transforming-growth-factor-beta and Scleroderma--Localized

Morphea and systemic sclerosis (SSc) are rare disorders of connective tissue characterized by increased skin thickness and fibrosis, with current treatment options having variable efficacies, many with limited therapeutic benefit. Janus kinase (JAK) inhibitors have been shown in preclinical studies to inhibit the fibrotic pathway in murine models of systemic sclerosis, by blocking TGF-beta mediated pathway of STAT protein activation. Additionally, case reports of the treatment of morphea and SSc with tofacitinib, a JAK 1/3 inhibitor, have shown improvement in skin sclerosis. Several JAK inhibitors have been developed and utilized in dermatologic and rheumatologic diseases. To date, tofacitinib has been by far the most commonly trialed JAK inhibitor in patients with SSc and morphea. Herein we review the preclinical studies reported in the literature supporting the use and efficacy of JAK inhibitors for the treatment of morphea and the cutaneous manifestations of SSc, as well as discuss the clinical cases published to date illustrating the benefits of JAK inhibitors in disease management. The pathogenesis and mechanism of action will be reviewed as it relates to the process of skin fibrosis in morphea and SSc, along with the murine models illustrating efficacy of JAK inhibitors in fibrotic disease. Based on available preclinical and clinical data as well as consideration of the mechanism of action of JAK inhibitors on the pathway for cutaneous fibrosis, there is promising evidence to support the use and further study of JAK inhibitors in the management of morphea and cutaneous fibrosis in SSc.

Topics: Animals; Fibrosis; Humans; Janus Kinase Inhibitors; Janus Kinases; Mice; Scleroderma, Localized; Scleroderma, Systemic; Transforming Growth Factor beta

Localized scleroderma (LoSc) is rare connective tissue disease that manifests with inflammation and fibrosis of the skin. Depending on the LoSc subtype, adjacent structures such as subcutaneous tissue, fascia, muscles, bones may be affected. The hallmark of fibrosis is tissue remodelling with excess deposition of extracellular matrix proteins (ECM), principally collagens. MicroRNAs (miRNAs) are small, noncoding RNA molecules that consist of 19-24 nucleotides and act as negative regulators of gene expression at the posttranscriptional level. Based on the current articles, approximately 40 microRNAs have been linked to fibrosis in different organs and diseases. The majority of these molecules promote or inhibit fibrosis by targeting connective tissue growth factor (CTGF), extracellular matrix proteins, TGF-β pathway and MAPK (mitogen-activated protein kinase) pathway. Further, particular microRNAs regulate fibrogenesis by altering epithelial-to-mesenchymal transition (EMT) or activating proliferation of myofibroblasts. MiRNAs are relatively stable, detectable in tissues and body fluids (serum, plasma) which suggest that they may serve as beneficial biomarkers to monitor the course of the disease and response to treatment. Herein, we report the present state of knowledge on microRNA expression in localized scleroderma.

Topics: Animals; Epithelial-Mesenchymal Transition; Fibrosis; Humans; MAP Kinase Signaling System; MicroRNAs; Myofibroblasts; Scleroderma, Localized; Skin; Transforming Growth Factor beta

IL-4 is potentially a major profibrogenic cytokine upregulating the expression of collagen genes. In vivo studies have shown that the disruption of STAT6, IL-4R, IL-4 or transforming growth factor-beta (TGF-beta) genes in TSK mice, which develop scleroderma-like syndrome, prevented the occurrence of skin sclerosis and of autoantibodies. Additionally, it is known that the homozygosity of TSK mutation is lethal and the embryos die by day 7-8 of pregnancy. The disruption of IL-4 gene rescued from death TSK/TSK mice suggesting a role for IL-4 in embryonic development. Since TGF-beta should compensate the lack of IL-4 on regulation of collagen gene expression, we have studied the effect of IL-4 on the expression of TGF-beta gene. Our results showed IL-4 dependence of the transcription of TGF-beta gene and that in both TSK/+ and TSK/TSK IL-4-/- mice the expression of TGF-beta gene is impaired.

Topics: Animals; Collagen; Fibrosis; Humans; Interleukin-4; Mice; Scleroderma, Localized; T-Lymphocytes; Transforming Growth Factor beta

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Disease Models, Animal; Interferon-gamma; Interleukins; Mice; Mice, Inbred BALB C; Scleroderma, Localized; Skin; Transforming Growth Factor beta

Localized scleroderma (LS) is a connective skin disease with marked sclerosis of the skin as the most prominent feature. Transforming growth factor beta (TGF-beta) plays a central role in the pathogenesis of sclerotic skin diseases. Recently, special attention was contributed to a family of transcription factor proteins involved in TGF-beta signal transduction from cell surface to the nucleus, the so-called SMADs. Ultraviolet (UV) irradiation has been reported to alter TGF-beta/SMAD pathway in human skin. We sought to investigate the effects of UVA1 on the gene and protein expressions of the TGF-beta/SMAD pathway in LS. UVA1 phototherapy was performed in eight LS patients five times weekly for 8 weeks resulting in a total of 40 treatment sessions (single dose 50 J/cm(2), cumulative dose 2,000 J/cm(2)). TGF-beta1, SMAD3, SMAD4, and SMAD7 mRNA expressions were determined by semiquantitative real-time reverse transcription polymerase chain reaction in lesional and unaffected skin of patients with LS. Additionally, immunohistochemical staining was performed in lesional skin before and after irradiation. Skin status markedly improved in all patients, resulting in a significant reduction of the clinical score from baseline to the end of treatment. Inhibitory SMAD7 mRNA was significantly higher in lesional skin as compared to unaffected skin, and significantly decreased after UVA1 phototherapy. In contrast, SMAD7 mRNA levels remained unchanged in irradiated, healthy skin after UVA1. Both TGF-beta and SMAD3 mRNA levels decreased after UVA1, whereas SMAD4 mRNA increased. However, changes in TGF-beta, SMAD3, and SMAD4 mRNA after UVA1 did not reach statistical significance. Immunohistochemical investigation did not reveal significant changes in the protein expression of SMADs after UVA1. Similar to scleroderma, SMAD7-mediated negative regulation seems to be impaired in LS. UVA1 phototherapy demonstrated the alteration of SMAD7 gene expression in LS, as SMAD7 mRNA levels normalized after UVA1. The pathogenetic relevance of SMAD7 levels with respect to clinical improvement needs further investigation.

Topics: Adult; Aged; Female; Gene Expression Regulation; Humans; Middle Aged; RNA, Messenger; Scleroderma, Localized; Signal Transduction; Skin; Smad3 Protein; Smad4 Protein; Smad7 Protein; Transforming Growth Factor beta; Ultraviolet Rays; Ultraviolet Therapy

Patients with scleroderma receiving Iloprost as a treatment for severe Raynaud's phenomenon report a reduction in skin tightness, suggesting that this drug inhibits skin fibrosis. Connective tissue growth factor (CTGF), a recently described profibrotic cytokine, acts downstream and in concert with TGF-beta to stimulate the fibrotic process and is involved in the fibrosis seen in scleroderma. Here we show that Iloprost, acting by elevation of cAMP, blocks the induction of CTGF and the increase in collagen synthesis in fibroblasts exposed to TGF-beta. The potency of Iloprost with respect to suppression of CTGF far exceeds that of other prostanoid receptor agonists, suggesting that its effect is mediated by the prostacyclin receptor IP. By sampling dermal interstitial fluid using a suction blister device, we show that CTGF levels are greatly elevated in the dermis of scleroderma patients compared with healthy controls and that Iloprost infusion causes a marked decrease in dermal CTGF levels. These studies suggest that Iloprost could be reducing the level of a key profibrotic cytokine in scleroderma patients and that endogenous production of eicosanoids may limit the fibrotic response to TGF-beta.

