transforming-growth-factor-beta and Scleroderma--Systemic

The management of systemic sclerosis (SSc) is complex, evolving, and requires a multidisciplinary approach. At diagnosis and throughout the disease course, clinical assessment and monitoring of skin involvement is vital using the modified Rodnan Skin Score, patient-reported outcomes, and new global composite scores (such as the Combined Response Index for Systemic Sclerosis, which also considers lung function). Immunomodulation is the mainstay of skin fibrosis treatment, with mycophenolate mofetil considered first line. Meanwhile vasculopathy-related manifestations (Raynaud's phenomenon, digital ulcers) and calcinosis, require general measures combined with specific pharmacologic (calcium-channel blockers, phosphodiesterase type 5 inhibitors, and prostanoids), nonpharmacologic (digital sympathectomy and botulinum toxin injections), and often multifaceted, management approaches. Patients should be screened at the time of diagnosis specifically for systemic manifestations and then regularly thereafter, with appropriate treatment. Numerous targeted therapeutic options for SSc, including skin fibrosis, are emerging and include B-cell depletion, anti-interleukin 6, Janus kinase, and transforming growth factor β inhibition. This second article in the continuing medical education series discusses these key aspects of SSc assessment and treatment, with particular focus on skin involvement. It is vital that dermatologists play a key role in the multidisciplinary approach to SSc management.

Topics: Adult; Botulinum Toxins; Calcium; Fibrosis; Humans; Janus Kinases; Mycophenolic Acid; Phosphodiesterase 5 Inhibitors; Prostaglandins; Raynaud Disease; Scleroderma, Systemic; Skin Ulcer; Transforming Growth Factor beta

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

Systemic sclerosis (SSc) is a disease of connective tissue with high rate of morbidity and mortality highlighted by extreme fibrosis affecting various organs such as the dermis, lungs, and heart. Until now, there is no specific cure for the fibrosis occurred in SSc disease. The SSc pathogenesis is yet unknown, but transforming growth factor beta (TGF-β), endothelin-1 (ET-1), and Ras-ERK1/2 cascade are the main factors contributing to the tissue fibrosis through extracellular matrix (ECM) accumulation. Several studies have hallmarked the association of ET-1 with or without TGF-β and Ras-ERK1/2 signaling in the development of SSc disease, vasculopathy, and fibrosis of the dermis, lungs, and several organs. Accordingly, different clinical and experimental studies have indicated the potential therapeutic role of ET-1 and Ras antagonists in these situations in SSc. In addition, ET-1 and connective tissue growth factor (CTGF) as a cofactor of the TGF-β cascade play a substantial initiative role in inducing fibrosis. Once initiated, TGF-β alone or in combination with ET-1 and CTGF can activate several kinase proteins such as the Ras-ERK1/2 pathway that serve as the fundamental factor for developing fibrosis. Furthermore, Salirasib is a synthetic small molecule that is able to inhibit all Ras forms. Therefore, it can be used as a potent therapeutic factor for fibrotic disorders. So, this review discusses the role of TGF-β/ET-1/Ras signaling and their involvement in SSc pathogenesis, particularly in its fibrotic situation.

Topics: Connective Tissue Growth Factor; Endothelin-1; Fibroblasts; Fibrosis; Humans; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) is a severe complication of connective tissue disease (CTD), causing death in systemic sclerosis (SSc). The past decade has yielded many scientific insights into microRNA (miRNAs) in PAH and SSc. This growth of knowledge has well-illustrated the complexity of microRNA (miRNA)-based regulation of gene expression in PAH. However, few miRNA-related SSc-PAH were elucidated. This review firstly discusses the role of transforming growth factor-beta (TGF-β) signaling and bone morphogenetic protein receptor type II (BMPR2) in PAH and SSc. Secondly, the miRNAs relating to TGF-β and BMPR2 signaling pathways in PAH and SSc or merely PAH were subsequently summarized. Finally, future studies might develop early diagnostic biomarkers and target-oriented therapeutic strategies for SSc-PAH and PAH treatment.

Topics: Bone Morphogenetic Protein Receptors, Type II; Humans; Hypertension, Pulmonary; MicroRNAs; Pulmonary Arterial Hypertension; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

The activation of fibroblasts is required for physiological tissue remodelling such as wound healing. However, when the regulatory mechanisms are disrupted and fibroblasts remain persistently activated, the progressive deposition of extracellular matrix proteins leads to tissue fibrosis, which results in dysfunction or even loss of function of the affected organ. Although fibrosis has been recognized as a major cause of morbidity and mortality in modern societies, there are only few treatment options available that directly disrupt the release of extracellular matrix from fibroblasts. Intensive research in recent years, however, identified several pathways as core fibrotic mechanisms that are shared across different fibrotic diseases and organs. We discuss herein selection of those core pathways, especially downstream of the profibrotic TGF-β pathway, which are druggable and which may be transferable from bench to bedside.

Topics: Animals; DNA Methylation; Ephrins; Fibroblast Growth Factor 9; Fibrosis; Guanylate Cyclase; Histone Code; Humans; Idiopathic Pulmonary Fibrosis; Janus Kinases; Myofibroblasts; Receptors, Cytoplasmic and Nuclear; Scleroderma, Systemic; Serotonin; Signal Transduction; Skin; STAT Transcription Factors; Transforming Growth Factor beta

Calcinosis cutis, defined as sub-epidermal deposition of calcium salts, is a major clinical problem in patients with SSc, affecting 20-40% of patients. A number of recognized factors associated with calcinosis have been identified, including disease duration, digital ischaemia and acro-osteolysis. Yet, to date, the pathogenesis of SSc-related calcinosis remains unknown, and currently there is no effective disease-modifying pharmacotherapy. Following onset of SSc, there are marked changes in the extracellular matrix (ECM) of the skin, notably a breakdown in the microfibrillar network and accumulation of type I collagen. Our hypothesis is that these pathological changes reflect a changing cellular phenotype and result in a primed microenvironment for soft tissue calcification, with SSc fibroblasts adopting a pro-osteogenic profile, and specific driving forces promoting tissue mineralization. Considering the role of the ECM in disease progression may help elucidate the mechanism(s) behind SSc-related calcinosis and inform the development of future therapeutic interventions.

Topics: Calcinosis; Cell Differentiation; Cell Hypoxia; Cellular Microenvironment; Collagen Type I; Disease Progression; Elastin; Extracellular Matrix; Fibrillin-1; Fibroblasts; Glucose Transporter Type 1; Humans; Mesenchymal Stem Cells; Myofibroblasts; Osteoblasts; Osteogenesis; Osteolysis; Phenotype; Phosphates; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Platelets are no longer recognized solely as cell fragments regulating hemostasis. They have pleiotropic functions and they are linked directly or indirectly with the three cornerstones of systemic sclerosis (SSc): vasculopathy, autoimmunity, and fibrosis. In this review, we summarize the current knowledge on the potential role of platelets in the pathogenesis of SSc.. Experimental evidence suggests that vasculopathy, a universal and early finding in SSc, may activate platelets which subsequently release several profibrotic mediators such as serotonin and transforming growth factor β (TGFβ). Platelets may also cross-react with the endothelium leading to the release of molecules, such as thymic stromal lymphopoietin (TSLP), that may trigger fibrosis or sustain vascular damage. Finally, activated platelets express CD40L and provide costimulatory help to B cells, something that may facilitate the breach in immune tolerance. Preclinical studies point to the direction that platelets are actively involved in SSc pathogenesis. Targeting platelets may be an attractive therapeutic approach in SSc.

Topics: Autoimmunity; Blood Platelets; Fibrosis; Humans; Inflammation; Scleroderma, Systemic; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Fibrosis is unregulated tissue repair that may cause impairment of organ function, especially in end-organ damage. Systemic sclerosis (SSc) is the prototype systemic fibrosing disorder. Classical targets for fibrosis in SSc like transforming growth factor Beta (TGF-β), Interleukin-6 (IL-6), and multiple tyrosine kinases, have not yielded therapeutic benefit. There is multitude of evidence from across different tissues like the heart, lung, skin, liver, colon, and, to some extent, the kidney, that interleukin-17 (IL-17) and its downstream pathways are strongly associated with the initiation and propagation of fibrosis. Data from scleroderma patients, as well as from animal models of SSc, mirror these findings. Interestingly, hitherto unknown to be related to IL-17, newer molecules like Programmed Death-protein1 (PD-1), the phosphatase SHP2, along with known signal transducers like signal transducer and activator of transcription (STAT3), have been recently shown to be involved in the pathogenesis of fibrosis. Related molecules include the intracellular signalling molecules Ras/Erk, mammalian target organ of rapamycin (mTOR), and complement components. The biology of these pathways has not yet been fully elucidated to predict regulatory mechanisms, redundancies, and potential off-target effects. All these need to be better understood in the context of each other, in an effort to arrive at the optimal target to modulate fibrosis.

Topics: Animals; Fibrosis; Humans; Interleukin-17; Lung; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

In systemic sclerosis (SSc) immuno-inflammatory events are central to disease development. Amongst other mediators of inflammation, interleukin 17 (IL-17) and Th17 cells have been reported to be increased in the peripheral blood and target organs including involved skin in SSc. They participate and amplify inflammatory responses by inducing the production of cytokines such as IL-6, chemokines such as CCL2 and CXCL8 (IL-8), matrix metalloproteinases-1, -2, -9 and the expression of adhesion molecules in stromal cells including fibroblasts and endothelial cells. In this respect, IL-17 and Th17 cells behave paradigmatically as documented in other autoimmune pathological conditions or infectious diseases. In experimental animal models of skin and lung fibrosis, IL-17 indirectly enhances the fibrotic process by favoring further inflammation by recruiting inflammatory cells, by activating and/or stimulating the production of TGF-β and other pro-fibrotic mediators, by inhibiting autophagy. Whether the findings generated in animal models of fibrosis can be translated to human SSc is unproven. Furthermore, it is controversial whether IL-17 directly promotes the transdifferentiation of human fibroblasts into myofibroblasts and enhances collagen production, with most of the available evidence against this possibility. The reductionist approach in which fibroblast in monolayers are cultured in plastic dishes under the influence of IL-17 limits the relevance of these findings. Further in vitro/ex vivo models with human tissues are being developed to investigate the real effect of IL-17 on extracellular matrix deposition, since agents blocking IL-17 are available for the clinic and it will be important to know whether their use in SSc would be beneficial or detrimental.

Topics: Animals; Fibroblasts; Fibrosis; Humans; Inflammation; Inflammation Mediators; Interleukin-17; Lung; Scleroderma, Systemic; Skin; Th17 Cells; Transforming Growth Factor beta

Systemic sclerosis (SSc), an autoimmune disease that is associated with a number of genetic and environmental risk factors, is characterized by progressive fibrosis and microvasculature damage in the skin, lungs, heart, digestive system, kidneys, muscles, joints, and nervous system. These abnormalities are associated with altered secretion of growth factor and profibrotic cytokines, such as transforming growth factor-beta (TGF-β), interleukin-4 (IL-4), platelet-derived growth factor (PDGF), and connective-tissue growth factor (CTGF). Among the cellular responses to this proinflammatory environment, the endothelial cells phenotypic conversion into activated myofibroblasts, a process known as endothelial to mesenchymal transition (EndMT), has been postulated. Reactive oxygen species (ROS) might play a key role in SSs-associated fibrosis and vascular damage by mediating and/or activating TGF-β-induced EndMT, a phenomenon that has been observed in other disease models. In this review, we identified and critically appraised published studies investigating associations ROS and EndMT and the presence of EndMT in SSc, highlighting a potential link between oxidative stress and EndMT in this condition.

Topics: Cytokines; Endothelial Cells; Epithelial-Mesenchymal Transition; Humans; Inflammation Mediators; Oxidative Stress; Reactive Oxygen Species; Scleroderma, Systemic; Transforming Growth Factor beta

With unknown etiology, scleroderma (SSc) is a multifaceted disease characterized by immune activation, vascular complications, and excessive fibrosis in internal organs. Genetic studies, including candidate gene association studies, genome-wide association studies, and whole-exome sequencing have supported the notion that while genetic susceptibility to SSc appears to be modest, SSc patients are genetically predisposed to this disease. The strongest genetic association for SSc lies within the MHC region, with loci in HLA-DRB1, HLA-DQB1, HLA-DPB1, and HLA-DOA1 being the most replicated. The non-HLA genes associated with SSc are involved in various functions, with the most robust associations including genes for B and T cell activation and innate immunity. Other pathways include genes involved in extracellular matrix deposition, cytokines, and autophagy. Among these genes, IRF5, STAT4, and CD247 were replicated most frequently while SNPs rs35677470 in DNASE1L3, rs5029939 in TNFAIP3, and rs7574685 in STAT4 have the strongest associations with SSc. In addition to genetic predisposition, it became clear that environmental factors and epigenetic influences also contribute to the development of SSc. Epigenetics, which refers to studies that focus on heritable phenotypes resulting from changes in chromatin structure without affecting the DNA sequence, is one of the most rapidly expanding fields in biomedical research. Indeed extensive epigenetic changes have been described in SSc. Alteration in enzymes and mediators involved in DNA methylation and histone modification, as well as dysregulated non-coding RNA levels all contribute to fibrosis, immune dysregulation, and impaired angiogenesis in this disease. Genes that are affected by epigenetic dysregulation include ones involved in autoimmunity, T cell function and regulation, TGFβ pathway, Wnt pathway, extracellular matrix, and transcription factors governing fibrosis and angiogenesis. In this review, we provide a comprehensive overview of the current findings of SSc genetic susceptibility, followed by an extensive description and a systematic review of epigenetic research that has been carried out to date in SSc. We also summarize the therapeutic potential of drugs that affect epigenetic mechanisms, and outline the future prospective of genomics and epigenomics research in SSc.

Topics: Epigenomics; Gene-Environment Interaction; Genetic Predisposition to Disease; Genomics; HLA Antigens; Humans; Interferon Regulatory Factors; Polymorphism, Genetic; Scleroderma, Systemic; Signal Transduction; STAT4 Transcription Factor; Transforming Growth Factor beta

Dickkopf-1 (Dkk-1) is a soluble inhibitor of the canonical Wnt pathway, which plays critical roles in embryonic development. Evidence suggests that this molecule regulates several aspects of both bone biology and fibrosis.. To provide an overview of our current knowledge of the role of Dkk-1 in joint remodeling and fibrosis.. We performed an electronic search (Medline) using the following key words: Dickkopf-1 (or Dkk-1), new bone formation, joint remodeling, ankylosing spondylitis, systemic sclerosis (or scleroderma), and fibrosis, supplemented by a manual search of references from retrieved articles.. Dkk-1 is a master regulator of joint remodeling in animal models of arthritis shifting the balance toward new bone formation when its expression is decreased and toward erosion/joint destruction when its expression is increased. In humans, evidence suggests that Dkk-1 may be dysfunctional in patients with ankylosing spondylitis, a prototype bone forming disease. Moreover, data from animal models indicate that Dkk-1 has a protective role against fibrosis in several organs. Recent data suggest that inhibiting the canonical Wnt pathway by overexpression of Dkk-1 could be a way to target TGF-β signaling in fibrotic diseases. Finally, B-cell depletion therapy in systemic sclerosis may exert its effects through TGF-β dependent upregulation of Dkk-1.. Dkk-1 appears to play a crucial role in both joint remodeling/ectopic ossification and fibrosis, and may be a prospective therapeutic modality for fibrotic diseases or diseases characterized by pathologic joint remodeling.

Topics: Animals; B-Lymphocytes; Bone Remodeling; Fibrosis; Humans; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Spondylarthropathies; Spondylitis, Ankylosing; Transforming Growth Factor beta; Wnt Signaling Pathway

Scar formation, with persistent alteration of the normal tissue structure, is an undesirable and significant result of both wound healing and fibrosing disorders. There are few strategies to prevent or to treat scarring. The transforming growth factor beta (TGF-β) superfamily is an important mediator of tissue repair. Each TGF-β isoform may exert a different effect on wound healing, which may be context-dependent. In particular, TGF-β1 may mediate fibrosis in adults' wounds, while TGF-β3 may promote scarless healing in the fetus and reduced scarring in adults. Thus, TGF-β3 may offer a scar-reducing therapy for acute and chronic wounds and fibrosing disorders.

Topics: Cicatrix; Fibrosis; Humans; Intercellular Signaling Peptides and Proteins; Protein Isoforms; Scleroderma, Systemic; Skin Physiological Phenomena; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta3; Wound Healing; Wounds and Injuries

Fibrosis is one of the leading causes of morbidity and mortality in the Western world. This disorder is characterised by an abnormal and increased rate of fibroblast proliferation and by an excessive deposition of connective tissue. The key player in fibrosis is the myofibroblast. Fibrosis leads to loss of organ structure and, eventually, to decrease in organ function. To date, there are hardly any effective therapies for the treatment of patients with fibrosis. Pirfenidone targets the myofibroblast and is effective in the treatment of idiopathic pulmonary fibrosis. Tyrosine kinase inhibitors are effective for the treatment of patients with some forms of systemic sclerosis. Here we describe various novel therapeutic targets, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), interleukin-13 (IL-13), lysyloxidase-2 and macrophage-fibroblast interactions. These new therapies are currently under investigation in pre-clinical and clinical studies.

Topics: Fibroblasts; Fibrosis; Humans; Idiopathic Pulmonary Fibrosis; Interleukin-13; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta; Treatment Outcome

Systemic sclerosis (SSc) is a multisystem fibrotic and autoimmune disease. Both genetic and epigenetic elements mediate SSc pathophysiology. This review summarizes the role of one epigenetic element, known as microRNAs (miRNAs), involved in different signaling pathways of SSc pathogenesis. The expression of key components in transforming growth factor-β (TGF-β) signaling pathway has been found to be regulated by miRNAs both upstream and downstream of TGF-β. We are specifically interested in the pathway components upstream of TGF-β, while miRNAs in other signaling pathways have not been extensively studied. The emerging role of miRNAs in vasculopathy of SSc suggests a promising new direction for future investigation. Elucidation of the regulatory role of miRNAs in the expression of signaling factors may facilitate the discovery of novel biomarkers in SSc and improve the understanding and treatment of this disease.

Topics: Autoimmune Diseases; Epigenesis, Genetic; Genetic Markers; Humans; MicroRNAs; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis (scleroderma, SSc) is a heterogeneous autoimmune connective tissue disease of unknown etiology. Interstitial lung disease (ILD) is a frequent complication, and a significant contributor to morbidity and mortality among SSc patients. SSc-ILD most commonly occurs within 10 years of diagnosis, and may be seen in patients with either the limited or diffuse cutaneous subset of SSc. SSc-ILD is a multifaceted disease process in which different factors and pathways are involved. Aberrant function of a variety of lung cells, cytokines, growth factors, peptides, and bioactive proteins, in combination with genetic and epigenetic regulators, have crucial functions in the pathogenesis of this disease. Here we present our view on recent advances regarding the pathogenesis of SSc-ILD.

Topics: beta Catenin; Epigenomics; Genetic Predisposition to Disease; HLA-D Antigens; Humans; Lung Diseases, Interstitial; Scleroderma, Systemic; Thrombin; Transforming Growth Factor beta

Transforming growth factor β (TGF-β) has long been implicated in fibrotic diseases, including the multisystem fibrotic disease systemic sclerosis (SSc). Expression of TGF-β-regulated genes in fibrotic skin and lungs of patients with SSc correlates with disease activity, which points to this cytokine as the central mediator of pathogenesis. Patients with SSc often develop pulmonary arterial hypertension (PAH), a particularly lethal complication caused by vascular dysfunction. Several genetic diseases with vascular features related to SSc, such as familial PAH and hereditary haemorrhagic telangiectasia, are caused by mutations in the TGF-β-sensing ALK-1 signalling pathway. These observations suggest that increased TGF-β signalling causes both vascular and fibrotic features of SSc. The question of how latent TGF-β becomes activated in local SSc tissues is, therefore, central to the understanding of SSc. Both TGF-β1 and TGF-β3 can be activated by integrins αvβ6 and αvβ8, whose upregulation in bronchial epithelial cells can activate TGF-β in SSc lungs. Other αv integrins, thrombospondin-1 or altered TGF-β sequestration by matrix proteins might be important in other target tissues. How the immune system triggers this process remains unclear, although links between inflammation and TGF-β activation are emerging. Together, these observations provide an increasingly secure framework for understanding TGF-β in SSc pathogenesis.

Topics: Humans; Myofibroblasts; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a multisystem connective tissue disease featured by immune abnormalities, vasculopathy and resultant fibrosis of the skin and various internal organs. Although the pathogenesis of SSc remains incompletely elucidated, it is currently accepted that this disease is caused by the complex interplay between hereditary and environmental factors. The deficiency of transcription factor Fli1, which is epigenetically suppressed in SSc dermal fibroblasts, potentially causes SSc-like phenotypical alteration in various cell types such as fibroblasts, endothelial cells, and macrophages, suggesting that Fli1 is a predisposing factor of SSc. KLF5 is another transcription factor which is suppressed in SSc dermal fibroblasts through an epigenetic mechanism. Importantly, double heterozygous mice for Fli1 and KLF5 develop three cardinal features of SSc, including immune abnormalities, vasculopathy and fibrosis. Therefore, these two transcription factors are likely to be critical predisposing factors regulating the development of SSc. Given that potential disease modifying drugs, bosentan and imatinib, reverse the expression and transcriptional activity of Fli1, the studies on the pathological process of double heterozygous mice and the impact of these transcription factors on various cell types may provide a new clue to further understand the pathogenesis of SSc leading to the development of new therapies.

Topics: Animals; Cells, Cultured; Disease Models, Animal; Epigenesis, Genetic; Fibroblasts; Fibrosis; Gene Expression Regulation; Genetic Predisposition to Disease; Kruppel-Like Transcription Factors; Mice; Mice, Transgenic; Proto-Oncogene Protein c-fli-1; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Fibrotic skin disorders may be debilitating and impair quality of life. There are few effective treatment options for cutaneous fibrotic diseases. In this review, we discuss our current understanding of the role of microRNAs (miRNAs) in skin fibrosis. miRNAs are a class of small, non-coding RNAs involved in skin fibrosis. These small RNAs range from 18 to 25 nucleotides in length and modify gene expression by binding to target messenger RNA (mRNA), causing degradation of the target mRNA or inhibiting the translation into proteins. We present an overview of the biogenesis, maturation and function of miRNAs. We highlight miRNA’s role in key skin fibrotic processes including: transforming growth factor-beta signaling, extracellular matrix deposition, and fibroblast proliferation and differentiation. Some miRNAs are profibrotic and their upregulation favors these processes contributing to fibrosis, while anti-fibrotic miRNAs inhibit these processes and may be reduced in fibrosis. Finally, we describe the diagnostic and therapeutic significance of miRNAs in the management of skin fibrosis. The discovery that miRNAs are detectable in serum, plasma, and other bodily fluids, and are relatively stable, suggests that miRNAs may serve as valuable biomarkers to monitor disease progression and response to treatment. In the treatment of skin fibrosis, antifibrotic miRNAs may be upregulated using mimics and viral vectors. Conversely, profibrotic miRNAs may be downregulated by employing anti-miRNAs, sponges, erasers and masks. We believe that miRNA-based therapies hold promise as important treatments and may transform the management of fibrotic skin diseases by physicians.

Topics: Cell Differentiation; Cell Proliferation; Humans; Keloid; MicroRNAs; Oligonucleotides; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

There are no effective treatments for fibrosis, an out-of-control wound-healing process in which excessive deposition of extracellular matrix (ECM) such as collagen, resulting in significant morbidity and mortality. Endostatin is a natural proteolytic fragment of collagen XVIII, which is known to possess potent antiangiogenic activity. Many clinical trials of endostatin have been conducted for anti-cancer therapy. In addition to antiangiogenic effects, recent studies have revealed that endostatin may suppress aberrant tissue remodeling and scarring. Neutralization of endogenous endostatin in rat myocardial infarction (MI) model worsened the outcomes of MI, indicating that endostatin may have protective role against left ventricular remodeling and heart failure after MI. Recently, we also reported inhibitory effects of peptides derived from endostatin on fibrosis. A peptide derived from the C-terminus of endostatin suppressed ECM production in fibroblasts in the presence of transforming growth factor-β (TGF-β), prevented TGF-β-induced dermal fibrosis ex vivo in human skin, and ameliorated skin and pulmonary fibrosis induced by bleomycin in vivo. The antifibrotic capacity was accompanied by reduced cell apoptosis and lower levels of lysyl oxidase and early growth response gene-1. Endostation may have the therapeutic potential for inhibiting fibrosis.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Bleomycin; Cicatrix; Early Growth Response Protein 1; Endostatins; Extracellular Matrix; Fibroblasts; Fibrosis; Heart Failure; Humans; Myocardial Infarction; Protein-Lysine 6-Oxidase; Pulmonary Fibrosis; Rats; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Ventricular Remodeling

Fibrosis is a key feature of systemic sclerosis (SSc) and arises from excessive release of collagens by pathologically activated fibroblasts. Affecting the skin and many internal organs, fibrosis represents a major cause for the high morbidity and mortality in SSc. So far, effective therapies to treat fibrosis in SSc and other fibrotic diseases are not available in clinical routine. Nevertheless, promising antifibrotic agents are emerging from translational studies with some having already entered clinical trials.. In this review, we focus on recent advances in the development of antifibrotic treatment strategies in SSc. We have selected for targeted therapeutic approaches that have proven high efficacy and tolerability in preclinical fibrosis models of SSc and/or are already in clinical evaluation. Applying these criteria, we discuss a large repertory of candidate antifibrotic therapies that block inflammatory pathways, inhibit profibrotic growth factors, modulate epigenetic signaling, and interfere with morphogenic pathways.. Many antifibrotic candidate therapies have proven efficacy and tolerability in preclinical models of SSc. So far, early clinical studies have tested only few of these agents. Besides discovering novel molecular treatment strategies, SSc research will now have to translate its findings into clinical practice.

Topics: Epigenesis, Genetic; Fibrosis; Humans; Immunosuppressive Agents; Molecular Targeted Therapy; Morphogenesis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Fibrosis plays a role in many pathological conditions, among which is the autoimmune disease systemic sclerosis (SSc). SSc is characterized by fibrosis in the skin and internal organs, but the etiology remains to be elucidated. Transforming growth factor-β (TGF-β) is a key player in the fibrotic process, also in SSc. TGF-β induces the production of several components of the extracellular matrix and induces differentiation of fibroblasts to myofibroblasts, which further worsens fibrosis. Although TGF-β has been extensively investigated in fibrosis, the roles of several components of its signaling pathway are still unknown. Endoglin is a coreceptor for TGF-β and is known to modulate TGF-β signaling. Therefore, endoglin could enhance the effects of TGF-β in fibrosis or act as an inhibitor. Multiple studies have been conducted that support either hypothesis. Elucidating the exact role of endoglin in TGF-β signaling during fibrosis is important in understanding the process of fibrosis and could lead to the development of better treatments.

Topics: Animals; Fibrosis; Humans; Models, Biological; Receptors, Cell Surface; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Autoimmune and inflammatory phenomena are characteristically present in systemic sclerosis (SSc) and impact on dysregulated fibroblast extracellular matrix deposition, hallmark of the disease in conjunction with fibroproliferative vasculopathy. Oligoclonal T helper 2-like cells are present in the skin and peripheral blood in early diffuse disease. Type 2 cytokines synergize with profibrotic cytokines including transforming growth factor beta, favoring collagen deposition and metalloproteinase inhibition by fibroblasts. Furthermore, chemokine with pro-fibrotic and pro-angiogenic properties are preferentially produced by fibroblasts under the influence of Th2-like cells. The profibrotic monocyte chemotactic protein 1 is also produced by fibroblasts, partially in response to Toll-like receptor 4 (TLR4) recognition, when autoantibodies (autoAb) bind to fibroblast surface. In addition, immune-complex formed by autoAb and ubiquitous antigens including topoisomerase-1 favor the production of interferon-alpha (IFN-α) possibly by interacting with intravesicular TLRs. Consistent with this findings, unbiased gene screening has revealed that SSc peripheral blood cells express genes induced by IFN-α, a characteristic shared with systemic lupus erythematosus and other autoimmune disorders. These findings highlight the complex relationship between adaptive and acquired immune responses, which may participate to the pathogenesis of SSc in manners until now unsuspected, which may help in identifying novel therapeutic targets.

Topics: Adaptive Immunity; Animals; Antigen-Antibody Complex; Autoantibodies; Chemokine CCL2; Collagen; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Expression; Humans; Inflammation; Interferon-alpha; Mice; Scleroderma, Systemic; Signal Transduction; Skin; Th1-Th2 Balance; Th2 Cells; Toll-Like Receptor 4; Transforming Growth Factor beta

Scleroderma (systemic sclerosis, SSc) is an autoimmune disease of unknown etiology characterized by organ fibrosis. There is no therapy for SSc. However, a recent body of evidence strongly implicates endothelin-1 (ET-1) in the pathogenesis of SSc. ET-1 is found in abundance in SSc patients. ET-1 directly induces fibrogenic effects in vitro, and is required for the ability of TGFβ to induce fibrogenic effects both in vitro and in vivo. Moreover, endothelin receptor antagonism reverses key features of the persistent fibrotic phenotype of fibroblasts isolated from lesions of SSc patients. However, clinically, endothelin receptor antagonism alone has had mixed results. This minireview summarizes these observations.

Topics: Animals; Endothelin-1; Fibrosis; Humans; Mice; Molecular Targeted Therapy; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Fibrosis is a deregulated and ultimately defective form of tissue repair that underlies a large number of chronic human diseases, as well as obesity and aging. The pathogenesis of fibrosis involves multiple cell types and extracellular signals, of which transforming growth factor-ß (TGF-ß) is pre-eminent. The prevalence of fibrosis is rising worldwide, and to date no agents has shown clinical efficacy in the attenuating or reversing the process. Recent studies implicate the immediate-early response transcription factor Egr-1 in the pathogenesis of fibrosis. Egr-1 couples acute changes in the cellular environment to sustained alterations in gene expression, and mediates a broad spectrum of biological responses to injury and stress. In contrast to other ligand-activated transcription factors such as NF-κB, c-jun and Smad2/3 that undergo post-translational modification such as phosphorylation and nuclear translocation, Egr-1 activity is regulated via its biosynthesis. Aberrant Egr-1 expression or activity is implicated in cancer, inflammation, atherosclerosis, and ischemic injury and recent studies now indicate an important role for Egr-1 in TGF-ß-dependent profibrotic responses. Fibrosis in various animal models and human diseases such as scleroderma (SSc) and idiopathic pulmonary fibrosis (IPF) is accompanied by aberrant Egr-1 expression. Moreover Egr-1 appears to be required for physiologic and pathological connective tissue remodeling, and Egr-1-null mice are protected from fibrosis. As a novel profibrotic mediator, Egr-1 thus appears to be a promising potential target for the development of anti-fibrotic therapies.

Topics: Animals; Connective Tissue; Early Growth Response Protein 1; Early Growth Response Transcription Factors; Fibroblasts; Fibrosis; Homeostasis; Humans; p300-CBP Transcription Factors; Proto-Oncogene Proteins c-abl; Repressor Proteins; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

Transforming growth factor-β (TGF-β) is a multifunctional cytokine, with important roles in maintaining tissue homeostasis. TGF-β signals via transmembrane serine/threonine kinase receptors and intracellular Smad transcriptional regulators. Perturbed TGF-β signaling has been implicated in a large variety of pathological conditions. Increased TGF-β levels have been found in patients with cancer, fibrosis, and systemic sclerosis, and were correlated with disease severity. In cancer, TGF-β mediates tumor invasion and metastasis by affecting both tumor cells and the tumor microenvironment including fibroblast activation and immune suppression. Furthermore, TGF-β is a strong stimulator of extracellular matrix deposition. On the basis of these observations, small molecule inhibitors of the TGF-β receptor kinases, neutralizing antibodies that interfere with ligand?receptor interactions, antisense oligonucleotides reducing TGF-β expression, and soluble receptor ectodomains that sequester TGF-β have been developed to intervene with excessive TGF-β signaling activity in the aforementioned disorders. Here, we review the current state of anti-TGF-β therapy in clinical trials.

Topics: Antibodies, Neutralizing; Fibrosis; Humans; Neoplasm Invasiveness; Neoplasm Metastasis; Neoplasms; Oligonucleotides, Antisense; Protein Serine-Threonine Kinases; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Heterogeneity in the clinical presentation and basic science findings of systemic sclerosis (SSc) has hindered the understanding of pathogenesis and development of effective treatments. Genome-wide profiling of SSc has measured this heterogeneity. Gene expression studies of diffuse SSc skin have shown reproducible, disease-specific gene expression signatures when compared with healthy controls and, surprisingly, disease-specific gene expression was found in both lesional and non-lesional skin. SSc-specific gene expression in peripheral blood cells and the lungs has also been demonstrated. Hypothesis-driven approaches that assess the contribution of individual pathways provide insight into the etiology of gene expression subsets.

Topics: Animals; Disease Models, Animal; Gene Expression Profiling; Gene Regulatory Networks; Genome-Wide Association Study; Humans; Leukocytes, Mononuclear; Lung; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Epigenetic modifications of gene expression comprise modifications of DNA by DNA methylation and modifications of the histone proteins by acetylation, methylation, SUMOylation or phosphorylation. DNA methylation in the promoter region of genes represses gene transcription. Histone modifications influence the structure of DNA and regulate gene expression by changing the availability of DNA for the transcriptional machinery or DNA-binding proteins. Histone modifications are mediated by enzymes and induce or repress gene expression. Aberrant expression of single enzymes disturb the normal balance of these modifiers leading to cancer or autoimmune diseases. We show in this article that epigenetic modifications contribute to the massive production of extracellular matrix proteins in systemic sclerosis skin fibroblasts. Both DNA methylation and histone modifications contribute to the activated phenotype of systemic sclerosis fibroblasts. In vitro and in vivo experiments demonstrate that the use of epigenetic-based drugs on these cells is able to reverse their activated phenotype.

Topics: Animals; Connective Tissue Growth Factor; DNA Modification Methylases; Epigenesis, Genetic; Fibroblasts; Fibrosis; Gene Expression Regulation; Histone Deacetylase Inhibitors; Humans; Molecular Targeted Therapy; PPAR gamma; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a chronic systemic disease characterized by autoimmunity, vascular lesions and progressive fibrosis. The fibrotic component is dominant in SSc compared with other vascular or autoimmune diseases and determines its prognosis and therapeutic refractoriness. Fibroblasts are responsible for abnormal extracellular matrix accumulation. Studies in cultured SSc skin fibroblasts have facilitated the identification of potential pathways involved in their profibrotic phenotype. Profibrotic fibroblasts characterized by abnormal growth and extracellular matrix synthesis may differentiate or expand from normal resident fibroblasts. Recruitment of bone marrow-derived progenitors and transdifferentiation of different cell lineages might also be involved. Multiple factors and signaling pathways appear to be involved in the development or persistence of the SSc fibroblast phenotype. Although their relative relevance and interplay are unclear, aberrant TGF-β signaling seems pivotal and constitutes the best characterized therapeutic target.

