1 knockout mice, reminiscent of SSc vasculopathy. Importantly, gene silencing of FLI1 induced CTSL mRNA expression and Fli1 occupied the CTSL promoter in human dermal microvascular endothelial cells. Collectively, these results suggest that endothelial CTSL up‐regulation partially due to Fli1 deficiency may contribute to the development of vasculopathy, while the decrease in dermal CTSL expression is likely associated with dermal fibrosis in SSc.     

Elevated plasma homocysteine level is possibly associated with skin sclerosis in a series of Japanese patients with systemic sclerosis

Homocysteine is a sulfhydryl‐containing amino acid that is derived from dietary methionine, and there has been increasing evidence that elevated plasma homocysteine levels are associated with increased risk of cardiovascular diseases, including carotid, coronary and peripheral arterial disease (PAD). The association of plasma homocysteine levels with peripheral vascular involvements, such as Raynaud phenomenon (RP), digital ulcers (DU) in systemic sclerosis (SSc) patients has not been well studied. The objective of this study was to examine plasma homocysteine levels and their clinical associations in patients with SSc. Plasma homocysteine levels in 151 Japanese patients with SSc and 20 healthy controls were examined. No significant differences were observed in plasma homocysteine levels between SSc patients and healthy individuals. Demographic and clinical features of the SSc patients revealed that severe skin sclerosis, anti‐topoisomerase I antibody positivity, complications of DU, acro‐osteolysis (AO) and interstitial lung disease (ILD) were significantly more prevalent among the patients with elevated plasma homocysteine levels. The plasma homocysteine levels were positively correlated with modified Rodnan total skin score. The plasma homocysteine levels in the SSc patients with DU, AO and ILD were significantly higher than those in the SSc without DU, AO and ILD, respectively. Plasma homocysteine levels did not correlate with either the mean or max intima‐media thickness (IMT) or plaque score, suggesting that plasma homocysteine levels might not be associated with carotid artery atherosclerosis in SSc patients. The measurement of plasma homocysteine levels in SSc patients might be useful for the risk stratifications of severe skin sclerosis, DU and AO.     

Gene expression of types I and III collagen, decorin, matrix metalloproteinases and tissue inhibitors of metalloproteinases in skin fibroblasts from patients with systemic sclerosis

Cultured fibroblasts from patients with systemic sclerosis (SSc) and normal individuals were examined for gene expression of types I and III collagen, decorin, matrix metalloproteinases (MMP) MMP-1, MMP-2, and MMP-3, tissue inhibitors of metalloproteinases (TIMP) TIMP-1 and TIMP-2, urokinase- and tissue-type plasminogen activators (u-PA and t-PA). Fibroblasts from patients with early stage SSC (less than 1 year duration of disease) exhibited higher levels of types I and III procollagen, decorin, MMP-1, MMP-3, TIMP-1, and PAs than those from normal individuals. The gene expression of procollagen α1(I) and TIMP-1 mRNAs were increased, but those of decorin, MMP-1, MMP-2, and MMP-3 were decreased, in fibroblasts from SSc patients with mid-stage SSc (2 to 4 years duration) as compared with those from normal individuals. In contrast, no significant difference in gene expression was found between fibroblasts from normal individuals and from patients with late-stage SSc (more than 6 years duration). These results suggest that gene expression of collagen, decorin, and degrading factors is dynamically modulated during fibrillogenesis. The responses of procollagen α1(I) mRNA to IL-1 and TGF-β were lower in fibroblasts from SSc patients with early and mid-stage disease, but not in those from patients with-late stage disease, than in control fibroblasts, which indicates that these cytokines may be involved in the earlier phases of fibrosis in SSc. Received: 30 August 1996     

体外培养毛囊真皮鞘细胞与皮肤成纤维细胞形态结构和bFGF、EGFR表达差异

Gharzi等[1]初步证实毛囊真皮鞘细胞参与皮肤损伤修复,但相关作用机制研究报道不多.成纤维细胞是皮肤创伤愈合主要效应细胞,毛囊真皮鞘细胞与皮肤成纤维细胞一样均属于间充质细胞.本研究旨在通过两种细胞形态结构及部分愈合相关蛋白表达差异研究,初步探讨毛囊真皮细胞在创伤愈合中的作用.     