Topics: Cells, Cultured; Collagen; Connective Tissue Growth Factor; Cyclic AMP; Down-Regulation; Drug Administration Schedule; Fibroblasts; Growth Substances; Humans; Iloprost; Immediate-Early Proteins; Infusions, Intravenous; Intercellular Signaling Peptides and Proteins; Prostaglandins; Receptors, Prostaglandin; RNA, Messenger; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Pansclerotic morphea (PSM) is a rare, devastating disease characterized by extensive soft tissue fibrosis, secondary contractions, and significant morbidity. PSM pathogenesis is unknown, and aggressive immunosuppressive treatments rarely slow disease progression. We aimed to characterize molecular mechanisms driving PSM and to identify therapeutically targetable pathways by performing single-cell and spatial RNA-Seq on 7 healthy controls and on lesional and nonlesional skin biopsies of a patient with PSM 12 months apart. We then validated our findings using immunostaining and in vitro approaches. Fibrotic skin was characterized by prominent type II IFN response, accompanied by infiltrating myeloid cells, B cells, and T cells, which were the main IFN-γ source. We identified unique CXCL9+ fibroblasts enriched in PSM, characterized by increased chemokine expression, including CXCL9, CXCL10, and CCL2. CXCL9+ fibroblasts were related to profibrotic COL8A1+ myofibroblasts, which had enriched TGF-β response. In vitro, TGF-β and IFN-γ synergistically increased CXCL9 and CXCL10 expression, contributing to the perpetuation of IFN-γ responses. Furthermore, cell-to-cell interaction analyses revealed cDC2B DCs as a key communication hub between CXCL9+ fibroblasts and COL8A1+ myofibroblasts. These results define PSM as an inflammation-driven condition centered on type II IFN responses. This work identified key pathogenic circuits between T cells, cDC2Bs, and myofibroblasts, and it suggests that JAK1/2 inhibition is a potential therapeutic option in PSM.

Topics: Chemokine CXCL10; Dendritic Cells; Fibroblasts; Humans; Interferon-gamma; Scleroderma, Localized; Transforming Growth Factor beta

Fibrosis is a common pathophysiological response of many tissues and organs subjected to chronic injury. Despite the diverse aetiology of keloid, lacaziosis and localized scleroderma, the process of fibrosis is present in the pathogenesis of all of these three entities beyond other individual clinical and histological distinct characteristics. Fibrosis was studied in 20 samples each of these three chronic cutaneous inflammatory diseases. An immunohistochemical study was carried out to explore the presence of α-smooth muscle actin (α-SMA) and vimentin cytoskeleton antigens, CD31, CD34, Ki67, p16; CD105, CD163, CD206 and FOXP3 antigens; and the central fibrotic cytokine TGF-β. Higher expression of vimentin in comparison to α-SMA in all three lesion types was found. CD31- and CD34-positive blood vessel endothelial cells were observed throughout the reticular dermis. Ki67 expression was low and almost absent in scleroderma. p16-positive levels were higher than ki67 and observed in reticular dermis of keloidal collagen in keloids, in collagen bundles in scleroderma and in the external layers of the granulomas in lacaziosis. The presence of α-actin positive cells and rarely CD34 positive cells, observed primarily in keloids, may be related to higher p16 antigen expression, a measure of cell senescence. Low FOXP3 expression was observed in all lesion types. CD105-positive cells were mainly found in perivascular tissue in close contact with the adventitia in keloids and scleroderma, while, in lacaziosis, these cells were chiefly observed in conjunction with collagen deposition in the external granuloma layer. We did not find high involvement of CD163 or CD206-positive cells in the fibrotic process. TGF-β was notable only in keloid and lacaziosis lesions. In conclusion, we have suggested vimentin to be the main myofibroblast general marker of the fibrotic process in all three studied diseases, while endothelial-to-mesenchymal transition (EndoMT) and mesenchymal stem cells (MSCs) and M2 macrophages may not play an important role.

Topics: Endothelial Cells; Fibroblasts; Fibrosis; Forkhead Transcription Factors; Humans; Keloid; Ki-67 Antigen; Lobomycosis; Scleroderma, Localized; Skin; Transforming Growth Factor beta; Vimentin

IL-17A is abundant in scleroderma but its role in fibrogenesis is controversial. We interrogated the role of IL-17A in extracellular matrix deposition and inflammation by investigating its effects on keratinocytes and fibroblasts cross-talk and in organotypic skin cultures. Keratinocyte-conditioned media of resting, IL-17A-, and/or transforming growth factor-β-primed primary keratinocytes were used to stimulate healthy donors and scleroderma fibroblasts. Alternatively, organotypic cultures of full human skin were challenged with these cytokines. Keratinocyte-conditioned media tilted the balance of col-I to matrix metalloproteinase-1 production by fibroblasts in favor of matrix metalloproteinase-1, significantly more so in healthy donors than in scleroderma, resulting in enhanced extracellular matrix turnover, further increased by IL-17A. In organotypic skin, transforming growth factor-β induced an extensive pro-fibrotic gene signature, including the enhanced expression of several collagen genes associated with Wnt signaling. IL-17A strongly promoted the expression of pro-inflammatory genes, with no direct effects on collagen genes, and attenuated Wnt signaling induced by transforming growth factor-β. In this model, at the protein level, IL-17A significantly decreased col-I production. Our data strongly support a pro-inflammatory and antifibrogenic activity of IL-17A in the context of keratinocyte-fibroblast interaction and in full skin. These data help in directing and interpreting targeted therapeutic approaches in scleroderma.

Topics: Cells, Cultured; Collagen Type I; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Expression Regulation; Gene Regulatory Networks; Humans; Inflammation; Interleukin-17; Keratinocytes; Matrix Metalloproteinase 1; Organ Culture Techniques; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Wnt Signaling Pathway

Clinical manifestations of skin fibrosis are very variable and ambiguous, making its management quite critical and challenging. The lack of appropriate established pharmacological interventions make its treatment even more complicated. Intricate details of the underlying pathogenesis are thus imperative to further explore different treatment possibilities. Of note, the TGF-β/Smad signaling axis and epithelial to mesenchymal transition (EMT) are the principal offenders in this fibrotic disorder.. Our current study is aimed at demonstrating the antifibrotic and anti-inflammatory potential of nimbolide, a triterpene derived from Indian traditional plant neem, in a murine model of Bleomycin-induced scleroderma.. Male C57BL/6 mice were administered with Bleomycin injections subcutaneously, daily for 28 days, at a constant site on the dorsum of the mice. Treatment with nimbolide lasted from day 1 to day 28. At the time of study termination, the injected sites were collected and stored suitably to conduct further molecular experiments and protein expression studies.. The results of our study show that nimbolide can significantly intervene in the TGF-β/Smad signaling axis and the consequent EMT process, thus attenuating deposition of extracellular matrix. Nimbolide also profoundly caused the regression of established inflammation-driven fibrosis, thus demonstrating both antifibrotic and anti-inflammatory activities. Another commendable finding of this study is that nimbolide was able to decrease the levels of LOXL2, a collagen cross-linker, which is aberrantly expressed in scleroderma. Although further mechanistic studies are required, our study displays nimbolide for the first time as a potent antifibrotic agent which can be used as a pharmacological intervention for the treatment of scleroderma.

Topics: Amino Acid Oxidoreductases; Animals; Anti-Inflammatory Agents; Bleomycin; Disease Models, Animal; Epithelial-Mesenchymal Transition; Fibrosis; Inflammation; Limonins; Male; Mice; Mice, Inbred C57BL; Scleroderma, Localized; Signal Transduction; Skin; Smad Proteins; Transforming Growth Factor beta

Nanoparticle-loaded delivery systems have attracted much attention recently. Poly(lactic-co-glycolic acid) (PLGA) is one of the most successful biodegradable polymers for biomedical applications. There are only a few studies on the treatment of dermal fibrosis with sustained-release drugs. Peroxisome proliferator-activated receptor-γ (PPAR-γ) plays an important role in endogenous anti-fibrotic defense mechanisms. Recent studies have suggested that pioglitazone, a synthetic PPAR-γ activator, has effects beyond reducing blood sugar and it can reduce fibrosis and inflammation when used systemically.. We aimed to assess the effects of local injections of pioglitazone-loaded PLGA nanoparticles (PGN-NP) on an experimental sclerosis and to demonstrate the in vivo pharmacokinetics of subcutaneously administered PLGA nanoparticles.. Locally injectable PGN-NP were prepared and subcutaneously administered to bleomycin (BLM)-induced scleroderma model mice. The effect of pioglitazone was also evaluated with cultured fibroblasts. Coumarin-6-loaded fluorescent PLGA nanoparticles (FL-NP) and silicon naphthalocyanine-loaded near-infrared PLGA nanoparticles (NIR-NP) were used to demonstrate in vitro cellular uptake by cultured fibroblasts and the in vivo pharmacokinetics of subcutaneously administered nanoparticles.. Weekly subcutaneous injections of PGN-NP attenuated skin fibrosis in BLM-induced scleroderma model mice. Pioglitazone significantly suppressed migration ability and TGF-β-mediated myofibroblast differentiation in cultured fibroblasts. FL-NP were internalized into cultured fibroblasts within 60 min, and PGN-NP-primed fibroblasts expressed anti-fibrotic phenotypes. Subcutaneously injected NIR-NP remained in the vicinity of the injection site more than non-particulate silicon naphthalocyanine.. These results provide a basis for the development of new treatments for dermal fibrosis and a better understanding of the potential of PLGA nanoparticles in dermatology.