Topics: Animals; Autoimmunity; Cell Growth Processes; Cell Transdifferentiation; Disease Models, Animal; Drug Evaluation, Preclinical; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Humans; Molecular Targeted Therapy; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a generalized connective tissue disorder, characterized by a wide spectrum of microvascular and immunological abnormalities, leading to a progressive thickening and fibrosis of the skin and other organs, such as the lungs, GI tract, heart and kidneys. SSc is thought to be an autoimmune disease owing to the presence of high affinity antibodies and possible clinical overlap with other autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. Autoimmune diseases arise because of a breakdown in immunological self tolerance. Self tolerance is maintained via multiple regulatory mechanisms within the immune system, including the thymic deletion of self-reactive T cells and mechanisms of peripheral tolerance. In recent years, the presence of CD4(+)CD25(+)FOXP3(+) Tregs has been identified as a major mechanism of peripheral tolerance, and accumulating evidence indicates that alterations in Treg frequencies and/or function may contribute to autoimmune diseases. Here, we will review recent data on the percentage, function and phenotype of CD4(+)CD25(+) Tregs in rheumatic disease, and discuss how recent developments may guide research in this area in SSc.

Topics: Animals; Autoantibodies; CD4 Antigens; Cell Communication; Fibrosis; Forkhead Transcription Factors; Humans; Interleukin-2 Receptor alpha Subunit; Rheumatic Diseases; Scleroderma, Systemic; Self Tolerance; Skin; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a chronic inflammatory autoimmune disease involving the connective tissue of the skin and various internal organs. In recent years research on SSc has evolved to provide a better understanding of the interdependence of the three major systems involved--namely, the vascular system, the immune system and the connective tissue. Hypoxia is increasingly recognised as a decisive factor in modulating the inflammatory process in SSc, activating fibroblasts and changing their phenotype. In addition, several mediators synthesised by immune cells, including cytokines such as transforming growth factor beta (TGFbeta) and platelet-derived growth factor (PDGF), cooperate in inducing the activation of fibroblasts and their differentiation into myofibroblasts. Therefore, a variety of intracellular and extracellular strategies to inhibit the activity of TGFbeta and PDGF are currently receiving intense investigation. To further improve our therapeutic strategies for this paradigmatic fibrotic disease, an improved understanding of connective tissue remodelling as it takes place in the different stages of SSc will be imperative.

Topics: Cell Differentiation; Connective Tissue; Extracellular Matrix; Fibroblasts; Humans; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

Phophodiesterases inhibitors (PDEis) act by inhibiting the catabolism of cyclic nucleotides, cAMP and cGMP, which are ubiquitously expressed in cells of the immune system. Increased levels of cAMP and/or cGMP have been reported to decrease the activity of pro-inflammatory TH1 cells, attenuate experimental autoimmune encephalomyelitis and experimental arthritis. PDE5i like Sildenafil improves endothelial dysfunction and vascular remodelling in patients with pulmonary artery hypertension and refractory secondary Raynaud's phenomenon, with a potential to cause disease modification in the former. Studies in animal models of fibrosis suggest that these drugs have anti-fibrotic effect and may be potentially useful in conditions like scleroderma. They also have been shown to have renoprotective effect in animal models. The emerging trends make it necessary to exploit the full therapeutic potential of this class of drugs in various autoimmune diseases like rheumatoid arthritis, scleroderma, profibrotic conditions and PAH.

Topics: Animals; Autoimmune Diseases; Clinical Trials as Topic; Cyclic AMP; Fibrosis; Humans; Kidney; Neovascularization, Pathologic; Phosphodiesterase Inhibitors; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) or scleroderma is an acquired disorder which typically results in fibrosis of the skin and internal organs. Skin fibrosis, the hallmark of this disease, is defined as excess deposition and accumulation of extracellular matrix, mainly type I collagen, in the dermis. Dermal fibroblasts isolated from lesional skin of SSc patients and cultured in vitro exhibit increased synthesis of collagen and decreased collagenase activity, consistent with the disease phenotype. This review focuses on the recent progress in the research for molecular mechanisms of skin fibrosis in SSc. The upregulated collagen production at transcriptional level in SSc fibroblasts involves various regulators including cytokines or transcription factors. Among them, transforming growth factor (TGF)-beta/Smad signaling is likely to play a key role in the pathogenesis of SSc, and the autocrine TGF-beta signaling hypothesis can explain intrinsic activation of collagen promoter in SSc fibroblasts. Imbalance between matrix metalloproteinases and tissue inhibitor of metalloproteinases might also contribute to the excess accumulation of collagen in the dermis.

Topics: Collagen; Collagenases; Cytokines; Fibroblasts; Fibrosis; Humans; Metalloendopeptidases; Scleroderma, Systemic; Skin; Smad Proteins; Tissue Inhibitor of Metalloproteinases; Transcription Factors; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a connective tissue disease characterized by autoimmunity and tissue fibrosis. In patients with SSc, there is a close association between the presence of specific autoantibodies and the development of clinical features. Although it is known that cytokines, including transforming growth factor-beta, can modulate the synthesis of extracellular matrix by fibroblasts, it is not clear how autoimmunity and tissue fibrosis are interrelated. Several recent lines of evidence indicate a potential role for B cells in the development of SSc. CD19 is a critical regulator of B-cell signaling thresholds, and B cells from SSc patients exhibit increased expression of CD19, a molecule that induces SSc-specific autoantibody production in transgenic mice. Both SSc patients and tight-skin mice, a genetic model of SSc, have intrinsic B-cell abnormalities characterized by chronic B-cell activation. Remarkably, CD19 loss or B-cell depletion using antimouse CD20 antibody suppresses the development of skin hyperplasia and autoimmunity in tight-skin mice. Additionally, a recent study revealed a possible beneficial effect of antihuman CD20 antibody (rituximab) therapy for SSc patients. As B cells have a variety of functions, further investigation into the pathogenic roles of B cells, as well as trials of B-cell-targeting therapies, may shed new light on the pathogenesis of SSc.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Antigens, CD19; Antigens, CD20; Autoantibodies; Autoimmune Diseases; B-Lymphocytes; Cytokines; Fibrosis; Humans; Lymphocyte Activation; Mice; Rituximab; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis (SSc) is an autoimmune disorder with clinical manifestations resulting from immune activation, fibrosis development and damage of small blood vessels. Although there have been no established treatments for SSc, lots of new treatments targeting organ and pathogenesis are in the process of development. Transforming growth factor (TGF)-beta is a major cytokine involved in the pathogenesis of fibrosis in SSc. The blockade of cell surface molecules capable of activating latent TGF-beta, blockade of ligand by the pan-isoform-specific antibody, soluble TGF-beta receptors and a recombinant latency associated peptide, as well as inhibitors for ALK5 and Smad3 are the potential strategies to abolish the pathological activation of TGF-beta signaling in SSc fibroblasts. Besides TGF-beta, connective tissue growth factor (CTGF)/CCN2, platelet-derived growth factor (PDGF) and endothelin-1 are the candidates for the new therapeutic targets. As for immune dysfunction in SSc, i.v. immunoglobulin infusion, stem cell transplantation and B-cell depletion are potential new therapies under or awaiting a randomized, double-blind, placebo-controlled trial, although their efficacies are still controversial. Phosphodiesterase-5 inhibitors, endothelin receptor antagonists and inhibitors for serotonin signaling are the new therapeutic targets for Raynaud's phenomenon, digital ulceration and pulmonary arterial hypertension in SSc. Imatinib mesylate may be a novel new therapy for fibrosis and vasculopathy in SSc because it reverses the expression levels of Fli1, which is a transcription factor downregulated in SSc through an epigenetic mechanism and is likely to be involved in the development of fibrosis and vasculopathy in this disease. Potential therapeutic targets other than those described above are also reviewed.

Topics: Animals; Autoimmune Diseases; B-Lymphocytes; Benzamides; Connective Tissue Growth Factor; Endothelin Receptor Antagonists; Endothelin-1; Humans; Imatinib Mesylate; Immunoglobulins; Mice; Phosphodiesterase 5 Inhibitors; Piperazines; Platelet-Derived Growth Factor; Pyrimidines; Randomized Controlled Trials as Topic; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Selective Serotonin Reuptake Inhibitors; Smad3 Protein; Transforming Growth Factor beta

Fibrillin-1 (FBN-1) is the main component of the 10-12 nm microfibrils found in the extracellular matrix (ECM). ECM displays a structural role in the tissue-specific organization and takes part in the regulation of various cytokines and growth factors. A growing body of evidences supports a narrow relationship between FBN-1 and TGF-beta. Homology between FBN-1 and latent TGF-beta (LTGF) allows microfibrills to be a reservoir for this cytokine. The Marfan syndrome (MFS), a prototypic fibrillinopathy, highlights these relationships as it relates to 2 major genes that are FBN1 and TGF-beta type II receptor (TGFBR2) genes. In a mouse model of MFS, an up-regulation of the TGF-beta pathway is partly responsible for the phenotype. This FBN-1/TGF-beta relationship may play also a role in systemic sclerosis (SSc), a multigenic disease characterized by excessive generalised ECM deposit. Indeed, two related animal models results from both gene mutations: the Tight Skin 1 mouse is due to a partial in-frame duplication of the Fbn1 gene and another model conditionally overexpresses TGF-beta type I receptor. A better understanding of FBN-1/TGF-beta relationship appears of great importance in fibrillinopathies: it may allow reconsidering the nosologic framework of these diseases including the TGF-beta signalopathies and could lead to innovative therapeutic strategies.

Topics: Extracellular Matrix; Fibrillin-1; Fibrillins; Homeostasis; Humans; Marfan Syndrome; Microfilament Proteins; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis (SSc), the focus of this review, is a generalized connective tissue disease that involves sclerotic changes in the skin and sometimes various other organ systems. Clinical outcomes have improved probably due to better management of the complications, but SSc is still considered to be incurable and diffuse cutaneous SSc carries high risk of fatality. Although the pathogenesis of SSc is still unknown, the basic mechanism appears to involve endothelial cell injury, overproduction of extracellular matrix (ECM), and aberrant immune activation. This review discusses recent studies that investigated the cellular and molecular mechanisms in the pathogenesis of SSc.

Topics: Antibodies, Antinuclear; Collagen; Endothelial Cells; Endothelin-1; Extracellular Matrix; Fibroblasts; Humans; Lymphocytes; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transcription Factors; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) is a pleiotropic cytokine with vital homeostatic functions. Aberrant TGF-beta expression is implicated in the pathogenesis of fibrosis in systemic sclerosis (SSc); thus, TGF-beta represents a molecular therapeutic target in this disease. Anti-TGF-beta monoclonal antibody has been evaluated in a small trial of early SSc, with disappointing results. Antibodies against the alphavbeta6 integrin that prevent latent TGF-beta activation, however, have shown promise in preclinical studies. Small-molecule inhibitors of TGF-beta-receptor activity are effective in animal models of fibrosis. Imatinib mesylate and related tyrosine kinase inhibitors also block TGF-beta pathways and abrogate fibrotic responses. The blocking of TGF-beta activity might lead to spontaneous immune activation, epithelial hyperplasia and impaired wound healing. Loss of immune tolerance is a potential concern in an autoimmune disease such as SSc. Novel insights from microarray-based gene expression analyses and studies of genetic polymorphisms in TGF-beta signaling could aid in identifying patients who are most likely to respond to anti-TGF-beta treatment. This intervention promises to have a major impact on the treatment of SSc. Concerns regarding efficacy and safety and whether biomarkers can indicate these features, questions regarding appropriate dosing and timing of therapy, and identification of potential responders are critical challenges ahead.

Topics: Animals; Humans; Ligands; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Fibrosis affects organs such as the skin, liver, kidney and lung and is a cause of significant morbidity. There is no therapy for fibrosis. Recent significant molecular insights into the signaling underlying fibrosis have been made. Transforming growth factor beta (TGF beta) signaling is a major contributor to fibrogenesis. The signaling mechanisms through which TGF beta induces fibrogenic responses have been under intense scrutiny. Moreover, the potent pro-fibrotic proteins endothelin-1 (ET-1) and CCN2 (connective tissue growth factor, CTGF) are believed to play an essential role in this process as downstream regulators or co-factors of TGF beta signaling. This review summarizes these recent crucial observations with emphasis on the disease scleroderma.

Topics: Animals; Connective Tissue Growth Factor; Endothelin-1; Fibrosis; Humans; Rats; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Deregulated transforming growth factor-beta (TGF-beta) activity and responses play prominent roles in the pathogenesis of systemic sclerosis (SSc), a chronic and progressive connective tissue disease characterized by fibrosis of the skin and internal organs. Systemic sclerosis has highly heterogeneous clinical manifestations, and patients can be classified into multiple subgroups on the basis of distinct molecular signatures defined by transcriptional profiling of gene expression in target organs. Current research to uncover how TGF-beta regulates fibroblast function opens the door for the discovery of targeted therapies. Anti-fibrotic treatments that selectively block TGF-beta expression, biological activity or intracellular signaling in SSc are currently under development.

Topics: Animals; Disease Models, Animal; Gene Expression Profiling; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Despite the heterogeneity of SSc, almost all patients have skin involvement. As such, skin manifestations are critical in the initial diagnosis of SSc and in the subsequent sub-classification into the different subsets of disease. The two principal subsets are lcSSc and dcSSc. The main difference between these two subsets is the speed of disease progression and the extent and severity of skin and visceral involvement; lcSSc has an insidious onset with skin involvement confined largely to the face and extremities. Whilst vascular manifestations of SSc such as pulmonary arterial hypertension are typically more common in lcSSc, patients in both subsets can develop ischaemic digital ulcers. In dcSSc, disease progression is very rapid, with skin thickening extending beyond the extremities and earlier, more widespread internal organ involvement. DcSSc is generally considered to be the more severe subset of the disease. Skin scores in SSc correlate inversely with survival and are considered a valuable marker of disease severity. Skin involvement is easily detectable and, using the modified Rodnan skin score, the degree of skin fibrosis can be quantified. As well as general management measures, a number of targeted therapies are commonly used for treatment of cutaneous manifestations of SSc. These include the intravenous prostanoid iloprost and the dual endothelin receptor antagonist bosentan, which is approved in Europe for the prevention of new digital ulcers.

Topics: Collagen; Connective Tissue Growth Factor; Diagnosis, Differential; Fibrosis; Humans; Scleroderma, Systemic; Skin; Skin Ulcer; Transforming Growth Factor beta

The hallmark of scleroderma is fibrosis by excessive extracellular matrix (ECM) deposition in the skin, lung, and other organs. Increasing evidence suggests that overexpression of transforming growth factor-beta (TGF-beta) and its receptors play a key pathogenic role in the development of tissue fibrosis in scleroderma. TGF-beta is known to induce the expression of ECM proteins in the pathogenesis of fibrosis in systemic sclerosis. Investigations into TGF-beta pathways will suggest new treatment strategies for fibrotic diseases.

Topics: Animals; Extracellular Matrix; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Humans; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Transforming Growth Factor beta

Persistent transforming growth factor beta (TGF-beta) signaling is the major factor contributing to scleroderma (SSc) fibrosis. This review will summarize recent progress on the noncanonical TGF-beta signaling pathways and their role in SSc fibrosis.. Canonical TGF-beta signaling involves activation of the TGF-beta receptors and downstream signal transducers Smad2/3. The term noncanonical TGF-beta signaling includes a variety of intracellular signaling pathways activated by TGF-beta independently of Smad2/3 activation. There is evidence that these pathways play important role in SSc fibrosis. In a subset of SSc fibroblasts, a multiligand receptor complex consisting of TGF-beta and CCN2 receptors drives constitutive activation of the Smad1 pathway. CCN2 is also a primary effector of this pathway, thus establishing an autocrine loop that amplifies TGF-beta signaling. SSc fibroblasts also demonstrate reduced expression of endogenous antagonists of TGF-beta signaling including transcriptional repressors, Friend leukemia integration-1 and perixosome proliferator-activated receptor-gamma, as well as inhibitor of Smad3 phosphorylation, PTEN. PTEN is a key mediator of the cross-talk between the sphingosine kinase and the TGF-beta pathways.. Discovery of the role of noncanonical TGF-beta signaling in fibrosis offers new molecular targets for the antifibrotic therapies. Due to the heterogeneous nature of SSc, knowledge of these pathways could help to tailor the therapy to the individual patient depending on the activation status of a specific profibrotic pathway.

Topics: Animals; Autocrine Communication; Collagen; Connective Tissue Growth Factor; Fibrosis; Humans; Models, Biological; Phosphotransferases (Alcohol Group Acceptor); PTEN Phosphohydrolase; Scleroderma, Systemic; Signal Transduction; Smad1 Protein; Transforming Growth Factor beta

To review evidence and best practice for current disease-modifying therapies for the treatment of systemic sclerosis.. Cyclophosphamide remains the treatment of choice for lung disease and severe skin disease associated with systemic sclerosis. Methotrexate is the treatment of choice for scleroderma overlap syndromes, whereas mycophenolate and azathioprine are also used for both skin and lung disease, alone or for maintenance therapy after cyclophosphamide induction. Haematopoietic stem cell transplantation and imatinib look promising, but trial results are awaited. Relaxin is contraindicated due to inefficacy and severe renal side effects on discontinuation of the drug. Tolerance to type I collagen may be a useful treatment in a carefully selected group of patients. Further trials are needed for biological agents such as infliximab, rituximab and intravenous immunoglobulin.. Although there is still no treatment that is well tolerated and unequivocally effective currently for systemic sclerosis, we have come a long way in the past number of years with respect to identifying possible treatments and new therapeutic targets. A number of novel agents including antiinterleukin-6, transforming growth factor-beta-directed therapies and other novel biological agents such as hyperimmune caprine serum are being developed based on new insights into the pathophysiology of disease.

Topics: Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Autoantigens; Azathioprine; Collagen Type I; Connective Tissue Growth Factor; Cyclophosphamide; Evidence-Based Practice; Fibrosis; Hematopoietic Stem Cell Transplantation; Humans; Immunoglobulins, Intravenous; Immunosuppressive Agents; Methotrexate; Mycophenolic Acid; Plasmapheresis; Rituximab; Scleroderma, Systemic; Transforming Growth Factor beta; Transplantation, Autologous; Tumor Necrosis Factor-alpha

The underlying pathogenesis of systemic sclerosis (SSc; scleroderma) involves a complex interplay of inflammation, fibrosis and vasculopathy that is incompletely understood. In this article, we highlight the important contributions that recent preclinical research has made to the knowledge base of pathogenesis and therapeutics in SSc, describe some of the newly developed models available for further investigation and discuss future research opportunities in this fascinating area.. Several well characterized SSc models are available for the study of fibrosis. However, recent study on transgenic and knockout models has advanced knowledge both in fibrosis research and in vascular disease in SSc. In the present review, we focus on models in which altered signalling, particularly transforming growth factor-beta (TGF-beta), is limited to fibroblasts. We discuss contemporary models of SSc vascular disease, transgenesis in fibrocyte research, the contribution to neurological signalling research and provide examples of how preclinical models have contributed to novel therapeutics development in SSc. We also look at how research from related disciplines impacts on the SSc knowledge base.. These new models represent exciting advances. However, none completely recapitulates the vasculopathic and inflammatory components of this disease. These advances help to delineate the relative contributions of specific ligands, receptors, their signalling pathways and feedback mechanisms, in fibrotic and inflammatory processes and this will provide new targets for potential therapies in SSc.

Topics: Animals; Collagen; Disease Models, Animal; Fibroblasts; Fibrosis; Hematopoietic Stem Cells; Mice; Mice, Knockout; Mice, Transgenic; Models, Biological; Neuropeptides; Platelet-Derived Growth Factor; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases

PDGF functions as a primary mitogen and chemoattractant for cells of mesenchymal origin. Members of the PDGF family play an important role during embryonic development and contribute to the maintenance of connective tissue in adults. Deregulation of PDGF signalling has been linked to atherosclerosis, pulmonary hypertension and organ fibrosis. Elevated expression of PDGF and its receptors has been found in scleroderma skin and lung tissues. There is evidence for a TGF-beta and IL-1alpha-dependent autocrine PDGF-A/PDGFRalpha signalling loop in scleroderma skin and lung fibroblasts, suggesting that a cross-talk between TGF-beta and PDGF pathways may regulate chronic fibrosis in scleroderma.

Topics: Animals; Female; Fibroblasts; Humans; Lung; Lung Diseases, Interstitial; Platelet-Derived Growth Factor; Pregnancy; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Scleroderma-associated interstitial lung disease (SSc-ILD) occurs frequently and for many patients SSc-ILD is a significant complication of their disease. SSc-ILD is now one of the leading causes of death among patients with SSc. SSc-ILD, classified most often as a non-specific interstitial pneumonia, may culminate in interstitial pulmonary fibrosis and end-stage lung disease. Fibrosis in the lung is the net result of fibroblast proliferation and deposition of excessive amounts of extracellular matrix proteins. Among the many cytokines and growth factors involved in the pathogenesis of SSc-ILD, ET-1 may be a central mediator. In vitro and in vivo studies of animals and SSc patients support the notion that ET-1 can enhance the proliferation of pulmonary mesenchymal cells and may also enhance the fibrogenic effects of TGF-beta. Two well-designed randomized controlled trials of the dual ET receptor blocker bosentan were negative in their primary (and for SSc also secondary) endpoints, although there might be explanations for this apparent lack of efficacy.

Topics: Animals; Antihypertensive Agents; Bosentan; Endothelin Receptor Antagonists; Endothelin-1; Humans; Lung Diseases, Interstitial; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Sulfonamides; Transforming Growth Factor beta; Treatment Failure

The University of California at Davis 200 and 206 (UCD-200/206) lines of chickens have proven to be the animal model that best reflects the situation in human SSc. We have demonstrated a misbalance of pro-fibrotic (TGF-beta1) and anti-fibrotic (TGF-beta2 and -beta3) TGF-beta isoforms as a possible cause for fibrotic alterations in this model. This opens new avenues for diagnosis and therapy for this still intractable condition.

Topics: Animals; Chickens; Fibrosis; Models, Animal; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a complex and heterogeneous chronic illness characterized by substantial patient to patient variability in clinical manifestations, internal organ involvement, and outcome. Genetic factors contribute to disease susceptibility, but environmental influences also play a significant role. The pathogenesis of SSc encompasses vascular, immunological, and fibrotic processes, which contribute to clinical manifestations and morbidity and must be addressed in the treatment plan. Although vascular interventions appear to reduce the frequency and severity of complications, such as scleroderma renal crisis and pulmonary hypertension, current therapies generally target the immune component of SSc in a non-selective fashion and have largely failed as diseases-modifying interventions. Newer insights into the mechanisms underlying autoimmunity, vascular injury and destruction, and particularly tissue fibrosis provide novel potential targets for therapy. Transforming growth factor-ss is a ubiquitous cytokine that appears to contribute to fibroblast activation, collagen overproduction, and pathological tissue fibrosis. Neutralizing antibodies and small molecules that block TGF-beta activation or function are effective in shutting down TGF-beta signaling and selectively inhibit the progression of fibrosis and may be entering clinical trials for the treatment of SSc.

Topics: Blood Vessels; Cardiovascular Agents; Fibrosis; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Immunosuppressive Agents; Inflammation; Kidney Diseases; Lung Diseases, Interstitial; Raynaud Disease; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Treatment Outcome

The endothelins (ET) are the family of 21 amino acid endogenous peptides with potent vasoconstriction function. There are 3 isoforms of the endothelin protein (ET-1, ET-2 and ET-3) encoded by separate genes and exhibit distinct tissue distribution and function. Endothelin 1 is the significant isoform in humans. Endothelin 1 is the most abundant, best characterized isoform with truly pluripotent properties. Endothelin 1 is involved in physiological processes of vascular tone and mitogenesis, whereas under pathological conditions fibrosis, vascular hypertension and inflammation are induced. In human body there are 2 separate ET receptors, ET(A)R and ET(B)R belonging to the G-protein family which produce differing, sometimes opposite effects. Both receptors are differentially expressed by different cell types as well as in different disease entities, In fibroblast cell culture in vitro ET-1 through its receptors modulates cell proliferation, differentiation, contraction and migration. Endothelin 1 is implicated in extracellular matrix (ECM) components synthesis. The dual regulatory role of ET-1 consist on stimulation of collagen I and III synthesis and simultaneously on inhibition of MMP-1 expression through inhibition of tissue inhibitors of metalloproteinase: TIMP-1 and TIMP-3. Endothelin 1 promotes the differentiation of fibroblasts into myofibroblast's phenotype via elevated expression of procontractile proteins alpha-SMA, ezrin, paxillin and moesin. The elevated level of endogenous ET-1 expression cause deficient of myofibroblast apoptosis and increased ECM components deposition. Endothelin 1 is a potent vasoconstrictor, a potent mitogen for fibroblast and smooth muscle cells, a strong stimulant of matrix biosynthesis and is a survival factor for myofibroblasts. Endothelin 1 plays a key role in inflammatory disease and in the connective tissue fibrosis. Elevated level of ET-1, TGF-beta and their receptors has been reported in the pathogenesis of systemic sclerosis.

Topics: Apoptosis; Biomarkers; Cell Differentiation; Cell Proliferation; Cells, Cultured; Connective Tissue; Endothelin-1; Endothelins; Fibrosis; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Transforming growth factor-beta (TGF-beta) is required for tissue homeostasis but is also implicated in disease processes including fibrosis, and thus represents a molecular target for therapy.. Multiple strategies for inhibiting excessive TGF-beta function exist. The three principal platforms are RNA-based technologies, monoclonal antibodies and small molecules. Monoclonal antibodies targeting TGF-beta have been used in a small clinical trial, with disappointing results to date. Antibodies to the alphavbeta6 integrin prevent local activation of latent TGF-beta and show promise in preclinical studies. Over a dozen small molecules inhibit the kinase activity of TGF-beta receptors. Several commonly used drugs appear to have unanticipated anti-TGF-beta activity and may therefore have a role in antifibrotic therapy. Because TGF-beta has important physiological functions, inhibiting its activity might potentially lead to aberrant immune activation, epithelial hyperplasia and impaired wound healing; spontaneous autoimmunity in particular is a concern in an autoimmune disease such as systemic sclerosis. Novel insights from DNA microarray analysis and genetic polymorphisms in TGF-beta signaling will aid in defining patient populations most likely to respond to anti-TGF-beta treatment.. Anti-TGF-beta therapies promise to have a major impact in systemic sclerosis. Significant concerns regarding efficacy and safety need to be addresed. The identification of optimal candidates for therapy, and of biomarkers of safety and efficacy, are critical challenges ahead.

Topics: Antibodies, Monoclonal; Fibrosis; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Excessive extracellular matrix deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc). Fibroblasts isolated from sclerotic lesions in patients with SSc and cultured in vitro are characterized by increased synthesis of collagen and other extracellular matrix components, consistent with the disease phenotype. Thus, cultured scleroderma fibroblasts serve as a principal experimental model for studying the mechanisms involved in extracellular matrix overproduction in SSc. The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that transforming growth factor-beta (TGF-beta) is a key mediator of tissue fibrosis as a consequence of extracellular matrix accumulation in the pathology of SSc. TGF-beta regulates diverse biological activities including cell growth, cell death or apoptosis, cell differentiation, and extracellular matrix synthesis. TGF-beta is known to induce the expression of extracellular matrix proteins in mesenchymal cells and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the extracellular matrix. This review focuses on the possible role of autocrine TGF-beta signaling in the pathogenesis of SSc.

Topics: Animals; Autocrine Communication; Connective Tissue Growth Factor; Extracellular Matrix; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Fibrosis affects organs such as the skin, liver, kidney and lung and is a cause of significant morbidity. There is no therapy for fibrosis. Recent significant molecular insights into the signaling underlying the fibrosis in the autoimmune connective tissue disease scleroderma (systemic sclerosis, SSc) have been made. Transforming growth factor beta (TGFbeta) signaling is a major contributor to fibrogenesis, including in SSc. However, it is now appreciated that TGFbeta-dependent and TGFbeta-independent mechanisms play key roles in the pathological fibrosis in SSc. In particular the potent pro-fibrotic proteins endothelin-1 (ET-1) and CCN2 (connective tissue growth factor, CTGF) are believed to play an essential role in this process. This review summarizes these recent crucial observations.

Topics: Connective Tissue Growth Factor; Endothelin-1; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

One of the growth factors that appear to play a crucial role in the pathogenesis of systemic sclerosis is TGF-beta. The three functionally and structurally similar human isoforms of TGF-beta play important roles in embryonic development, in the regulation of tissue repair following injury and in immune responses. Systemic sclerosis fibroblasts express increased levels of TGF-beta receptors on their surface which in turn results in increased signalimg of TGF-beta induced collagen gene expression. Some of the patents and intricate pathways that mediate the stimulation of collagen gene expression by TGF-beta have recently been described and a potential inhibition of these pathways may lead to novel therapeutic targets for systemic sclerosis.

Topics: Animals; Collagen; Drug Delivery Systems; Fibroblasts; Gene Expression Regulation; Humans; Patents as Topic; Protein Isoforms; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis is a complex multi-systemic disease with a mostly unresolved pathogenesis. Following an inflammatory reaction, overproduction of collagen and other extra-cellular matrix components leads to a characteristic fibrosis. It remains unclear why this overproduction by fibroblasts and myofibroblasts occurs. Micro-vascular disturbances and endothelial cells, as well as immunomodulation and inflammation are central factors. Besides intrinsic influences, such as genetic polymorphisms, multiple mediators with fibrotic effects such as Platelet Derived Growth Factor, Transforming Growth Factor-beta and Connective Tissue Growth Factor have been characterized. These have become targets for innovative therapeutic strategies that might lead to specific treatments for systemic sclerosis.

Topics: Animals; Antibodies, Antinuclear; Autoantibodies; Collagen Diseases; Disease Models, Animal; Endothelial Cells; Extracellular Matrix; Fibroblasts; Fibrosis; Humans; Mice; Platelet-Derived Growth Factor; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Vasoconstriction

The autoimmune disease scleroderma (systemic sclerosis (SSc)) is characterized by extensive tissue fibrosis, causing significant morbidity. There is no therapy for the fibrosis observed in SSc; indeed, the underlying cause of the scarring observed in this disease is unknown. Transforming growth factor-beta (TGFbeta) has long been hypothesized to be a major contributor to pathological fibrotic diseases, including SSc. Recently, the signaling pathways through which TGFbeta activates a fibrotic program have been elucidated and, as a consequence, several possible points for anti-fibrotic drug intervention in SSc have emerged.

Topics: Cicatrix; Cytokines; Drug Synergism; Fibrosis; Humans; Scleroderma, Systemic; Smad Proteins; Transforming Growth Factor beta

Idiopathic pulmonary fibrosis (IPF) is an under-recognised, rare, progressive disease of the lungs with unknown aetiology and high mortality. The currently advocated pathogenic mechanism is represented by progressive multifocal fibrosis. It is diagnosed based on clinical, radiographic, physiological and histopathological criteria. Existing therapeutic guidelines recommend anti-inflammatory and immunosuppressive combinations, despite proven limited efficacy. There is no therapy approved specifically for IPF, but several antifibrotic agents are currently under development for this indication. Pirfenidone is an antifibrotic agent potentially effective for IPF therapy, and preclinical and available clinical data support its use in IPF. Future clinical studies are expected to provide more consistent information on survival benefit, lung function and health-related quality of life.

Topics: Administration, Oral; Amiodarone; Animals; Anti-Inflammatory Agents, Non-Steroidal; Bleomycin; Cell Line; Clinical Trials, Phase II as Topic; Connective Tissue Growth Factor; Cricetinae; Cyclophosphamide; Drug Evaluation, Preclinical; Drugs, Investigational; Fibroblasts; Hermanski-Pudlak Syndrome; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Lung Transplantation; Mice; Orphan Drug Production; Photosensitivity Disorders; Pulmonary Fibrosis; Pyridones; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Transforming Growth Factor beta; Treatment Outcome; Tumor Necrosis Factor-alpha

During the past two decades, the human pro-alpha2(I) collagen gene (COL1A2) has emerged as an informative model in which to study the general principles that govern the transcriptional control of extracellular matrix deposition in normal and fibrotic conditions. Multiple studies have in fact delineated the genomic regions, cis-acting elements and trans-acting factors implicated in constitutive, cytokine-modulated and tissue-specific expression of COL1A2. These functional components are integrated into a regulatory network that consists of the proximal promoter, far-upstream enhancer and downstream repressor, and which operates according to two mechanisms. The first mechanism is one in which combinatorial interactions among promoter-bound proteins determine transcriptional outcome in different cellular and experimental contexts. The other mechanism is one whereby cooperative assembly of protein complexes at distantly located DNA elements directs spatiotemporal specificity. These transcriptional studies have also an additional value in translational research, in that they are providing the conceptual means to develop new animal models of and therapeutic strategies for fibrotic diseases.

Topics: Animals; Chromatin; Collagen; Collagen Type I; Cytokines; Extracellular Matrix; Fibrosis; Gene Expression Regulation; Humans; Interferon-gamma; Models, Biological; Promoter Regions, Genetic; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Transforming growth factor-beta (TGF-beta) plays a critical role in the development of tissue fibrosis. Its expression is consistently elevated in affected organs and correlates with increased extracellular matrix deposition. During the last few years, tremendous progress has been made in understanding the molecular aspects of intracellular signaling downstream of the TGF-beta receptors. In particular, Smad proteins, TGF-beta receptor kinase substrates that translocate into the cell nucleus to act as transcription factors, have been studied extensively. Their role in the transcriptional regulation of type I collagen and other extracellular matrix (ECM) genes expression, and in the development of fibrosis is of critical importance because it may lead to novel therapeutic strategies for the treatment of these multi-organ tissue reactions to injury. Systemic sclerosis (SSc) is a complex autoimmune disease characterized by pathological remodelling of connective tissues correlated to the activation of TGF-beta/Smad signaling pathway. This review focuses on the mechanisms underlying Smad modulation of gene expression and how they relate to fibrotic process. Potential implications for the development of therapeutic approaches against tissue fibrosis during SSc are discussed.

Topics: Gene Expression Regulation; Humans; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Important recent understandings of fibrillins and fibrillin-associated microfibril proteins suggest new ways these proteins might contribute to tissue fibrosis seen in systemic sclerosis by regulating latent transforming growth factor-beta. This review discusses mutant-fibrillin mouse models of Marfan syndrome and SSc (Tsk mice), and studies suggesting that alterations in microfibrils might contribute to human SSc.. Fibrillin-1 mutations associated with Marfan syndrome have recently been shown to induce genes activated by TGF-beta. The inhibition of TGF-beta in these mouse models largely reverses phenotypic and pathologic disease manifestations. Recent studies suggest that alterations in the fibrillin-1 structure from mutant Tsk fibrillin cause hypodermal fibrosis and associated changes in dermal gene expression, suggesting stimulation of cytokine-mediating signals. Genetic mutations in fibrillin-1, in a higher frequency in SSc patient populations, and autoantibodies to fibrillin provide potential links to human SSc.. Fibrillin is placed centrally not only as the primary structural component of microfibrils, but also a key regulator of cytokines in the TGF-beta superfamily. Fibrillin may thus communicate alterations in matrix to fibroblast gene expression. These observations complement emerging understandings of the effects of Tsk fibrillin, and genetic and autoimmune studies of human fibrillin on dermal fibrosis.