Leukotriene B4 enhances adherence of human polymorphonuclear leukocytes to dermal microvascular endothelial cells in vitro

Summary Adherence of polymorphonuclear leukocytes (PMNs) to endothelial cells (ECs) is a crucial step in the diapedesis of inflammatory cells to the site of inflammation. We have demonstrated that leukotriene B₄ (LTB₄), a metabolite of the arachidonic acid cascade, and N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) significantly enhance the binding of human PMNs to selected populations of human dermal microvascular endothelial cells (MECs) in vitro. MECs were isolated from the vascular-rich portion of foreskins of newborns. MECs were grown in Iscove's modified Dulbecco's media with 2% prepartum serum and 8% newborn calf serum on 1% gelatin-coated plastic dishes. PMNs isolated from five human donors were added to the culture dishes for varying time intervals (usually 30 min) in the presence and absence of the chemotactic stimuli LTB₄ and FMLP. Addition of PMNs to MECs in the absence of chemotactic stimuli results in baseline binding to the MEC monolayer. About one in every 150 ECs binds more than five PMNs. These selected ECs are randomly distributed throughout the monolayer. LTB₄ from 10^-10 to 10^-7 M increases the number of MECs which selectively bind PMNs by 260% at 10^-7 M. FMLP also increases adherence in qualitatively and quantitatively similar fashion. These data support a role for LTB₄ in the mediation of adherence of neutrophils to dermal MECs. In contrast to other endothelial cells from the large blood vessels, such as from umbilical veins or calf thoracic aortae, PMNs bind only to selected MECs in culture, even when stimulated with LTB₄ or FMLP. These findings suggest a specific subpopulation of MECs which can be induced to express specific finding sites by LTB₄.Part of this work has been presented at the Meeting of the Western Society of Investigative Dermatology in Carmel, February 3–6, 1986     

Cold atmospheric plasma (CAP) activates angiogenesis-related molecules in skin keratinocytes,fibroblasts and endothelial cells and improves wound angiogenesis in an autocrine and paracrine mode

Background

Cold atmospheric plasma (CAP) emerged as a novel therapeutic field with applications developed for bacterial sterilization, wound healing and cancer treatment. For clinical implementation it is important to know how CAP works and which molecular changes occur after the CAP treatment. Vascularization is an important step during wound healing, however, the effects of CAP on wound angiogenesis are not well examined so far. Furthermore, it has not been investigated, whether CAP primarily affects endothelial cells directly or via paracrine mechanisms to modulate the vasculature.

Knockdown of paraoxonase 1 expression influences the ageing of human dermal microvascular endothelial cells

Skin is one of the most commonly studied tissues for microcirculation research owing to its close correlation of cutaneous vascular function, ageing and age‐related cardiovascular events. To elucidate proteins that determine this correlation between endothelial cell function and ageing in the vascular environment of the skin, we performed a proteomic analysis of plasma samples from six donors in their 20s (young) and six donors in their 60s (old). Among identified proteins, paraoxonase 1 (PON1) was selected in this study. To elucidate the role of PON1 on skin ageing and determine how it controls cellular senescence, the characteristics of PON1 in human dermal microvascular endothelial cells (HDMECs) were determined. When the expression of endogenous PON1 was knocked‐down by small interfering RNA (siRNA) targeting PON1, HDMECs showed characteristic features of cellular senescence such as increases in senescence‐associated β‐galactosidase stained cells and enlarged and flattened cell morphology. At 48 h post‐transfection, the protein expression of p16 in PON1 siRNA‐treated HDMECs was higher than that in scrambled siRNA‐treated HDMECs. In addition, the expressions of moesin and rho GTP dissociation inhibitor, additional age‐related candidate biomarkers, were decreased by PON1 knock‐down in HDMECs. In conclusion, these results suggest that PON1 functions as an ageing‐related protein and plays an important role in the cellular senescence of HDMECs.     

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.     