Topics: Animals; Bleomycin; Cell Differentiation; Delayed-Action Preparations; Disease Models, Animal; Drug Carriers; Fibroblasts; Fibrosis; Humans; Injections, Subcutaneous; Mice; Mice, Inbred BALB C; Nanoparticles; Pioglitazone; Polylactic Acid-Polyglycolic Acid Copolymer; PPAR gamma; Scleroderma, Localized; Skin; Transforming Growth Factor beta; Treatment Outcome

Scleroderma is an inflammatory autoimmune disease which begins with inflammation due to tissue injury and advances to progressive accumulation of extracellular matrix resulting in scarring and hardening of the skin. Inflammation is a salutary response to tissue injury caused by varied factors. While inflammation is required for systematic wound healing, dysregulated chronic inflammation often leads to tissue scarring. Prominent role of inflammation in pathology and physiology makes it a double edge sword. The objective of this study was to investigate the role of Withaferin A (WFA), a steroidal lactone from Withania somnifera in a 28-day murine model of bleomycin-induced experimental scleroderma. Withaferin A was administered at two doses 2 and 4 mg/kg intraperitoneally for 28 days. At the time of study termination, we observed significant reduction in dorsal skin thickness. Our results indicate that WFA was able to sufficiently suppress pro-inflammatory phase of fibrosis, TGF-β/Smad signaling and also significantly repressed fibroblast conversion to myofibroblasts. Additionally, our study also demonstrated that WFA modulates FoxO3a-Akt-dependent NF-κβ/IKK-mediated inflammatory cascade, which is a prime signaling pathway in fibrogenesis. The findings of this study are persuasive of WFA as an antifibrotic agent with promising therapeutic effects in scleroderma. © 2018 BioFactors, 44(6):507-517, 2018.

Topics: Animals; Anti-Inflammatory Agents; Bleomycin; Cell Differentiation; Dermatologic Agents; Disease Models, Animal; Extracellular Matrix; Fibroblasts; Forkhead Box Protein O3; Gene Expression Regulation; Humans; I-kappa B Kinase; Male; Mice; Mice, Inbred C57BL; Myofibroblasts; NF-kappa B; Proto-Oncogene Proteins c-akt; Scleroderma, Localized; Signal Transduction; Skin; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Withania; Withanolides

SSc is a devastating disease that results in fibrosis of the skin and other organs. Fibroblasts are a key driver of the fibrotic process through deposition of extracellular matrix. The mechanisms by which fibroblasts are induced to become pro-fibrotic remain unclear. Thus, we examined the ability of SSc keratinocytes to promote fibroblast activation and the source of this effect.. Keratinocytes were isolated from skin biopsies of 9 lcSSc, 10 dcSSc and 13 control patients. Conditioned media was saved from the cultures. Normal fresh primary fibroblasts were exposed to healthy control and SSc keratinocyte conditioned media in the presence or absence of neutralizing antibodies for TGF-β. Gene expression was assessed by microarrays and real-time PCR. Immunocytochemistry was performed for α-smooth muscle actin (α-SMA), collagen type 1 (COL1A1) and CCL5 expression.. SSc keratinocyte conditioned media promoted fibroblast activation, characterized by increased α-SMA and COL1A1 mRNA and protein expression. This effect was independent of TGF-β. Microarray analysis identified upregulation of nuclear factor κB (NF-κB) and downregulation of peroxisome proliferator-activated receptor γ (PPAR-γ) pathways in both SSc subtypes. Scleroderma keratinocytes exhibited increased expression of NF-κB-regulated cytokines and chemokines and lesional skin staining confirmed upregulation of CCL5 in basal keratinocytes.. Scleroderma keratinocytes promote the activation of fibroblasts in a TGF-β-independent manner and demonstrate an imbalance in NF-κB1 and PPAR-γ expression leading to increased cytokine and CCL5 production. Further study of keratinocyte mediators of fibrosis, including CCL5, may provide novel targets for skin fibrosis therapy.

Topics: Actins; Adult; Aged; Cell Differentiation; Cells, Cultured; Chemokine CCL5; Collagen Type I; Collagen Type I, alpha 1 Chain; Culture Media, Conditioned; Down-Regulation; Female; Fibroblasts; Fibrosis; Gene Expression Profiling; Humans; Immunohistochemistry; Keratinocytes; Male; Middle Aged; NF-kappa B; PPAR gamma; Real-Time Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Diffuse; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Up-Regulation

Wnt signalling has been implicated in activating a fibrogenic programme in fibroblasts in systemic sclerosis (SSc). Porcupine is an O-acyltransferase required for secretion of Wnt proteins in mammals. Here, we aimed to evaluate the antifibrotic effects of pharmacological inhibition of porcupine in preclinical models of SSc.. The porcupine inhibitor GNF6231 was evaluated in the mouse models of bleomycin-induced skin fibrosis, in tight-skin-1 mice, in murine sclerodermatous chronic-graft-versus-host disease (cGvHD) and in fibrosis induced by a constitutively active transforming growth factor-β-receptor I.. Treatment with pharmacologically relevant and well-tolerated doses of GNF6231 inhibited the activation of Wnt signalling in fibrotic murine skin. GNF6231 ameliorated skin fibrosis in all four models. Treatment with GNF6231 also reduced pulmonary fibrosis associated with murine cGvHD. Most importantly, GNF6231 prevented progression of fibrosis and showed evidence of reversal of established fibrosis.. These data suggest that targeting the Wnt pathway through inhibition of porcupine provides a potential therapeutic approach to fibrosis in SSc. This is of particular interest, as a close analogue of GNF6231 has already demonstrated robust pathway inhibition in humans and could be available for clinical trials.

Topics: Acyltransferases; Aminopyridines; Animals; Bleomycin; Disease Models, Animal; Disease Progression; Female; Fibrosis; Graft vs Host Disease; Membrane Proteins; Mice, Inbred BALB C; Piperazines; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Wnt Signaling Pathway

Bleomycin-induced scleroderma in mice is an established model for human scleroderma. Making use of this, we have established a new model for wound retardation. After inducing dermal sclerosis by local bleomycin treatment in nude mice, a full-thickness wound was made by punch excision on the bleomycin application site. Mice pretreated with bleomycin showed a significant delay in wound closure, as compared with mice pretreated with phosphate-buffered saline. Proliferation of keratinocytes was significantly inhibited and the number of Ki-67-positive keratinocytes was significantly lower in the bleomycin-pretreated skin. Also, the number of CD31-positive blood vessels was markedly reduced in the bleomycin-treated skin. The topical daily application of basic fibroblast growth factor (bFGF) significantly promoted wound closure, while increasing blood vessel formation and reducing transforming growth factor-β and alpha-smooth muscle actin mRNA levels. Furthermore, only two applications of PG-FGF1, a fusion protein of FGF1 with heparan sulfate proteoglycan, overcame the delay in wound closure. Wound delay in this model mainly occurred as a result of decreased vessel formation and keratinocyte migration following bleomycin treatment. It is expected that this model will provide novel insights into the pathogenesis of wound healing and the exploration of possible candidate drugs for refractory or chronic wounds in the clinical setting.

Topics: Actins; Animals; Bleomycin; Cell Proliferation; Disease Models, Animal; Female; Fibroblast Growth Factor 1; Heparan Sulfate Proteoglycans; Keratinocytes; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Physiologic; RNA, Messenger; Scleroderma, Localized; Transforming Growth Factor beta; Wound Healing

Scleroderma is a progressive autoimmune disease affecting multiple organs. Fibrosis, the hallmark of scleroderma, represents transformation of self-limited wound healing into a deregulated self-sustaining process. The factors responsible for maintaining persistent fibroblast activation in scleroderma and other conditions with chronic fibrosis are not well understood. Toll-like receptor 4 (TLR4) and its damage-associated endogenous ligands are implicated in immune and fibrotic responses. We now show that fibronectin extra domain A (Fn(EDA)) is an endogenous TLR4 ligand markedly elevated in the circulation and lesional skin biopsies from patients with scleroderma, as well as in mice with experimentally induced cutaneous fibrosis. Synthesis of Fn(EDA) was preferentially stimulated by transforming growth factor-β in normal fibroblasts and was constitutively up-regulated in scleroderma fibroblasts. Exogenous Fn(EDA) was a potent stimulus for collagen production, myofibroblast differentiation, and wound healing in vitro and increased the mechanical stiffness of human organotypic skin equivalents. Each of these profibrotic Fn(EDA) responses was abrogated by genetic, RNA interference, or pharmacological disruption of TLR4 signaling. Moreover, either genetic loss of Fn(EDA) or TLR4 blockade using a small molecule mitigated experimentally induced cutaneous fibrosis in mice. These observations implicate the Fn(EDA)-TLR4 axis in cutaneous fibrosis and suggest a paradigm in which aberrant Fn(EDA) accumulation in the fibrotic milieu drives sustained fibroblast activation via TLR4. This model explains how a damage-associated endogenous TLR4 ligand might contribute to converting self-limited tissue repair responses into intractable fibrogenesis in chronic conditions such as scleroderma. Disrupting sustained TLR4 signaling therefore represents a potential strategy for the treatment of fibrosis in scleroderma.