Topics: Animals; Disease Models, Animal; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Gene Expression Regulation; Humans; Marfan Syndrome; Mice; Mice, Mutant Strains; Microfibrils; Microfilament Proteins; Mutation; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

The aim of this article is to review studies which support the hypothesis that allograft inflammatory factor-1, a protein initially identified in chronically rejected cardiac allografts, may be involved in the pathogenesis of the progressive fibroproliferative vasculopathy which is a hallmark of systemic sclerosis.. Recent findings demonstrated elevated allograft inflammatory factor-1 expression both in systemic sclerosis affected tissues and peripheral blood mononuclear cells. A detailed immunohistopathologic study examined the tissue and cellular localization of the protein in affected systemic sclerosis tissues and demonstrated its expression in the endothelium of dermal and pulmonary vessels, in the pulmonary parenchyma, and in relevant inflammatory cells including T cells and macrophages. Furthermore, functional studies showed specific allograft inflammatory factor-1 isoform expression stimulation by transforming growth factor-beta.. This review summarizes recent findings suggesting that allograft inflammatory factor-1 may play an important role in systemic sclerosis vasculopathy and provides supporting evidence to consider the molecule as a novel therapeutic target.

Topics: Biomarkers; Calcium-Binding Proteins; DNA-Binding Proteins; Endothelium, Vascular; Gene Expression; Humans; Leukocytes, Mononuclear; Microfilament Proteins; Polymorphism, Genetic; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis is a rare disease characterized by vascular hyperreactivity and collagen deposition. Endothelial cell, fibroblast and lymphocyte abnormalities have been reported in systemic sclerosis. Fibroblast dysfunction is characterized by uncontrolled activation of the transforming growth factor-beta (TGF-beta) pathway and excess synthesis of both connective tissue growth factor (CTGF) and free radicals. These promote the accumulation of extracellular matrix. Endothelial cells produce excess quantities of endothelin 1 and inducible NO synthase. They also undergo early apoptosis. Oxidative stress appears to play a major role in disease progression. Increased levels of interleukin 4, a profibrotic cytokine, have been detected in plasma and skin of systemic sclerosis patients. Autoantibodies are detectable in the serum of almost all systemic sclerosis patients. Some are directed against well-identified ubiquitous nuclear proteins and have no demonstrated pathogenic role. Other autoantibodies bind to endothelial cells or fibroblasts and may have a pathogenic role.

Topics: Adult; Animals; Apoptosis; Autoantibodies; Cells, Cultured; Disease Models, Animal; Disease Progression; Endothelium, Vascular; Female; Fibroblasts; Free Radicals; Humans; Interleukin-4; Male; Mice; Mice, Inbred BALB C; Oxidative Stress; Phenotype; Pregnancy; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Topics: Angiotensin-Converting Enzyme Inhibitors; Antigens, CD19; Autoantibodies; B-Lymphocytes; Biomarkers; Centromere; Collagen; Connective Tissue Growth Factor; Cyclophosphamide; DNA Topoisomerases, Type I; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Prognosis; Pulse Therapy, Drug; Scleroderma, Systemic; Transforming Growth Factor beta

The pathological changes in systemic sclerosis are characterised by severe organ fibrosis, obliteration of arteries and arterioles and disturbance of immunological functions. Recent research has yielded new insight into the complex pathogenesis of systemic sclerosis, with new therapeutic options as a possible consequence.. Relevant medical literature has been reviewed.. The pathological deposition of extracellular matrix in systemic sclerosis is most probably caused by changes in the regulation of dermal fibroblasts. The review focuses on three of the many molecules involved in the regulation of fibrosis; Transforming growth factor beta, Connective tissue growth factor and Endothelin-1.. Treatment of organ-specific disease complication has for long remained the only therapeutic option in systemic sclerosis. Based on current knowledge of the process of fibrosis, new therapeutic trials employing substances directed towards pro-fibrotic molecules are now in progress.

Topics: Connective Tissue Growth Factor; Endothelin-1; Endothelins; Fibroblasts; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: Animals; Disease Models, Animal; Humans; Mice; Microtubules; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis (SSc), or scleroderma, is a connective tissue disorder characterized by progressive fibrosis of the skin and internal organs. It has significance for gastroenterologists because the gastrointestinal tract is involved in 90% of SSc patients, who often present with esophageal dysfunction. Though the exact pathogenesis of SSc is unknown, there is increasing evidence supporting an immune mechanism. Cytokines are the soluble mediators of immune activation, altered fibroblast proliferation and extracellular matrix accumulation in SSc and thereby provide important therapeutic targets. In the present review, the involvement of cytokines in SSc is discussed with particular emphasis on cytokines and growth factors that have been implicated in the disease process and likely play an important role in the gastrointestinal manifestations of scleroderma. The role of cytokines as therapeutic targets in scleroderma forms the basis of this timely review.

Topics: Chemokine CCL2; Connective Tissue Growth Factor; Cytokines; Gastrointestinal Diseases; Growth Substances; Humans; Immediate-Early Proteins; Insulin-Like Growth Factor Binding Proteins; Intercellular Signaling Peptides and Proteins; Interleukin-4; Interleukin-6; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

Platelet-derived growth factor (PDGF) isoforms play a major role in stimulating the replication, survival, and migration of myofibroblasts during the pathogenesis of fibrotic diseases. During fibrogenesis, PDGF is secreted by a variety of cell types as a response to injury, and many pro-inflammatory cytokines mediate their mitogenic effects via the autocrine release of PDGF. PDGF action is determined by the relative expression of PDGF alpha-receptors (PDGFRalpha) and beta-receptors (PDGFRbeta) on the surface of myofibroblasts. These receptors are induced during fibrogenesis, thereby amplifying biological responses to PDGF isoforms. PDGF action is also modulated by extracellular binding proteins and matrix molecules. This review summarizes the literature on the role of PDGF and its receptors in the development of fibrosis in a variety of organ systems, including lung, liver, kidney, and skin.

Topics: Animals; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Inflammation; Interleukin-1; Kidney; Liver; Lung; Models, Biological; Muscles; Platelet-Derived Growth Factor; Protein Isoforms; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Tissue Distribution; Transforming Growth Factor beta; Transforming Growth Factor beta1

Modern molecular genetic methods have allowed better understanding of established mouse models of scleroderma and also facilitated the development of new and better defined mouse strains for investigating the pathogenesis of the disease. The best characterized scleroderma animal model is the type 1 tight skin mouse (Tsk1). Backcrossing these animals with other mutant strains has been informative. These experiments implicate the IL-4 ligand-receptor axis in the development of skin fibrosis. Parallel expression analysis of genes using microarrays has provided insight into novel mediators of fibrosis including the C-C chemokine MCP-3. Other experiments suggest that embryonically defined fibroblast-specific regulatory elements may be targets for activation in this model. The same lineage-specific elements have been used to selectively activate TGF beta signaling pathways in fibrosis to generate a novel model for scleroderma and also have been used to develop systems for ligand-dependent fibroblast-specific genetic recombination that will allow further analysis key candidate genes implicated in scleroderma pathogenesis. Better mouse models will improve understanding of this intractable rheumatic disease and can be expected to ultimately lead to improved treatments and outcome.

Topics: Animals; Chemokine CCL7; Crosses, Genetic; Cytokines; Disease Models, Animal; Fibrosis; Forecasting; Gene Expression; Genes, Reporter; Humans; Mice; Mice, Inbred Strains; Mice, Mutant Strains; Mice, Transgenic; Models, Biological; Monocyte Chemoattractant Proteins; Receptors, Interleukin-4; Recombination, Genetic; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

The cause of fibrotic disease is unknown. We have undertaken transcriptional profiling of dermal fibroblasts cultured from patients with the fibrotic disease scleroderma (systemic sclerosis, SSc) to identify genes overexpressed in fibrosis and have explored their contribution to the fibrotic phenotype. Connective tissue growth factor (CTGF, CCN2), a member of the CCN family of proteins, is overexpressed in SSc fibroblasts. In adult skin, CTGF is not normally expressed in dermal fibroblasts. However, CTGF is induced during the wound healing response and is constitutively overexpressed by fibroblasts present in fibrotic lesions. The overexpression of CTGF present in fibrotic lesions contributes to the phenotype of scleroderma in that CTGF promotes matrix deposition, and fibroblast adhesion and proliferation. In animal models, whereas either TGF beta or CTGF alone produce only a transient fibrotic response, CTGF and TGF beta act together to promote sustained fibrosis. Thus the constitutive overexpression of CTGF by fibroblasts present in fibrotic lesions would be expected to directly contribute to chronic, persistent fibrosis.

Topics: Animals; Connective Tissue Growth Factor; Disease Models, Animal; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; NIH 3T3 Cells; Phenotype; Protein Structure, Tertiary; Response Elements; RNA, Messenger; Scleroderma, Systemic; Skin; Sp1 Transcription Factor; Transcription, Genetic; Transforming Growth Factor beta

A hypothesis for the pathogenesis of systemic sclerosis (SSc) is proposed. Transforming growth factor-beta (TGF-beta) has received attention as an essential factor in the pathogenesis of various fibrotic disorders, including SSc, although some unknown additional factor has been sought as the second mediator of fibrotic disorders. Connective tissue growth factor (CTGF) has been shown to be closely related to the pathogenesis of SSc as follows: (1) CTGF mRNA expression was observed in the fibrotic lesions but not in the early nonfibrotic lesions or atrophic lesions. (2) Serum CTGF protein concentrations were significantly elevated, and correlated with skin sclerosis and lung fibrosis. (3) In our animal model, TGF-beta-induced subcutaneous fibrosis and subsequent CTGF application caused persistent fibrosis. Based on these data, we hypothesize that a 2-step process of fibrosis occurs in SSc: that is, TGF-beta induces fibrosis in the early stage and afterwards CTGF acts to maintain tissue fibrosis.

Topics: Connective Tissue Growth Factor; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: Humans; Ligases; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Systemic sclerosis (SSc) is an autoimmune disorder of unknown etiology characterized by severe and often progressive cutaneous and visceral fibrosis, pronounced alterations in the microvasculature, and numerous cellular and humoral immune abnormalities. Clinically, SSc is very heterogeneous, encompassing a spectrum ranging from mild limited forms of skin sclerosis with minimal internal organ involvement to severe skin and multiple internal organ fibrosis. Mortality and morbidity in SSc are very high and are directly related to the extent of the fibrotic and microvascular alterations. A better understanding of the pathogenesis of this incurable disorder will help to better target and design effective therapy in the future.

Topics: Autoimmune Diseases; Chimera; Collagen; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Immune System; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a connective tissue disorder characterized by vascular abnormalities and excessive collagen synthesis. Extracellular matrix overproduction by fibroblasts results from abnormal interactions among endothelial cells, mononuclear cells (lymphocytes and monocytes) and fibroblasts, in a setting of vascular hyperreactivity and tissue hypoxia. Many autoantibodies have been identified in the sera of SSc patients; some of them are specific to the disease, such as anti-centromere antibodies in limited SSc, anti-topoisomerase 1 and anti-RNA polymerase I/III antibodies in diffuse SSc. Their pathogenetic role(s) remains uncertain. However, genetic, environmental and possibly alloreactive factors might also contribute to disease susceptibility.

Topics: Autoantibodies; CD8-Positive T-Lymphocytes; Chimera; Endothelium, Vascular; Fibroblasts; Interleukin-4; Leukocytes, Mononuclear; Models, Biological; Scleroderma, Systemic; Transforming Growth Factor beta

The triad of pathologic changes that defines systemic sclerosis (scleroderma) includes immune system activation with autoimmunity; an obliterative, proliferative small vessel vasculopathy; and fibrosis. Available data suggest that several cytokines, including chemokines, contribute to the development of scleroderma complications. This review focuses on chemokines and their contribution to tissue fibrosis and pulmonary hypertension in scleroderma.. Proteins and mRNAs for monocyte chemoattractant protein-1; pulmonary and activation-regulated chemokine; macrophage inflammatory protein-1, regulated upon activation normal T cell expressed and secreted; interleukin-8; and transforming growth factor-beta have been found in increased amounts in blood or involved tissue from scleroderma patients. These factors are likely to contribute directly to tissue damage in scleroderma through several pathways, including stimulation of extracellular matrix production, induction of TGF-beta production and activation, and chemoattraction of T cells and nonspecific inflammatory cells into tissues.. Multiple chemokines are part of the pathologic network that causes tissue damage in scleroderma, and, as such, may provide therapeutic targets in scleroderma.

Topics: Animals; Biomarkers; Bronchoalveolar Lavage Fluid; Chemokine CCL2; Chemokine CCL5; Chemokines; Chemokines, CC; Female; Humans; Interleukin-8; Male; Scleroderma, Systemic; Sensitivity and Specificity; Severity of Illness Index; T-Lymphocytes; Transforming Growth Factor beta

Topics: Collagen; Fibrosis; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: DNA-Binding Proteins; Fibrosis; Humans; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Excessive extracellular matrix (ECM) deposition in the skin, lung, and other organs is a hallmark of systemic sclerosis (SSc). The pathogenesis of SSc is still poorly understood, but increasing evidence suggests that transforming growth factor (TGF)-beta is a key mediator of tissue fibrosis as a consequence of ECM accumulation in pathological states such as systemic sclerosis. TGF-beta regulates diverse biological activities including cell growth, cell death or apoptosis, cell differentiation, and ECM synthesis. TGF-beta is known to induce the expression of ECM proteins in mesenchymal cells and to stimulate the production of protease inhibitors that prevent enzymatic breakdown of the ECM. This review focuses on the possible role of TGF-beta in the pathogenesis of fibrosis in SSc.

Topics: Connective Tissue Growth Factor; DNA-Binding Proteins; Extracellular Matrix; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mitogen-Activated Protein Kinases; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Trans-Activators; Transforming Growth Factor beta

Tissue fibrosis is the result of a complex series of events focusing on regulation of fibroblast proliferation, synthesis of extracellular matrix, and apoptosis. Transforming growth factor-beta is important for the stimulation of the fibrotic response by promoting the production of extracellular matrix proteins, by promoting the differentiation of the myofibroblast cell morphology, and by protecting these cells against apoptotic stimuli. Other cytokines such as interleukin-1 may have stimulatory and counter-regulatory effects on fibrosis. The effects of these signaling molecules depend on cellular environment and are organ specific. Furthermore, intercellular interactions and cell-matrix interactions can stimulate or inhibit the apoptotic pathway. Through selective inhibition of apoptosis in myofibroblasts, fibrosis can become dysregulated and lead to diseases such as systemic sclerosis.

Topics: Animals; Apoptosis; Fibroblasts; Fibrosis; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

While the biology of the pathogenesis of scleroderma is continually being better understood, there still is no single agent or therapeutic combination that has a clear impact on the disease process. Traditional medications (colchicine, potassium aminobenzoate (potaba), D-penicillamine) are disappointing in clinical practice despite anecdotal evidence of benefit. Furthermore, the most popular traditional drug, D-penicillamine, failed to clearly show benefit when tested in a well-designed clinical trial comparing conventional high dose with a very low dose (125 mg po. every other day [corrected]) [1]. Currently, most success in managing scleroderma and improving quality of life is secondary to organ-specific therapy, such as management of a renal crisis with an ACE inhibitor, treatment of Raynaud's phenomenon with calcium channel blockers, or control of serious gastrointestinal reflux disease with a proton pump inhibitor. In this review we will focus on novel therapies that are currently being tested in the treatment of scleroderma and have the potential of modifying the disease process and overall clinical outcome. We have attempted to review the rationale for each agent, recognising that its true biological effect will only be determined in clinical trials.

Topics: Autoimmune Diseases; Bone Marrow Transplantation; Cysteine; Humans; Interferon-gamma; Nitric Oxide; Piperidines; Prostaglandins; Quinazolines; Quinazolinones; Relaxin; Scleroderma, Systemic; Tetracycline; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Alprostadil; Collagen; Cytokines; Diagnosis, Differential; Fibrillins; Humans; Microfilament Proteins; Mutation; Penicillamine; Prognosis; Scleroderma, Systemic; Transforming Growth Factor beta

Evidence for a role for members of the transforming growth factor beta (TGF-beta) family of cytokines in the pathogensis of systemic sclerosis and other fibrotic conditions is provided from studies of TGF-beta protein and gene expression in lesional biopsy specimens, from altered responses of explanted fibroblasts to TGF-beta stimulation which are associated with increased receptor expression on these cells and from genetic data linking TGF-beta gene loci to the disease. Of the many effects of TGF-beta on fibroblast properties induction of the connective tissue growth factor/Cyr61/NOV (CCN) family members, connective tissue growth factor (CTGF) may be particularly relevant to fibrosis. Moreover, systemic sclerosis (SSc) fibroblasts demonstrate constitutive over expression of CTGF that promotes migration, proliferation and matrix production. Studies of mechanisms regulating constitutive expression of CTGF by SSc fibroblasts are currently being undertaken and indicate that a TGF-beta responsive element in the CTGF promoter is involved, although this appears to function independent of the Smad proteins, suggesting that other TGF-beta-regulated pathways may be involved. TGF-neutralizing strategies have now been shown to abrogate many animal models of fibrosis, and will soon reach the clinical arena for SSc. These agents will further clarify the role of this ligand in initiating or sustaining fibrosis and offer the exciting possibility of targeted therapy for this disease.

Topics: Connective Tissue Growth Factor; DNA-Binding Proteins; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Multigene Family; Nephroblastoma Overexpressed Protein; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Smad3 Protein; Trans-Activators; Transforming Growth Factor beta

Topics: Collagen; Cytokines; Dermatology; Esophagus; Humans; Pulmonary Fibrosis; Scleroderma, Systemic; Severity of Illness Index; Skin; Transforming Growth Factor beta

The regulation of matrix biosynthesis in systemic sclerosis has been the focus of many studies, because excessive matrix synthesis causes pathologic changes, and because this would seem to be a good target for therapies aimed at ameliorating the disease. Possible targets for antifibrotic therapies include both matrix gene stimulatory and inhibitory pathways. Much recent progress has been made in understanding the mechanism of action of transforming growth factor-beta (TGF-beta), an important profibrotic cytokine with pleiotropic effects on fibroblasts. It appears that TGF-beta may use multiple signal transduction pathways in fibroblasts and it is possible that defects in any of these pathways may result in an abnormal response to TGF-beta, resulting in fibrosis. Studies on negative regulation of matrix gene expression have singled out the antifibrotic cytokines tumor necrosis factor-alpha and interferon-gamma. Finally, a new approach that compares mRNA expression in normal versus diseased fibroblasts has already lead to the discovery of genes that may play a role in the development of fibrosis. This represents an important advance because genes can be identified that have not previously been implicated in the control of matrix synthesis, and thus might not otherwise have been studied in this context.

Topics: Connective Tissue Growth Factor; DNA Methylation; Extracellular Matrix Proteins; Fibroblasts; Gene Expression Regulation; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Scleroderma, Systemic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Topics: Animals; Controlled Clinical Trials as Topic; Dose-Response Relationship, Drug; Drug Administration Schedule; Female; Humans; Interferon-gamma; Interferons; Male; Prognosis; Pulmonary Fibrosis; Scleroderma, Systemic; Sensitivity and Specificity; Severity of Illness Index; Transforming Growth Factor beta; Treatment Outcome

Topics: Animals; Connective Tissue Growth Factor; Fibrosis; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; Mitogens; Scleroderma, Systemic; Skin Diseases; Transforming Growth Factor beta

Endothelial injury, obliterative microvascular lesions, and increased vascular wall thickness are present in all involved organs in scleroderma. The vascular pathology is associated with altered vascular function with increased vasospasm, reduced vasodilatory capacity and increased adhesiveness of the blood vessels to platelets and lymphocytes. The extent of injury and dysfunction is reflected by changes in the circulating levels of vascular markers. The initial triggers for the vascular pathology are not known. Possible viral triggers are visited here, including cytomegalovirus in view of increased levels of anti-CMV antibodies in scleroderma, and the remarkable similarities between CMV vasculopathies and scleroderma vascular disease. Endothelial apoptosis in scleroderma may be related to viral infection, immune reactions to viral or environmental factors, reperfusion injury or to anti-endothelial antibodies. The impact of the vascular pathology on the evolution of tissue fibrosis is not known; still, cytokines (TGFbeta, IL4), vascular factors (endothelin), and growth factors (PDGF) are possibly crucial signals that link the vascular disease to tissue fibrosis. Knowledge of the regulation of these and other factors will provide the opportunity to develop more rational therapeutic approaches to the disease.

Topics: Animals; Autoimmunity; Endothelium, Vascular; Fibroblasts; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a multisystem connective tissue disorder in which there is progressive fibrosis. Transforming growth factor beta (TGF beta) has wide-ranging cellular actions. It is a potent chemoattractant for human dermal fibroblasts, from which it may induce synthesis of collagen, which suggests that it may have a central role to play in the pathogenesis of SSc. This is supported to some extent by in vitro studies. SSc fibroblasts produce more collagens and fibronectin than normal fibroblasts and elevated TIMP levels have been observed, all of which could be explained on the basis of TGF beta stimulation of fibroblasts. Some studies have suggested that fibroblasts are the source of TGF beta. However, the serum of patients with SSc is cytotoxic to endothelial cells, which could culminate in TGF beta synthesis by them, with secondary fibroblast stimulation. The role of TGF beta remains elusive, although it would seem an ideal candidate as a mediator of fibrosis in systemic sclerosis.

Topics: Cell Culture Techniques; Fibroblasts; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Topics: Animals; Endothelium, Vascular; Scleroderma, Systemic; Transforming Growth Factor beta

Although there is no cure for systemic sclerosis, the disease is certainly treatable. And the more that is understood of its pathogenesis, the better the prospects for treatment. Excessive collagen deposition in the skin and viscera is responsible for the morbidity and mortality. The immune system plays a critical role in setting up the fibrotic process, but impaired regulation of collagen gene expression probably underlies the persistence of fibrosis. The development of effective antifibrotic drugs, in particular agents that inhibit transcription factors involved in the activation of type I collagen genes, is likely to be the way forward for treatment of the later stages of diffuse systemic sclerosis, with immunomodulatory therapy for the earlier stages.

Topics: Collagen; Cyclophosphamide; Female; Gene Expression; Humans; Middle Aged; Prednisolone; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

It is now evident that persistent overproduction of collagen and other connective tissue macromolecules results in excessive tissue deposition, and is responsible for the progressive nature of fibrosis in SSc. Up-regulation of collagen gene expression in SSc fibroblasts appears to be a critical event in the development of tissue fibrosis. The coordinate transcriptional activation of a number of extracellular matrix genes suggests a fundamental alteration in the regulatory control of gene expression in SSc fibroblasts. Trans-acting nuclear factors that bind to cis-acting elements in enhancer and promoter regions of the genes modulate the basal and inducible transcriptional activity of the collagen genes. The identity of the nuclear transcriptional factors that regulate normal collagen gene expression remains to be firmly established, and to date, no alterations in the level or in the activity of such DNA binding factors has been demonstrated in SSc fibroblasts. In addition to important interactions between fibroblasts and the extracellular matrix, cytokines and other cellular mediators can positively and negatively influence fibroblast collagen synthesis. Some of these signaling molecules may have physiologic roles, and their aberrant expression, or altered responsiveness of SSc fibroblasts to them, may result in the acquisition of the activated phenotype. The rapid expansion of knowledge regarding the effects of cytokines on extracellular matrix synthesis has led to an appreciation of the enormous complexity of regulatory networks that operate in the physiologic maintenance of connective tissue and which may be responsible for the occurrence of pathologic fibrosis. The ubiquitous growth factor TGF beta is the most potent inducer of collagen gene expression and connective tissue accumulation yet discovered. The expression of TGF beta in activated infiltrating mononuclear cells suggests a role for this cytokine as a mediator of fibroblast activation in SSc. Furthermore, the recognition that TGF beta is capable of inducing its own expression in a variety of cell types, coupled with the demonstration that a subpopulation of SSc dermal fibroblasts produces TGF beta, indicates the existence of a possible autocrine loop whereby lymphocyte-derived TGF beta in early SSc not only signals biosynthetic activation of fibroblasts in a paracrine manner, but autoinduces endogenous TGF beta production by the target fibroblasts themselves. Such an autocrine loop involving

Topics: Animals; Collagen; Cytokines; Fibroblasts; Gene Expression Regulation; Growth Substances; Humans; Interferons; Interleukin-1; Retroviridae; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Viral Proteins

In summary, adhesion molecules are likely to play a prominent role in scleroderma pathogenesis and evolution. Endothelial adhesion molecules required for leukocyte extravasation are upregulated in affected tissue, though the mechanism is unclear. Certainly, endothelial adhesion molecule expression is seen in the context of other diseases not characterized by fibrosis. Adhesion molecules on the fibroblast, particularly those that play a role in fibroblast collagen interactions, may be very important. The ability of fibroblasts to organize collagen fibrils, and to exert forces across collagenous tissue, is likely to involve a prominent role of alpha 2 beta 1 integrin. Enhanced organization and contraction of newly formed collagen, as well as unregulated procollagen production, may be intimately linked in this disease process. At least two factors that strongly enhance fibroblast force generation could potentially influence other aspects of scleroderma. TGF beta is a potent stimulus for collagen production and has been found to be elevated in lesional scleroderma. Endothelin 1 is also a potent vasoconstrictor and is elevated in scleroderma patient serum as well [60,62-65]. Its apparent role in other fibrocontractive diseases suggests that its potential role in the pathogenesis of scleroderma deserves additional attention.

Topics: Cell Adhesion Molecules; Collagen; Fibroblasts; Humans; Integrin beta1; Integrins; Leukocytes; Scleroderma, Systemic; Transforming Growth Factor beta

Excessive connective tissue deposition in the skin and other organs is the pathologic hallmark of systemic sclerosis (SSc), and fibrosis accounts for much of the chronic morbidity of this disease. Unregulated production of collagen in SSc skin fibroblasts has been demonstrated both in vivo and in vitro, and is considered to be a critical process in the development of fibrosis. In addition to collagen, other components of the extracellular matrix are also overexpressed in SSc fibroblasts, and may be important in the functional alterations of connective tissue. The transcriptional activity of several genes coding for matrix macromolecules is upregulated in SSc fibroblasts. The pleiotropic signaling molecule transforming growth factor-beta (TGF beta) is likely to be intimately involved in initiating and perhaps perpetuating the fibrotic response in SSc. TGF beta, a potent profibrotic cytokine, is highly expressed in endothelial cells, in fibroblasts near blood vessels, and in perivascular inflammatory cells in involved tissues in SSc. The apparent failure of SSc fibroblasts to down-regulate their collagen synthesis when cultured in a three-dimensional matrix suggest an additional mechanism for the maintenance of unregulated collagen production in these cells. Finally, subpopulations of fibroblasts with an activated biosynthetic phenotype may become dominant in SSc. Therefore, persistent elevation of extracellular matrix gene expression in SSc tissues may be the result of a series of events representing the interaction of genetic and hormonal factors. A precise delineation of the mechanisms of fibroblast activation is needed for understanding the pathogenesis of SSc, and for the rational design of therapeutic interventions aimed at interfering with the fibrotic process.

Topics: Collagen; Connective Tissue; Extracellular Matrix Proteins; Fibroblasts; Humans; Scleroderma, Systemic; Sclerosis; Transforming Growth Factor beta

Fibrosis is the pathologic hallmark of many common diseases. Much information has recently emerged about the cellular and biochemical events that result in its development and progression. It is now known that in affected tissues, chronic inflammation generally precedes fibrosis and that inflammatory cell-derived cytokines are crucial mediators of fibrogenesis. Several cytokines have been identified that influence wound healing and tissue repair processes in vivo and that modulate the production of collagen in vitro. Of these, transforming growth factor-beta is of the most interest because this pleiotropic cytokine is expressed at high levels during tissue remodeling and greatly affects the formation of connective tissue. Furthermore, it has been recently shown that transforming growth factor-beta can stimulate the transcription of collagen genes through the production or activation of specific DNA-binding trans-acting factors. A precise understanding of the molecular mechanisms responsible for the effects of this cytokine on collagen gene expression may allow the design of selective therapeutic interventions aimed at retarding or preventing the development of fibrosis.

Topics: Collagen; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

Systemic sclerosis (scleroderma, SSc) is a progressive and often fatal disorder characterized clinically by sclerotic changes in the skin, joints and internal organ systems such as the gastrointestinal tract, heart, lungs and kidneys, moreover pathologically by abnormalities of mucopolysaccharides, fibrous tissue deposition, atrophy of parenchymal structures in skin and various internal organs, and by vascular insufficiency. Little is known of its etiology and pathogenesis. Transforming growth factor-beta (TGF-beta), platelet derived growth factor-AA (PDGF-AA) and PDGF-alpha receptor interaction may play an important role in the pathogenesis of scleroderma. Furthermore, many of the proteoglycans act as modulators of growth factor activities. Dermatomyositis is also a complex connective tissue disease of unknown etiology, in which inflammatory change in the skin, muscle, and lung in association with vascular insufficiency and internal malignancy.

Topics: Blood Vessels; Dermatomyositis; Humans; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic scleroderma is an acquired disorder which typically results in fibrosis of the skin and internal organs. The pathogenesis of systemic scleroderma is characterized by three distinct processes: microvascular alterations including capillary endothelial cell injury, perivascular inflammatory reaction in dermis, and excessive accumulation of collagen in the dermal layer of lesional skin. In this review, molecular mechanisms resulting in activation of collagen synthesis by dermal fibroblasts in scleroderma are discussed. Specifically, the role of inflammatory cells and the cytokines/growth factors produced by these cells in the pathogenesis of scleroderma is emphasized. The possibilities for prevention and resolution of tissue fibrosis on the basis of these observations are also discussed. Understanding the pathogenetic mechanisms of scleroderma at a molecular level is likely to provide possibilities for development of more specific therapeutic modalities for this and other fibrotic disorders.

Topics: Cell Adhesion Molecules; Chemotaxis, Leukocyte; Collagen; Connective Tissue; Cyclosporine; Cytokines; Female; Fibroblasts; Fibrosis; Humans; Integrins; Interferon-gamma; Male; Penicillamine; Photopheresis; Platelet-Derived Growth Factor; Procollagen-Proline Dioxygenase; Scleroderma, Systemic; Sex Ratio; Transcription, Genetic; Transforming Growth Factor beta

Scleroderma fibrotic lesions demonstrate vascular disease, mononuclear cell infiltrates, and increased collagen. Fibroblasts in these lesions are activated to synthesize increased extracellular matrix substances, a phenotype that continues when these cells are removed and grown in tissue culture. Levels of messenger RNA for connective-tissue substances, measured directly in biopsies of scleroderma skin, show increased message for type I collagen, but not type III collagen or fibronectin. Increased procollagen type I in scleroderma skin occurs in the papillary dermis, perivascular areas, and deep interstitium, even in skin areas that are not yet fibrotic. Scleroderma fibroblasts express more intercellular adhesion molecule 1 on their surfaces than do normal cells, and this molecule is increased in endothelial cells, mononuclear cells, and fibroblasts. In vitro scleroderma fibroblasts adhere more frequently to extracellular matrix substances and retract collagen lattices to a greater extent. Peripheral blood lymphocytes from scleroderma patients produce excessive amounts of interleukin-2 when incubated with type I collagen, and circulating basophils release more histamine than do normal cells. There is evidence for activated eosinophils both in the dermis and pulmonary lesions in scleroderma, which may play a role in fibrosis. Transforming growth factor-beta is overexpressed by alveolar macrophages from patients with fibrotic pulmonary disease. Scleroderma fibroblasts, when exposed to transforming growth factor-beta, overexpress the alpha-type receptor for platelet-derived growth factor. Scleroderma sera more frequently contain measurable quantities of interleukin-4, interleukin-6, and interleukin-2. Interleukin-4 causes adult dermal fibroblasts to proliferate and to make interleukin-6. Interleukin-6 has been shown to stimulate fibroblast synthesis of collagen and glycosaminoglycans.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Cell Adhesion Molecules; Connective Tissue; Cytokines; Endothelins; Fibrosis; Humans; Interleukins; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

The cause of systemic sclerosis remains unknown, but cellular and molecular mechanisms possibly responsible for the characteristic clinical manifestations of fibrosis and vascular damage (Raynaud's phenomenon, telangiectasis, digital infection, and renal arteriopathy) are becoming understood in greater detail. One possibly important cytokine is transforming growth factor-beta (TGF-beta); its involvement is reviewed here. With regard to vascular lesions, TGF-beta has variably been shown to inhibit endothelial cell growth in vitro but to promote angiogenesis in vivo, a paradox that remains unresolved. Nonetheless, an injurious activity of TGF-beta on microvascular endothelial cells could help to explain the intimal proliferation and microvascular obliteration seen. Whether as a result of or as a cause of endothelial cell damage, platelet activation has been well documented in systemic sclerosis and the platelet alpha granule pool contains a large quantity of TGF-beta. TGF-beta is also produced by activated macrophages and T cells, both of which are known to occur within systemic sclerosis lesions. An important effect of TGF-beta is its stimulation of fibroblast collagen and fibronectin synthesis and their deposition into the extracellular matrix. Stimulation by TGF-beta may therefore account for the fibrosis seen in the dermis and in the internal organs. Direct evidence of TGF-beta involvement in systemic sclerosis is scanty, and awaits discovery of either an abnormal expression of or response to TGF-beta. The biologic effects of TGF-beta appear to be regulated at the level of activation from a latent polypeptide precursor form. Descriptions of the importance of this cytokine in pathologic conditions will need to account for this activation and its regulation. Nonetheless, the physiologic effects so far attributed to TGF-beta make its involvement in systemic sclerosis an attractive possibility to explain some of the manifestations of this enigmatic disease.

Topics: Chemotactic Factors; Fibroblasts; Humans; Monocytes; Procollagen; RNA, Messenger; Scleroderma, Systemic; Skin; T-Lymphocytes; Transforming Growth Factor beta

Topics: Animals; Atrophy; Bone Marrow Transplantation; Disease Models, Animal; Edema; Graft vs Host Disease; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred Strains; Radiation Chimera; Scleroderma, Systemic; Sclerosis; Spleen; T-Lymphocytes; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Fibroblasts are key effector cells in tissue remodeling. They remain persistently activated in fibrotic diseases, resulting in progressive deposition of extracellular matrix. Although fibroblast activation may be initiated by external factors, prolonged activation can induce an "autonomous," self-maintaining profibrotic phenotype in fibroblasts. Accumulating evidence suggests that epigenetic alterations play a central role in establishing this persistently activated pathologic phenotype of fibroblasts. We demonstrated that in fibrotic skin of patients with systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease, TGF-β induced the expression of DNA methyltransferase 3A (DNMT3A) and DNMT1 in fibroblasts in a SMAD-dependent manner to silence the expression of suppressor of cytokine signaling 3 (SOCS3) by promoter hypermethylation. Downregulation of SOCS3 facilitated activation of STAT3 to promote fibroblast-to-myofibroblast transition, collagen release, and fibrosis in vitro and in vivo. Reestablishment of the epigenetic control of STAT3 signaling by genetic or pharmacological inactivation of DNMT3A reversed the activated phenotype of SSc fibroblasts in tissue culture, inhibited TGF-β-dependent fibroblast activation, and ameliorated experimental fibrosis in murine models. These findings identify a pathway of epigenetic imprinting of fibroblasts in fibrotic disease with translational implications for the development of targeted therapies in fibrotic diseases.