IL-10 inhibits ICAM-1 expression on human Langerhans cells but not on keratinocytes, dermal endothelial cells or fibroblasts

Abstract Intercellular adhesion molecule-1 (ICAM-1) is regularly expressed or inducible on all major cutaneous cell populations including Langerhans cells, keratinocytes, endothelial cells and dermal fibroblasts. ICAM-1 is induced in the skin under inflammatory conditions and plays an important role in the activation of T cells. Interleukin-10 (IL-10) is a pluripotent immunosuppressive cytokine that inhibits proliferation of T cells via inhibition of antigen-presenting cells including Langerhans cells. We demonstrates that IL-10 inhibits baseline and also cytokine-stimulated ICAM-1 expression on human Langerhans cells, which has previously been shown in the murine system. No effect of IL-10 was seen on human dermal vascular endothelial cells, which like Langerhans cells are also able to present antigen. Additionally, no inhibitory effect of IL-10 was observed on the ICAM-1 expression of keratinocytes and dermal fibroblasts. As IL-10 only weakly suppresses MCH II on human Langerhans cells, inhibition of ICAM-1 and other accessory molecules by IL-10 seems to be an important mechanism inhibiting the antigen-presenting function of human Langerhans cells. Received: 9 December 1997     

Cultivation of human dermal microvascular endothelial cells in vitro: Immunocytochemical and ultrastructural characterization and effect of treatment with three synthetic retinoids

Summary A new reliable and reproducible technique to culture endothelial cells from the small vessels and capillaries of human skin is introduced, and proliferation and differentiation of the growing cells are characterized. The endothelial origin of the culture cells was confirmed by light- and electron microscopy and by labelling with Ulex europaeus Agglutinin I and an antibody against Factor VIII-related antigen. Further immunocytochemical characterization showed that 92–100% of the cells were positive for ₂-microglobulin and the entire cell population expressed vimentin, whereas cytokeratins, desmin, HLA-DR antigen, Leu 6 and S 100 protein, could not be detected. As vascular endothelium is a common site of inflammation and retinoids have been shown to be of good clinical efficacy in some chronic inflammatory skin diseases, we investigated the influence of etretinate, etretin and isotretinoin on proliferation and antigen expression of our culture cells. All retinoids applied inhibited proliferation of endothelial cells in a dose- and time-dependent manner whereas they induced neither HLA-DR nor intercellular adhesion molecule-1 (ICAM-1). Furthermore, none of the retinoids applied influenced the -interferon-induced expression of these surface antigens on endothelial cells. Our results suggest that the action of retinoids in skin inflammation is not mediated by modulation of HLA-DR or ICAM-1. The cell culture technique described here is an interesting and reliable model for studying the influence of drugs on endothelial cell growth and differentiation in vitro.This work was partly presented at the ESDR-JSID-SID Tri-continental Meeting, Washington D. C., April 26–30, 1989     

Fli1 deficiency contributes to the suppression of endothelial CXCL5 expression in systemic sclerosis

CXCL5 is a member of CXC chemokines with neutrophilic chemoattractant and pro-angiogenic properties, which has been implicated in the pathological angiogenesis of rheumatoid arthritis and inflammatory bowel diseases. Since aberrant angiogenesis is also involved in the developmental process of systemic sclerosis (SSc), we herein measured serum CXCL5 levels in 63 SSc and 18 healthy subjects and investigated their clinical significance and the mechanism explaining altered expression of CXCL5 in SSc. Serum CXCL5 levels were significantly lower in SSc patients than in healthy subjects. In diffuse cutaneous SSc (dcSSc), serum CXCL5 levels were uniformly decreased in early stage (<1 year) and positively correlated with disease duration in patients with disease duration of <6 years. In non-early stage dcSSc (≥1 year), decreased serum CXCL5 levels were linked to the development of digital ulcers. Consistently, the expression levels of CXCL5 proteins were decreased in dermal blood vessels of early stage dcSSc. Importantly, Fli1 bound to the CXCL5 promoter and its gene silencing significantly suppressed the CXCL5 mRNA expression in human dermal microvascular endothelial cells. Furthermore, endothelial cell-specific Fli1 knockout mice, an animal model of SSc vasculopathy, exhibited decreased CXCL5 expression in dermal blood vessels. Collectively, these results indicate that CXCL5 is a member of angiogenesis-related genes, whose expression is suppressed at least partially due to Fli1 deficiency in SSc endothelial cells. Since Fli1 deficiency is deeply related to aberrant angiogenesis in SSc, it is plausible that serum CXCL5 levels inversely reflect the severity of SSc vasculopathy.