Topics: Adult; Animals; Biopsy; Bleomycin; Cell Differentiation; Chronic Disease; Collagen; Female; Fibronectins; Fibrosis; Fluorescent Antibody Technique; Humans; Ligands; Male; Mice, Inbred C57BL; Middle Aged; Myofibroblasts; Scleroderma, Localized; Signal Transduction; Skin; Toll-Like Receptor 4; Transforming Growth Factor beta

Systemic plasma cell dyscrasias have diverse manifestations in the skin and include an inflammatory paraneoplastic process. We encountered cases of scleroderma and eosinophilic fasciitis in the setting of an underlying plasma cell dyscrasia.. Ten cases of scleroderma-like tissue reactions in the setting of an underlying plasma cell dyscrasia were encountered. The biopsies were stained for Transforming growth factor (Transforming growth factor) beta, IgG4, kappa, and lambda.. Patients presented with a sclerodermoid reaction represented by eosinophilic fasciitis (5 cases), morphea (3 cases), and systemic scleroderma (2 cases). The mean age of presentation was 70 years with a striking female predominance (4:1). Acral accentuation was noted in 8 cases. In 6 of the cases, the cutaneous sclerosis antedated (4 cases) by weeks to 2 years or occurred concurrently (2 cases) with the initial diagnosis of the plasma cell. The biopsies showed changes typical of eosinophilic fasciitis and/or scleroderma. In 5 cases, light chain-restricted plasma cells were present on the biopsy. There was staining of the plasma cells for Transforming growth factor beta in 3 out of 5 cases tested.. In any older patient presenting with a sudden onset of eosinophilic fasciitis or scleroderma especially with acral accentuation, investigations should be conducted in regards to an underlying plasma cell dyscrasia.

Topics: Aged; Aged, 80 and over; Biomarkers, Tumor; Biopsy; Eosinophilia; Fasciitis; Female; Humans; Immunoglobulin G; Immunoglobulin kappa-Chains; Immunoglobulin lambda-Chains; Immunohistochemistry; Male; Middle Aged; Paraneoplastic Syndromes; Paraproteinemias; Prognosis; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Overexpression of vascular endothelial growth factor (VEGF) in scleroderma (SSc) skin may play a role in the pathogenesis of the disease. Our study was undertaken to evaluate whether dermal fibroblasts function as one of the sources of the increased VEGF in SSc, and to clarify its mechanism.. Protein and mRNA levels of VEGF were analyzed using immunoblotting, enzyme-linked immunosorbent assay, and real-time PCR. The DNA-binding ability of Smad3 was evaluated by DNA affinity precipitation.. VEGF mRNA expression in vivo was increased in SSc skin compared to skin with other collagen diseases. Expression of VEGF protein and mRNA in cultured SSc dermal fibroblasts was constitutively and significantly upregulated. Ectopic TGF-β stimulation induced VEGF synthesis in normal fibroblasts, and TGF-β knockdown normalized the upregulated VEGF levels in SSc fibroblasts. Furthermore, Smad3 overexpression induced VEGF levels. We found that bp -532 to -521 on the VEGF promoter is a putative binding site for Smads, and that the binding activity of Smad3 to VEGF promoter was constitutively increased in SSc fibroblasts as well as in normal fibroblasts treated with exogenous TGF-β1.. We demonstrated that VEGF were overexpressed due to autocrine TGF-β/Smad signaling in SSc. TGF-β signaling may contribute to the pathogenesis of angiopathy as well as tissue fibrosis.

Topics: Adult; Autocrine Communication; Female; Fibroblasts; Humans; Promoter Regions, Genetic; Scleroderma, Localized; Skin; Smad3 Protein; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

A noncanonical pathway of transforming growth factor-β signalling, the c-Abl/protein kinase C-δ (PKC-δ)/Friend leukemia virus integration 1 (Fli1) axis, is a powerful regulator of collagen synthesis in dermal fibroblasts.. To investigate the significance of the c-Abl/PKC-δ/Fli1 pathway for the establishment of the profibrotic phenotype in lesional dermal fibroblasts from patients with localized scleroderma (LSc).. The activation status of the c-Abl/PKC-δ/Fli1 pathway was evaluated by immunoblotting and chromatin immunoprecipitation using cultured dermal fibroblasts from patients with LSc and closely matched healthy controls and by immunostaining on skin sections. The effects of a platelet-derived growth factor receptor inhibitor AG1296 and gene silencing of c-Abl on the expression levels of type I collagen were evaluated by immunoblotting.. The phosphorylation levels of Fli1 at threonine 312 were increased, while the total Fli1 levels and the binding of Fli1 to the COL1A2 promoter were decreased, in cultured LSc fibroblasts compared with cultured normal fibroblasts. Furthermore, in cultured LSc fibroblasts, the expression levels of c-Abl were elevated compared with cultured normal fibroblasts and PKC-δ was preferentially localized in the nucleus. These findings were also confirmed in vivo by immunohistochemistry using skin sections. Moreover, gene silencing of c-Abl, but not AG1296, significantly suppressed the expression of type I collagen in cultured LSc fibroblasts.. Constitutive activation of the c-Abl/PKC-δ/Fli1 pathway at least partially contributes to the establishment of the profibrotic phenotype in LSc dermal fibroblasts, which provides a novel molecular basis to explain the efficacy of imatinib against skin sclerosis in a certain subset of LSc.

Topics: Adolescent; Adult; Benzamides; Case-Control Studies; Cells, Cultured; Child; Child, Preschool; Female; Fibroblasts; Humans; Imatinib Mesylate; Male; Middle Aged; Phosphorylation; Piperazines; Protein Kinase C-delta; Protein Kinase Inhibitors; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins c-abl; Pyrimidines; Scleroderma, Localized; Transforming Growth Factor beta

microRNAs (miRNAs) play a part in various cellular activities. However, the role of miRNA in SSc is not fully understood. This study investigated the expression and role of miR-92a in SSc patients and evaluated the possibility that miR-92a is involved in the pathogenesis of this disease.. Serum samples were obtained from 61 SSc patients. mRNAs were purified from serum and levels of miR-92a and miR-135 were measured with quantitative real-time PCR. miR-92a expression in dermal fibroblasts was also determined by quantitative real-time PCR. Immunoblotting was performed to detect MMP-1 protein.. The median serum levels of miR-92a, not miR-135, were significantly higher in SSc patients than normal subjects. The constitutive up-regulated miR-92a expression was also found in cultured dermal fibroblasts from SSc skin, which was decreased by the transfection with siRNA of TGF-β. Furthermore, the forced overexpression of miR-92a in normal dermal fibroblasts using miR-92a mimic resulted in the down-regulation of MMP-1 expression.. The increase of miR-92a in SSc may be due to the stimulation of intrinsic TGF-β activation seen in this disease. There is also a possibility that MMP-1 is the target of miR-92a and that increased miR-92a expression therefore plays a role in excessive collagen accumulation in SSc via the down-regulation of MMP-1. Clarifying the role of miRNAs in SSc may result in a better understanding of this disease and the development of new therapeutic approaches.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Cells, Cultured; Dermatomyositis; Dermis; Female; Fibroblasts; Gene Expression Regulation; Gene Silencing; Humans; Integrin alphaVbeta3; Lupus Erythematosus, Systemic; Male; Matrix Metalloproteinase 1; MicroRNAs; Middle Aged; RNA, Small Interfering; Scleroderma, Diffuse; Scleroderma, Localized; Transforming Growth Factor beta

Dermal wound healing depends on highly complex interplay among various cytokines and cell types. Disruption of this process can result in impaired healing in the form of excessive scarring, as is the case in fibrotic diseases such as keloid and scleroderma. In the present study, we found Fussel-15, a new member of the Ski/Sno family of TGF-β/BMP signaling repressors, to be expressed in early wound healing and constantly overexpressed in keloid-derived and scleroderma-derived fibroblasts. Comparing the results of three-dimensional free-floating and attached-released in vitro wound healing assays, we observed that Fussel-15 is expressed during the migratory phase in the free-floating assay, indicating that Fussel-15 might play a role during fibroblast migration. Fussel-15-transfected fibroblasts showed greater migration ability in a scratch wound healing assay, compared with control-transfected cells. This migratory phenotype due to Fussel-15 was confirmed by increased peripheral F-actin localization and modifications in size, amount, and distribution of focal adhesion complexes, which were observed using F-actin and focal adhesion kinase (FAK) immunofluorescence staining, respectively. The present results suggest that expression of Fussel-15 during wound healing might promote fibroblast migration. Permanent expression of Fussel-15 in keloid and skin sclerosis fibroblasts could be involved in the pathogenesis of these conditions, but the molecular mechanism underlying this up-regulation remains to be determined.