Topics: Animals; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methyltransferase 3A; Epigenesis, Genetic; Female; Fibrosis; Gene Expression Regulation, Enzymologic; Humans; Male; Mice; Myofibroblasts; Scleroderma, Systemic; Signal Transduction; STAT3 Transcription Factor; Suppressor of Cytokine Signaling 3 Protein; Transforming Growth Factor beta

The aim of the study was to determine whether plasma levels of total glycosaminoglycans (GAGs), matrix metalloproteinases (MMPs) (MMP-3, MMP-10), and their tissue inhibitors (TIMPs) (TIMP-1, TIMP-2) as well as transforming growth factor

Topics: Adult; Female; Glycosaminoglycans; Humans; Male; Matrix Metalloproteinase 10; Matrix Metalloproteinase 3; Middle Aged; Scleroderma, Systemic; Tissue Inhibitor of Metalloproteinase-1; Tissue Inhibitor of Metalloproteinase-2; Transforming Growth Factor beta

Systemic sclerosis (SSc) is characterized by autoimmunity, vasculopathy and fibrosis. Fibrosis is due to an activation of fibroblasts by the transforming growth factor-ß (TGF-ß). This study investigates the proteomic response of SSc fibroblasts to TGF-ß.. Skin fibroblasts from diffuse SSc patients and healthy controls (HC) are cultured with or without TGF-ß. Two-dimensional differential in-gel electrophoresis and mass spectrometry (MS) combined with Ingenuity Pathway analysis (IPA) and Panther/David software analyze proteins differentially expressed between groups. Real-time cell analyzer (RTCA) assesses fibroblast proliferation and viability.. Two-hundred-and-seventy-nine proteins are differentially expressed between groups. Principal component analysis shows significant differences between groups. IPA shows specific process networks such as actin cytoskeleton and integrin signaling. Panther and David software show predominant biological processes such as cellular and metabolic processes. TGF-ß enhances protein synthesis and protein pathways. IPA and RTCA suggest the involvement of epidermal growth factor receptor (EGFR) and phosphatidylinositol 3 kinase (Pi3K).. That the proteome of fibroblasts differs between SSc patients and HC is confirmed, and it is demonstrated that fibroblasts exacerbate their proteomic phenotype upon stimulation with TGF-ß. EGFR and Pi3K are highlighted as proteins of interest in SSc fibroblasts.

Topics: Adult; ErbB Receptors; Female; Fibroblasts; Humans; Male; Mass Spectrometry; Middle Aged; Phosphatidylinositol 3-Kinases; Proteomics; Scleroderma, Systemic; Transforming Growth Factor beta; Two-Dimensional Difference Gel Electrophoresis

Skin fibrosis mediated by activated dermal fibroblasts is a hallmark of systemic sclerosis (SSc), especially in the subset of patients with diffuse disease. Transforming growth factor-beta (TGFβ) and interleukin-6 (IL-6) are key candidate mediators in SSc. Our aim was to elucidate the specific effect of IL-6 pathway blockade on the biology of SSc fibroblasts in vivo by using samples from a unique clinical experiment-the faSScinate study-in which patients with SSc were treated for 24 weeks with tocilizumab (TCZ), an IL-6 receptor-α inhibitor.. We analysed the molecular, functional and genomic characteristics of explant fibroblasts cultured from matched skin biopsy samples collected at baseline and at week 24 from 12 patients receiving placebo (n=6) or TCZ (n=6) and compared these with matched healthy control fibroblast strains.. The hallmark functional and molecular-activated phenotype was defined in SSc samples and was stable over 24 weeks in placebo-treated cases. RNA sequencing analysis robustly defined key dysregulated pathways likely to drive SSc fibroblast activation in vivo. Treatment with TCZ for 24 weeks profoundly altered the biological characteristics of explant dermal fibroblasts by normalising functional properties and reversing gene expression profiles dominated by TGFβ-regulated genes and molecular pathways.. We demonstrated the exceptional value of using explant dermal fibroblast cultures from a well-designed trial in SSc to provide a molecular framework linking IL-6 to key profibrotic pathways. The profound impact of IL-6R blockade on the activated fibroblast phenotype highlights the potential of IL-6 as a therapeutic target in SSc and other fibrotic diseases.. NCT01532869; Post-results.

Topics: Adult; Antibodies, Monoclonal, Humanized; Biopsy; Cells, Cultured; Double-Blind Method; Female; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Immunosuppressive Agents; Interleukin-6; Male; Middle Aged; Molecular Targeted Therapy; Receptors, Interleukin-6; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Rituximab (RTX) may favorably affect skin and lung fibrosis in patients with systemic sclerosis (SSc); however, the underlying molecular mechanisms remain unknown. We aimed to explore the hypothesis that RTX may mediate its antifibrotic effects by regulating the expression of Dickkopf-1 (Dkk-1), an inhibitor of the Wnt pathway.. Fourteen patients with SSc and five healthy subjects were recruited. Dkk-1 expression was immunohistochemically assessed in skin biopsies obtained from 11 patients with SSc (8 treated with RTX and 3 with standard treatment), whereas DKK1 gene expression was assessed in 3 patients prior to and following RTX administration.. In baseline biopsies obtained from all patients with SSc but not in healthy subjects, Dkk-1 was undetectable in skin fibroblasts. Following RTX treatment, four out of eight patients had obvious upregulation of Dkk-1 skin expression. Similarly, RTX treatment correlated with a significant 4.8-fold upregulation of DKK1 gene expression (p = 0.030). In contrast, TGFβ expression in the upper dermis was significantly attenuated following treatment. Moreover, this decreased expression of TGFβ in the skin was significantly more pronounced in the subgroup of patients with Dkk-1 upregulation. In this subgroup TGFβ was downregulated by 50.88 % in contrast to only 15.98 % in patients who did not have Dkk-1 upregulation (p = 0.022).. This is the first study demonstrating a link between B cell depletion and skin Dkk-1 upregulation in patients with SSc. RTX-mediated B cell depletion may mechanistically function via the recently established TGFβ-Dkk-1 axis in improving skin fibrosis.

Topics: Adult; Female; Fibroblasts; Fibrosis; Humans; Immunoassay; Immunohistochemistry; Immunologic Factors; Intercellular Signaling Peptides and Proteins; Lymphocyte Depletion; Male; Middle Aged; Rituximab; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Up-Regulation

TGF-β has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-β-regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-β may benefit these patients.. Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-β isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-β-regulated biomarker genes thrombospondin-1 (THBS1) and cartilage oligomeric protein (COMP) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS).. In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks and P = 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P < 0.001 at all time points). Expression levels of other TGF-β-regulated genes, including SERPINE1 and CTGF, declined (P = 0.049 and P = 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P < 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment.. The rapid inhibition of TGF-β-regulated gene expression in response to fresolimumab strongly implicates TGF-β in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis.. Clinicaltrials.gov NCT01284322.

Topics: Adult; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Cartilage Oligomeric Matrix Protein; Female; Gene Expression; Genetic Markers; Humans; Male; Middle Aged; Myofibroblasts; Scleroderma, Systemic; Thrombospondin 1; Transforming Growth Factor beta; Treatment Outcome

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

Systemic sclerosis (SSc) is characterized by chronic inflammation and fibrosis, two processes associated with transforming growth factor β (TGF-β) functions. In the present study, we investigated the expression of TGF-β isoforms in serum and the skin distribution of TGF-β and two receptors (TGF-βR1 and TGF-βR2) and their relationship with some clinical, inflammatory, autoimmune (autoantibodies), and vascular (platelets) biomarkers in SSc patients. A total of 56 SSc patients and 120 control subjects (CS) were included. The serum levels of TGF-β isoforms were quantified by immunoassay with magnetic microspheres, and the skin biopsies were processed by immunohistochemistry. The soluble levels of the three active TGF-β isoforms were lower in SSc patients than in CS (p < 0.0001). However, sTGF-β1 and sTGF-β3 levels were positively correlated with C-reactive protein levels in SSc patients. Additionally, sTGF-β2 and sTGF-β3 levels were positively correlated with the number of platelets in SSc patients. In skin biopsies, TGF-β1, TGF-βR1, and TGF-βR2 expression levels were higher in SSc patients than CS. In conclusion, this is the first study showing a joint decrease of the 3 active TGF-β isoforms in SSc patients. However, TGF-β1, TGF-βR1, and TGF-βR2 are possibly increased in clinically involved skin. Therefore, it is likely that a distinct role is played by TGF-β at the local (skin lesions) and systemic levels in SSc patients.

Topics: Biomarkers; Humans; Protein Isoforms; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Lung fibrosis is the leading cause of death in SSc, with no cure currently available. Antifibrotic Endostatin (ES) production does not reach therapeutic levels in SSc patients, suggesting a deficit in its release from Collagen XVIII by the main cleavage enzyme, Cathepsin L (CTSL). Thus, elucidating a potential deficit in CTSL expression and activity unravels an underlying molecular cause for SSc-driven lung fibrosis.. Fibrosis was induced experimentally using TGF-β in vitro, in primary human lung fibroblasts (pLFs), and ex vivo, in human lung tissues. ES and CTSL expression was quantified using ELISA, RT-qPCR, immunoblotting or immunofluorescence. Recombinant NC1-FLAG peptide was used to assess CTSL cleavage activity. CTSL expression was also compared between SSc vs normal (NL)-derived pLFs and lung tissues.. ES levels were significantly reduced in media conditioned by TGF-β-induced pLFs. TGF-β-stimulated pLFs significantly reduced expression and secretion of CTSL into the extracellular matrix (ECM). CTSL was also sequestered in its inactive form into extracellular vesicles, further reducing its availability in the ECM. Media conditioned by TGF-β-induced pLFs showed reduced cleavage of NC1-Flag and reduced release of the antifibrotic ES fragment. SSc-derived pLFs and lung tissues expressed significantly lower levels of CTSL compared with NL.. Our findings identify CTSL as a protein protective against lung fibrosis via its activation of antifibrotic ES, and whose expression in SSc pLFs and lung tissues is suppressed. Identifying strategies to boost CTSL endogenous levels in SSc patients could serve as a viable therapeutic strategy.

Topics: Cathepsin L; Cells, Cultured; Fibroblasts; Fibrosis; Humans; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a chronic, heterogeneous disease of connective tissue characterized by organ fibrosis together with vascular injury and autoimmunity. TGF-β plays a central role in generating fibrosis, including SSc. Periostin is a matricellular protein playing a key role in the generation of fibrosis by amplifying the TGF-β signals. SOX11 is a transcription factor playing several important roles in organ development in embryos. We have previously shown that SOX11 induces periostin expression. However, the roles of the interactions among the TGF-β signals, periostin, and SOX11 remain unknown in the pathogenesis of SSc. In this study, we found that most clones of dermal fibroblasts derived from patients with SSc showed constitutive, high expression of SOX11, which is significantly induced by TGF-β1. SOX11 forms a positive loop with periostin to activate the TGF-β signals in SSc dermal fibroblasts. Genetic deletion of Sox11 in Postn-expressing fibroblasts impairs dermal fibrosis by bleomycin. Moreover, using the DNA microarray method, we identified several fibrotic factors dependent on the TGF-β/SOX11/periostin pathway in SSc dermal fibroblasts. Our findings, taken together, show that a positive loop formed by SOX11 and periostin in fibroblasts upregulates the TGF-β signals, leading to skin fibrosis.

Topics: Fibroblasts; Fibrosis; Humans; Scleroderma, Systemic; Signal Transduction; Skin; SOXC Transcription Factors; Transforming Growth Factor beta

Transport and Golgi Organization protein 1 (TANGO1) is a protein that regulates the export of procollagen from the endoplasmic reticulum and has a role in the organization of exit sites for general protein export. What regulates the expression of TANGO1 and the role of TANGO1 in fibrosis is poorly understood and has never been studied in the setting of systemic sclerosis (SSc). We undertook this study to determine the role of TANGO1 in SSc fibrosis.. SSc (n = 15) and healthy (n = 12) primary fibroblast lung cell lines were investigated for the expression of TANGO1. Histologic analyses for TANGO1 were performed on lung biopsy samples (n = 12 SSc patient samples and n = 8 healthy control samples).. SSc fibroblasts showed increased expression of TANGO1 protein in cultured fibroblasts. TANGO1 colocalizes with α-smooth muscle actin (α-SMA)-positive cells in SSc lung tissue and is highly up-regulated in the neointima of SSc vessels. TANGO1 expression was dependent on the inflammasome activation of caspase 1. It was also dependent on signaling from the interleukin-1 (IL-1) and transforming growth factor β (TGFβ) receptors. The decrease in TANGO1 down-regulated export of larger cargos including collagen and laminin. Reduced TANGO1 protein had no effect on smaller molecular weight cargoes; however, the secretion of elastin was significantly reduced.. TANGO1 is markedly increased in SSc fibroblasts and was found to be elevated in lung tissue in association with α-SMA-positive cells. TANGO1 expression is driven by inflammasome-dependent caspase 1 activation and is mediated by IL-1 and TGFβ downstream signaling. These observations suggest that during fibrosis, caspase 1 promotes the up-regulation of TANGO1 and the organization of endoplasmic reticulum exits sites, ultimately contributing to procollagen export and fibrosis.

Topics: Caspase 1; Endoplasmic Reticulum; Fibroblasts; Fibrosis; Humans; Inflammasomes; Interleukin-1; Procollagen; Scleroderma, Systemic; Transforming Growth Factor beta

Skin fibrosis is a common pathological manifestation in systemic sclerosis (SSc), keloid, and localized scleroderma (LS) characterized by fibroblast activation and excessive extracellular matrix (ECM) deposition. However, few effective drugs are available to treat skin fibrosis due to its unclear mechanisms. In our study, we reanalyzed skin RNA-sequencing data of Caucasian, African, and Hispanic SSc patients from the Gene Expression Omnibus (GEO) database. We found that the focal adhesion pathway was up-regulated and Zyxin appeared to be the primary focal adhesion protein involved in skin fibrosis, and we further verified its expression in Chinese skin tissues of several fibrotic diseases, including SSc, keloid, and LS. Moreover, we found Zyxin inhibition could significantly alleviate skin fibrosis using Zyxin knock-down and knock-out mice, nude mouse model and skin explants of human keloid. Double immunofluorescence staining showed that Zyxin was highly expressed in fibroblasts. Further analysis revealed pro-fibrotic gene expression and collagen production increased in Zyxin over-expressed fibroblasts, and decreased in Zyxin interfered SSc fibroblasts. In addition, transcriptome and cell culture analyses revealed Zyxin inhibition could effectively attenuate skin fibrosis by regulating the FAK/PI3K/AKT and TGF-β signaling pathways via integrins. These results suggest Zyxin appears a potential new therapeutic target for skin fibrosis.

Topics: Animals; Fibroblasts; Fibrosis; Humans; Integrins; Keloid; Mice; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Zyxin

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of the skin and internal organs. Key players mediating fibrosis are myofibroblasts (MF) that, following transforming growth factor β (TGFβ) exposure, produce a collagen-rich extracellular matrix (ECM) that induces myofibroblast differentiation. Myofibroblasts express αvβ3 integrin (a membrane receptor for thyroid hormones) and miRNA-21 that promotes deiodinase-type-3 expression (D3), causing the degradation of triiodothyronine (T3) that attenuates fibrosis. We hypothesized that αvβ3 affects the fibrotic processes through its thyroid hormones (THs) binding site. To test this, dermal fibroblasts (DF) were cultured with/without TGFβ and removed with a base, leaving only normal/fibrotic ECMs in wells. Then, DF were cultured on the ECMs with/without tetrac (αvβ3 ligand, T4 antagonist), and evaluated for pro-fibrotic characteristics, αvβ3, miRNA-21, and D3 levels. Blood free-T3 (fT3), miRNA-21 levels, and the modified Rodnan skin score (MRSS) were evaluated in SSc patients. We found that the "fibrotic-ECM" significantly increased the pro-fibrotic characteristics of DF and the levels of miRNA-21, D3, and αvβ3, compared to the "normal-ECM." Tetrac significantly inhibited the effects of the "fibrotic-ECM" on the cells. In accordance with tetrac's effect on D3/miRNA-21, a negative correlation was found between the patients' fT3 to miRNA-21 levels, and to the development of pulmonary arterial hypertension (PAH). We conclude that occupying the THs binding site of αvβ3 may delay the development of fibrosis.

Topics: Fibroblasts; Fibrosis; Humans; Integrin alphaVbeta3; MicroRNAs; Scleroderma, Systemic; Skin; Thyroid Hormones; Transforming Growth Factor beta

Systemic sclerosis (SSc) has the highest mortality rate among the rheumatic diseases, with lung fibrosis leading as the cause of death. A characteristic of severe SSc-related lung fibrosis is its progressive nature. Although most research has focused on the pathology of the fibrosis, the mechanism mediating the fibrotic spread remains unclear. We hypothesized that extracellular vesicle (EV) communication drives the propagation of SSc lung fibrosis.. EVs were isolated from normal (NL) or SSc-derived human lungs and primary lung fibroblasts (pLFs). EVs were also isolated from human fibrotic lungs and pLFs induced experimentally with transforming growth factor-β (TGFβ). Fibrotic potency of EVs was assessed using functional assays in vitro and in vivo. Transmission electron microscopy, nanoparticle tracking analysis, real-time quantitative polymerase chain reaction (RT-qPCR), immunoblotting, and immunofluorescence were used to analyze EVs, their cargo, extracellular matrix (ECM) fractions, and conditioned media.. SSc lungs and pLFs released significantly more EVs than NL lungs, and their EVs showed increased fibrotic content and activity. TGFβ-stimulated NL lung cores and pLFs increased packaging of fibrotic proteins, including fibronectin, collagens, and TGFβ, into released EVs. The EVs induced a fibrotic phenotype in recipient pLFs and in vivo in mouse lungs. Furthermore, EVs interacted with and contributed to the ECM. Finally, suppressing EV release in vivo reduced severity of murine lung fibrosis.. Our findings highlight EV communication as a novel mechanism for propagation of SSc lung fibrosis. Identifying therapies that reduce EV release, activity, and/or fibrotic cargo in SSc patient lungs may be a viable therapeutic strategy to improve fibrosis.

Topics: Animals; Extracellular Vesicles; Fibroblasts; Fibrosis; Humans; Lung; Mice; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Fibroblasts (Fb) are key effector cells in systemic sclerosis (SSc). Fb stimulation with transforming growth factor beta 1 (TGF-β1) is considered as a positive control in studies assessing fibrogenesis. The lack of standardization of TGF-β1 stimulation might be responsible for discrepancies in experiments performed in different conditions. Using quantitative proteomics analysis, we evaluated the impact of changes in experimental conditions on proteomic profiles of primary Fb. Principal component analysis (PCA) identified several groups of differentially expressed proteins influenced by cell passage, culture medium, and both concentration and duration of exposure to TGF-β1 stimulation. Bioinformatics analysis revealed that late passages expressed proteins involved in senescence. TGF-β1 concentration and time of stimulation were correlated with the expression of proteins involved in the fibrogenesis and inflammatory processes. These data underline the need for standardization of culture conditions to allow inter-data comparisons in future in vitro studies, especially when using "omics" approaches.

Topics: Cells, Cultured; Computational Biology; Fibroblasts; Humans; Proteomics; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Mitochondrial transcription factor A (TFAM) controls the transcription of core proteins required for mitochondrial homeostasis. This study was undertaken to investigate changes in TFAM expression in systemic sclerosis (SSc), to analyze mitochondrial function, and to evaluate the consequences for fibroblast activation.. TFAM expression was analyzed by immunofluorescence and Western blotting. The effects of TFAM knockout were investigated in cultured fibroblasts and in murine models of bleomycin-induced skin fibrosis, bleomycin-induced lung fibrosis, and skin fibrosis induced by overexpression of constitutively active transforming growth factor β type I receptor (TGFβRΙ).. TFAM expression was down-regulated in fibroblasts in SSc skin and in cultured SSc fibroblasts. The down-regulation of TFAM was associated with decreased mitochondrial number and accumulation of damaged mitochondria with release of mitochondrial DNA (mtDNA), accumulation of deletions in mtDNA, metabolic alterations with impaired oxidative phosphorylation, and release of the mitokine GDF15. Normal fibroblasts subjected to long-term, but not acute, exposure to TGFβ mimicked SSc fibroblasts, with down-regulation of TFAM and accumulation of mitochondrial damage. Down-regulation of TFAM promoted fibroblast activation with up-regulation of fibrosis-relevant Gene Ontology terms in RNA-Seq, partially in a reactive oxygen species-dependent manner. Mice with fibroblast-specific knockout of Tfam were prone to fibrotic tissue remodeling, with fibrotic responses even to NaCl instillation and enhanced sensitivity to bleomycin injection and overexpression of constitutively active TGFβRI. TFAM knockout fostered Smad3 signaling to promote fibroblast activation.. Alterations in the key mitochondrial transcription factor TFAM in response to prolonged activation of TGFβ and associated mitochondrial damage induce transcriptional programs that promote fibroblast-to-myofibroblast transition and drive tissue fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; DNA-Binding Proteins; DNA, Mitochondrial; Fibroblasts; Fibrosis; Mice; Mitochondrial Proteins; Scleroderma, Systemic; Skin; Skin Diseases; Transcription Factors; Transforming Growth Factor beta

The development of precision therapeutics for systemic sclerosis (SSc) has been hindered by the lack of models that accurately mimic the disease in vitro. This study was undertaken to design and test a self-assembled skin equivalent (saSE) system that recapitulates the cross-talk between macrophages and fibroblasts in cutaneous fibrosis.. SSc-derived dermal fibroblasts (SScDFs) and normal dermal fibroblasts (NDFs) were cultured with CD14+ monocytes from SSc patients or healthy controls to allow de novo stroma formation. Monocyte donor-matched plasma was introduced at week 3 prior to seeding keratinocytes to produce saSE with a stratified epithelium. Tissue was characterized by immunohistochemical staining, atomic force microscopy, enzyme-linked immunosorbent assay, and quantitative reverse transcriptase-polymerase chain reaction.. Stroma synthesized de novo from NDFs and SScDFs supported a fully stratified epithelium to form saSE. A thicker and stiffer dermis was generated by saSE with SScDFs, and more interleukin-6 and transforming growth factor β (TGFβ) was secreted by saSE with SScDFs compared to saSE with NDFs, regardless of the inclusion of monocytes. Tissue with SSc monocytes and plasma had amplified dermal thickness and stiffness relative to control tissue. Viable CD163+ macrophages were found within the stroma of saSE 5 weeks after seeding. Additionally, SSc saSE contained greater numbers of CD163+ and CD206+ macrophages compared to control saSE. TGFβ blockade inhibited stromal stiffness to a greater extent in SSc saSE compared to control saSE.. These data suggest reciprocal activation between macrophages and fibroblasts that increases tissue thickness and stiffness, which is dependent in part on TGFβ activation. The saSE system may serve as a platform for preclinical therapeutic testing and for molecular characterization of SSc skin pathology through recapitulation of the interactions between macrophages and fibroblasts.

Topics: Cells, Cultured; Fibroblasts; Fibrosis; Humans; Macrophage Activation; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a collagen disease that exhibits intractable fibrosis and vascular injury of the skin and internal organs. Transforming growth factor-β (TGF-β)/Smad signaling plays a central role in extracellular matrix (ECM) production by α-SMA-positive myofibroblasts. Myofibroblasts may be partially derived from various precursor cells in addition to resident fibroblasts. Recently, our high-throughput in vitro screening discovered a small compound, LG283, that may disrupt the differentiation of epithelial cells into myofibroblasts. This compound was originally generated as a curcumin derivative.. In this study, we investigated the effect of LG283 on inhibiting fibrosis and its mechanism. The action of LG283 on TGF-β-dependent fibrogenic activity and epithelial-mesenchymal transition (EMT) was analyzed in vitro. The effects of LG283 were also examined in a bleomycin-induced skin fibrosis mouse model.. LG283 suppressed TGF-β-induced expression of ECM, α-SMA, and transcription factors Snail 1 and 2, and Smad3 phosphorylation in cultured human dermal fibroblasts. LG283 was also found to block EMT induction in cultured human epithelial cells. During these processes, Smad3 phosphorylation and/or expression of Snail 1 and 2 were inhibited by LG283 treatment. In the bleomycin-induced skin fibrosis model, oral administration of LG283 efficiently protected against the development of fibrosis and decrease of capillary vessels without significantly affecting cell infiltration or cytokine concentrations in the skin. No apparent adverse effects of LG283 were found. LG283 treatment remarkably inhibited the enhanced expression of α-SMA and phosphorylated Smad3, as well as those of Snail 1 and 2, in the bleomycin-injected skin.. The LG283 compound exhibits antagonistic activity on fibrosis and vascular injury through inhibition of TGF-β/Smad/Snail mesenchymal transition pathways and thus, may be a candidate therapeutic for the treatment of SSc. Although the involvement of EMT in the pathogenesis of SSc remains unclear, the screening of EMT regulatory compounds may be an attractive approach for SSc therapy.

Topics: Animals; Bleomycin; Fibrosis; Humans; Mice; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Vascular System Injuries

Topics: Cholecalciferol; Cytokines; Fibroblast Growth Factor 2; Fibrosis; Humans; Interleukin-17; Leukocytes, Mononuclear; Scleroderma, Systemic; Transforming Growth Factor beta

Activation of melanocortin 1 receptor (MC1R) is known to exert broad anti-inflammatory and anti-fibrotic effects. The purpose of this study is to investigate the potential of dersimelagon, a novel oral MC1R agonist, as a therapeutic agent for systemic sclerosis (SSc).. The effects of dersimelagon phosphoric acid (MT-7117) on skin fibrosis and lung inflammation were evaluated in bleomycin (BLM)-induced SSc murine models that were optimized for prophylactic and therapeutic evaluation. Microarray-based gene expression analysis and serum protein profiling were performed in the BLM-induced SSc models. The effect of MT-7117 on transforming growth factor-β (TGF-β)-induced activation of human dermal fibroblasts was evaluated in vitro. Immunohistochemical analyses of MC1R expression in the skin of SSc patients were performed.. Prophylactic treatment with MT-7117 (≥ 0.3 mg/kg/day p.o.) significantly inhibited skin fibrosis and lung inflammation, and therapeutic treatment with MT-7117 (≥ 3 mg/kg/day p.o.) significantly suppressed the development of skin fibrosis in the BLM-induced SSc models. Gene array analysis demonstrated that MT-7117 exerts an anti-inflammatory effect via suppression of the activation of inflammatory cells and inflammation-related signals; additionally, vascular dysfunction was extracted as the pathology targeted by MT-7117. Serum protein profiling revealed that multiple SSc-related biomarkers including P-selectin, osteoprotegerin, cystatin C, growth and differentiation factor-15, and S100A9 were suppressed by MT-7117. MT-7117 inhibited the activation of human dermal fibroblasts by suppressing TGF-β-induced ACTA2 (encoding α-smooth muscle actin) mRNA elevation. MC1R was expressed by monocytes/macrophages, neutrophils, blood vessels (endothelial cells), fibroblasts, and epidermis (keratinocytes) in the skin of SSc patients, suggesting that these MC1R-positive cells could be targets for MT-7117.. MT-7117 demonstrates disease-modifying effects in preclinical models of SSc. Investigations of its mechanism of action and target expression analyses indicate that MT-7117 exerts its positive effect by affecting inflammation, vascular dysfunction, and fibrosis, which are all key pathologies of SSc. The results of the present study suggest that MT-7117 is a potential therapeutic agent for SSc. A phase 2 clinical trial investigating the efficacy and tolerability of MT-7117 in patients with early, progressive diffuse cutaneous SSc is currently in progress.

Topics: Animals; Bleomycin; Blood Proteins; Disease Models, Animal; Endothelial Cells; Fibroblasts; Fibrosis; Humans; Inflammation; Mice; Pneumonia; Receptor, Melanocortin, Type 1; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation, microangiopathy, and progressive fibrosis in the skin and internal organs. To evaluate the pathophysiologic mechanisms and efficacies of potential therapeutics for SSc, a preclinical model recapitulating the disease phenotypes is needed. Here, we introduce a novel animal model for SSc using immunodeficient mice injected with peripheral blood mononuclear cells (PBMCs) from SSc patients. Human PBMCs acquired from SSc patients and healthy controls were transferred into NOD.Cg-Prkdc

Topics: Animals; Bleomycin; Disease Models, Animal; Fibrosis; Humans; Inflammation; Interleukin-17; Leukocytes, Mononuclear; Lung; Mice; Mice, Inbred NOD; Mice, SCID; Scleroderma, Systemic; Skin; Th17 Cells; Transforming Growth Factor beta

In early systemic sclerosis (Scleroderma, SSc), the vasculature is impaired. Although the exact etiology of endothelial cell damage in SSc remains unclear, it is hypothesized that endothelial to mesenchymal transition (EndoMT) plays a key role. To perform physiologically relevant angiogenic studies, we set out to develop an angiogenesis-on-a-chip platform that is suitable for assessing disease parameters that are relevant to SSc and other vasculopathies. In the model, we substituted Fetal Bovine Serum (FBS) with Human Serum without impairing the stability of the culture. We showed that 3D microvessels and angiogenic factor-induced sprouts exposed to key pro-inflammatory and pro-fibrotic cytokines (TNFα and TGFβ) undergo structural alterations consisting of destructive vasculopathy (loss of small vessels). We also showed that these detrimental effects can be prevented by compound-mediated inhibition of TGFβ-ALK5 signaling or addition of a TNFα neutralizing antibody to the 3D cultures. This demonstrates that our in vitro model is suitable for compound testing and identification of new drugs that can protect from microvascular destabilization or regression in disease-mimicking conditions. To support this, we demonstrated that sera obtained from SSc patients can exert an anti-angiogenic effect on the 3D vessel model, opening the doors to screening for potential SSc drugs, enabling direct patient translatability and personalization of drug treatment.

Topics: Angiogenesis Inducing Agents; Antibodies, Neutralizing; Humans; Lab-On-A-Chip Devices; Microvessels; Neovascularization, Pathologic; Scleroderma, Systemic; Serum Albumin, Bovine; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In addition to autoimmune and inflammatory diseases, variants of the TNFAIP3 gene encoding the ubiquitin-editing enzyme A20 are also associated with fibrosis in systemic sclerosis (SSc). However, it remains unclear how genetic factors contribute to SSc pathogenesis, and which cell types drive the disease due to SSc-specific genetic alterations. We therefore characterize the expression, function, and role of A20, and its negative transcriptional regulator DREAM, in patients with SSc and disease models. Levels of A20 are significantly reduced in SSc skin and lungs, while DREAM is elevated. In isolated fibroblasts, A20 mitigates ex vivo profibrotic responses. Mice haploinsufficient for A20, or harboring fibroblasts-specific A20 deletion, recapitulate major pathological features of SSc, whereas DREAM-null mice with elevated A20 expression are protected. In DREAM-null fibroblasts, TGF-β induces the expression of A20, compared to wild-type fibroblasts. An anti-fibrotic small molecule targeting cellular adiponectin receptors stimulates A20 expression in vitro in wild-type but not A20-deficient fibroblasts and in bleomycin-treated mice. Thus, A20 has a novel cell-intrinsic function in restraining fibroblast activation, and together with DREAM, constitutes a critical regulatory network governing the fibrotic process in SSc. A20 and DREAM represent novel druggable targets for fibrosis therapy.

Topics: Animals; Bleomycin; Cells, Cultured; Disease Models, Animal; Fibroblasts; Fibrosis; Mice; Mice, Knockout; Receptors, Adiponectin; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Ubiquitins

Systemic sclerosis (SSc) is a connective tissue disorder characterized by the development of fibrosis in the skin and internal organs. Increasing evidence suggests that mesenchymal stem cells (MSCs) can be used to a treatment for fibrotic diseases. Recent studies have demonstrated that some of the biological effects of MSCs are due to the secretion of exosomes. However, the precise mechanisms underlying MSCs-derived exosomes in skin fibrosis are not well understood.. We aimed to elucidate the effect of MSCs-derived exosomes on skin fibrosis in SSc and the mechanism underlying their inhibitory action on fibrosis.. Exosome was collected from MSCs by ultracentrifugation method. We examined the suppressive effect of MSCs-derived exosome on skin fibrosis in bleomycin-induced SSc mouse model. Skin samples from the injected site were collected for further examination, and micro-RNA analysis of MSCs-derived exosome was performed.. Injection of MSCs-derived exosomes significantly inhibited bleomycin-induced dermal fibrosis in mice. MSCs-derived exosomes significantly reduced the amount of collagen and the number of α-SMA. This study demonstrated that inhibition of collagen type I expression by miR-196b-5p in exosomes might be one of the mechanisms by which MSCs suppress skin fibrosis in an SSc mouse model.

Topics: Animals; Bleomycin; Cells, Cultured; Collagen Type I; Disease Models, Animal; Exosomes; Female; Fibroblasts; Fibrosis; Humans; Mesenchymal Stem Cells; Mice; MicroRNAs; Receptor, Transforming Growth Factor-beta Type I; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

The TβRII∆k-fib transgenic (TG) mouse model of scleroderma replicates key fibrotic and vasculopathic complications of systemic sclerosis through fibroblast-directed upregulation of TGFβ signalling. We have examined peroxisome proliferator-activated receptor (PPAR) pathway perturbation in this model and explored the impact of the pan-PPAR agonist lanifibranor on the cardiorespiratory phenotype.. PPAR pathway gene and protein expression differences from TG and WT sex-matched littermate mice were determined at baseline and following administration of one of two doses of lanifibranor (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. The prevention of bleomycin-induced lung fibrosis and SU5416-induced pulmonary hypertension by lanifibranor was explored.. Gene expression data were consistent with the downregulation of the PPAR pathway in the TβRII∆k-fib mouse model. TG mice treated with high-dose lanifibranor demonstrated significant protection from lung fibrosis after bleomycin and from right ventricular hypertrophy following induction of pulmonary hypertension by SU5416, despite no significant change in right ventricular systolic pressure.. In the TβRII∆k-fib mouse strain, treatment with 100 mg/kg lanifibranor reduces the development of lung fibrosis and right ventricular hypertrophy induced by bleomycin or SU5416, respectively. Reduced PPAR activity may contribute to the exaggerated fibroproliferative response to tissue injury in this transgenic model of scleroderma and its pulmonary complications.

Topics: Animals; Benzothiazoles; Mice; Mice, Transgenic; PPAR gamma; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Sulfonamides; Transforming Growth Factor beta

Systemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood.. Immunohistochemistry analysis showed accumulation of CD14

Topics: Adult; Aged; Biomarkers; Case-Control Studies; Cell Differentiation; Cytokines; Disease Susceptibility; Female; Fibroblasts; Fibronectins; Humans; Lipopolysaccharide Receptors; Macrophages; Male; Middle Aged; Monocytes; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Myofibroblasts are the key effector cells responsible for the exaggerated tissue fibrosis in Systemic Sclerosis (SSc). Despite their importance to SSc pathogenesis, the specific transcriptome of SSc myofibroblasts has not been described. The purpose of this study was to identify transcriptome differences between SSc myofibroblasts and non-myofibroblastic cells. Alpha smooth muscle actin (α-SMA) expressing myofibroblasts and α-SMA negative cells were isolated employing laser capture microdissection from dermal cell cultures from four patients with diffuse SSc of recent onset. Total mRNA was extracted from both cell populations, amplified and analyzed employing microarrays. Results for specific genes were validated by Western blots and by immunohistochemistry. Transcriptome analysis revealed 97 differentially expressed transcripts in SSc myofibroblasts compared with non-myofibroblasts. Annotation clustering of the SSc myofibroblast-specific transcripts failed to show a TGF-β signature. The most represented transcripts corresponded to several different genes from the Neuroblastoma Breakpoint Family (NBPF) of genes. NBPF genes are highly expanded in humans but are not present in murine or rat genomes. In vitro studies employing cultured SSc dermal fibroblasts and immunohistochemistry of affected SSc skin confirmed increased NBPF expression in SSc. These results indicate that SSc myofibroblasts represent a unique cell lineage expressing a specific transcriptome that includes very high levels of transcripts corresponding to numerous NBPF genes. Elevated expression of NBPF genes in SSc myofibroblasts suggests that NBPF gene products may play a role in SSc pathogenesis and may represent a novel therapeutic target.

Topics: Adult; Blotting, Western; Case-Control Studies; Female; Gene Expression Profiling; Genes, Neoplasm; Humans; Laser Capture Microdissection; Male; Myofibroblasts; Neoplasm Proteins; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

To examine the potential therapeutic effect of mesenchymal stem cells (MSCs) for experimental scleroderma.. Fifty-four mice six-week-old (30-35 g) were studied. Hypochlorous acid (HOCl) induced scleroderma was considered. Mice were divided into 3 groups: (I) Control: Six mice did not receive any treatment and were sacrificed at the end of the experiment; (II) HOCl mice (induced scleroderma as a positive control): (III) MSCs-treated HOCl mice: Thirty six HOCl-induced mice were injected with MSCs (7.5 × 105) intravenous every week for 3 weeks. Skin pieces were taken from the backs of mice and lung tissue pieces. a smooth muscle actin (α-SMA) and transforming growth factor-β (TGF-β1) were analysed or fixed in 10 % formalin for skin and lung tissue histopathological analysis. Plasma nitric oxide (NO) was also assayed.. There was a significant rise in the NO level and of the cutaneous and lung tissue α-SMA and TGF-β1 in untreated scleroderma-induced mice. The values significantly normalized after MSC therapy over the 7 weeks duration of the study. The altered histopathology of the skin and lung tissues in the scleroderma-induced mice showed a remarkable tendency to normalization of the skin and lung parenchyma and vasculature.. There was a significant rise in the level of NO and skin and lung tissue α-SMA and TGF-β1 in untreated scleroderma-induced mice and values were significantly normalized after MSC therapy over the 7 weeks duration of the study. Altered histopathology of the skin and lung appeared nearly normal after MSC therapy.

Topics: Animals; Disease Models, Animal; Humans; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Nitric Oxide; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Skin fibrosis is one of the major features of scleroderma. WNT/β-catenin signaling is associated with the progression of skin fibrosis. In this study, we aimed to determine the effect of icaritin (IT), a natural compound, on scleroderma-related skin fibrosis and its mechanisms. We found that IT could reduce the expression of COL1A1, COL1A2, COL3A1, CTGF, and α-SMA in human foreskin fibroblasts (HFF-1 cells), scleroderma skin fibroblasts (SSF cells), and TGF-β-induced HFF-1 cells. Wnt/β-catenin signaling was shown to be suppressed by IT. Additionally, IT activated AMPK signaling in HFF-1 cells. In conclusion, IT has an anti-skin fibrotic effect through activation of AMPK signaling and inhibition of WNT/β-catenin signaling. Our findings indicate the potential role of IT in the treatment of scleroderma and provide novel insight for the selection of drug therapy for scleroderma.

Topics: Actins; AMP-Activated Protein Kinases; beta Catenin; Cell Line; Cell Survival; Collagen Type I; Collagen Type I, alpha 1 Chain; Fibroblasts; Flavonoids; Humans; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Wnt Proteins

Systemc sclerosis (SSc) is an autoimmune disorder characterized by fibrosis of the skin and internal organs. Recently, it has been shown that leucine-rich α-2 glycoprotein (LRG) functions as a modulator of transforming growth factor-β (TGF-β) signaling in fibrosis. We aimed to characterize the effect of LRG in SSc model and SSc patients.. Histological analysis was performed on LRG knockout (KO) and wild type (WT) mouse in the skin and the lung after bleomycin administration. Serum LRG levels were measured during the injection period. Gene expression analysis of the skin and lung tissue from LRG KO and WT mice was performed. In addition, serum LRG levels were determined in SSc patients and healthy controls.. LRG KO mice display an inhibition of fibrosis in the skin in association with a decrease of dermal thickness, collagen deposition, and phospho-Smad3 expression after bleomycin. Serum LRG concentration significantly increased in WT mice after bleomycin. There was also a suppression of inflammation and fibrosis in the LRG KO mouse lung indicated by a reduction of lung weight, collagen content, and phospho-Smad3 expression after bleomycin. Gene expressions of TGF-β and Smad2/3 were significantly reduced in LRG KO mice. Serum LRG levels in SSc patients were significantly higher than those in controls.. LRG promotes fibrotic processes in SSc model through TGF-β-Smad3 signaling, and LRG can be a biomarker for SSc in humans and also a potential therapeutic target for SSc.

Topics: Animals; Bleomycin; Disease Models, Animal; Fibroblasts; Fibrosis; Glycoproteins; Humans; Mice; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Both TGFβ and estradiol (E2), a form of estrogen, are pro-fibrotic in the skin. In the connective tissue disease, systemic sclerosis (SSc), both TGFβ and E2 are likely pathogenic. Yet the regulation of TGFβ in E2-induced dermal fibrosis remains ill-defined. Elucidating those regulatory mechanisms will improve the understanding of fibrotic disease pathogenesis and set the stage for developing potential therapeutics. Using E2-stimulated primary human dermal fibroblasts in vitro and human skin tissue ex vivo, we identified the important regulatory proteins for TGFβ and investigated the extracellular matrix (ECM) components that are directly stimulated by E2-induced TGFβ signaling.. We used primary human dermal fibroblasts in vitro and human skin tissue ex vivo stimulated with E2 or vehicle (ethanol) to measure TGFβ1 and TGFβ2 levels using quantitative PCR (qPCR). To identify the necessary cell signaling proteins in E2-induced TGFβ1 and TGFβ2 transcription, human dermal fibroblasts were pre-treated with an inhibitor of the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway, U0126. Finally, human skin tissue ex vivo was pre-treated with SB-431542, a TGFβ receptor inhibitor, and ICI 182,780, an estrogen receptor α (ERα) inhibitor, to establish the effects of TGFβ and ERα signaling on E2-induced collagen 22A1 (Col22A1) transcription.. We found that expression of TGFβ1, TGFβ2, and Col22A1, a TGFβ-responsive gene, is induced in response to E2 stimulation. Mechanistically, Col22A1 induction was blocked by SB-431542 and ICI 182,780 despite E2 stimulation. Additionally, inhibiting E2-induced ERK/MAPK activation and early growth response 1 (EGR1) transcription prevents the E2-induced increase in TGFβ1 and TGFβ2 transcription and translation.. We conclude that E2-induced dermal fibrosis occurs in part through induction of TGFβ1, 2, and Col22A1, which is regulated through EGR1 and the MAPK pathway. Thus, blocking estrogen signaling and/or production may be a novel therapeutic option in pro-fibrotic diseases.

Topics: Cells, Cultured; Fibroblasts; Fibrosis; Humans; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

X-linked inhibitor of apoptosis protein (XIAP) is a multifunctional protein with important functions in apoptosis, cellular differentiation and cytoskeletal organisation and is emerging as potential target for the treatment of various cancers. The aim of the current study was to investigate the role of XIAP in the pathogenesis of systemic sclerosis (SSc).. The expression of XIAP in human skin samples of patients with SSc and chronic graft versus host disease (cGvHD) and healthy individuals was analysed by quantitative PCR, immunofluorescence (IF) and western blot. XIAP was inactivated by siRNA-mediated knockdown and pharmacological inhibition. The effects of XIAP inactivation were analysed in cultured fibroblasts and in the fibrosis models bleomycin-induced and topoisomerase-I-(topoI)-induced fibrosis and in Wnt10b-transgenic mice.. The expression of XIAP, but not of other inhibitor of apoptosis protein family members, was increased in fibroblasts in SSc and sclerodermatous cGvHD. Transforming growth factor beta (TGF-β) induced the expression of XIAP in a SMAD3-dependent manner. Inactivation of XIAP reduced WNT-induced fibroblast activation and collagen release. Inhibition of XIAP also ameliorated fibrosis induced by bleomycin, topoI and overexpression of Wnt10b in well-tolerated doses. The profibrotic effects of XIAP were mediated via WNT/β-catenin signalling. Inactivation of XIAP reduces binding of β-catenin to TCF to in a TLE-dependent manner to block WNT/β-catenin-dependent transcription.. Our data characterise XIAP as a novel link between two core pathways of fibrosis. XIAP is overexpressed in SSc and cGvHD in a TGF-β/SMAD3-dependent manner and in turn amplifies the profibrotic effects of WNT/β-catenin signalling on fibroblasts via transducin-like enhancer of split 3. Targeted inactivation of XIAP inhibits the aberrant activation of fibroblasts in murine models of SSc.

Topics: Animals; beta Catenin; Bleomycin; Disease Models, Animal; Fibroblasts; Fibrosis; Humans; Mice; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; X-Linked Inhibitor of Apoptosis Protein

Transforming growth factor-β (TGFβ) is a key mediator of fibroblast activation in fibrotic diseases, including systemic sclerosis. Here we show that Engrailed 1 (EN1) is reexpressed in multiple fibroblast subpopulations in the skin of SSc patients. We characterize EN1 as a molecular amplifier of TGFβ signaling in myofibroblast differentiation: TGFβ induces EN1 expression in a SMAD3-dependent manner, and in turn, EN1 mediates the profibrotic effects of TGFβ. RNA sequencing demonstrates that EN1 induces a profibrotic gene expression profile functionally related to cytoskeleton organization and ROCK activation. EN1 regulates gene expression by modulating the activity of SP1 and other SP transcription factors, as confirmed by ChIP-seq experiments for EN1 and SP1. Functional experiments confirm the coordinating role of EN1 on ROCK activity and the reorganization of cytoskeleton during myofibroblast differentiation, in both standard fibroblast culture systems and in vitro skin models. Consistently, mice with fibroblast-specific knockout of En1 demonstrate impaired fibroblast-to-myofibroblast transition and are partially protected from experimental skin fibrosis.

Topics: Adult; Aged; Animals; Case-Control Studies; Cell Differentiation; Cytoskeleton; Female; Gene Expression Regulation; Homeodomain Proteins; Humans; Male; Mice, Knockout; Middle Aged; Myofibroblasts; rho-Associated Kinases; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Young Adult

Innate lymphoid cells-2 (ILC2) were shown to be involved in the development of lung or hepatic fibrosis. We sought to explore the functional and phenotypic heterogeneity of ILC2 in skin fibrosis within systemic sclerosis (SSc).. Blood samples and skin biopsies from healthy donor or patients with SSc were analysed by immunostaining techniques. The fibrotic role of sorted ILC2 was studied in vitro on dermal fibroblast and further explored by transcriptomic approach. Finally, the efficacy of a new treatment against fibrosis was assessed with a mouse model of SSc.. We found that ILC2 numbers were increased in the skin of patients with SSc and correlated with the extent of skin fibrosis. In SSc skin, KLRG1. Our results demonstrate a novel role for natural ILC2 and highlight their inter-relationships with TGFβ and IL10 in the development of skin fibrosis, thereby opening up new therapeutic approaches in SSc.

Topics: Adult; Animals; Anti-Inflammatory Agents, Non-Steroidal; Biopsy; Cell Differentiation; Collagen; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Gene Expression Profiling; Humans; Interleukin-10; Lectins, C-Type; Lymphocytes; Male; Mice; Middle Aged; Myofibroblasts; Pyridones; Receptors, Immunologic; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Activation of fibroblasts is essential for physiological tissue repair. Uncontrolled activation of fibroblasts, however, may lead to tissue fibrosis with organ dysfunction. Although several pathways capable of promoting fibroblast activation and tissue repair have been identified, their interplay in the context of chronic fibrotic diseases remains incompletely understood. Here, we provide evidence that transforming growth factor-β (TGFβ) activates autophagy by an epigenetic mechanism to amplify its profibrotic effects. TGFβ induces autophagy in fibrotic diseases by SMAD3-dependent downregulation of the H4K16 histone acetyltransferase MYST1, which regulates the expression of core components of the autophagy machinery such as ATG7 and BECLIN1. Activation of autophagy in fibroblasts promotes collagen release and is both, sufficient and required, to induce tissue fibrosis. Forced expression of MYST1 abrogates the stimulatory effects of TGFβ on autophagy and re-establishes the epigenetic control of autophagy in fibrotic conditions. Interference with the aberrant activation of autophagy inhibits TGFβ-induced fibroblast activation and ameliorates experimental dermal and pulmonary fibrosis. These findings link uncontrolled TGFβ signaling to aberrant autophagy and deregulated epigenetics in fibrotic diseases and may contribute to the development of therapeutic interventions in fibrotic diseases.

Topics: Adult; Aged; Animals; Autophagy; Autophagy-Related Protein 7; Biopsy; Case-Control Studies; Disease Models, Animal; Down-Regulation; Epigenesis, Genetic; Female; Fibroblasts; Fibrosis; Gene Knockout Techniques; Healthy Volunteers; Histone Acetyltransferases; Humans; Male; Mice; Mice, Transgenic; Middle Aged; NIH 3T3 Cells; Primary Cell Culture; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Transforming Growth Factor beta; Young Adult

Systemic sclerosis primarily affects women. This sex bias raises the question on the role female hormones could play in the development of fibrosis, which is largely unknown. Our aim was to evaluate the effects of estrogens in the development of experimental dermal fibrosis, in the mouse models of bleomycin-induced dermal fibrosis and tight skin (Tsk-1) mice, and on the activation of dermal fibroblasts by transforming growth factor-β (TGF-β). Estrogen inhibition, obtained through gene inactivation for the estrogen receptor-αknockout or treatment with tamoxifen, exacerbated skin fibrosis in the bleomycin model and in the Tsk-1 mice. In the dermal fibroblasts, treatment with 17-β-estradiol significantly decreased the stimulatory effects of TGF-β on collagen synthesis and myofibroblast differentiation, decreased the activation of canonical TGF-β signaling, and markedly reduced the expression of the TGF-β target genes. Tamoxifen reversed the inhibitory effects of estrogens by restoring Smad2/3 phosphorylation and TGF-β-induced collagen synthesis. Our results demonstrate a beneficial effect of estrogens in dermal fibrosis. Estrogens reduce the TGF-β-dependent activation of dermal fibroblasts, and estrogen inhibition leads to a more severe experimental dermal fibrosis. These findings are consistent with the prominent development of systemic sclerosis in postmenopausal women and the greater severity of the disease in men.

Topics: Animals; Biopsy; Bleomycin; Cells, Cultured; Collagen; Disease Models, Animal; Estrogen Antagonists; Estrogen Receptor alpha; Estrogens; Female; Fibroblasts; Humans; Male; Mice; Mice, Knockout; Mice, Transgenic; Primary Cell Culture; Scleroderma, Systemic; Severity of Illness Index; Sex Factors; Signal Transduction; Skin; Tamoxifen; Transforming Growth Factor beta

Galectin-9 is a β-galactoside-binding protein with two carbohydrate recognition domains. Recent studies have revealed that galectin-9 regulates cellular biological reactions and plays a pivotal role in fibrosis. The aim of this study was to determine the role of galectin-9 in the pathogenesis of bleomycin-induced systemic sclerosis (SSc).. Human galectin-9 levels in the serum of patients with SSc and mouse sera galectin-9 levels were measured by a Bio-Plex immunoassay and enzyme-linked immunosorbent assay. Lung fibrosis was induced using bleomycin in galectin-9 wild-type and knockout mice. The effects of galectin-9 on the fibrosis markers and signaling molecules in the mouse lung tissues and primary lung fibroblast cells were assessed with western blotting and quantitative polymerase chain reaction.. Galectin-9 levels in the serum were significantly higher (9-fold) in patients compared to those of healthy individuals. Galectin-9 deficiency in mice prominently ameliorated epithelial proliferation, collagen I accumulation, and α-smooth muscle actin expression. In addition, the galectin-9 knockout mice showed reduced protein expression levels of fibrosis markers such as Smad2/3, connective tissue growth factor, and endothelin-1. Differences between the wild-type and knockout groups were also observed in the AKT, mitogen-activated protein kinase, and c-Jun N-terminal kinase signaling pathways. Galectin-9 deficiency decreased the signal activation induced by transforming growth factor-beta in mouse primary fibroblasts, which plays a critical role in fibroblast activation and aberrant catabolism of the extracellular matrix.. Our findings suggest that lack of galectin-9 protects against bleomycin-induced SSc. Moreover, galectin-9 might be involved in regulating the progression of fibrosis in multiple pathways.

Topics: Animals; Biomarkers; Bleomycin; Fibroblasts; Galectins; Lung; Mice; Mice, Knockout; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta

Systemic scleroderma (SSc) is an autoimmune disease that affects over 2.5 million people globally. SSc results in dysfunctional connective tissues with excessive profibrotic signaling, affecting skin, cardiovascular, and particularly lung tissue. Over three-quarters of individuals with SSc develop pulmonary fibrosis within 5 years, the main cause of SSc mortality. No approved medicines to manage lung SSc currently exist. Recent research suggests that profibrotic signaling by transforming growth factor β (TGF-β) is directly tied to SSc. Previous studies have also shown that ubiquitin E3 ligases potently control TGF-β signaling through targeted degradation of key regulatory proteins; however, the roles of these ligases in SSc-TGF-β signaling remain unclear. Here we utilized primary SSc patient lung cells for high-throughput screening of TGF-β signaling via high-content imaging of nuclear translocation of the profibrotic transcription factor SMAD family member 2/3 (SMAD2/3). We screened an RNAi library targeting ubiquitin E3 ligases and observed that knockdown of the E3 ligase Kelch-like protein 42 (KLHL42) impairs TGF-β-dependent profibrotic signaling. KLHL42 knockdown reduced fibrotic tissue production and decreased TGF-β-mediated SMAD activation. Using unbiased ubiquitin proteomics, we identified phosphatase 2 regulatory subunit B'ϵ (PPP2R5ϵ) as a KLHL42 substrate. Mechanistic experiments validated ubiquitin-mediated control of PPP2R5ϵ stability through KLHL42. PPP2R5ϵ knockdown exacerbated TGF-β-mediated profibrotic signaling, indicating a role of PPP2R5ϵ in SSc. Our findings indicate that the KLHL42-PPP2R5ϵ axis controls profibrotic signaling in SSc lung fibroblasts. We propose that future studies could investigate whether chemical inhibition of KLHL42 may ameliorate profibrotic signaling in SSc.

Topics: Fibroblasts; Fibrosis; Humans; Lung; Protein Phosphatase 2; Proteolysis; Proteomics; Scleroderma, Systemic; Signal Transduction; Smad2 Protein; Transforming Growth Factor beta; Ubiquitin-Protein Ligases

Genome-wide gene expression studies implicate macrophages as mediators of fibrosis in systemic sclerosis (SSc), but little is known about how these cells contribute to fibrotic activation in SSc. We undertook this study to characterize the activation profile of SSc monocyte-derived macrophages and assessed their interaction with SSc fibroblasts.. Plasma and peripheral blood mononuclear cells (PBMCs) were obtained from whole blood from SSc patients (n = 24) and age- and sex-matched healthy controls (n = 12). Monocytes were cultured with autologous or allogeneic plasma to differentiate cells into macrophages. For reciprocal activation studies, macrophages were cocultured with fibroblasts using Transwell plates.. The gene expression signature associated with blood-derived human SSc macrophages was enriched in SSc skin in an independent cohort and correlated with skin fibrosis. SSc macrophages expressed surface markers associated with activation and released CCL2, interleukin-6, and transforming growth factor β under basal conditions (n = 8) (P < 0.05). Differentiation of healthy donor monocytes in plasma from SSc patients conferred the immunophenotype of SSc macrophages (n = 13) (P < 0.05). Transwell experiments demonstrated that coculture of SSc macrophages with SSc fibroblasts induced fibroblast activation (n = 3) (P < 0.05).. These data demonstrate that the activation profile of SSc macrophages is profibrotic. SSc macrophages are activated under basal conditions and release mediators and express surface markers associated with both alternative and inflammatory macrophage activation. These findings also suggest that activation of SSc macrophages arises from soluble factors in local microenvironments. These studies implicate macrophages as likely drivers of fibrosis in SSc and suggest that therapeutic targeting of these cells may be beneficial in ameliorating disease in SSc patients.

Topics: Adult; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Cell Differentiation; Chemokine CCL2; Coculture Techniques; Female; Fibroblasts; Fibrosis; HLA-DR Antigens; Humans; Immunophenotyping; Interleukin-6; Lectins, C-Type; Leukocytes, Mononuclear; Macrophage Activation; Macrophages; Male; Mannose Receptor; Mannose-Binding Lectins; Middle Aged; Monocytes; Phosphorylation; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Cell Surface; RNA, Messenger; Scleroderma, Systemic; Skin; STAT3 Transcription Factor; Transcriptome; Transforming Growth Factor beta

The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFβ signaling. TGFβ is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFβ in serum of SSc patients and investigated if this correlates with integrin expression on monocytes.. A TGFβ/SMAD3- or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFβ and BMP levels respectively. A pan-specific TGFβ1/2/3 neutralizing antibody was used to confirm TGFβ signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFβ1 or a small molecule inhibitor of TGFBR1: SB-505124.. SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFβ levels were not different. Addition of a pan-specific TGFβ1/2/3 neutralizing antibody fully inhibited SMAD3-reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFβ1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFβ receptor inhibitor SB-505124 had the opposite effect.. Total TGFβ serum levels are not different between SSc patients and controls, but TGFβ activity is. This coincides with a reduced expression of TGFβ-activating integrins in monocytes of SSc patients. Because TGFβ regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.

Topics: Adult; Aged; Cells, Cultured; Female; Fibroblasts; Gene Expression; Humans; Integrins; Male; Middle Aged; Monocytes; Receptor, Transforming Growth Factor-beta Type I; Scleroderma, Systemic; Smad Proteins; Transforming Growth Factor beta

SSc is a systemic fibrotic disease affecting skin, numerous internal organs and the microvasculature. The molecular pathogenesis of SSc tissue fibrosis has not been fully elucidated, although TGF-β1 plays a crucial role. The Hic-5 protein encoded by the TGF-β1-inducible HIC-5 gene participates in numerous TGF-β-mediated pathways, however, the role of Hic-5 in SSc fibrosis has not been investigated. The aim of this study was to examine HIC-5 involvement in SSc tissue fibrosis.. Affected skin from three patients with diffuse SSc and dermal fibroblasts cultured from affected and non-affected SSc skin were examined for HIC-5 and COL1A1 gene expression. Real-time PCR, IF microscopy, western blotting and small interfering RNA-mediated HIC-5 were performed.. HIC-5 and COL1A1 transcripts and Hic-5, type 1 collagen (COL1) and α-smooth muscle actin (α-SMA) protein levels were increased in clinically affected SSc skin compared with normal skin and in cultured dermal fibroblasts from affected SSc skin compared with non-affected skin fibroblasts from the same patients. HIC-5 knockdown caused a marked reduction of COL1 production in SSc dermal fibroblasts.. HIC-5 expression is increased in affected SSc skin compared with skin from normal individuals. Affected SSc skin fibroblasts display increased HIC-5 and COL1A1 expression compared with non-affected skin fibroblasts from the same patients. Hic-5 protein was significantly increased in cultured SSc dermal fibroblasts. HIC-5 mRNA knockdown in SSc fibroblasts caused >50% reduction of COL1 production. Although these are preliminary results owing to the small number of skin samples studied, they indicate that Hic-5 plays a role in the profibrotic activation of SSc dermal fibroblasts and may represent a novel molecular target for antifibrotic therapy in SSc.

Topics: Actins; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Fibroblasts; Gene Expression; Gene Knockdown Techniques; Humans; Intracellular Signaling Peptides and Proteins; LIM Domain Proteins; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Coactivators are a heterogeneous family of transcriptional regulators that are essential for modulation of transcriptional outcomes and fine-tune numerous cellular processes. The aim of the present study was to evaluate the role of the coactivator peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in the pathogenesis of systemic sclerosis (SSc).. Expression of PGC-1α was analysed by real-time PCR, western blot and immunofluorescence. Modulation of autophagy was analysed by reporter studies by expression of autophagy-related genes. The effects of PGC-1α knockdown on collagen production and myofibroblast differentiation were analysed in cultured human fibroblasts and in two mouse models with fibroblast-specific knockout of PGC-1α.. The expression of PGC-1α was induced in dermal fibroblasts of patients with SSc and experimental murine fibrosis. Transforming growth factor beta (TGFβ), hypoxia and epigenetic mechanisms regulate the expression of PGC-1α in fibroblasts. Knockdown of PGC-1α prevented the activation of autophagy by TGFβ and this translated into reduced fibroblast-to-myofibroblast differentiation and collagen release. Knockout of PGC-1α in fibroblasts prevented skin fibrosis induced by bleomycin and by overexpression of a constitutively active TGFβ receptor type I. Moreover, pharmacological inhibition of PGC-1α by SR18292 induced regression of pre-established, bleomycin-induced skin fibrosis.. PGC-1α is upregulated in SSc and promotes autophagy to foster TGFβ-induced fibroblast activation. Targeting of PGC-1α prevents aberrant autophagy, inhibits fibroblast activation and tissue fibrosis and may over therapeutic potential.

Topics: Animals; Autophagy; Bleomycin; Blotting, Western; Collagen; Disease Models, Animal; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Humans; Mice; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Polymerase Chain Reaction; Receptor, Transforming Growth Factor-beta Type I; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Up-Regulation

Scleroderma (SSc) is an autoimmune connective tissue disease characterized by immune dysregulation, vasculopathy, and fibrosis. We have previously demonstrated that low Fli1 expression in SSc fibroblasts and endothelial cells plays an important role in SSc pathogenesis. Cells of myeloid and lymphoid origin also express Fli1 and are dysregulated in patients with SSc, playing key roles in disease pathogenesis. However, the role for immune Fli1 in SSc is not yet clear. Our aim was to elucidate whether Fli1 contributes to the immune dysregulation seen in SSc. Comparison of the expression of Fli1 in monocytes, B- and T-cell fractions of PBMCs isolated from SSc patients and healthy controls (HC), showed an increase in Fli1 levels in monocytes. We used siRNA transfected human myeloid cells and mouse peritoneal macrophages obtained from

Topics: Animals; Autoimmune Diseases; Cells, Cultured; Coculture Techniques; Disease Models, Animal; Down-Regulation; Fibroblasts; Fibrosis; Gene Expression; Healthy Volunteers; Humans; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Monocytes; Myeloid Cells; Proto-Oncogene Protein c-fli-1; RNA, Small Interfering; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Aberrant activation of fibroblasts with progressive deposition of extracellular matrix is a key feature of systemic sclerosis (SSc), a prototypical idiopathic fibrotic disease. Here, we demonstrate that the profibrotic cytokine transforming growth factor β selectively up-regulates fibroblast growth factor receptor 3 (FGFR3) and its ligand FGF9 to promote fibroblast activation and tissue fibrosis, leading to a prominent FGFR3 signature in the SSc skin. Transcriptome profiling, in silico analysis and functional experiments revealed that FGFR3 induces multiple profibrotic pathways including endothelin, interleukin-4, and connective tissue growth factor signaling mediated by transcription factor CREB (cAMP response element-binding protein). Inhibition of FGFR3 signaling by fibroblast-specific knockout of FGFR3 or FGF9 or pharmacological inhibition of FGFR3 blocked fibroblast activation and attenuated experimental skin fibrosis in mice. These findings characterize FGFR3 as an upstream regulator of a network of profibrotic mediators in SSc and as a potential target for the treatment of fibrosis.

Topics: Animals; Cells, Cultured; Fibroblasts; Fibrosis; Mice; Receptor, Fibroblast Growth Factor, Type 3; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

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

Expression of dipeptidylpeptidase 4 (DPP-4) identifies a dermal fibroblast lineage involved in scarring during wound healing. The role of DDP-4 in tissue fibrosis is, however, unknown. The aim of the present study was to evaluate DPP-4 as a potential target for the treatment of fibrosis in patients with systemic sclerosis (SSc).. Expression of DPP-4 in skin biopsy samples and dermal fibroblasts was analyzed by real-time polymerase chain reaction, immunofluorescence, and Western blot analyses. The activity of DPP-4 was modulated by overexpression, knockdown, and pharmacologic inhibition of DPP4 using sitagliptin and vildagliptin. The effects of DPP4 inhibition were analyzed in human dermal fibroblasts and in different mouse models of SSc (each n = 6).. The expression of DPP-4 and the number of DPP-4-positive fibroblasts were increased in the fibrotic skin of SSc patients, in a transforming growth factor β (TGFβ)-dependent manner. DPP-4-positive fibroblasts expressed higher levels of myofibroblast markers and collagen (each P < 0.001 versus healthy controls). Overexpression of DPP4 promoted fibroblast activation, whereas pharmacologic inhibition or genetic inactivation of DPP4 reduced the proliferation, migration, and expression of contractile proteins and release of collagen (each P < 0.001 versus control mice) by interfering with TGFβ-induced ERK signaling. DPP4-knockout mice were less sensitive to bleomycin-induced dermal and pulmonary fibrosis (P < 0.0001 versus wild-type controls). Treatment with DPP4 inhibitors promoted regression of fibrosis in mice that had received bleomycin challenge and mice with chronic graft-versus-host disease, and ameliorated fibrosis in TSK1 mice (each P < 0.001 versus untreated controls). These antifibrotic effects were associated with a reduction in inflammation.. DPP-4 characterizes a population of activated fibroblasts and shows that DPP-4 regulates TGFβ-induced fibroblast activation in the fibrotic skin of SSc patients. Inhibition of DPP4 exerts potent antifibrotic effects when administered in well-tolerated doses. As DPP4 inhibitors are already in clinical use for diabetes, these results may have direct translational implications for the treatment of fibrosis in patients with SSc.

Topics: Adult; Aged; Animals; Bleomycin; Cell Movement; Cell Proliferation; Collagen; Dipeptidyl Peptidase 4; Dipeptidyl-Peptidase IV Inhibitors; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Gene Knockdown Techniques; Graft vs Host Disease; Humans; Lung; Male; MAP Kinase Signaling System; Mice; Mice, Knockout; Middle Aged; Myofibroblasts; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Sitagliptin Phosphate; Skin; Transforming Growth Factor beta; Vildagliptin

There is considerable evidence that implicates dysregulation of type I interferon signalling (or type I IFN signature) in the pathogenesis of systemic sclerosis (SSc). Interferon regulatory factor 7 (IRF7) has been recognised as a master regulator of type I IFN signalling. The objective of this study was to elucidate the role of IRF7 in dermal fibrosis and SSc pathogenesis.. SSc and healthy control skin biopsies were investigated to determine IRF7 expression and activation. The role of IRF7 in fibrosis was investigated using IRF7 knockout (KO) mice in the bleomycin-induced and TSK/+mouse models. In vitro experiments with dermal fibroblasts from patients with SSc and healthy controls were performed.. IRF7 expression was significantly upregulated and activated in SSc skin tissue and explanted SSc dermal fibroblasts compared with unaffected, matched controls. Moreover, IRF7 expression was stimulated by IFN-α in dermal fibroblasts. Importantly, IRF7 co-immunoprecipitated with Smad3, a key mediator of transforming growth factor (TGF)-β signalling, and IRF7 knockdown reduced profibrotic factors in SSc fibroblasts. IRF7 KO mice demonstrated attenuated dermal fibrosis and inflammation compared with wild-type mice in response to bleomycin. Specifically, hydroxyproline content, dermal thickness as well as Col1a2, ACTA2 and interleukin-6 mRNA levels were significantly attenuated in IRF7 KO mice skin tissue. Furthermore, IRF7 KO in TSK/+mice attenuated hydroxyproline content, subcutaneous hypodermal thickness, Col1a2 mRNA as well as α-smooth muscle actin and fibronectin expression.. IRF7 is upregulated in SSc skin, interacts with Smad3 and potentiates TGF-β-mediated fibrosis, and therefore may represent a promising therapeutic target in SSc.

Topics: Animals; Bleomycin; Disease Models, Animal; Fibroblasts; Fibrosis; Humans; Inflammation; Interferon Regulatory Factor-7; Mice; Mice, Knockout; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Systemic sclerosis is a multisystem inflammatory and vascular lesion leading to extensive tissue fibrosis. A reversible S-adenosyl-l-homocysteine hydrolase (SAHH) inhibitor, DZ2002, modulates the pathologic processes of various inflammatory diseases and autoimmune diseases. This study is designed to investigate the therapeutic potentiality of DZ2002 for experimental systemic sclerosis models.. The anti-inflammatory and anti-fibrotic features of DZ2002 and its mechanisms were investigated in a bleomycin (BLM)-induced dermal fibrosis mice model. The effects of DZ2002 on expression of extracellular matrix components and TGF-β signaling in human dermal fibroblasts were analyzed. Simultaneously, the effects of DZ2002 on macrophage activation and endothelial cell adhesion molecule expression were also evaluated.. DZ2002 significantly attenuated dermal fibrosis in BLM-induced mice. Consistently, DZ2002 inhibited the expression of various molecules associated with dermal fibrosis, including transforming growth factor β1, connective tissue growth factor, tumor necrosis factor-α, interferon-γ, IL-1β, IL-4, IL-6, IL-10, IL-12p40, IL-17A, and monocyte chemotactic protein 1 in the lesional skin of BLM-induced mice. Furthermore, DZ2002 decreased the proportion of macrophages, neutrophils, and T cells (especially T helper cells) in the skin tissue of BLM-induced mice. In addition, DZ2002 attenuated both M1 macrophage and M2 macrophage differentiation in vivo and in vitro. Importantly, DZ2002 directly reversed the profibrotic phenotype of transforming growth factor-β1-treated dermal fibroblasts and suppressed ICAM-1, VCAM-1, VEGF, bFGF, and ET-1 expression in endothelial cells. Finally, our investigations showed that DZ2002 relieved systemic sclerosis by regulating fibrosis TGF-β/Smad signaling pathway.. DZ2002 prevents the development of experimental dermal fibrosis by reversing the profibrotic phenotype of various cell types and would be a potential drug for the treatment of systemic sclerosis.