Topics: Actins; Biological Assay; Cell Adhesion; Cell Line; Cell Movement; Cell Proliferation; Co-Repressor Proteins; Collagen; Dermis; Extracellular Matrix; Fibroblasts; Focal Adhesions; Gene Expression Regulation; Humans; Keloid; Protein Transport; Repressor Proteins; Scleroderma, Localized; Transforming Growth Factor beta; Wound Healing

Systemic sclerosis (SSc) and morphoea are connective tissue diseases characterized by fibrosis of the skin. Although to date their pathogenesis has not been clearly defined, it is thought that autoimmunity may play a role in the development of the skin lesions observed in both these diseases. As regulatory T cells (Tregs) play a key role in the modulation of immune responses, it has recently been suggested that Treg impairment may lead to the development of autoimmune diseases.. To investigate the presence of Tregs and their immunomodulatory cytokines, transforming growth factor (TGF)-beta and interleukin (IL)-10, in patients with SSc and morphoea.. Fifteen patients with SSc and 15 with morphoea were enrolled. Immunohistochemistry was applied to identify FoxP3+ (forkhead/winged helix transcription factor) Tregs, TGF-beta+ cells and IL-10+ cells in the skin, cytofluorometry to detect CD4+CD25+FoxP3+ Tregs in the blood, and enzyme-linked immunosorbent assays to analyse TGF-beta and IL-10 serum levels.. Fewer FoxP3+ Tregs and TGF-beta+ and IL-10+ cells were found in the skin of patients with scleroderma than in controls. Similarly, there were reduced TGF-beta and IL-10 serum levels and fewer circulating CD4+CD25brightFoxP3+ cells in patients with SSc or morphoea, than in controls.. The quantitative reduction of Tregs, together with that of TGF-beta and IL-10 serum levels, may be responsible for the loss of tolerance observed in both SSc and morphoea.

Topics: Adult; CD4 Lymphocyte Count; Female; Forkhead Transcription Factors; Humans; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Male; Middle Aged; Scleroderma, Localized; Scleroderma, Systemic; Skin; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.

Topics: Cell Adhesion; Cell Hypoxia; Cells, Cultured; Culture Media, Serum-Free; Extracellular Matrix; Fibrillin-2; Fibrillins; Fibroblasts; Fluorescent Antibody Technique; Gene Expression; Humans; Immunohistochemistry; Interleukin-4; Microfilament Proteins; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Localized; Sclerosis; Skin; Tenascin; Transforming Growth Factor beta; Wound Healing

Despite several investigations, the transcriptional mechanisms which regulate the expression of both type I collagen genes (COL1A1 and COL1A2) in either physiological or pathological situations, such as scleroderma, are not completely known. In this study, we determined the effects of both native ichtyan chondroïtin sulphate (CS) and its derived hydrolytic fragments (CSf) on human normal (NF) and scleroderma (SF) fibroblasts. Here, we demonstrate for the first time that CS and CSf exert an inhibitory effect on type I collagen protein synthesis and decrease the corresponding mRNA steady-state levels of COL1A1 and COL1A2 in NF and SF. These glycosaminoglycan molecules repress COL1A1 gene transcription through a -112/-61 bp sequence upstream the start site of transcription and imply hc-Krox and Sp1 transcription factors. In addition, CS and CSf induced a down-regulation of TbetaRI expression. As a conclusion, our findings highlight a possible new role for CS and CSf as anti-fibrotic molecules and could help in elucidating the mechanisms of action by which CS and CSf exert their inhibitory effect on type I collagen synthesis.

Topics: Base Pairing; Base Sequence; Chondroitin Sulfates; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Collagen Type III; DNA-Binding Proteins; Fibroblasts; Gene Expression Regulation; Humans; Molecular Sequence Data; Peptide Fragments; Promoter Regions, Genetic; Protein Binding; RNA, Messenger; Scleroderma, Localized; Smad Proteins; Sp1 Transcription Factor; Sp3 Transcription Factor; Transcription Factors; Transforming Growth Factor beta

Localized scleroderma (LSc) is a connective tissue disorder limited to skin and subcutaneous tissue, which may share pathogenic processes with systemic sclerosis (SSc). We previously demonstrated that upregulated expression of integrin alphavbeta5 might contribute to autocrine TGF-beta signaling in SSc fibroblasts. Based on these data, we presently focused on alphavbeta5 and assessed its involvement in pathogenesis of LSc. We initially demonstrated that LSc fibroblasts might be activated by the stimulation of autocrine TGF-beta. Consistent with SSc fibroblasts, expression levels of alphavbeta5 were elevated in LSc fibroblasts in vitro and in vivo. Anti-alphavbeta5 antibody partially reversed expression levels of type I procollagen and MMP-1 and constitutive DNA-Smad3 binding in LSc fibroblasts. In LSc fibroblasts pretreated with antisense TGF-beta1, exogenous latent TGF-beta1 stimulation increased expression of type I procollagen in an alphavbeta5-dependent manner. The luciferase activities of TMLC cells, Mv1Lu cells stably expressing a portion of the plasminogen activator inhibitor 1 promoter, co-cultured with LSc fibroblasts were significantly elevated compared with those co-cultured with normal fibroblasts and were significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of LSc fibroblasts. These results indicate that upregulated expression of alphavbeta5 contributes to autocrine TGF-beta signaling in LSc fibroblasts.

Topics: Antibodies; Autocrine Communication; Biopsy; Cells, Cultured; Collagen Type I; Dermis; DNA-Binding Proteins; Female; Fibroblasts; Fluorescent Antibody Technique; Humans; Integrins; Male; Matrix Metalloproteinase 1; Membrane Proteins; Receptors, Vitronectin; Scleroderma, Localized; Smad3 Protein; Transforming Growth Factor beta; Transforming Growth Factor beta1

Systemic sclerosis (SSc) is a connective tissue disorder with an unknown etiology. There are currently no effective therapies for SSc. (In this study, working with a bleomycin(BLM)-induced scleroderma model mice, we performed two transfections of human hepatocyte growth factor (HGF) cDNA into the skeletal muscle and showed that this treatment not only helped to prevent the dermal sclerosis simultaneously injected BLM but also improved the symptoms of dermal sclerosis induced by BLM 4 weeks previously.) RT-PCR, ELISA and an immunohistochemical analysis revealed that both mRNA and protein of human HGF as well as murine HGF were enhanced in the skin, lung, muscle and the serum after two transfections of human HGF cDNA. These analyses also revealed that this treatment significantly reduced both the expression of the TGF-beta1 mRNA and the production of TGF-beta1 on macrophage-like cells that infiltrated the dermis and the fibroblastic cells in BLM-induced scleroderma. Furthermore, HGF-gene transfection both prevented and ameliorated the symptoms of not only dermal sclerosis but also of lung fibrosis induced by a subcutaneous BLM injection. These results indicated that gene therapy by the transfection of the human HGF cDNA may thus be a useful therapy for SSc and lung fibrosis involved with SSc.