Topics: Adenine; Animals; Bleomycin; Butyrates; Cell Line; Cells, Cultured; Dermis; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Inflammation; Macrophages; Mice, Inbred C57BL; Scleroderma, Systemic; THP-1 Cells; Transforming Growth Factor beta; Vascular Diseases; Vascular Endothelial Growth Factor A

Several studies have demonstrated that the secreted glycoprotein and integrin ligand milk fat globule-associated protein with epidermal growth factor- and factor VIII-like domains (MFG-E8) negatively regulates fibrosis in the liver, lungs, and respiratory tract. However, the mechanisms and roles of MFG-E8 in skin fibrosis in systemic sclerosis (SSc) have not been characterized. We undertook this study to elucidate the role of MFG-E8 in skin fibrosis in SSc.. We assessed expression of MFG-E8 in the skin and serum in SSc patients. We examined the effect of recombinant MFG-E8 (rMFG-E8) on latent transforming growth factor β (TGFβ)-induced gene/protein expression in SSc fibroblasts. We examined the effects of deficiency or administration of MFG-E8 on fibrosis mouse models.. We demonstrated that MFG-E8 expression around dermal blood vessels and the serum MFG-E8 level in SSc patients (n = 7 and n = 44, respectively) were lower than those in healthy individuals (n = 6 and n = 28, respectively). Treatment with rMFG-E8 significantly inhibited latent TGFβ-induced expression of type I collagen, α-smooth muscle actin, and CCN2 in SSc fibroblasts (n = 3-8), which suggested that MFG-E8 inhibited activation of latent TGFβ as well as TGFβ signaling via binding to αv integrin. In a mouse model of bleomycin-induced fibrosis (n = 5-8) and in a TSK mouse model (a genetic model of SSc) (n = 5-10), deficient expression of MFG-E8 significantly enhanced both pulmonary and skin fibrosis, and administration of rMFG-E8 significantly inhibited bleomycin-induced dermal fibrosis.. These results suggest that vasculopathy-induced dysfunction of pericytes and endothelial cells, the main cells secreting MFG-E8, may be associated with the decreased expression of MFG-E8 in SSc and that the deficient inhibitory regulation of latent TGFβ-induced skin fibrosis by MFG-E8 may be involved in the pathogenesis of SSc and may be a therapeutic target for fibrosis in SSc patients.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, Surface; Bleomycin; Collagen Type I; Female; Fibroblasts; Fibrosis; Humans; Integrin alphaV; Lung; Male; Mice; Mice, Knockout; Middle Aged; Milk Proteins; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Ubiquitination of proteins leads to their degradation by the proteasome, and is regulated by ubiquitin ligases and substrate-specific ubiquitin-specific peptidases (USPs). The ubiquitination process also plays important roles in the regulation of cell metabolism and cell cycle. Here, we found that the expression of several USPs is increased in SSc tenosynovial and skin biopsies, and we demonstrated that USP inhibition decreases TGF-β signalling in primary fibroblast cell lines.. High-density transcriptomic studies were performed using total RNA obtained from SSc tenosynovial samples. Confirmatory immunostaining experiments were performed on tenosynovial and skin samples. In vitro experiments were conducted in order to study the influence of USP modulation on responses to TGF-β stimulation.. Tenosynovial biopsies from SSc patients overexpressed known disease-associated gene pathways: fibrosis, cytokines and chemokines, and Wnt/TGF-β signalling, but also several USPs. Immunohistochemistry experiments confirmed the detection of USPs in the same samples, and in SSc skin biopsies. Exposure of primary fibroblast cell lines to TGF-β induced USP gene expression. The use of a pan-USP inhibitor decreased SMAD3 phosphorylation, and expression of COL1A1, COL3A1 and fibronectin gene expression in TGF-β-stimulated fibroblasts. The effect of the USP inhibitor resulted in increased SMAD3 ubiquitination, and was blocked by a proteasome inhibitor, thereby confirming the specificity of its action.. Overexpression of several USPs, including USP15, amplifies fibrotic responses induced by TGF-β, and is a potential therapeutic target in SSc.

Topics: Fibroblasts; Humans; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Ubiquitin-Specific Proteases

Topics: Actins; Cells, Cultured; Collagen; Fibrosis; Humans; Myofibroblasts; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis is a connective tissue disease characterized by tissue fibrosis leading to interstitial lung disease. Transforming growth factor-β (TGF-β) has been of interest as a potential diagnostic marker and also as a drug target in systemic sclerosis. The aim of this study was to assess the serum content of TGF-β1 in patients with systemic sclerosis and to assess its potential role in tissue fibrosis. The study included 30 patients, 5 men and 25 women, of the mean age of 46.9 ± 12.8 years, diagnosed with systemic sclerosis. The control group consisted of 19 women of the mean age of 28.4 ± 7.8 years, diagnosed with primary Raynaud's disease. TGF-β1 serum levels were measured, chest imaging examinations were performed, and fibrotic tissue changes were assessed using the modified Rodnan Skin Score. We found that the mean serum TGF-β1 content in patients with systemic sclerosis was 598.7 ± 242.6 pg/mL, whereas it was 568.4 ± 322.2 pg/mL in the control group (p = 0.378). We also failed to substantiate any significant relationship between TGF-β1 serum levels and the severity of pulmonary and skin fibrosis in systemic sclerosis. In conclusion, systemic sclerosis does not seem a disease that would be accompanied by a specific enhancement of serum TGF-β1. Thus, this cytokine is rather unlikely to play an essential role in the development and course of the disease, nor can it be considered diagnostic or prognostic marker.

Topics: Adult; Female; Fibrosis; Humans; Male; Middle Aged; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factors; Young Adult

We previously identified CYR61 as a histone deacetylase 5 (HDAC-5)-repressed gene in systemic sclerosis (SSc; scleroderma) endothelial cells (ECs). When overexpressed, cysteine-rich angiogenic inducer 61 (CYR-61) promoted angiogenesis in SSc ECs. This study was undertaken to examine the role of CYR-61 in fibrosis and determine the mechanisms involved in CYR-61-mediated angiogenesis in SSc.. Dermal ECs and fibroblasts were isolated from biopsy specimens from healthy subjects and patients with SSc. CYR-61 level was determined by quantitative polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. CYR-61 was overexpressed using a CYR61 vector or knocked down using small interfering RNA, and functional and mechanistic studies were then conducted in fibroblasts and ECs.. Lower CYR61 messenger RNA levels were observed in dermal fibroblasts and ECs from SSc patients than in those from healthy controls. In SSc fibroblasts, overexpression of CYR-61 led to significant reduction in the expression of profibrotic genes, including COL1A1 (P = 0.002) and ACTA2 (P = 0.04), and an increase in the expression of matrix-degrading genes, including MMP1 (P = 0.002) and MMP3 (P =0.004), and proangiogenic VEGF (P = 0.03). The antifibrotic effect of CYR-61 was further demonstrated by delay in wound healing, inhibition of gel contraction, inactivation of the transforming growth factor β pathway, and early superoxide production associated with senescence in SSc fibroblasts. In SSc ECs, overexpression of CYR-61 led to increased production of vascular endothelial cell growth factor. The proangiogenic effects of CYR-61 were mediated by signaling through αvβ3 receptors and downstream activation of AMP-activated protein kinase, AKT, and the endothelial cell nitric oxide synthase/nitric oxide pathway system.. CYR-61, which is epigenetically regulated by HDAC-5, is a potent antifibrotic and proangiogenic mediator in SSc. Therapeutic intervention to promote CYR-61 activity or increase CYR-61 levels might be of benefit in SSc.

Topics: Cells, Cultured; Cysteine-Rich Protein 61; Dermis; Endothelial Cells; Fibroblasts; Histone Deacetylases; Humans; Neovascularization, Pathologic; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Progranulin (PGRN) is an autocrine growth factor with numerous physiological and pathologic roles. Previous reports demonstrated PGRN could increase dermal fibroblasts in wound healing and activate cancer-associated fibroblasts in some cancers. Because systemic sclerosis (SSc) is a prototypical fibrosis-related disorder, here, the aim was to clarify the role and mechanism of PGRN in bleomycin (BLM)-induced model of SSc for the first time. It was observed that the serum PGRN levels were increased in SSc patients compared with healthy controls. Immunohistology and quantitative RT-PCR demonstrated that PGRN was also elevated in the lesion from the mice model of BLM-induced dermal fibrosis. In addition, in BLM-treated mice, PGRN deficiency not only attenuated dermal fibrosis but also decreased the differentiation of myofibroblasts. The reduced progression of skin sclerosis in PGRN-deficient mice was associated with down-regulation of transforming growth factor (TGF)-β receptor I (TβR I) and decreased level of phosphorylated Smad3, with correspondingly impaired expression of its downstream target gene connective tissue growth factor (CTGF) in skin lesion. In contrast, exogenous PGRN significantly increased the level of TβR I and phosphorylated Smad3 in cultured mouse fibroblasts. This study demonstrates that PGRN plays a promoting role in the development of dermal fibrosis through the activation of the TGF-β/Smad3 signaling via up-regulation of TβR I. PGRN may be a new therapeutic target in SSc.

Topics: Animals; Bleomycin; Female; Humans; Mice; Mice, Knockout; Middle Aged; Progranulins; Receptor, Transforming Growth Factor-beta Type I; Scleroderma, Systemic; Signal Transduction; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Systemic sclerosis (SSc) is characterised by aberrant hedgehog signalling in fibrotic tissues. The hedgehog acyltransferase (HHAT) skinny hedgehog catalyses the attachment of palmitate onto sonic hedgehog (SHH). Palmitoylation of SHH is required for multimerisation of SHH proteins, which is thought to promote long-range, endocrine hedgehog signalling. The aim of this study was to evaluate the role of HHAT in the pathogenesis of SSc.. Expression of HHAT was analysed by real-time polymerase chain reaction(RT-PCR), immunofluorescence and histomorphometry. The effects of HHAT knockdown were analysed by reporter assays, target gene studies and quantification of collagen release and myofibroblast differentiation in cultured human fibroblasts and in two mouse models.. The expression of HHAT was upregulated in dermal fibroblasts of patients with SSc in a transforming growth factor-β (TGFβ)/SMAD-dependent manner. Knockdown of HHAT reduced TGFβ-induced hedgehog signalling as well as myofibroblast differentiation and collagen release in human dermal fibroblasts. Knockdown of HHAT in the skin of mice ameliorated bleomycin-induced and topoisomerase-induced skin fibrosis.. HHAT is regulated in SSc in a TGFβ-dependent manner and in turn stimulates TGFβ-induced long-range hedgehog signalling to promote fibroblast activation and tissue fibrosis. Targeting of HHAT might be a novel approach to more selectively interfere with the profibrotic effects of long-range hedgehog signalling.

Topics: Acyltransferases; Adult; Aged; Animals; Blotting, Western; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Female; Fibroblasts; Gene Expression Regulation; Humans; Male; Mice; Middle Aged; RNA; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Young Adult

In systemic sclerosis (SSc), previously healthy adults develop an inflammatory prodrome with subsequent progressive fibrosis of the skin and viscera. SSc has a weak signature for genetic contribution, and there are few pathogenic insights or targeted treatments for this condition. Here, chromatin accessibility and transcriptome profiling coupled with targeted epigenetic editing revealed constitutive activation of a previously unannotated transforming growth factor-β2 (

Topics: Cell Cycle Proteins; Epigenesis, Genetic; Fibroblasts; Fibrosis; Histone Acetyltransferases; Humans; NF-kappa B; Scleroderma, Systemic; Skin; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta2; Tumor Necrosis Factor-alpha

Systemic sclerosis (scleroderma, SSc) is a devastating fibrotic disease with few treatment options. Fumaric acid esters, including dimethyl fumarate (DMF, Tecfidera; Biogen, Cambridge, MA), have shown therapeutic effects in several disease models, prompting us to determine whether DMF is effective as a treatment for SSc dermal fibrosis. We found that DMF blocks the profibrotic effects of transforming growth factor-β (TGFβ) in SSc skin fibroblasts. Mechanistically, we found that DMF treatment reduced nuclear localization of transcriptional coactivator with PDZ binding motif (TAZ) and Yes-associated protein (YAP) proteins via inhibition of the phosphatidylinositol 3 kinase/protein kinase B (Akt) pathway. In addition, DMF abrogated TGFβ/Akt1 mediated inhibitory phosphorylation of glycogen kinase 3β (GSK3β) and a subsequent β-transducin repeat-containing proteins (βTRCP) mediated proteasomal degradation of TAZ, as well as a corresponding decrease of TAZ/YAP transcriptional targets. Depletion of TAZ/YAP recapitulated the antifibrotic effects of DMF. We also confirmed the increase of TAZ/YAP in skin biopsies from patients with diffuse SSc. We further showed that DMF significantly diminished nuclear TAZ/YAP localization in fibroblasts cultured on a stiff surface. Importantly, DMF prevented bleomycin-induced skin fibrosis in mice. Together, our work demonstrates a mechanism of the antifibrotic effect of DMF via inhibition of Akt1/GSK3β/TAZ/YAP signaling and confirms a critical role of TAZ/YAP in mediating the profibrotic responses in dermal fibroblasts. This study supports the use of DMF as a treatment for SSc dermal fibrosis.

Topics: Adaptor Proteins, Signal Transducing; Adult; Animals; Biopsy; Bleomycin; Cell Cycle Proteins; Cell Nucleus; Cells, Cultured; Dimethyl Fumarate; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Humans; Intracellular Signaling Peptides and Proteins; Male; Mice; Mice, Inbred C57BL; Middle Aged; Phosphatidylinositol 3-Kinase; Phosphoproteins; Proteolysis; Proto-Oncogene Proteins c-akt; Scleroderma, Systemic; Signal Transduction; Skin; Trans-Activators; Transcription Factors; Transcriptional Coactivator with PDZ-Binding Motif Proteins; Transforming Growth Factor beta; Treatment Outcome; YAP-Signaling Proteins

SSc is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs. IL-6 and related cytokines that signal through STAT3 have been implicated in the pathogenesis of SSc and mouse models of fibrosis. The aim of this study was to investigate the efficacy of inhibiting STAT3 in the development of fibrosis in two mouse models of skin fibrosis.. Biopsy samples of skin from SSc patients and healthy control subjects were used to determine the expression pattern of phosphotyrosyl (pY705)-STAT3. C188-9, a small molecule inhibitor of STAT3, was used to treat fibrosis in the bleomycin-induced fibrosis model and Tsk-1 mice. In vitro studies were performed to determine the extent to which STAT3 regulates the fibrotic phenotype of dermal fibroblasts.. Increased STAT3 and pY705-STAT3 was observed in SSc skin biopsies and in both mouse models of SSc. STAT3 inhibition with C188-9 resulted in attenuated skin fibrosis, myofibroblast accumulation, pro-fibrotic gene expression and collagen deposition in both mouse models of skin fibrosis. C188-9 decreased in vitro dermal fibroblast production of fibrotic genes induced by IL-6 trans-signalling and TGF-β. Finally, TGF-β induced phosphotyrosylation of STAT3 in a SMAD3-dependent manner.. STAT3 inhibition decreases dermal fibrosis in two models of SSc. STAT3 regulates dermal fibroblasts function in vitro and can be activated by TGF-β. These data suggest that STAT3 is a potential therapeutic target for dermal fibrosis in diseases such as SSc.

Topics: Animals; Female; Fibrosis; Humans; Mice; Mice, Inbred C57BL; Naphthols; Scleroderma, Systemic; Signal Transduction; Skin; Skin Diseases; STAT3 Transcription Factor; Sulfonamides; Transforming Growth Factor beta

Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family growth factors, which affects multiple aspects of the wound healing process such as epithelialization, wound contraction and angiogenesis. In our study, we measured the serum HB-EGF levels of 51 systemic sclerosis (SSc) patients, which showed a significant increase compared with those of 20 normal subjects. Further analysis revealed a positive correlation between the HB-EGF level and pulmonary ground-glass score but no correlation between the former and pulmonary fibrosis score. Other findings include: a significant increase of serum sialylated carbohydrate antigen KL-6 levels and significant shortness of disease duration in the diffuse cutaneous SSc patients with elevated HB-EGF levels; and significantly higher HB-EGF levels in the presence of Raynaud's phenomenon, in that of telangiectasia, and in the absence of contracture of phalanges in all SSc patients. We then evaluated HB-EGF mRNA levels of fibroblasts harvested from skin samples of the SSc patients and those of foreskin-derived fibroblasts treated with transforming growth factor-β, both of which were significantly higher than each control. In conclusion, we speculate that HB-EGF plays a pro-inflammatory role in the active skin and lung lesions of SSc.

Topics: Adult; Aged; Biopsy; Cells, Cultured; Female; Fibroblasts; Fibrosis; Heparin-binding EGF-like Growth Factor; Humans; Lung; Male; Middle Aged; Mucin-1; Pulmonary Fibrosis; Respiratory Function Tests; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis.. PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast-endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis.. PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density.. Our data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

Topics: Animals; Caveolin 1; Cells, Cultured; Endothelial Cells; Eye Proteins; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Mice; Mice, Transgenic; Neovascularization, Pathologic; Nerve Growth Factors; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Serpins; Skin; Transforming Growth Factor beta

Stimulators of soluble guanylate cyclase (sGC) are currently investigated in clinical trials for the treatment of fibrosis in systemic sclerosis (SSc). In this study, we aim to investigate the role of protein kinases G (PKG) as downstream mediators of sGC-cyclic guanosine monophosphate (cGMP) in SSc.. Mice with combined knockout of PKG1 and 2 were challenged with bleomycin and treated with the sGC stimulator BAY 41-2272. Fibroblasts were treated with BAY 41-2272 and with the PKG inhibitor KT 5823.. PKG1 and 2 are upregulated in SSc in a transforming growth factor-β1 (TGFβ1)-dependent manner, as an attempt to compensate for the decreased signalling through the sGC-cGMP-PKG pathway. Inhibition or knockout of PKG1 and 2 abrogates the inhibitory effects of sGC stimulation on fibroblast activation in a SMAD-independent, but extracellular signal-regulated kinase (ERK)-dependent manner. In vivo, sGC stimulation fails to prevent bleomycin-induced fibrosis in PKG1 and 2 knockout mice.. Our data provide evidence that PKGs are essential mediators of the antifibrotic effects of sGC stimulators through interfering with non-canonical TGFβ signalling. TGFβ1 promotes its profibrotic effects through inhibition of sGC-cGMP-PKG signalling, sGC stimulation exerts its antifibrotic effects by inhibition of TGFβ1-induced ERK phosphorylation.

Topics: Adult; Aged; Animals; Bleomycin; Blotting, Western; Carbazoles; Cell Culture Techniques; Cyclic GMP-Dependent Protein Kinases; Female; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Humans; Male; Mice; Mice, Knockout; Middle Aged; Pyrazoles; Pyridines; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Signal Transduction; Soluble Guanylyl Cyclase; Transforming Growth Factor beta

Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor β (TGFβ) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin-2), a critical regulator of the Wnt canonical pathway.. Canonical Wnt signaling activation was analyzed by TOPflash T cell factor/lymphoid enhancer factor promoter assays. Axin-2 was evaluated in vitro by analysis of Axin-2 primary/mature transcript expression and decay, TGFβ receptor type I (TGFβRI) blockade, small interfering RNA-mediated depletion of tristetraprolin 1, and XAV-939-mediated Axin-2 stabilization. In vivo, Axin-2 messenger RNA (mRNA) and protein expression was determined in skin and lung biopsy samples from mice that express a kinase-deficient TGFβRII specifically on fibroblasts (TβRIIΔk-fib-transgenic mice) and from littermate controls.. SSc fibroblasts displayed an increased response to canonical Wnt ligands despite basal levels of Wnt signaling that were comparable to those in healthy control fibroblasts in vitro. Notably, we showed that SSc fibroblasts had reduced basal expression of Axin-2, which was caused by an endogenous TGFβ-dependent increase in Axin-2 mRNA decay. Accordingly, we observed that TGFβ decreased Axin-2 expression both in vitro in healthy control fibroblasts and in vivo in TβRIIΔk-fib-transgenic mice. Additionally, using Axin-2 gain- and loss-of-function experiments, we demonstrated that the TGFβ-induced increased response to Wnt activation characteristic of SSc fibroblasts depended on reduced bioavailability of Axin-2.. This study highlights the importance of reduced bioavailability of Axin-2 in mediating the increased canonical Wnt response observed in SSc fibroblasts. This novel mechanism extends our understanding of the processes involved in Wnt/β-catenin-driven pathology and supports the rationale for targeting the TGFβ pathway to regulate the aberrant Wnt signaling observed during fibrosis.

Topics: Animals; Axin Protein; Down-Regulation; Fibroblasts; Humans; Lung; Mice; Mice, Transgenic; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Wnt Signaling Pathway

The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) transfers negatively charged ADP-ribose units to target proteins. This modification can have pronounced regulatory effects on target proteins. Recent studies showed that PARP-1 can poly(ADP-ribosyl)ate (PARylate) Smad proteins. However, the role of PARP-1 in the pathogenesis of systemic sclerosis (SSc) has not been investigated.. The expression of PARP-1 was determined by quantitative PCR and immunohistochemistry. DNA methylation was analysed by methylated DNA immunoprecipitation assays. Transforming growth factor-β (TGFβ) signalling was assessed using reporter assays, chromatin immunoprecipitation assays and target gene analysis. The effect of PARP-1 inactivation was investigated in bleomycin-induced and topoisomerase-induced fibrosis as well as in tight-skin-1 (Tsk-1) mice.. PARP-1 negatively regulates canonical TGFβ signalling in experimental skin fibrosis. The downregulation of PARP-1 in SSc fibroblasts may thus directly contribute to hyperactive TGFβ signalling and to persistent fibroblast activation in SSc.

Topics: Adult; Aged; Animals; Disease Models, Animal; DNA Methylation; Down-Regulation; Female; Fibroblasts; Fibrosis; Humans; Male; Mice; Middle Aged; Poly (ADP-Ribose) Polymerase-1; Protein Serine-Threonine Kinases; Scleroderma, Systemic; Signal Transduction; Skin; Skin Diseases; Smad3 Protein; Transforming Growth Factor beta; Young Adult

Periostin is a matricellular protein that belongs to a class of extracellular matrix (ECM)-related molecules defined by their ability to modulate cell-matrix interactions. We previously reported an elevated level of circulating periostin in patients with systemic sclerosis (SSc) and its association with the severity of skin sclerosis.. To examine the role of periostin in transforming growth factor (TGF)-β signaling involved in fibrosis.. Levels of periostin were examined in skin and lung fibroblasts obtained from SSc patients. Levels of ECM proteins and pro-fibrotic factors were evaluated in periostin-expressing human skin fibroblasts in the presence or absence of TGF-β. Effects of periostin on the Smad proteins were also evaluated following stimulation with TGF-β by immunoblotting, immunofluorescence staining, and RNA interference.. Periostin was strongly expressed in skin and lung fibroblasts from SSc patients. Although recombinant periostin alone did not affect ECM protein levels, TGF-β and recombinant periostin treatment or periostin overexpression in skin fibroblasts significantly enhanced the production of ECM proteins. Overexpression of periostin in the presence of TGF-β also augmented expressions of α-smooth muscle actin and early growth response-1 but decreased the level and activity of matrix metalloproteinase 1. Interestingly, the level of Smad 7, a TGF-β-inducible inhibitor of TGF-β signaling, was reduced in periostin-expressing fibroblasts but increased in periostin-silenced fibroblasts. In addition, Smad 7 reduction induced by periostin was partially inhibited in integrin α. Periostin contributes to fibrosis by enhancing TGF-β signaling via Smad 7 inhibition, which may lead to ECM deposition and periostin generation.

Topics: Biopsy; Cell Adhesion Molecules; Cell Line; Extracellular Matrix Proteins; Fibroblasts; Fibrosis; Humans; Lung; Primary Cell Culture; Recombinant Proteins; RNA Interference; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Skin; Smad7 Protein; Transforming Growth Factor beta; Up-Regulation

Retinoic acid (RA) is an active metabolite of vitamin A and has been reported to improve the clinical symptoms of patients with systemic sclerosis (SSc). However, the mechanism of RA in the prevention of SSc remains unclear. Regulatory T cells (Tregs) are a subpopulation of T cells with immunosuppressive activity. The quantitative and functional defects of Tregs may mediate the immune dysfunction in SSc. The addition of all-trans retinoic acid (ATRA) to human naïve CD4+ cells could promote the maturation of Tregs and increase the stable expression of Foxp3. In this study, we explored the role of RA on Tregs in SSc CD4+ T cells and its possible epigenetic mechanisms, so as to further understand the mechanisms of RA on SSc.. CD4+ T cells were isolated from peripheral blood of SSc and treated with or without ATRA and/or transforming growth factor-. The expression of Tregs and FOXP3 in CD4+ T cells from patients with SSc increased in response to ATRA. Moreover, combined stimulation with ATRA and TGF-. ATRA acts as an inducer of Treg response in SSc CD4+ T cells via demethylation of the FOXP3 promoter and activation of FOXP3 expression. This may be one of the molecular mechanisms for ATRA, and therefore, RA can be used for the treatment of SSc.

Topics: CD4 Antigens; Cell Differentiation; Cells, Cultured; Demethylation; DNA Methylation; Epigenesis, Genetic; Flow Cytometry; Forkhead Transcription Factors; Humans; Interleukin-2 Receptor alpha Subunit; Scleroderma, Systemic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tretinoin; Up-Regulation

Scleroderma or systemic sclerosis (SSc) is a clinically heterogeneous rheumatological autoimmune disease affecting the skin, internal organs and blood vessels. There is at present no effective treatment for this condition. Our study investigated the effects of 5-aminolevulinic acid (5-ALA), which is a precursor of haem synthesis, on graft-vs-host disease (GvHD)-induced SSc murine model. Lymphocytes were intravenously injected from donor mice (B10.D2) into recipient BALB/c mice (recombination-activating gene 2 (Rag-2)-null mice) deficient in mature T and B cells to induce sclerodermatous GvHD (scl-GvHD). To investigate the effect of 5-ALA on scl-GvHD, combination of 5-ALA and sodium ferrous citrate (SFC) was orally administered to the recipient mice for 9 weeks. 5-ALA/SFC treatment significantly reduced progressive inflammation and fibrosis in the skin and ears. Furthermore, 5-ALA/SFC suppressed mRNA expression of transforming growth factor-β, type I collagen and inflammatory cytokines. These results indicate that the 5-ALA/SFC combination treatment has a protective effect against tissue fibrosis and inflammation in a murine scl-GvHD-induced skin and ear inflammation and fibrosis. Furthermore, the efficacy of 5-ALA/SFC suggests important implications of HO-1 protective activity in autoimmune diseases, and therefore, 5-ALA/SFC may have promising clinical applications. These findings suggested that the 5-ALA/SFC treatment may be the potential strategies for SSc.

Topics: Aminolevulinic Acid; Animals; Collagen Type I; Cytokines; Disease Models, Animal; Drug Therapy, Combination; Female; Ferrous Compounds; Fibrosis; Gene Expression; Graft vs Host Disease; Heme Oxygenase-1; Inflammation; Membrane Proteins; Mice; Mice, Inbred BALB C; NF-E2-Related Factor 2; Photosensitizing Agents; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Uncontrolled activation of TGFβ signaling is a common denominator of fibrotic tissue remodeling. Here we characterize the tyrosine phosphatase SHP2 as a molecular checkpoint for TGFβ-induced JAK2/STAT3 signaling and as a potential target for the treatment of fibrosis. TGFβ stimulates the phosphatase activity of SHP2, although this effect is in part counterbalanced by inhibitory effects on SHP2 expression. Stimulation with TGFβ promotes recruitment of SHP2 to JAK2 in fibroblasts with subsequent dephosphorylation of JAK2 at Y570 and activation of STAT3. The effects of SHP2 on STAT3 activation translate into major regulatory effects of SHP2 on fibroblast activation and tissue fibrosis. Genetic or pharmacologic inactivation of SHP2 promotes accumulation of JAK2 phosphorylated at Y570, reduces JAK2/STAT3 signaling, inhibits TGFβ-induced fibroblast activation and ameliorates dermal and pulmonary fibrosis. Given the availability of potent SHP2 inhibitors, SHP2 might thus be a potential target for the treatment of fibrosis.

Topics: Adult; Aged; Animals; Cell Line; Down-Regulation; Female; Fibroblasts; Fibrosis; Humans; Janus Kinase 2; Male; Mice, Knockout; Middle Aged; Organ Specificity; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Quinolines; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; STAT3 Transcription Factor; Transforming Growth Factor beta; Young Adult

Systemic sclerosis (SSc) is a multisystemic fibrotic disease characterized by excessive collagen deposition and extracellular matrix synthesis. Though transforming growth factor-β (TGF-β) plays a fundamental role in the pathogenesis of SSc, the mechanism by which TGF-β signaling acts in SSc remains largely unclear. Here, we showed that TGF-β type II receptor (TGFBR2) was significantly upregulated in both human SSc dermal tissues and primary fibroblasts. In fibroblasts, siRNA-induced knockdown of TGFBR2 resulted in a reduction of p-SMAD2/3 levels and reduced production of type I collagen. Additionally, functional experiments revealed that downregulation of TGFBR2 yielded an anti-growth effect on fibroblasts through inhibiting cell cycle progression. Further studies showed that miR-3606-3p could directly target the 3'-UTR of TGFBR2 and significantly decrease the levels of both TGFBR2 mRNA and protein. Furthermore, SSc dermal tissues and primary fibroblasts contain significantly reduced amounts of miR-3606-3p, and the overexpression of miR-3606-3p in fibroblasts replicates the phenotype of TGFBR2 downregulation. Collectively, our findings demonstrated that increased TGFBR2 could be responsible for the hyperactive TGF-β signaling observed in SSc. Moreover, we identified a pivotal role for miR-3606-3p in SSc, which acts, at least partly, through the attenuation of TGF-β signaling via TGFBR2 repression, suggesting that the regulation of miR-3606-3p/TGFBR2 could be a promising therapeutic target that could improve the treatment strategy for fibrosis.

Topics: 3' Untranslated Regions; Apoptosis; Base Sequence; Cell Cycle; Cells, Cultured; Collagen; Dermis; Fibroblasts; Humans; MicroRNAs; Receptor, Transforming Growth Factor-beta Type II; Scleroderma, Systemic; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Inhibition of transforming growth factor (TGF)-β1 signalling may be one of the most reliable approaches to treat skin fibrosis of scleroderma. Although there have been many basic researches of TGF-β blockade reagents, few of them were proved to have inhibitory effects on fibrosis both in vitro and in vivo. In this study, we randomly chose four commercially available low molecular weight compounds (Repsox, LY2109761, LY364947 and K02288) from TGF-β1 inhibitor library, and compared their antifibrotic effects in vitro and in vivo. We demonstrated that Repsox has the most potent inhibitory effects on TGF-β-induced expression of CTGF and collagen of cultured normal dermal fibroblasts in vitro and their constitutive overexpression of scleroderma fibroblast in vitro. In addition, Repsox could attenuate skin fibrosis by bleomycin in vivo, via the downregulation of CTGF or collagen. Our results may facilitate clinical trial of Repsox against fibrotic diseases in future.

Topics: Actins; Amino Acids; Aminopyridines; Animals; Bleomycin; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Fibroblasts; Fibrosis; Humans; Inhibitory Concentration 50; Mice; Phenols; Pyrazoles; Pyridines; Pyrroles; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Up-Regulation; Xanthenes

Janus kinase 2 (JAK2) has recently been described as a novel downstream mediator of the pro-fibrotic effects of transforming growth factor-β. Although JAK2 inhibitors are in clinical use for myelodysplastic syndromes, patients often rapidly develop resistance. Tumour cells can escape the therapeutic effects of selective JAK2 inhibitors by mutation-independent transactivation of JAK2 by JAK1. Here, we used selective JAK2 inhibition as a model to test the hypothesis that chronic treatment may provoke resistance by facilitating non-physiological signalling pathways in fibroblasts.. The antifibrotic effects of long-term treatment with selective JAK2 inhibitors and reactivation of JAK2 signalling by JAK1-dependent transphosphorylation was analysed in cultured fibroblasts and experimental dermal and pulmonary fibrosis. Combined JAK1/JAK2 inhibition and co-treatment with an HSP90 inhibitor were evaluated as strategies to overcome resistance.. The antifibrotic effects of selective JAK2 inhibitors on fibroblasts decreased with prolonged treatment as JAK2 signalling was reactivated by JAK1-dependent transphosphorylation of JAK2. This reactivation could be prevented by HSP90 inhibition, which destabilised JAK2 protein, or with combined JAK1/JAK2 inhibitors. Treatment with combined JAK1/JAK2 inhibitors or with JAK2 inhibitors in combination with HSP90 inhibitors was more effective than monotherapy with JAK2 inhibitors in bleomycin-induced pulmonary fibrosis and in adTBR-induced dermal fibrosis.. Fibroblasts can develop resistance to chronic treatment with JAK2 inhibitors by induction of non-physiological JAK1-dependent transactivation of JAK2 and that inhibition of this compensatory signalling pathway, for example, by co-inhibition of JAK1 or HSP90 is important to maintain the antifibrotic effects of JAK2 inhibition with long-term treatment.

Topics: Adult; Animals; Antibiotics, Antineoplastic; Benzoquinones; Bleomycin; Blotting, Western; Disease Models, Animal; Fibroblasts; Fibrosis; HSP90 Heat-Shock Proteins; Humans; Immunohistochemistry; Janus Kinase 1; Janus Kinase 2; Lactams, Macrocyclic; Lung; Male; Mice; Middle Aged; Nitriles; Phosphorylation; Protein Kinase Inhibitors; Pulmonary Fibrosis; Pyrazoles; Pyrimidines; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Sulfonamides; Transforming Growth Factor beta

Treg abnormalities have been implicated in the pathogenesis of systemic sclerosis (SSc). Treg subpopulations and their cytokines, IL-10 and TGF-β in the peripheral blood of early stage SSc patients were investigated. We hypothesized that epigenetically regulated methylation of the FOXP3 promoter and enhancer regions are altered in Tregs of SSc patients, which might be involved in the T cell imbalance. CD4+CD25+Foxp3+CD127- Treg cells were significantly elevated in patients with diffuse cutaneous SSc and in patients with anti-Scl-70/RNA-Pol-III autoantibody positivity and with lung fibrosis. Increased CD62L+ Treg cells were present in all SSc subgroups. The production of immunosuppressive cytokines by both CD127- and CD62L+ Tregs was diminished. We observed reduced methylation of Treg specific FOXP3 enhancer regions, and elevated FOXP3 gene expression in active SSc cases with negative correlation in the frequency of CD62L+IL-10+ Tregs. Our data indicate an inappropriate distribution and cytokine production of Treg cells in early form SSc.