Topics: Animals; Antimetabolites; Bleomycin; Female; Fibrosis; Gene Expression; Genetic Therapy; Genetic Vectors; Hepatocyte Growth Factor; Humans; Injections, Intramuscular; Liposomes; Lung; Mice; Mice, Inbred C3H; Models, Animal; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Localized; Scleroderma, Systemic; Sendai virus; Transfection; Transforming Growth Factor beta

Ultraviolet A (UVA) phototherapy proved to be an efficient line of treatment of scleroderma. The mechanism through which it acts is still not clear.. To detect the mechanism of action of UVA phototherapy in morphea through measuring its effect on the levels of different parameters related to collagen metabolism.. Twenty-one cases of morphea were treated with low-dose broad-band UVA for 20 sessions. Twelve cases received 20 J/cm(2)/session with a cumulative dose of 400 J/cm(2) and nine cases received 10 J/cm(2)/session with a cumulative dose of 200 J/cm(2). The response was assessed clinically every week. Two skin biopsies were taken from the lesional skin of each patient before starting and after the end of therapy. Paraffin sections were examined for quantitative polymerase chain reaction measurement of collagen I, collagen III, collagenase, transforming growth factor-beta (TGF-beta) and interferon gamma (IFNgamma).. Eighteen patients reported remarkable softening of the skin lesions, with variable degrees ranging from moderate in 57.1% of them good in 19% to very good response in 9.5%. After treatment, all the studied parameters revealed statistically significant changes. There was a significant decrease in collagen I, collagen III and TGF-beta and a significant increase in collagenase (MMP-1) and IFNgamma. The relative change was found to be greatest in collagenase, followed by IFNgamma then TGF-beta and finally collagen I. The changes in collagen I, collagenase, IFNgamma and TGF-beta were found to increase gradually with the degree of clinical response. In all the parameters studied the relative change was significantly higher in cases treated with 20 J/cm(2)/session in contrast to those treated with 10 J/cm(2)/session although no statistically significant difference could be detected in the clinical response to those doses.. The efficacy of low-dose UVA phototherapy in the treatment of localized scleroderma is mainly obtained by the increased production of MMP-1 and IFNgamma, and to a lesser extent by decreasing TGF-beta and collagen production. Concerning the use of 10 or 20 J/cm(2)/session those effects are dose dependent, but the clinical response does not significantly differ.

Topics: Adolescent; Adult; Aged; Analysis of Variance; Child; Collagen; Collagenases; Female; Humans; Interferon-gamma; Male; Middle Aged; Polymerase Chain Reaction; Scleroderma, Localized; Transforming Growth Factor beta; Treatment Outcome; Ultraviolet Therapy

Pericytes (PCs) are smooth muscle-like mural cells of capillaries and venules, which can synthesize matrix components and fibroblast-activating cytokines, and are thus potential mediators of pathological changes in scleroderma. In this study, alterations in microvessels were quantitatively imaged, taking PC into account for the first time.. Skin biopsies from systemic (12) and localized (14) scleroderma forms as well as age-, sex-, and body location-matched controls were examined with respect to capillary and venular densities as well as endothelial cell (EC) and PC counts using a newly developed (in respect of PC and EC) indirect collagen IV immunostaining-based method.. Hyperplasia of the PC that doubled the microvascular PC density was the most conspicuous characteristic. In the capillaries of the upper dermal plexus of the periphery of the sclerotic zones, median ratios of PC : EC were 0.23 (controls 0.10) or 0.18 (controls 0.11) in systemic or localized scleroderma, respectively. Furthermore, an increase in capillary density in the upper dermal plexus could be demonstrated in the marginal zones of both types of disease.. The observed PC increase in the peripheral zones of active disease supports the hypothesis of a vascular pathogenesis of scleroderma and directs the focus to microvascular PC.

Topics: Adolescent; Adult; Aged; Capillaries; Cell Count; Collagen Type IV; Endothelial Cells; Female; Humans; Hyperplasia; Immunohistochemistry; Male; Middle Aged; Pericytes; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic scleroderma is a chronic disease, which leads to fibrosis of the skin and internal organs. Fibroblasts obtained from patients with this disease demonstrate an activated state in culture. We, in this study, report strong, constitutive overexpression of plasminogen activator inhibitor type-2 (PAI-2) in scleroderma fibroblasts and demonstrate that this induction observed at the mRNA and protein level is dependent on serum addition. Induced PAI-2 protein levels were restricted to the non-glycosylated 47-kDa form, which is located intracellularly. Induction was stable for at least 12 passages. No modulation by fibrogenic cytokines--for example, transforming growth factor-beta1 or connective tissue growth factor--or by antagonizing IL-1 receptors was observed. The data indicate that scleroderma fibroblasts are more sensitive to the induction of PAI-2 expression than control fibroblasts by a presently unknown factor in serum.

Topics: Adult; Biopsy; Blotting, Northern; Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Glycosylation; Humans; Male; Microscopy, Fluorescence; Middle Aged; Plasminogen Activator Inhibitor 2; Receptors, Interleukin-1; RNA; RNA, Messenger; Scleroderma, Localized; Scleroderma, Systemic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

A 62-year-old Japanese man presented with multiple small atrophic macules on the trunk and extremities. The lesions were discrete, oval in shape and enclosed by lilac ring. They were distributed in a Christmas tree distribution, reminiscent of pityriasis rosea. Skin biopsy showed increased collagen fibres in the dermis and invading subcutaneous tissue. The clinico-pathological features were consistent with guttate morphoea, a rare variant of localized scleroderma. Serological tests revealed a positive reaction to human T-cell lymphoma/lymphotropic virus type-1 infection.

Topics: Biopsy; Collagen Type I; Human T-lymphotropic virus 1; Humans; Leukemia-Lymphoma, Adult T-Cell; Male; Middle Aged; Scleroderma, Localized; Serologic Tests; Skin Diseases, Viral; Transforming Growth Factor beta; Transforming Growth Factor beta1

Scleroderma is a connective tissue disorder characterised by excessive accumulation of collagen in the skin and internal organs. The most likely explanation for this process is local activation of collagen synthesis from fibroblasts. Our intention was to elucidate whether TGF-beta3 and mast cells play a pathogenic role in abnormal connective tissue formation in scleroderma. In this study, skin biopsies from 20 patients with scleroderma and five from healthy individuals were studied by an indirect immunoperoxidase technique to determine the immunoreactivity of TGF-beta3 in the dermis. In addition, skin samples were stained with toluidine blue to count the number of mast cells in scleroderma, and tissues were examined under the electron microscope to evaluate the ultrastructural changes. Increased TGF-beta3 immunoreactivities were detected in the dermis in the patient's skin, suggesting the presence of a subpopulation responsible for the increased collagen production. Mast cell counts in the skin of patients with scleroderma were significantly greater (19.2 +/- 4.1/unit) than those of normal controls (4.4 +/- 1.2/unit). Ultrastructural observations indicated that there is a close relationship between the mast cells and fibroblasts. These results suggest that fibrosis in scleroderma could evolve through the activation of fibroblasts and the regulatory mechanisms that appear to modulate the behavior of these cells with respect to collagen production.

Topics: Adult; Biomarkers; Biopsy, Needle; Case-Control Studies; Cell Movement; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Mast Cells; Microscopy, Electron; Middle Aged; Photomicrography; Prognosis; Reference Values; Scleroderma, Localized; Sensitivity and Specificity; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta3

Linear scleroderma (LS) is a localized form of scleroderma characterized by mononuclear cell infiltration and fibroblast proliferation. In the later stages of the disease, excessive collagen is deposited with concomitant skin and appendage atrophy. These symptoms suggest a breakdown of fibroblast cell function, and consequently, growth factors have been thought to play a role in the pathogenesis of LS. The present study examined the expression of TGF-beta and PDGF in skin biopsies obtained from patients with LS and from normal subjects. Samples were prepared for immunohistochemistry. To identify TGF-beta, two polyclonal antibodies were used: TGF-beta1 (RaB4) and TGF-beta2 (CL-B1/29) and, to identify PDGF, two monoclonal antibodies were used: PDGF-AA (3E-205) and PDGF-BB (1F-133). Staining for TGF-beta1 and TGF-beta2 was observed around blood vessels (endothelial cells), and sweat glands in both LS and normal skin. Staining for PDGF-AA and PDGF-BB was intense in endothelial cells and sweat glands in LS and normal skin. Mononuclear cell infiltrates and abnormal collagen bundles did not stain for TGF-beta or PDGF. The strength and extent of staining was evaluated in tissues using a scale from zero (no staining) to four (strong staining). The amount of TGF-beta1, TGF-beta2, PDGF-AA and PDGF-BB was found similar in LS and normal skin. These results do not support the hypothesis that the excessive fibroblast cell activity and abnormal collagen deposition observed in LS are associated with downregulation of TGF-beta or PDGF.

Topics: Adolescent; Adult; Biopsy; Child; Female; Humans; Middle Aged; Platelet-Derived Growth Factor; Scleroderma, Localized; Transforming Growth Factor beta

Scleroderma is a connective tissue disorder with unknown etiology. Myofibroblasts appear during fibrotic processes such as scleroderma, hypertrophic scarring, and wound healing. We previously established a mouse model for scleroderma by local injections of bleomycin. To determine the phenotype of the fibroblasts in sclerotic skin after bleomycin treatment, we examined the expression of alpha-smooth muscle actin (alpha-SMA), a marker for myofibroblasts, in lesional skin as well as in fibrous lung in this model. Dermal sclerosis was induced by daily local injections of bleomycin (100 microg/ml) for 3 weeks in C3H mice. Immunohistochemical examination showed that alpha-SMA-reactive cells were detectable on fibroblastic cells in bleomycin-injected skin at 1 week. There was a significant increase in the immunoreactive fibroblastic cells for alpha-SMA in lesional skin in parallel with the induction of dermal sclerosis. After 3 weeks' treatment with bleomycin, the number of alpha-SMA-reactive fibroblasts showed an 11-fold increase compared with that in control PBS-treated mice. alpha-SMA-positive cells were also detected in lung parenchyma after bleomycin treatment. Following concomitant treatment with anti-transforming growth factor-beta (TGF-beta) antibody with bleomycin, the number of alpha-SMA-positive fibroblastic cells was significantly reduced up to 50%, along with the reduction of dermal sclerosis. To confirm the protein level of alpha-SMA, immunoblotting was carried out. Results showed an increase of alpha-SMA expression in lesional skin at 3 weeks of bleomycin treatment, which was reduced following anti-TGF-beta antibody treatment. These data suggest that fibroblastic cells are phenotypically altered into myofibroblasts during the fibrotic process in the experimental model of bleomycin-induced scleroderma, which was considered mediated, for the most part, by TGF-beta. Blockade of TGF-beta may be a therapeutic intervention for scleroderma.

Topics: Actins; Animals; Bleomycin; Disease Models, Animal; Female; Fibroblasts; Mice; Mice, Inbred C3H; Muscle, Smooth; Scleroderma, Localized; Scleroderma, Systemic; Sclerosis; Transforming Growth Factor beta

Topics: Adult; Anti-Inflammatory Agents; Female; Humans; Scleroderma, Localized; Steroids; Transforming Growth Factor beta; Transforming Growth Factor beta3; Treatment Outcome; Up-Regulation

Interleukin-10 (IL-10) is a cytokine with many regulatory functions. In particular, IL-10 exerts neutralizing effect on other cytokines, and therefore IL-10 is thought to have important therapeutic implications. Recent reports suggest that IL-10 regulates not only immunocytes but also collagen and collagenase gene expression in fibroblasts. In this study, we investigated the effect of IL-10 on gene expression of extracellular matrix (ECM) proteins, such as type I collagen, fibronectin, and decorin, in human skin fibroblasts. Results of Northern blot analysis showed that both collagen I and fibronectin mRNAs were downregulated, while decorin gene expression was enhanced by IL-10 (10 ng/ml) time-dependently (6-24 h). alpha1(I) collagen and fibronectin mRNAs were decreased to one-third and one-fourth, respectively, by 50 ng/ml IL-10, whereas decorin mRNA was increased up to 2.7-fold by 50 ng/ml IL-10. Response to IL-10 by scleroderma fibroblasts was similar to that in normal dermal fibroblasts, with decreased expression levels of collagen and fibronectin and induced decorin mRNA levels. Transforming growth factor-beta (TGF-beta) is a crucial fibrogenic cytokine which upregulates the mRNA expression of collagen and fibronectin, whereas it downregulates decorin mRNA expression in fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) has recently been shown to upregulate the type I collagen mRNA expression in cultured fibroblasts. We therefore examined whether IL-10 alters gene expression of ECM elicited by TGF-beta and MCP-1. Our results demonstrated that IL-10 downregulated the TGF-beta-elicited increase of mRNA expression of type I collagen and fibronectin, while partially recovering TGF-beta-elicited decrease of decorin expression in normal skin fibroblasts. By contrast, IL-10 did not alter the MCP-1-elicited upregulation of mRNA expression of either alpha1(I) collagen and decorin. Our data indicate that IL-10 differentially regulates TGF-beta and MCP-1 in the modulation of ECM proteins and therefore suggest that IL-10 plays a role in the regulation of tissue remodeling.

Topics: Blotting, Northern; Cells, Cultured; Chemokine CCL2; Collagen; Decorin; Dose-Response Relationship, Drug; Down-Regulation; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Interleukin-10; Proteoglycans; RNA, Messenger; Scleroderma, Localized; Skin; Time Factors; Transforming Growth Factor beta; Up-Regulation

To study the distribution of transforming growth factor beta (TGF-beta) isoforms TGF-beta 1, TGF-beta 2 and TGF-beta 3 and vascular endothelial growth factor (VEGF) in vulvar lichen sclerosus.. Biopsies were obtained from 10 patients with vulvar lichen sclerosus, snap frozen and stained with polyclonal antibodies to TGF-beta 1, TGF-beta 2, TGF-beta 3 and VEGF. Control tissues used were uninvolved thigh tissue from two of the lichen sclerosus patients and normal vulvar tissue obtained from eight patients during gynecologic procedures. Two specimens of morphea were also examined.. Weak TGF-beta 1 staining was demonstrated in the epidermis of all the lichen sclerosus, morphea, thigh and five of the eight normal vulvar specimens. Slight increase in TGF-beta 1 staining was seen in the upper and middermis in 6 of the 10 lichen sclerosus specimens and in the morphea specimens as compared to the control tissue, and this staining was localized within cells. TGF-beta 2 staining was present throughout the epidermis in all the normal thigh, normal vulva, lichen sclerosus and morphea specimens. TGF-beta 2 staining was increased within cells in the upper and middermis of the lichen sclerosus and morphea specimens. TGF-beta 3 staining occurred in the basal half of the epidermis of all the control, lichen sclerosus and morphea specimens, and only slight upper dermal staining of a few individual cells was seen in 3 of the 10 lichen sclerosus specimens. VEGF staining was similar in the normal tissues, lichen sclerosus and morphea.. These results suggest that TGF-beta may.

Topics: Biopsy; Endothelial Growth Factors; Female; Humans; Immunohistochemistry; Lichen Sclerosus et Atrophicus; Lymphokines; Protein Isoforms; Scleroderma, Localized; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors; Vulvar Diseases

Recent studies have suggested that dermal dendritic cells (DDCs) may play a part in maintaining the structure of the dermis and in dermal immune modulation. Alteration in the population of DDCs has been noted in localized and systemic scleroderma, particularly a decline in the number of CD34+ DDCs. Objectives To define the alteration of the DDC populations with respect to the histological stage of morphoea.. We examined 33 biopsies of morphoea, categorized into four histological stages, and examined the DDC population (CD34+ DDCs and factor XIIIa+ DDCs), the lymphocytic infiltrate, and tenascin (extracellular matrix glycoprotein) and transforming growth factor (TGF)-beta1 expression in each biopsy.. As the dermis became less inflammatory and more sclerotic, there was a significant decline in the number of CD34+ DDCs and an increase in the number of factor XIIIa+ DDCs. The pan-T-cell infiltrate (UCHL-1/CD45RO) and tenascin deposition exhibited a similar pattern, with elevated expression in inflammatory stages and a decrease in expression as the dermis became sclerotic. TGF-beta1 was significantly elevated in three of the four histological stages of morphoea, in both the inflammatory and sclerotic stages. The proposed four-stage histological analysis of morphoea biopsies was a useful basis for studying dendritic cells and mediators in cutaneous sclerosis.. Our study indicates that there is a reciprocal relationship between CD34+ DDCs and factor XIIIa+ DDCs in morphoea that correlates with the relative degrees of inflammation and sclerosis.