Topics: Adult; Aged; Antibodies, Antinuclear; DNA Methylation; DNA Topoisomerases, Type I; Epigenesis, Genetic; Female; Forkhead Transcription Factors; Gene Expression; Gene Expression Regulation; Humans; Interleukin-10; Middle Aged; Nuclear Proteins; Promoter Regions, Genetic; Pulmonary Fibrosis; RNA Polymerase III; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

T helper 9 (Th9) cells and interleukin (IL)-9 are involved in the pathogenesis of several autoimmune diseases. The exact role of IL-9 and Th9 cells in patients with systemic sclerosis (SSc) have not yet been studied adequately. IL-9, IL-9R, transcription factor PU.1 (PU.1), IL-4, thymic stromal lymphopoietin (TSLP) and transforming growth factor (TGF)-β expression were assessed in skin and kidney biopsies of SSc patients and healthy controls (HC) by immunohistochemistry (IHC). The cellular source of IL-9 was also analysed by confocal microscopy analysis. Peripheral IL-9-producing cells were also studied by flow cytometry. The functional relevance of IL-9 increased expression in SSc was also investigated. Our results demonstrated a strong expression of IL-9, IL-9R, IL-4, TSLP and TGF-β in skin tissues of patients with both limited and diffuse SSc. IL-9 expression was observed mainly in the context of skin infiltrating mononuclear cells and keratinizing squamous epithelium. IL-9 over-expression was also observed in renal biopsies of patients with SSc. IL-9 producing cells in the skin were identified as Th9 cells. Similarly, Th9 cells were expanded and were the major source of IL-9 among SSc peripheral blood mononuclear cells (PBMC), their percentage being correlated directly with the modified Rodnan skin score. Infiltrating mononuclear cells, mast cells and neutrophils expressed IL-9R. In in-vitro studies stimulation with rIL-9 significantly induced NET (neutrophil extracellular traps) release by dying cells (NETosis) in neutrophils, expansion of mast cells and increase of anti-systemic scleroderma 70 (Scl70) production by B cells. Our findings suggest that Th9 cells and IL-9 could be implicated in the pathogenesis of SSc.

Topics: Adult; Autoantibodies; B-Lymphocytes; CD4-Positive T-Lymphocytes; Cell Differentiation; Cytokines; Extracellular Traps; Female; Humans; Interleukin-4; Interleukin-9; Male; Mast Cells; Middle Aged; Neutrophils; Proto-Oncogene Proteins; Receptors, Interleukin-9; Scleroderma, Systemic; Skin; Thymic Stromal Lymphopoietin; Trans-Activators; Transforming Growth Factor beta

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

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Protein tyrosine phosphatases have received little attention in the study of SSc or fibrosis. Here, we show that the tyrosine phosphatase PTP4A1 is highly expressed in fibroblasts from patients with SSc. PTP4A1 and its close homolog PTP4A2 are critical promoters of TGFβ signaling in primary dermal fibroblasts and of bleomycin-induced fibrosis in vivo. PTP4A1 promotes TGFβ signaling in human fibroblasts through enhancement of ERK activity, which stimulates SMAD3 expression and nuclear translocation. Upstream from ERK, we show that PTP4A1 directly interacts with SRC and inhibits SRC basal activation independently of its phosphatase activity. Unexpectedly, PTP4A2 minimally interacts with SRC and does not promote the SRC-ERK-SMAD3 pathway. Thus, in addition to defining PTP4A1 as a molecule of interest for TGFβ-dependent fibrosis, our study provides information regarding the functional specificity of different members of the PTP4A subclass of phosphatases.

Topics: Animals; Cell Line; Dermis; Extracellular Signal-Regulated MAP Kinases; Female; Fibroblasts; Humans; Immediate-Early Proteins; MAP Kinase Signaling System; Mice, Inbred C57BL; Mice, Knockout; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins pp60(c-src); Scleroderma, Systemic; Smad3 Protein; Transforming Growth Factor beta

Signal transducer and activator of transcription 3 (STAT3) is phosphorylated by various kinases, several of which have been implicated in aberrant fibroblast activation in fibrotic diseases including systemic sclerosis (SSc). Here we show that profibrotic signals converge on STAT3 and that STAT3 may be an important molecular checkpoint for tissue fibrosis. STAT3 signaling is hyperactivated in SSc in a TGFβ-dependent manner. Expression profiling and functional studies in vitro and in vivo demonstrate that STAT3 activation is mediated by the combined action of JAK, SRC, c-ABL, and JNK kinases. STAT3-deficient fibroblasts are less sensitive to the pro-fibrotic effects of TGFβ. Fibroblast-specific knockout of STAT3, or its pharmacological inhibition, ameliorate skin fibrosis in experimental mouse models. STAT3 thus integrates several profibrotic signals and might be a core mediator of fibrosis. Considering that several STAT3 inhibitors are currently tested in clinical trials, STAT3 might be a candidate for molecular targeted therapies of SSc.

Topics: Adolescent; Adult; Aged; Aminosalicylic Acids; Animals; Benzenesulfonates; Biopsy; Bleomycin; Collagen; Enzyme Activation; Female; Fibroblasts; Fibrosis; Humans; Inflammation; Male; Mice; Microscopy, Confocal; Middle Aged; Phosphorylation; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; STAT3 Transcription Factor; Transforming Growth Factor beta; Young Adult

Osteopontin a matricellular protein has pro-fibrotic effects and binds integrin such as αvβ1 and αvβ3. Vitronectin is one of the integrin αvβ3 ligands and is a multifunctional glycoprotein.. The aim of the present study was to evaluate serum osteopontin and vitronectin levels in a cohort of patients with systemic sclerosis (SSc).. Eighty-six patients with SSc, 46 patients with systemic lupus erythematosus (SLE), and 38 healthy controls (HC) were enrolled in the study. Serum osteopontin, vitronectin, IL-6, and TGF-β levels were analyzed.. Serum osteopontin levels were higher in the SSc and SLE groups compared to the HC group (p < 0.01 and p < 0.001, respectively). However, it was not correlated with disease activity and severity scores in the SSc group. On the other hand, serum vitronectin levels were lower in the SSc group than in the SLE and HC groups (p < 0.001 for both).. These results may suggest that osteopontin levels may be increased due to the inflammatory process and osteopontin has not a specific role on fibrosis in SSc. On the other hand, serum vitronectin levels decrease in SSc in contrast to SLE. It may be concluded that the one cause of decreased serum vitronectin levels in SSc may be its accumulation in fibrotic area.

Topics: Adult; Aged; Female; Humans; Interleukin-6; Male; Middle Aged; Osteopontin; Scleroderma, Systemic; Transforming Growth Factor beta; Vitronectin

TWIST1 is a member of the class B of basic helix-loop-helix transcription factors that regulates cell lineage determination and differentiation and has been implicated in epithelial-to-mesenchymal transition. Here, we aimed to investigate the role of TWIST1 for the activation of resident fibroblasts in systemic sclerosis (SSc).. The expression of Twist1 in fibroblasts was modulated by forced overexpression or siRNA-mediated knockdown. Interaction of Twist1, E12 and inhibitor Of differentiation (Id) was analysed by co-immunoprecipitation. The role of Twist1 in vivo was evaluated using inducible, conditional knockout mice with either ubiquitous or fibroblast-specific depletion of Twist1. Mice were either challenged with bleomycin or overexpressing a constitutively active transforming growth factor (TGF)β receptor I.. The expression of TWIST1 was increased in fibroblasts in fibrotic human and murine skin in a TGFβ/SMAD3-dependent manner. TWIST1 in turn enhanced TGFβ-induced fibroblast activation in a p38-dependent manner. The stimulatory effects of TWIST1 on resident fibroblasts were mediated by TWIST1 homodimers. TGFβ promotes the formation of TWIST1 homodimers by upregulation of TWIST1 and by induction of inhibitor of DNA-binding proteins, which have high affinity for E12/E47 and compete against TWIST1 for E12/E47 binding. Mice with selective depletion of Twist1 in fibroblasts are protected from experimental skin fibrosis in different murine models to a comparable degree as mice with ubiquitous depletion of Twist1.. Our data identify TWIST1 as a central pro-fibrotic factor in SSc, which facilitates fibroblast activation by amplifying TGFβ signalling. Targeting of TWIST1 may thus be a novel approach to normalise aberrant TGFβ signalling in SSc.

Topics: Animals; Case-Control Studies; Female; Fibroblasts; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Male; Mice, Knockout; Nuclear Proteins; Protein Multimerization; RNA, Messenger; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Twist-Related Protein 1

Although there is increasing evidence that oxidative stress is involved in collagen synthesis and myofibroblast activation, the NADPH oxidase (Nox) system is incompletely investigated in the context of human dermal fibroblasts (HDFs) and skin fibrosis. Using the pan-Nox inhibitor diphenyleneiodonium (DPI) as an initial tool, we show that gene expression of collagen type I, α-smooth muscle actin (α-SMA) and fibronectin 1 is suppressed in HDFs. Detailed expression analysis of all Nox isoforms and adaptors revealed expression of RNA and protein expression of Nox4, p22

Topics: Actins; Adult; Animals; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Computer Simulation; Cytokines; Disease Models, Animal; Enzyme Inhibitors; Female; Fibroblasts; Fibronectins; Fibrosis; Gene Expression; Gene Expression Profiling; Gene Silencing; Humans; Infant, Newborn; Isoenzymes; Male; Mice; Middle Aged; Multienzyme Complexes; Myofibroblasts; NADH, NADPH Oxidoreductases; NADPH Oxidase 1; NADPH Oxidase 2; NADPH Oxidase 4; Onium Compounds; Primary Cell Culture; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Young Adult

Systemic sclerosis (SSc) is a multisystem inflammatory and vascular disease resulting in extensive tissue fibrosis. Glycyrrhizin, clinically used for chronic hepatic diseases and itching dermatitis, modulates the pathological processes of inflammation, vasculopathy, and fibrosis in human diseases and their animal models. Therefore, we investigated a potential impact of glycyrrhizin on the key pathological manifestations of SSc, including inflammation, vasculopathy, and tissue fibrosis, with bleomycin-treated mice mimicking the fibrotic and inflammatory components of SSc and endothelial cell-specific Fli1-knockout mice recapitulating SSc vasculopathy. Glycyrrhizin significantly ameliorated dermal fibrosis in bleomycin-treated mice, which was partly attributable to blockade of transforming growth factor-β signaling in dermal fibroblasts through the down-regulation of thrombospondin 1, a latent transforming growth factor-β receptor, and transcription factors Smad3 and Ets1. Furthermore, bleomycin-dependent induction of T helper type 2-skewed immune polarization, M2 macrophage infiltration, and endothelial-to-mesenchymal transition were greatly suppressed in mice administered glycyrrhizin. Glycyrrhizin also improved vascular permeability of endothelial cell-specific Fli1-knockout mice by increasing the expression of molecules regulating vascular integrity. These results indicate that glycyrrhizin ameliorates bleomycin-induced dermal fibrosis through the inhibition of fibroblast activation, T helper type 2-skewed immune polarization, M2 macrophage infiltration, and endothelial-to-mesenchymal transition and improves endothelial Fli1 deficiency-dependent vascular disintegrity, implying its potential as a disease-modifying drug for SSc.

Topics: Animals; Bleomycin; Disease Models, Animal; Fibroblasts; Fibrosis; Glycyrrhizic Acid; Humans; Inflammation; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Scleroderma, Systemic; Skin; Th2 Cells; Transforming Growth Factor beta; Vascular Diseases

Hedgehog signalling plays a critical role during the pathogenesis of fibrosis in systemic sclerosis (SSc). Besides canonical hedgehog signalling with smoothened (SMO)-dependent activation of GLI transcription factors, GLI can be activated independently of classical hedgehog ligands and receptors (so-called non-canonical pathways). Here, we aimed to evaluate the role of non-canonical hedgehog signalling in SSc and to test the efficacy of direct GLI inhibitors that target simultaneously canonical and non-canonical hedgehog pathways.. The GLI inhibitor GANT-61 was used to inhibit canonical as well as non-canonical hedgehog signalling, while the SMO inhibitor vismodegib was used to selectively target canonical hedgehog signalling. Furthermore, GLI2 was selectively depleted in fibroblasts using the Cre-LoxP system. The effects of pharmacological or genetic of GLI2 on transforming growth factor-β (TGF-β) signalling were analysed in cultured fibroblasts, in bleomycin-induced pulmonary fibrosis and in mice with overexpression of a constitutively active TGF-β receptor I.. TGF-β upregulated GLI2 in a Smad3-dependent manner and induced nuclear accumulation and DNA binding of GLI2. Fibroblast-specific knockout of GLI2 protected mice from TBR. Our data demonstrate that hedgehog pathways and TGF-β signalling both converge to GLI2 and that GLI2 integrates those signalling to promote tissue fibrosis. These findings may have translational implications as non-selective inhibitors of GLI2 are in clinical use and selective molecules are currently in development.

Topics: Adult; Aged; Anilides; Animals; Cells, Cultured; Collagen Type I; Connective Tissue Growth Factor; Female; Fibroblasts; Fibrosis; Gene Knockout Techniques; Hedgehog Proteins; Humans; Kruppel-Like Transcription Factors; Male; Mice; Mice, Knockout; Mice, Transgenic; Middle Aged; Plasminogen Activator Inhibitor 1; Protein Serine-Threonine Kinases; Pteridines; Pulmonary Fibrosis; Pyridines; Pyrimidines; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Recombinant Proteins; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Smoothened Receptor; Transforming Growth Factor beta; Young Adult; Zinc Finger Protein Gli2

The efficacy and feasibility of targeting transforming growth factor-β (TGFβ) in pulmonary fibrosis and lung vascular remodeling in systemic sclerosis (SSc) have not been well elucidated. In this study we analyzed how blocking TGFβ signaling affects pulmonary abnormalities in Fos-related antigen 2 (Fra-2) transgenic (Tg) mice, a murine model that manifests three important lung pathological features of SSc: fibrosis, inflammation, and vascular remodeling. To interrupt TGFβ signaling in the Fra-2 Tg mice, we used a pan-TGFβ-blocking antibody, 1D11, and Tg mice in which TGFβ receptor type 2 (Tgfbr2) is deleted from smooth muscle cells and myofibroblasts (α-SMA-Cre

Topics: Actins; Animals; Bone Morphogenetic Protein Receptors, Type II; Disease Models, Animal; Fos-Related Antigen-2; Gene Deletion; Granulocytes; Mice, Transgenic; Myocytes, Smooth Muscle; Pneumonia; Protein Serine-Threonine Kinases; Pulmonary Alveoli; Pulmonary Fibrosis; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases; Vascular Remodeling

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) governs antioxidant, innate immune and cytoprotective responses and its deregulation is prominent in chronic inflammatory conditions. To examine the hypothesis that Nrf2 might be implicated in systemic sclerosis (SSc), we investigated its expression, activity, and mechanism of action in SSc patient samples and mouse models of fibrosis and evaluated the effects of a novel pharmacologic Nrf2 agonist. We found that both expression and activity of Nrf2 were significantly reduced in SSc patient skin biopsies and showed negative correlation with inflammatory gene expression. In skin fibroblasts, Nrf2 mitigated fibrotic responses by blocking canonical transforming growth factor-β (TGF-β)-Smad signaling, whereas silencing Nrf2 resulted in constitutively elevated collagen synthesis, spontaneous myofibroblast differentiation, and enhanced TGF-ß responses. Bleomycin treatment of Nrf2-null mice resulted in exaggerated fibrosis. In wild-type mice, treatment with a novel pharmacologic Nrf2 agonist 2-trifluoromethyl-2'-methoxychalcone prevented dermal fibrosis induced by TGF-β. These findings are the first to identify Nrf2 as a cell-intrinsic antifibrotic factor with key roles in maintaining extracellular matrix homeostasis and a pathogenic role in SSc. Pharmacologic reactivation of Nrf2, therefore, represents a novel therapeutic strategy toward effective treatment of fibrosis in SSc.

Topics: Animals; Bleomycin; Cells, Cultured; Chalcones; Down-Regulation; Fibroblasts; Fibrosis; Gene Expression Regulation; Genetic Predisposition to Disease; Humans; Mice; Mice, Knockout; NF-E2-Related Factor 2; Oleanolic Acid; RNA, Messenger; Scleroderma, Systemic; Transforming Growth Factor beta

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

Scleroderma is a fibrosis-related disorder characterized by cutaneous and internal organ fibrosis, and excessive collagen deposition in extracellular matrix (ECM) is a major cause of fibrosis. Transforming growth factor-β (TGF-β)/SMAD signaling has a central role in the pathogenesis of fibrosis by inducing abnormal collagen accumulation in ECM, and latent TGF-β-binding protein 4 (LTBP-4) affects the secretion of latent TGF-β to ECM. A previous study indicated that bleomycin (BLM) treatment increased LTBP-4 expression in lung fibroblasts of Thy-1 knockout mice with lung fibrosis, and LTBP-4 further promoted TGF-β bioavailability as well as SMAD3 phosphorylation. However, the expression and function of LTBP-4 in human scleroderma remain unclear. We aimed to investigate the potential role of LTBP-4 in scleroderma through clinical, in vivo and in vitro studies. LTBP-4 and TGF-β expressions were significantly upregulated in systemic scleroderma (SSc) patients' plasma compared with normal controls (LTBP-4, 1,215±100.2 vs 542.8±41.7 ng/ml, P<0.0001; TGF-β, 1.5±0.2 vs 0.7±0.1 ng/ml, P=0.0031), while no significant difference was found between localized scleroderma (LSc) and normal controls. The plasma concentrations of LTBP-4 and TGF-β were even higher in SSc patients with lung fibrosis (LTBP-4, 1462± 137.3 vs 892.8±113.4 ng/ml, P=0.0037; TGF-β, 2.0±0.4 vs 0.9±0.2 ng/ml, P=0.0212) and esophagus involvement (1390±134.4 vs 940.7±127.0 ng/ml, P=0.0269; TGF-β, 1.9±0.3 vs 0.9±0.2 ng/ml, P=0.0426). The area under receiver operating characteristics (ROC) curve of LTBP-4 was 0.86. Immunohistochemistry measurement also demonstrated a higher LTBP-4 expression in sclerotic skin tissue of LSc and SSc compared with normal controls. More positive fibroblasts were also found in BLM-induced scleroderma mouse model than the saline-treated group. In in vitro studies, knockdown of LTBP-4 in SSc skin fibroblasts prominently reduced downstream COL1A1, COL1A2, and COL3A1 mRNA level by 84%, 82%, and 43%, respectively, and other fibrosis-related genes' expression were also decreased. Furthermore, extracellular TGF-β level and the SMAD2/3 phosphorylation were inhibited through LTBP-4 knockdown treatment, suggesting that the knockdown of LTBP-4 reduced the collagen expression through TGF-β/SMAD signaling pathway. Taken together, these data suggest that LTBP-4 affects fibrotic process in scleroderma, and the high expression of LTBP-4 in SSc plasma may serve as a clinical biomarker in diagn

Topics: Adult; Animals; Collagen; Female; Fibrosis; Gene Knockdown Techniques; Humans; Latent TGF-beta Binding Proteins; Male; Mice; Mice, Inbred C57BL; Middle Aged; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins, Receptor-Regulated; Transforming Growth Factor beta

Sirt1 is a member of the sirtuin family of proteins. Sirt1 is a class III histone deacetylase with important regulatory roles in transcription, cellular differentiation, proliferation and metabolism. As aberrant epigenetic modifications have been linked to the pathogenesis of systemic sclerosis (SSc), we aimed to investigate the role of Sirt1 in fibroblast activation.. Sirt1 expression was analysed by real-time PCR, western blot and immunohistochemistry. Sirt1 signalling was modulated with the Sirt1 agonist resveratrol and by fibroblast-specific knockout. The role of Sirt1 was evaluated in bleomycin-induced skin fibrosis and in mice overexpressing a constitutively active transforming growth fac-tor-β (TGF-β) receptor I (TBRIact).. The expression of Sirt1 was decreased in patients with SSc and in experimental fibrosis in a TGF-β-dependent manner. Activation of Sirt1 potentiated the profibrotic effects of TGF-β with increased Smad reporter activity, elevated transcription of TGF-β target genes and enhanced release of collagen. In contrast, knockdown of Sirt1 inhibited TGF-β/SMAD signalling and reduced release of collagen in fibroblasts. Consistently, mice with fibroblast-specific knockdown of Sirt1 were less susceptible to bleomycin- or TBRIact-induced fibrosis.. We identified Sirt1 as a crucial regulator of TGF-β/Smad signalling in SSc. Although Sirt1 is downregulated, this decrease is not sufficient to counterbalance the excessive activation of TGF-β signalling in SSc. However, augmentation of this endogenous regulatory mechanism, for example, by knockdown of Sirt1, can effectively inhibit TGF-β signalling and exerts potent antifibrotic effects. Sirt1 may thus be a key regulator of fibroblast activation in SSc.

Topics: Adult; Aged; Animals; Bleomycin; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Down-Regulation; Female; Fibroblasts; Fibrosis; Humans; Male; Mice, Knockout; Middle Aged; Scleroderma, Systemic; Signal Transduction; Sirtuin 1; Skin; Transforming Growth Factor beta

Activating transcription factor 3 (ATF3), a member of the ATF/cAMP-responsive element binding (CREB) family of transcription factors, regulates cellular response to stress including oxidative stress. The aim of this study was to analyse the role of ATF3 in fibroblast activation in systemic sclerosis (SSc).. ATF3 was analysed by reverse transcription quantitative PCR, western blot and immunohistochemistry. ATF3 knockout fibroblasts and mice were used to study the functional role of ATF3. Knockdown experiments, reporter assays and coimmunoprecipitation were performed to study the effects of ATF3 on Smad and activation protein 1 (AP-1) signalling. The role of c-Jun was analysed by costaining, specific inactivation and coimmunoprecipitation.. Transforming growth factor-β (TGFβ) upregulates the expression of ATF3 in SSc fibroblasts. ATF3-deficient fibroblasts were less sensitive to TGFβ, whereas ectopic expression of ATF3 enhanced the profibrotic effects of TGFβ. Mechanistically, ATF3 interacts with Smad3 directly on stimulation with TGFβ and regulates Smad activity in a c-Jun-dependent manner. Knockout of ATF3 protected mice from bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active TGFβ receptor I. Reporter assays and analyses of the expression of Smad target genes demonstrated that binding of ATF3 regulates the transcriptional activity of Smad3.. We demonstrate for the first time a key role for ATF3 in fibrosis. Knockout of the ATF3 gene reduced the stimulatory effect of TGFβ on fibroblasts by interfering with canonical Smad signalling and protected the mice from experimental fibrosis in two different models. ATF3 might thus be a candidate for molecular targeted therapies for SSc.

Topics: Activating Transcription Factor 3; Adult; Aged; Animals; Blotting, Western; Case-Control Studies; Dermis; Female; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Gene Expression Profiling; Gene Expression Regulation; Humans; Immunohistochemistry; Male; Mice; Mice, Knockout; Middle Aged; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-jun; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Signal Transduction; Smad3 Protein; Transcription Factor AP-1; Transforming Growth Factor beta; Young Adult

Tribbles homologue 3 (TRB3) is a pseudokinase that modifies the activation of various intracellular signalling pathways to control fundamental processes extending from mitosis and cell activation to apoptosis and modulation of gene expression. Here, we aimed to analyse the role of TRB3 in fibroblast activation in systemic sclerosis (SSc).. The expression of TRB3 was quantified by quantitative PCR, western blot and immunohistochemistry. The role of TRB3 was analysed in cultured fibroblasts and in experimental fibrosis using small interfering RNA (siRNA)-mediated knockdown and overexpression of TRB3.. TRB3 expression was increased in fibroblasts of patients with SSc and in murine models of SSc in a transforming growth factor-β (TGF-β)/Smad-dependent manner. Overexpression of TRB3 stimulated canonical TGF-β signalling and induced an activated phenotype in resting fibroblasts. In contrast, knockdown of TRB3 reduced the profibrotic effects of TGF-β and decreased the collagen synthesis. Moreover, siRNA-mediated knockdown of TRB3 exerted potent antifibrotic effects and ameliorated bleomycin as well as constitutively active TGF-β receptor I-induced fibrosis with reduced dermal thickening, decreased hydroxyproline content and impaired myofibroblast differentiation.. The present study characterises TRB3 as a novel profibrotic mediator in SSc. TGF-β induces TRB3, which in turn activates canonical TGF-β/Smad signalling and stimulates the release of collagen, thereby inducing a positive feedback loop that may contribute to aberrant TGF-β signalling in SSc.

Topics: Adult; Aged; Animals; Antibiotics, Antineoplastic; Bleomycin; Case-Control Studies; Cell Cycle Proteins; Cells, Cultured; Collagen; Dermis; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Gene Knock-In Techniques; Gene Knockdown Techniques; Humans; Immunohistochemistry; Male; Mice; Middle Aged; Protein Serine-Threonine Kinases; Real-Time Polymerase Chain Reaction; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Repressor Proteins; Scleroderma, Systemic; Signal Transduction; Skin Diseases; Smad3 Protein; Transforming Growth Factor beta; Young Adult

Long non-coding RNAs (lncRNAs) are thought to have various functions other than RNA silencing. We tried to evaluate the expression of lncRNAs in patients with systemic sclerosis (SSc) and determined whether lncRNAs controls collagen expression in dermal fibroblasts. lncRNA expression was determined by real-time PCR and in situ hybridization. Protein and mRNA levels of collagen were analysed using immunoblotting and real-time PCR. We found TSIX, one of the lncRNAs, was overexpressed in SSc dermal fibroblasts both in vivo and in vitro, which was inhibited by the transfection of transforming growth factor (TGF)-β1 siRNA. TSIX siRNA reduced the mRNA expression of type I collagen in normal and SSc dermal fibroblasts, but not the levels of major disease-related cytokines. In addition, TSIX siRNA significantly reduced type I collagen mRNA stability, but not protein half-lives. Furthermore, we first investigated serum lncRNA levels in patients with SSc, and serum TSIX levels were significantly increased in SSc patients. TSIX is a new regulator of collagen expression which stabilizes the collagen mRNA. The upregulation of TSIX seen in SSc fibroblasts may result from activated endogenous TGF-β signalling and may play a role in the constitutive upregulation of collagen in these cells. Further studies on the regulatory mechanism of tissue fibrosis by lncRNAs in SSc skin lead to a better understanding of the pathogenesis, new diagnostic methods by their serum levels and new therapeutic approaches using siRNAs.

Topics: Adult; Aged; Aged, 80 and over; Collagen Type I; Dermis; Female; Fibroblasts; Humans; Lupus Erythematosus, Systemic; Male; Middle Aged; RNA Interference; RNA Stability; RNA, Long Noncoding; RNA, Messenger; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Young Adult

Although transforming growth factor β (TGFβ) is recognized as being a key trigger of fibroblast activation in systemic sclerosis (SSc), prominent innate immunity suggests that additional pathways contribute to disease persistence. Toll-like receptor 9 (TLR9) is implicated in autoimmunity and fibrosis; however, the expression, mechanism of action, and pathogenic role of TLR9 signaling in SSc remain uncharacterized. The aim of this study was to explore the expression, activity, and potential pathogenic role of TLR9 in the context of skin fibrosis in SSc and in mouse models of experimental fibrosis.. Expression and localization of TLR9 were evaluated in SSc skin biopsy specimens and explanted skin fibroblasts. Fibrotic responses elicited by type A CpG oligonucleotide and mitochondrial DNA (mtDNA) were examined in human skin fibroblasts by a combination of real-time quantitative polymerase chain reaction, Western blot analysis, transient transfection, immunofluorescence microscopy, and functional assays. Expression of TLR9 was examined in 2 distinct mouse models of experimental fibrosis.. Skin biopsy specimens obtained from 2 independent cohorts of SSc patients showed up-regulation of TLR9, and myofibroblasts were the major cellular source. Moreover, SSc skin biopsy specimens showed evidence of TLR9 pathway activation. CpG induced robust TLR9-dependent fibrotic responses in explanted normal fibroblasts that could be blocked by bortezomib and were mediated through the action of endogenous TGFβ. Mice with experimental fibrosis showed a time-dependent increase in TLR9 localized primarily to myofibroblasts in the dermis.. In isolated fibroblasts, TLR9 elicits fibrotic responses mediated via endogenous TGFβ. In patients with SSc, mtDNA and other damage-associated TLR9 ligands in the skin might trigger localized activation of TLR9 signaling, TGFβ production, and consequent fibroblast activation. Disrupting this fibrotic process with inhibitors targeting TLR9 or its downstream signaling pathways might therefore represent a novel approach to SSc therapy.

Topics: Animals; Cells, Cultured; Female; Fibroblasts; Fibrosis; Mice; Mice, Inbred C57BL; Scleroderma, Systemic; Signal Transduction; Skin; Toll-Like Receptor 9; Transforming Growth Factor beta

The pathogenesis of systemic sclerosis (SSc) involves a distinctive triad of autoimmune, vascular and inflammatory alterations resulting in fibrosis. Evidence suggests that peroxisome proliferator-activated receptors (PPARs) play an important role in SSc-related fibrosis and their agonists may become effective therapeutic targets.. To determine the expression of PPARs in human fibrotic skin and investigate the effects of IVA337, a pan PPAR agonist, in in vitro and in vivo models of fibrosis.. The antifibrotic effects of IVA337 were studied using a bleomycin-induced mouse model of dermal fibrosis. The in vivo effect of IVA337 on wound closure and inflammation were studied using an excisional model of wound healing.. Low levels of PPARα and PPARγ were detected in the skin of patients with SSc compared with controls. In mice, IVA337 was associated with decreased extracellular matrix (ECM) deposition and reduced expression of phosphorylated SMAD2/3-intracellular effector of transforming growth factor (TGF)-β1. Although the antifibrotic effect of pan PPAR was similar to that of PPARγ agonist alone, a significant downregulation of several markers of inflammation was associated with IVA337. Despite its anti-inflammatory and antifibrotic properties, IVA337 did not interfere with wound closure. In vitro effects of IVA337 included attenuation of transcription of ECM genes and alteration of canonical and non-canonical TGF-β signalling pathways.. These findings indicate that simultaneous activation of all three PPAR isoforms exerts a dampening effect on inflammation and fibrosis, making IVA337 a potentially effective therapeutic candidate in the treatment of fibrotic diseases including SSc.

Topics: Animals; Anti-Inflammatory Agents; Benzothiazoles; Bleomycin; Dermatologic Agents; Disease Models, Animal; Extracellular Matrix; Fibrosis; Humans; Mice; PPAR alpha; PPAR gamma; Scleroderma, Systemic; Signal Transduction; Smad2 Protein; Sulfonamides; Transforming Growth Factor beta; Transforming Growth Factor beta1; Wound Healing

Systemic Sclerosis (SSc) is an autoimmune disease characterized by vascular and immune dysfunction. A hallmark of SSc is the excessive accumulation of extracellular matrix in the skin and in internal organs. There is a high and unmet medical need for novel therapies in this disease. The pathogenesis of SSc is complex and still poorly understood, but the innate immune system has emerged as an important factor in the disease. SSc patients show increased numbers of macrophages/monocytes in the blood and in the skin compared to healthy individuals and these cells are important sources of profibrotic cytokines and chemokines. Paquinimod belongs to a class of orally active quinoline-3-carboxamide (quinoline) derivatives with immunomodulatory properties and has shown effects in several models of autoimmune/inflammatory disorders. Paquinimod is currently in clinical development for treatment of SSc. The immunomodulatory effects of paquinimod is by targeting the myeloid cell compartment via the S100A9 protein.. In this study we investigate whether targeting of myeloid cells by paquinimod can effect disease development in an experimental model of SSc, the tight skin 1 (Tsk-1) mouse model.. Seven weeks old female B6.Cg-Fbn1(Tsk)/J (Tsk-1) mice were treated with vehicle or paquinimod at the dose of 5 or 25mg/kg/day in the drinking water for 8 weeks. The effect of paquinimod on the level of skin fibrosis and on different subpopulations within the myeloid cell compartment in skin biopsies were evaluated by using histology, immunohistochemisty, a hydroxyproline assay and real-time PCR. Furthermore, the level of IgG in serum from treated animals was also analysed. The statistical analyses were performed using Mann-Whitney nonparametric two tailed rank test.. The results show that treatment with paquinimod reduces skin fibrosis measured as reduction of skin thickness and decreased number of myofibroblasts and total hydroxyproline content. The effect on fibrosis was associated with a polarization of macrophages in the skin from a pro-fibrotic M2 to a M1 phenotype. Paquinimod treatment also resulted in a reduced TGFβ-response in the skin and an abrogation of the increased auto-antibody production in this SSc model.. Paquinimod reduces skin fibrosis in an experimental model of SSc, and this effect correlates with local and systemic effects on the immune system.

Topics: Animals; Autoimmune Diseases; Calgranulin B; Disease Models, Animal; Female; Fibrosis; Humans; Immunoglobulin G; Immunosuppressive Agents; Macrophages; Mice; Quinolines; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

The ubiquitin-editing cytosolic enzyme A20, the major negative regulator of toll-like receptor (TLR)-mediated cellular inflammatory responses, has tight genetic linkage with systemic sclerosis (SSc). Because recent studies implicate endogenous ligand-driven TLR signaling in SSc pathogenesis, we sought to investigate the regulation, role and mechanism of action of A20 in skin fibroblasts.. A20 expression and the effects of forced A20 expression or siRNA-mediated A20 knockdown on fibrotic responses induced by transforming growth factor-ß (TGF-ß) were evaluated was evaluated in explanted human skin fibroblasts. Additionally, A20 regulation by TGF-ß, and by adiponectin, a pleiotropic adipokine with anti-fibrotic activity, was evaluated.. In normal fibroblasts, TGF-ß induced sustained downregulation of A20, and abrogated its TLR4-dependent induction. Forced expression of A20 aborted the stimulation of collagen gene expression and myofibroblast transformation induced by TGF-ß, and disrupted canonical Smad signaling and Smad-dependent transcriptional responses. Conversely, siRNA-mediated knockdown of A20 enhanced the amplitude of fibrotic responses elicited by TGF-ß. Adiponectin, previously shown to block TLR-dependent fibrotic responses, elicited rapid and sustained increase in A20 accumulation in fibroblasts.. These results identify the ubiquitin-editing enzyme A20 as a novel endogenous mechanism for negative regulation of fibrotic response intensity. Systemic sclerosis-associated genetic variants of A20 that cause impaired A20 expression or function, combined with direct suppression of A20 by TGF-ß within the fibrotic milieu, might play a significant functional role in persistence of fibrotic responses, while pharmacological augmentation of A20 inhibitory pathway activity might represent a novel therapeutic strategy.