Topics: Adolescent; Adult; Aged; Antigens, CD34; Biopsy; Child; Dendritic Cells; Female; HLA-DR Antigens; Humans; Immunohistochemistry; Male; Middle Aged; Scleroderma, Localized; Tenascin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Excessive accumulation of fibrillar collagens is a hallmark of the cutaneous fibrosis in both systemic and localized scleroderma. Turnover of the collagenous extracellular matrix is dependent on the balance between collagenolytic matrix metalloproteinases and their specific inhibitors. We have examined the expression of the novel, matrix associated tissue inhibitor of metalloproteinases-3 (TIMP-3) in normal and scleroderma skin fibroblasts in culture and in vivo. The levels of TIMP-3 mRNA were elevated up to 2.5-fold in five of seven systemic sclerosis fibroblast strains, whereas TIMP-1 mRNA expression was elevated up to 1.8-fold in two and TIMP-2 mRNA expression up to 1.8-fold in two systemic sclerosis strains. Using in situ hybridization, TIMP-3 mRNA was detected in seven of 12 localized scleroderma skin samples, specifically in fibroblasts within fibrotic collagen fibers or in the vicinity of inflammatory cells. TIMP-1 mRNA was detected in three of eight scleroderma skin samples in fibroblasts adjacent to inflammatory cells. The expression of TIMP-3 mRNA by systemic sclerosis and normal skin fibroblasts was enhanced to a similar extent (by 8.6- and 8.1-fold, respectively) by transforming growth factor-beta, and suppressed down to 34 and 54%, respectively, by tumor necrosis factor-alpha. Specific activation of TIMP-3 gene expression in scleroderma skin fibroblasts in culture and in vivo suggests a role for TIMP-3 in the pathogenesis of dermal fibrosis via inhibition of turnover of fibrotic dermal extracellular matrix, possibly due to upregulation of TIMP-3 expression by transforming growth factor-beta.

Topics: Adult; Cells, Cultured; Enzyme Activation; Female; Fibroblasts; Humans; Male; Middle Aged; RNA, Messenger; Scleroderma, Localized; Skin; Tissue Inhibitor of Metalloproteinase-3; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We recently reported that the serum concentration of procollagen type I carboxyterminal propeptide (P1CP) in patients with systemic sclereosis (SSc) was elevated. In the present study, we investigated collagen metabolism in in vitro cultured scleroderma fibroblasts by measuring P1CP levels in the culture medium.. Spontaneous P1CP production was 4.2 times higher in fibroblast cultures from patients with SSc (n = 11) than in those from healthy controls (n = 10). P1CP production in fibroblasts derived from diffuse cutaneous SSc patients was significantly greater than that from limited cutaneous SSc patients. The serum P1CP level in SSc patients was correlated with the P1CP production of cultured fibroblasts (r = 0.815, p < 0.005). Transforming growth factor beta increased P1CP production, and gamma-interferon decreased P1CP production similarly in both SSc and normal fibroblasts. In contrast, histamine dihydrochloride increased P1CP production only in SSc fibroblasts but not in controls.. These findings suggest that P1CP production in SSc fibroblasts is relevant to in vivo collagen synthesis in SSc patients.

Topics: Adolescent; Adult; Aged; Case-Control Studies; Cells, Cultured; Chlorpheniramine; Cimetidine; Collagen; Dose-Response Relationship, Drug; Female; Fibroblasts; Histamine; Humans; Interferon-gamma; Male; Middle Aged; Peptide Fragments; Procollagen; Scleroderma, Localized; Scleroderma, Systemic; Transforming Growth Factor beta

To determine the presence of transforming growth factor beta 1 (TGF beta 1) and inflammatory cell markers (HLA-DR and Factor XIIIa) and to compare these with the presence of type I procollagen, in clinically uninvolved and involved skin from patients with different subsets of systemic sclerosis (SSc), and to analyze circulating levels of TGF beta 1 in SSc patients.. TGF beta 1, HLA-DR, Factor XIIIa, and type I procollagen were detected in skin biopsy sections using a biotin-streptavidin-peroxidase system. Levels of circulating TGF beta 1 were measured using a capture enzyme-linked immunosorbent assay technique.. Patients with active diffuse cutaneous SSc (dcSSc) showed minimal TGF beta 1 but significant type I procollagen staining in involved skin, while the clinically uninvolved skin of these patients showed moderate extracellular and intra-epidermal TGF beta 1 immunoreactivity. Patients with limited cutaneous SSc (lcSSc) showed elevated TGF beta 1 staining in both involved and uninvolved skin, as well as procollagen staining. Significant TGF beta 1 reactivity, HLA-DR and Factor XIIIa immunoreactivity, numerous inflammatory cells, and procollagen staining were seen in specimens from patients with morphea. Sequential biopsies suggested the presence of cytokine activity at the earliest stages of disease, which was not maintained with progression of sclerosis. Among the disease groups studied, elevated levels of circulating TGF beta 1 were seen only in patients with morphea.. The pattern of TGF beta 1 staining in dermal sections from patients with dcSSc, lcSSc, and morphea suggests that this cytokine is important in the pathogenesis of scleroderma. Furthermore, the presence of TGF beta 1 prior to the onset of fibrosis indicates an early involvement of this growth factor, possibly in the inflammatory stage of the disease.

Topics: Biomarkers; Female; Humans; Immunohistochemistry; Inflammation; Male; Procollagen; Raynaud Disease; Scleroderma, Localized; Scleroderma, Systemic; Serology; Staining and Labeling; Tissue Distribution; Transforming Growth Factor beta

The ability of transforming growth factor-beta (TGF-beta) to induce synthesis of extracellular matrix proteins stimulated this study in which we address the hypothesis that TGF-beta can induce, in normal fibroblasts, the sustained, elevated collagen synthesis characteristic of the scleroderma fibroblast.. Fibroblasts were studied for synthesis of and responsiveness to TGF-beta. Secreted TGF-beta levels were determined in a bioassay and at the transcriptional level in a series of scleroderma (SSc) and normal fibroblasts. The ability of cells to interact functionally with a 3-dimensional collagen matrix after TGF-beta treatment was examined. The kinetics of TGF-beta-induced fibrosis in fibroblasts was studied.. SSc fibroblasts were not characterized by elevated TGF-beta synthesis. There was no evidence of coordinate regulation of TGF-beta and collagen over passage number. Repeated pulses of 200 pM of TGF-beta did not significantly induce sustained procollagen alpha 1(I) mRNA synthesis in normal fibroblasts, and this treatment did not significantly alter the characteristics of normal fibroblasts in a collagen gel. mRNA for both collagen and TGF-beta type II receptor was induced by TGF-beta in both SSc and control cells. SSc fibroblasts were found to have an impaired ability to activate the small latent complex of TGF-beta.. Our data give no support to the hypothesis that TGF-beta can maintain the SSc phenotype in vitro or that it is able to induce this phenotype. The inducibility of TGF-beta receptor mRNA in SSc fibroblasts after exposure to TGF-beta suggests that the lack of sustained elevation in collagen synthesis is not due to lack of responsiveness by the fibroblasts but is rather a reflection of the transient nature of TGF-beta-induced fibrosis.

Topics: Cell Division; Cell Line; Collagen; Female; Fibroblasts; Humans; Male; Procollagen; Receptors, Transforming Growth Factor beta; RNA, Messenger; Scleroderma, Localized; Transforming Growth Factor beta

Topics: Adenocarcinoma; Antibodies, Anti-Idiotypic; Female; Humans; Lung Neoplasms; Middle Aged; Scleroderma, Localized; Transforming Growth Factor beta

A characteristic feature of fibroblasts cultured from affected skin areas of patients with systemic sclerosis (SSc) and localized scleroderma (morphea) is excessive activation of collagen biosynthesis. To elucidate the nature of fibroblast activation in scleroderma we have studied the expression of 3 noncollagenous connective tissue components, osteonectin, small dermatan sulfate proteoglycan (proteoglycan II, decorin), and transforming growth factor-beta 1 (TGF-beta 1), by measuring their mRNA levels in fibroblast cultures from 6 patients with SSc and 3 with morphea. A clear correlation was observed between the increase in type I collagen and osteonectin mRNA in these cell lines. The apparent overproduction of osteonectin by scleroderma fibroblasts is in accordance with the suggested activation of osteonectin expression during tissue remodeling. The levels of decorin mRNA showed marked variation in the cell lines, but were in no correlation with collagen or osteonectin mRNA. The levels of TGF-beta 1 mRNA were found to be slightly elevated in fibroblasts grown from affected scleroderma skin. This may suggest that this potent activator of collagen production has a role during the initial activation of dermal fibroblasts both in SSc and morphea.

Topics: Adolescent; Adult; Blotting, Northern; Cells, Cultured; Collagen; Decorin; Extracellular Matrix; Extracellular Matrix Proteins; Female; Fibroblasts; Gene Expression; Humans; Male; Middle Aged; Osteonectin; Proteoglycans; RNA, Messenger; Scleroderma, Localized; Scleroderma, Systemic; Skin; Transforming Growth Factor beta