Topics: Blotting, Western; Cells, Cultured; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Gene Knockdown Techniques; Humans; Microscopy, Confocal; Polymerase Chain Reaction; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins; Transforming Growth Factor beta; Tumor Necrosis Factor alpha-Induced Protein 3

To investigate the effects and mechanisms of adipose-derived stem cells (ADSCs) on bleomycin-induced mice of scleroderma.. In the study, 24 C57BL/6J female mice were randomly divided into control group, bleomycin(BLM)group, ADSCs (hypodermic injection) group and ADSCs (intravenous injection) group . BLM [2 mg/(kg×d)] was injected into the mice to establish the model of scleroderma. There were 6 mice in each group .The control group mice were injected with normal saline 2 mL/(kg×d) by subcutaneously. The rest of the three groups were injected with BLM. ADSCs groups were injected with ADSCs (2×10. The proportion of Th17 and Treg increased in BLM group than in control group(15.30%±1.29% vs.4.32%±0.79%; 9.90%±1.95% vs.5.18%±1.35%, P<0.05), the expression of Th17 significantly decreased (5.02%±0.83%, 6.00%±0.82% vs.15.30%±1.29%, P<0.05) and the expression of Treg increased after the ADSCs therapy (14.32%±1.59%, 11.09%±4.31% vs. 9.90%±1.95%, P<0.05). The expression levels of IL-17,IL-6,tumor necrosis factor-α (TNF-α)mRNA in the lung tissue and IL-6 in the serum increased in BLM group than in control group [3.54±0.30, 10.65±0.66, 5.37±0.52 vs. 1.00±0.00; (21.2±1.74) ng/L vs. (16.87±1.09) ng/L, P<0.05]. The expression of these cytokines significant decreased after the ADSCs therapy [1.63±0.45,1.50±0.29 vs.3.54±0.30; 3.11±0.85, 2.98±0.76 vs.10.65±0.66;1.45±0.47, 1.59±0.41 vs. 5.37±0.52; (17.87±1.45) ng/L, (17.61±1.16) ng/L vs. (21.2±1.74) ng/L, P<0.05]. But there was no obvious difference between ADSCs (hypodermic injection) group and ADSCs (intravenous injection) group(P>0.05). The expression of TGF-β in the serum increased in BLM group than in control group[(33.95±2.49) ng/L vs. (28.8± 2.29) ng/L, P<0.05], however, the expression of TGF-β mRNA had no significant differences than that of control group (1.17±0.11 vs.1.00±0.00, P>0.05). The expression of TGF-β mRNA and protein had no significant differences than that of BLM group [1.25±0.11,1.26±0.12 vs.1.17±0.11; (31.84±2.04) ng/L, (31.25±2.36) ng/L vs. (33.95±2.49) ng/L, P>0.05]. HE staining showed that the inflammation of lung tissue was relieved and the dermal thickness and collagen deposition were decreased after the ADSCs therapy.. ADSCs could effectively alleviate inflammation of the lungs and fibrosis of skin; the effects of anti-inflammatory and anti-fibrosis were associated with immune regulating function.

Topics: Adipose Tissue; Adult Stem Cells; Animals; Bleomycin; Cytokines; Female; Fibrosis; Inflammation; Interleukin-17; Interleukin-6; Lung; Mice; Mice, Inbred C57BL; RNA, Messenger; Scleroderma, Systemic; Skin; T-Lymphocytes, Regulatory; Th17 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Casein kinase II (CK2) is a constitutively active serine/threonine protein kinase that plays a key role in cellular transformation and tumorigenesis. The purpose of the study was to characterise whether CK2 contributes to the pathologic activation of fibroblasts in patients with SSc and to evaluate the antifibrotic potential of CK2 inhibition.. Activation of CK2, JAK2 and STAT3 in human skin and in experimental fibrosis was analysed by immunohistochemistry. CK2 signalling was inhibited by the selective CK2 inhibitor 4, 5, 6, 7-Tetrabromobenzotriazole (TBB). The mouse models of bleomycin-induced and TGFβ receptor I (TBR)-induced dermal fibrosis were used to evaluate the antifibrotic potential of specific CK2 inhibition in vivo.. Increased expression of CK2 was detected in skin fibroblasts of SSc patients. Inhibition of CK2 by TBB abrogated the TGFβ-induced activation of JAK2/STAT3 signalling and prevented the stimulatory effects of TGFβ on collagen release and myofibroblasts differentiation in cultured fibroblasts. Inhibition of CK2 prevented bleomycin-induced and TBR-induced skin fibrosis with decreased dermal thickening, lower myofibroblast counts and reduced accumulation of collagen. Treatment with TBB also induced regression of pre-established fibrosis. The antifibrotic effects of TBB were accompanied by reduced activation of JAK2/STAT3 signalling in vivo.. We provide evidence that CK2 is activated in SSc and contributes to fibroblast activation by regulating JAK2/STAT3 signalling. Inhibition of CK2 reduced the pro-fibrotic effects of TGFβ and inhibited experimental fibrosis. Targeting of CK2 may thus be a novel therapeutic approach for SSc and other fibrotic diseases.

Topics: Adult; Aged; Animals; Casein Kinase II; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Humans; Janus Kinase 2; Male; Mice; Middle Aged; Scleroderma, Systemic; Signal Transduction; Skin; STAT3 Transcription Factor; Transforming Growth Factor beta; Triazoles; Young Adult

Leri's pleonosteosis (LP) is an autosomal dominant rheumatic condition characterised by flexion contractures of the interphalangeal joints, limited motion of multiple joints, and short broad metacarpals, metatarsals and phalanges. Scleroderma-like skin thickening can be seen in some individuals with LP. We undertook a study to characterise the phenotype of LP and identify its genetic basis.. Whole-genome single-nucleotide polymorphism genotyping in two families with LP defined microduplications of chromosome 8q22.1 as the cause of this condition. Expression analysis of dermal fibroblasts from affected individuals showed overexpression of two genes, GDF6 and SDC2, within the duplicated region, leading to dysregulation of genes that encode proteins of the extracellular matrix and downstream players in the transforming growth factor (TGF)-β pathway. Western blot analysis revealed markedly decreased inhibitory SMAD6 levels in patients with LP. Furthermore, in a cohort of 330 systemic sclerosis cases, we show that the minor allele of a missense SDC2 variant, p.Ser71Thr, could confer protection against disease (p<1×10(-5)).. Our work identifies the genetic cause of LP in these two families, demonstrates the phenotypic range of the condition, implicates dysregulation of extracellular matrix homoeostasis genes in its pathogenesis, and highlights the link between TGF-β/SMAD signalling, growth/differentiation factor 6 and syndecan-2. We propose that LP is an additional member of the growing 'TGF-β-pathies' group of musculoskeletal disorders, which includes Myhre syndrome, acromicric dysplasia, geleophysic dysplasias, Weill-Marchesani syndromes and stiff skin syndrome. Identification of a systemic sclerosis-protective SDC2 variant lays the foundation for exploration of the role of syndecan-2 in systemic sclerosis in the future.

Topics: Adult; Aged; Child, Preschool; Chromosomes, Human, Pair 8; Extracellular Matrix; Facies; Female; Fibroblasts; Gene Duplication; Gene Expression Profiling; Growth Differentiation Factor 6; Hand Deformities, Congenital; Humans; Infant; Joint Diseases; Male; Middle Aged; Ossification, Heterotopic; Phenotype; Scleroderma, Systemic; Signal Transduction; Syndecan-2; Transforming Growth Factor beta; Young Adult

Vitamin D receptor (VDR) is a member of the nuclear receptor superfamily. Its ligand, 1,25-(OH)2D, is a metabolically active hormone derived from vitamin D3. The levels of vitamin D3 are decreased in patients with systemic sclerosis (SSc). Here, we aimed to analyse the role of VDR signalling in fibrosis.. VDR expression was analysed in SSc skin, experimental fibrosis and human fibroblasts. VDR signalling was modulated by siRNA and with the selective agonist paricalcitol. The effects of VDR on Smad signalling were analysed by reporter assays, target gene analyses and coimmunoprecipitation. The effects of paricalcitol were evaluated in the models of bleomycin-induced fibrosis and fibrosis induced by overexpression of a constitutively active transforming growth factor-β (TGF-β) receptor I (TBRI(CA)).. VDR expression was decreased in fibroblasts of SSc patients and murine models of SSc in a TGF-β-dependent manner. Knockdown of VDR enhanced the sensitivity of fibroblasts towards TGF-β. In contrast, activation of VDR by paricalcitol reduced the stimulatory effects of TGF-β on fibroblasts and inhibited collagen release and myofibroblast differentiation. Paricalcitol stimulated the formation of complexes between VDR and phosphorylated Smad3 in fibroblasts to inhibit Smad-dependent transcription. Preventive and therapeutic treatment with paricalcitol exerted potent antifibrotic effects and ameliorated bleomycin- as well as TBRI(CA)-induced fibrosis.. We characterise VDR as a negative regulator of TGF-β/Smad signalling. Impaired VDR signalling with reduced expression of VDR and decreased levels of its ligand may thus contribute to hyperactive TGF-β signalling and aberrant fibroblast activation in SSc.

Topics: Adult; Aged; Animals; Bleomycin; Disease Models, Animal; Ergocalciferols; Female; Fibroblasts; Fibrosis; Humans; Male; Mice; Middle Aged; Receptors, Calcitriol; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins; Transforming Growth Factor beta; Young Adult

We have previously described the antifibrotic role of the soluble guanylate cyclase (sGC). The mode of action, however, remained elusive. In the present study, we describe a novel link between sGC signalling and transforming growth factor β (TGFβ) signalling that mediates the antifibrotic effects of the sGC.. Human fibroblasts and murine sGC knockout fibroblasts were treated with the sGC stimulator BAY 41-2272 or the stable cyclic guanosine monophosphate (cGMP) analogue 8-Bromo-cGMP and stimulated with TGFβ. sGC knockout fibroblasts were isolated from sGCI(fl/fl) mice, and recombination was induced by Cre-adenovirus. In vivo, we studied the antifibrotic effects of BAY 41-2272 in mice overexpressing a constitutively active TGF-β1 receptor.. sGC stimulation inhibited TGFβ-dependent fibroblast activation and collagen release. sGC knockout fibroblasts confirmed that the sGC is essential for the antifibrotic effects of BAY 41-2272. Furthermore, 8-Bromo-cGMP reduced TGFβ-dependent collagen release. While nuclear p-SMAD2 and 3 levels, SMAD reporter activity and transcription of classical TGFβ target genes remained unchanged, sGC stimulation blocked the phosphorylation of ERK. In vivo, sGC stimulation inhibited TGFβ-driven dermal fibrosis but did not change p-SMAD2 and 3 levels and TGFβ target gene expression, confirming that non-canonical TGFβ pathways mediate the antifibrotic sGC activity.. We elucidated the antifibrotic mode of action of the sGC that increases cGMP levels, blocks non-canonical TGFβ signalling and inhibits experimental fibrosis. Since sGC stimulators have shown excellent efficacy and tolerability in phase 3 clinical trials for pulmonary arterial hypertension, they may be further developed for the simultaneous treatment of fibrosis and vascular disease in systemic sclerosis.

Topics: Animals; Case-Control Studies; Cells, Cultured; Collagen; Cyclic GMP; Disease Models, Animal; Fibroblasts; Fibrosis; Guanylate Cyclase; Humans; In Vitro Techniques; MAP Kinase Signaling System; Mice; Mice, Knockout; Pyrazoles; Pyridines; Receptors, Cytoplasmic and Nuclear; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins; Soluble Guanylyl Cyclase; Transforming Growth Factor beta

S100A4 is a calcium binding protein with regulatory functions in cell homeostasis, proliferation and differentiation that has been shown to promote cancer progression and metastasis. In the present study, we evaluated the role of S100A4 in fibroblast activation in systemic sclerosis (SSc).. The expression of S100A4 was analysed in human samples, murine models of SSc and in cultured fibroblasts by real-time PCR, immunohistochemistry and western blot. The functional role of S100A4 was evaluated using siRNA, overexpression, recombinant protein and S100A4 knockout (S100A4(-/-)) mice. Transforming growth factor β (TGF-β) signalling was assessed by reporter assays, staining for phosphorylated Smad2/3 and analyses of target genes.. The expression of S100A4 was increased in SSc skin and in experimental fibrosis in a TGF-β/Smad-dependent manner. Overexpression of S100A4 or stimulation with recombinant S100A4 induced an activated phenotype in resting normal fibroblasts. In contrast, knockdown of S100A4 reduced the pro-fibrotic effects of TGF-β and decreased the release of collagen. S100A4(-/-) mice were protected from bleomycin-induced skin fibrosis with reduced dermal thickening, decreased hydroxyproline content and lower myofibroblast counts. Deficiency of S100A4 also ameliorated fibrosis in the tight-skin-1 (Tsk-1) mouse model.. We characterised S100A4 as a downstream mediator of the stimulatory effects of TGF-β on fibroblasts in SSc. TGF-β induces the expression of S100A4 to stimulate the release of collagen in SSc fibroblasts and induce fibrosis. Since S100A4 is essentially required for the pro-fibrotic effects of TGF-β and neutralising antibodies against S100A4 are currently evaluated, S100A4 might be a candidate for novel antifibrotic therapies.

Topics: Adult; Aged; Animals; Disease Models, Animal; Female; Fibroblasts; Humans; Male; Mice; Mice, Knockout; Middle Aged; S100 Calcium-Binding Protein A4; S100 Proteins; Scleroderma, Systemic; Skin; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Young Adult

Fli-1, a potential predisposing factor for systemic sclerosis (SSc), is constitutively down-regulated in the lesional skin of patients with SSc by an epigenetic mechanism. To investigate the impact of Fli-1 deficiency on the induction of an SSc phenotype in various cell types, we generated bleomycin-induced skin fibrosis in Fli-1(+/-) mice and investigated the molecular mechanisms underlying its phenotypic alterations.. Messenger RNA (mRNA) levels and protein expression of target molecules were examined by quantitative reverse transcription-polymerase chain reaction and immunostaining. Transforming growth factor β (TGFβ) bioassay was used to evaluate the activation of latent TGFβ. The binding of Fli-1 to the target gene promoters was assessed with chromatin immunoprecipitation.. Bleomycin induced more severe dermal fibrosis in Fli-1(+/-) mice than in wild-type mice. Fli-1 haploinsufficiency activated dermal fibroblasts via the up-regulation of αvβ3 and αvβ5 integrins and activation of latent TGFβ. Dermal fibrosis in Fli-1(+/-) mice was also attributable to endothelial-to-mesenchymal transition, which is directly induced by Fli-1 deficiency and amplified by bleomycin. Th2/Th17-skewed inflammation and increased infiltration of mast cells and macrophages were seen, partly due to the altered expression of cell adhesion molecules in endothelial cells as well as the induction of the skin chemokines. Fli-1(+/-) mouse macrophages preferentially differentiated into an M2 phenotype upon stimulation with interleukin-4 (IL-4) or IL-13.. Our findings provide strong evidence for the fundamental role of Fli-1 deficiency in inducing SSc-like phenotypic alterations in dermal fibroblasts, endothelial cells, and macrophages in a manner consistent with human disease.

Topics: Animals; Bleomycin; Cell Movement; Cytokines; Disease Models, Animal; Endothelium, Vascular; Epithelial-Mesenchymal Transition; Female; Fibrosis; Haploinsufficiency; Immune System; Integrins; Macrophages; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Proto-Oncogene Protein c-fli-1; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

The objective was to survey the expression and localization of Th17-related cytokines and their correlation with skin lesion severity in early systemic sclerosis (SSc).. The mRNA expression was detected by real-time quantitative polymerase chain reaction (RT-qPCR) from 21 SSc patients and 12 healthy controls (HC). The protein expression was examined by immunohistochemistry (IHC) and Western blotting.. The RT-qPCR analysis showed a significantly higher expression of IL-17A, IL-21, IL-22, IL-26, IL-17RA, IL-21R, and IL-22R1 mRNA; consistently, the IHC analysis showed an over-expression of IL-17RA, IL-21R and IL-22R1 and the Western blotting analysis showed an over-expression of IL-17A, IL-21, IL-21R and IL-22R1 in early SSc skin lesions. The mRNA levels of IL-21 were higher in diffuse cutaneous than limited cutaneous SSc lesions. The mRNA expression of IL-26, IL-22, IL-22R1, mRNA and protein expression of IL-17A, IL-21, IL-21R were positively correlated with the modified Rodnan skin score of SSc. In addition, the mRNA levels of ICAM-1 were positively correlated with IL-17A/IL-17RA, and VEGFA and IL-4 were both positively correlated with IL-21/IL-21R, while TGF-β were moderately negatively correlated with IL-22/IL-22R1.. Th17 cytokines contribute to progression in early SSc skin lesions. IL-21/IL-21R could act as potential biomarkers presenting early SSc skin lesions severity.

Topics: Adult; Aged; Early Diagnosis; Female; Gene Expression; Humans; Intercellular Adhesion Molecule-1; Interleukin-17; Interleukin-21 Receptor alpha Subunit; Interleukin-22; Interleukin-4; Interleukins; Male; Middle Aged; Receptors, Interleukin; Receptors, Interleukin-17; RNA, Messenger; Scleroderma, Systemic; Severity of Illness Index; Skin; Th17 Cells; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Fibrillin-1 (FBN1) deficiency-induced systemic sclerosis is attributed to elevation of interleukin-4 (IL4) and TGF-β, but the mechanism underlying FBN1 deficiency-associated osteopenia is not fully understood. We show that bone marrow mesenchymal stem cells (BMMSCs) from FBN1-deficient (Fbn1(+/-)) mice exhibit decreased osteogenic differentiation and increased adipogenic differentiation. Mechanistically, this lineage alteration is regulated by IL4/IL4Rα-mediated activation of mTOR signaling to down-regulate RUNX2 and up-regulate PPARγ2, respectively, via P70 ribosomal S6 protein kinase (P70S6K). Additionally, we reveal that activation of TGF-β/SMAD3/SP1 signaling results in enhancement of SP1 binding to the IL4Rα promoter to synergistically activate mTOR pathway in Fbn1(+/-) BMMSCs. Blockage of mTOR signaling by osteoblastic-specific knockout or rapamycin treatment rescues osteopenia phenotype in Fbn1(+/-) mice by improving osteogenic differentiation of BMMSCs. Collectively, this study identifies a previously unrecognized role of the FBN1/TGF-β/IL4Rα/mTOR cascade in BMMSC lineage selection and provides experimental evidence that rapamycin treatment may provide an anabolic therapy for osteopenia in Fbn1(+/-) mice.

Topics: Adipogenesis; Animals; Bone Diseases, Metabolic; Cell Differentiation; Cells, Cultured; Female; Fibrillin-1; Fibrillins; Flow Cytometry; Immunoblotting; Immunosuppressive Agents; Male; Mesenchymal Stem Cells; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Microfilament Proteins; Microscopy, Fluorescence; Osteogenesis; Receptors, Cell Surface; RNA Interference; Scleroderma, Systemic; Signal Transduction; Sirolimus; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

Mesenchymal responses are an essential aspect of tissue repair. Failure to terminate this repair process correctly, however, results in fibrosis and organ dysfunction. Therapies that block fibrosis and restore tissue homeostasis are not yet available for clinical use. Here we characterize the nuclear receptor NR4A1 as an endogenous inhibitor of transforming growth factor-β (TGF-β) signaling and as a potential target for anti-fibrotic therapies. NR4A1 recruits a repressor complex comprising SP1, SIN3A, CoREST, LSD1, and HDAC1 to TGF-β target genes, thereby limiting pro-fibrotic TGF-β effects. Even though temporary upregulation of TGF-β in physiologic wound healing induces NR4A1 expression and thereby creates a negative feedback loop, the persistent activation of TGF-β signaling in fibrotic diseases uses AKT- and HDAC-dependent mechanisms to inhibit NR4A1 expression and activation. Small-molecule NR4A1 agonists can overcome this lack of active NR4A1 and inhibit experimentally-induced skin, lung, liver, and kidney fibrosis in mice. Our data demonstrate a regulatory role of NR4A1 in TGF-β signaling and fibrosis, providing the first proof of concept for targeting NR4A1 in fibrotic diseases.

Topics: Adolescent; Adult; Aged; Animals; Case-Control Studies; Cells, Cultured; Co-Repressor Proteins; Female; Fibroblasts; Fibrosis; Histone Deacetylase 1; Histone Demethylases; Humans; Idiopathic Pulmonary Fibrosis; Liver; Liver Cirrhosis, Alcoholic; Lung; Male; Mice; Mice, Knockout; Middle Aged; Nuclear Receptor Subfamily 4, Group A, Member 1; Repressor Proteins; Scleroderma, Systemic; Signal Transduction; Sin3 Histone Deacetylase and Corepressor Complex; Skin; Sp1 Transcription Factor; Transforming Growth Factor beta; Wound Healing; Young Adult

Up to 10% of patients with systemic sclerosis (SSc) develop pulmonary arterial hypertension (PAH). This risk persists throughout the disease and is time dependent, suggesting that SSc is a susceptibility factor. Outcome for SSc-PAH is poor compared with heritable or idiopathic forms, despite clinical and pathological similarities. Although susceptibility in heritable PAH and idiopathic PAH is strongly associated with gene mutations leading to reduced expression of bone morphogenetic protein receptor (BMPR) II, these mutations have not been observed in SSc-PAH.. To explore BMPRII expression and function in a mouse model of SSc (TβRIIΔk-fib) that is susceptible to developing pulmonary hypertension and in SSc lung.. BMPRII and downstream signaling pathways were profiled in lung tissue and fibroblasts from the TβRIIΔk-fib model, which develops pulmonary vasculopathy with pulmonary hypertension that is exacerbated by SU5416. Complementary studies examined SSc or control lung tissue and fibroblasts.. Our study shows reduced BMPRII, impaired signaling, and altered receptor turnover activity in a transforming growth factor (TGF)-β-dependent mouse model of SSc-PAH. Similarly, a significant reduction in BMPRII expression is observed in SSc lung tissue and fibroblasts. Increased proteasomal degradation of BMPRII appears to underlie this and may result from heightened TGF-β activity.. We found reduced BMPRII protein in patients with SSc-PAH and a relevant mouse model associated with increased proteasomal degradation of BMPRII. Collectively, these results suggest that impaired BMP signaling, resulting from TGF-β-dependent increased receptor degradation, may promote PAH susceptibility in SSc and provide a unifying mechanism across different forms of PAH.

Topics: Animals; Blotting, Western; Bone Morphogenetic Protein Receptors, Type II; Disease Models, Animal; Fibroblasts; Humans; Hypertension, Pulmonary; Lung; Mice; Mice, Transgenic; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Persistent fibroblast activation underlies skin fibrosis in systemic sclerosis (SSc), but the transcriptional and epigenetic mechanisms controlling this process are not well understood. In view of the potent influence of acetylation status governing tissue fibrosis, we undertook this study to investigate the expression of the antiaging deacetylase enzyme sirtuin 1 (SIRT1) in SSc and its effects on fibrotic responses in vitro and in vivo.. Tissue expression of SIRTs was interrogated from publicly available genome-wide expression data sets and by immunohistochemistry. The effects of SIRT1 on modulating fibrotic responses, as well as the underlying mechanisms, were examined in human and mouse fibroblasts in culture and in an experimental fibrosis model in the mouse.. Analysis of transcriptome data revealed a selective reduction of SIRT1 messenger RNA (mRNA) levels in SSc skin biopsy samples as well as a negative correlation of SIRT1 mRNA with the skin score. Cellular SIRT1 levels were suppressed in normal fibroblasts exposed to hypoxia or platelet-derived growth factor and were constitutively down-regulated in SSc fibroblasts. Activation of SIRT1 attenuated fibrotic responses in skin fibroblasts and skin organ cultures, while genetic or pharmacologic inhibition of SIRT1 had profibrotic effects. The antifibrotic effects of SIRT1 were due in part to decreased expression and function of the acetyltransferase p300. In mice, experimentally induced skin fibrosis was accompanied by reduced SIRT1 expression in lesional tissue fibroblasts, and both fibrosis and loss of SIRT1 in these mice were mitigated by treatment with a SIRT1 activator.. SIRT1 has antifibrotic effects, and its reduced tissue expression in patients with SSc might have a direct causal role in progression of fibrosis. Pharmacologic modulation of SIRT1 in these patients therefore might represent a potential treatment strategy.

Topics: Animals; Case-Control Studies; Cells, Cultured; Disease Models, Animal; Enzyme Inhibitors; Fibroblasts; Humans; Mice; p300-CBP Transcription Factors; Platelet-Derived Growth Factor; Real-Time Polymerase Chain Reaction; Resveratrol; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Sirtuin 1; Skin; Smad Proteins; Stilbenes; Transforming Growth Factor beta

Topics: Animals; Female; Fibroblasts; Humans; Idiopathic Pulmonary Fibrosis; Liver; Liver Cirrhosis, Alcoholic; Lung; Male; Nuclear Receptor Subfamily 4, Group A, Member 1; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is an autoimmune disease characterized by skin and internal organ fibrosis, caused by microvascular dysfunction. In recent years, the hypothesis that anti-endothelial cell antibodies (AECA) play a key role in microvascular damage seems to be increasingly convincing. In fact, AECA can induce antibody-dependent cellular apoptosis and stimulate the microvasculature to release pro-inflammatory and pro-fibrotic cytokines. Human-microvascular-endothelial-cells (MVECs) were stimulated with SSc sera (with and without AECA) and with sera from healthy donors. The conditioned MVEC culture media were then added to fibroblast cultures obtained from control skin (CTR), non-affected skin of SSc patients (NA), and affected skin of the same sclerodermic (SSc) patients, respectively. AECA contributed to the MVEC increased release of endothelin-1 (ET-1) in the culture medium and to MVEC apoptosis. Fibroblast (CTR, NA, and SSc) proliferation was increased after treatment with AECA-positive conditioned media, compared to AECA-negative and control conditioned media. Furthermore, both AECA-positive (in major contribution) and AECA-negative conditioned media were responsible for alpha-smooth-muscle-actin (αSMA) over-expression in all fibroblast cultures, compared to control conditioned media. Fibroblast type I collagen synthesis was upregulated by both SSc conditioned media (with and without AECA). Finally, the synthesis of fibroblast transforming-growth-factor-beta (TGF-β) was statistically higher in AECA-positive conditioned media, compared to AECA-negative and control conditioned media. These findings support the concept that AECA may mediate the crosstalk between endothelial damage and dermal-fibroblast activation in SSc.

Topics: Actins; Autoantibodies; Cells, Cultured; Collagen Type I; Culture Media, Conditioned; Endothelial Cells; Endothelin-1; Endothelium; Female; Fibroblasts; Humans; Microvessels; Middle Aged; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Pulmonary arterial hypertension (PAH) can be idiopathic or secondary to autoimmune diseases, and it represents one of the most threatening complications of systemic sclerosis (SSc). Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine with proinflammatory functions that appears to be involved in the pathogenesis of hypoxia-induced PH. In SSc patients, high serum levels of MIF have been associated with the development of ulcers and PAH. Stem cell growth factor β (SCGF β) is a human growth factor that, together with MIF, is involved in the pathogenesis of chronic spinal cord injury. The aim of our study was to measure serum levels of MIF in patients with idiopathic and SSc-associated PAH. We enrolled 13 patients with idiopathic PAH and 15 with SSc-associated PAH. We also selected 14 SSc patients without PAH and 12 normal healthy controls, matched for sex and age. PAH was confirmed by right hearth catheterism (mPAP>25 mmHg). MIF and SCGF β levels were measured by ELISA. We found significantly higher circulating levels of MIF and of SCGF β in patients with idiopathic PAH (P=0.03 and P=0.004) and with PAH secondary to SSc (P=0.018 and P=0.023) compared to SSc patients without PAH. Higher levels of MIF were found in those patients with an higher New York Heart Association (NYHA) class (P=0.03). We can hypothesize that MIF and SCGF β are able to play a role in PAH, both idiopathic or secondary, and in the future they may be evaluated as useful biomarkers and prognostic factors for this serious vascular disease.

Topics: Aged; Biomarkers; Case-Control Studies; Female; Humans; Hypertension, Pulmonary; Macrophage Migration-Inhibitory Factors; Middle Aged; Predictive Value of Tests; Prognosis; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Sensitivity and Specificity; Transforming Growth Factor beta

The most prominent feature of systemic sclerosis (SSc) and other diseases associated with fibrosis is the prolonged activation of fibroblasts not eliminated by apoptosis, hence characterized by accumulation of more extra cellular matrix (ECM). We tend to verify if microRNA-29a (miR-29a) as an anti-fibrotic factor could induce apoptosis in SSc fibroblasts. We did not detect apoptosis in SSc fibroblasts. We found that Bcl-2 expression was upregulated in SSc fibroblasts and the ratio of Bax:Bcl-2 in these cells was significantly lower (p = 0.02) compared to normal fibroblasts. Transfection of both SSc and transforming growth factor-β (TGF-β) stimulated fibroblasts by miR-29a mimic, significantly decreased the expression of two anti-apoptotic members of the Bcl-2 family, Bcl-2 (p = 0.0005, p = 0.01) and Bcl-XL (p = 0.0001, p = 0.006), resulted in enhanced Bax:Bcl-2 ratio and induced a high rate of apoptosis. Recently, miR-29 has been introduced as an anti-fibrotic factor with potential therapeutic effect on SSc. Until now, it has not been proposed whether there is a relationship between miR-29a and apoptosis in SSc. According to our results, it seems that miR-29a is a potent inducer of apoptosis in SSc fibroblasts and an attenuator of ECM production in these cells. MiR-29a disrupted the expression profiling of Bcl-2 family proteins (Bax, Bcl-2 and Bcl-XL) which is the central point of dynamic life-death rheostat in many apoptotic pathways. Furthermore, dermal fibroblasts from patients with SSc showed elevation in TNF-α mRNA levels, while restoration of miR-29a decreases TNF-α production in these cells. Although further molecular studies are necessary to investigate the underlying apoptotic pathways, the present findings suggest that anti-fibrotic and pro-apoptotic properties of miR-29a could provide novel benefits toward the development of fibroblast-specific anti-fibrotic therapies.

Topics: Apoptosis; bcl-2-Associated X Protein; Cells, Cultured; Dermis; Fibroblasts; Gene Expression Regulation; Humans; MicroRNAs; Proto-Oncogene Proteins c-bcl-2; Scleroderma, Systemic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The precise mechanisms of tissue fibrosis have not yet been elucidated in systemic sclerosis (SSc). However, studies of the regulation of DNA methylation, the most widely studied epigenetic mechanism, have confirmed the involvement of the TET family proteins, recently identified DNA demethylases, in the pathogenesis of SSc. The mRNA levels of TET family members were compared in normal and SSc fibroblasts. The effects of hypoxia and siRNA specific to HIF-1α on TET expression were also examined. Global methylation status was analysed by LUMA. The presence of 5-hydroxymethylcytosine (5hmC) in SSc was examined by immunohistochemistry. The level of TET1 mRNA in SSc fibroblasts was elevated by 1.68 fold compared with that of normal fibroblasts, but the expression levels of TET2 and TET3 were comparable between both cell types. The expression levels of DNMT1 and DNMT3B mRNA have a tendency to elevate in SSc fibroblasts. Among TET family members, the expression of TET1 was exclusively induced by hypoxia via HIF-1α-independent pathways in SSc fibroblasts, but not in normal fibroblasts. The methylation level was decreased in SSc fibroblasts relative to normal fibroblasts, and 5hmC was present in dermal fibroblasts of skin sections from patients with SSc. TET1 expression in SSc fibroblasts was abnormally regulated in the hypoxic environment and accompanied by global DNA hypomethylation, suggesting the involvement of aberrant DNA methylation in the pathogenesis of SSc.

Topics: Case-Control Studies; Cells, Cultured; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; DNA Methyltransferase 3B; Fibroblasts; Humans; Hypoxia; Mixed Function Oxygenases; Proto-Oncogene Proteins; Scleroderma, Systemic; Transforming Growth Factor beta

Uncontrolled fibrosis of skin and internal organs is the main characteristic of scleroderma, and collagen is a major extracellular matrix protein that deposits in the fibrotic organs. As the chaperone of collagen, heat shock protein 47 (HSP47) is closely related with the development of fibrosis. To explore the potential function of HSP47 in the pathogenesis of scleroderma, the clinical, in vivo and in vitro studies were performed. In clinical, the increased mRNA level of HSP47 was observed in the skin fibroblasts and PBMC from scleroderma patients, and the enhanced protein level of HSP47 was also detected in the skin biopsy and plasma of the above patients. Unexpectedly, the enhanced levels of HSP47 were positively correlated with the presence of anti-centromere antibody in scleroderma patients. Moreover, a high expression of HSP47 was found in the skin lesion of BLM-induced scleroderma mouse model. Further in vitro studies demonstrated that HSP47 knockdown could block the intracellular and extracellular collagen over-productions induced by exogenous TGF-β. Therefore, the results in this study provide direct evidence that HSP47 is involved in the pathogenesis of scleroderma. The high expression of HSP47 can be detected in the circulatory system of scleroderma patients, indicating that HSP47 may become a pathological marker to assess the progression of scleroderma, and also explain the systemic fibrosis of scleroderma. Meanwhile, collagen over-expression is blocked by HSP47 knockdown, suggesting the possibility that HSP47 can be a potential therapeutic target for scleroderma.

Topics: Adolescent; Adult; Animals; Biopsy; Blotting, Western; Cells, Cultured; Collagen; Female; Fibroblasts; Fibrosis; HSP47 Heat-Shock Proteins; Humans; Leukocytes, Mononuclear; Male; Mice; Mice, Inbred C3H; Middle Aged; NIH 3T3 Cells; Protein Binding; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Young Adult

Transforming growth factor (TGF)-β and interleukin (IL)-13 play a crucial role in the pathogenesis of systemic sclerosis (SSc), partly through activation of collagen production that leads to fibrosis. The aim of the present study was to determine whether TFG-β alters IL-13 production in T lymphocytes from patients with SSc from that seen in those of healthy donors.. IL-13 mRNA and protein synthesis under TFG-β exposure was measured in circulating T lymphocytes from healthy donors and patients with SSc and also in the Jurkat Th2 T-cell line, using quantitative real-time PCR and fluorescence-activated cell sorting analysis, respectively. The involvement of Smad and GATA-3 transcription factors was assessed by using specific inhibitors and small interfering RNA, and the binding capacity of GATA-3 to the IL-13 gene promoter was evaluated by chromatin immunoprecipitation assay.. TGF-β induced a significant decrease in IL-13 mRNA and protein levels in lymphocytes from healthy donors (mean [±SD] inhibition of 30% ± 10% and 20% ± 7%, respectively; p < 0.05). In contrast, TGF-β promoted a significant increase in IL-13 mRNA levels and IL-13 synthesis by CD4(+) and CD8(+) T-cell subtypes from patients with SSc, with respective increases of 2.4 ± 0.3-fold, 1.6 ± 0.05-fold and 2.7 ± 0.02-fold. The involvement of the Smad signaling pathway and upregulation of GATA-3 binding capacity on the IL-13 promoter in lymphocytes from patients with SSc contributed to the effect of TGF-β on IL-13 production.. These results demonstrate that TGF-β upregulates IL-13 synthesis through GATA-3 expression in the T lymphocytes of patients with SSc, confirming that the GATA-3 transcription factor can be regarded as a novel therapeutic target in patients with SSc.

Topics: Adult; Aged; Biomarkers; Female; GATA3 Transcription Factor; Humans; Interleukin-13; Jurkat Cells; Male; Middle Aged; Scleroderma, Systemic; T-Lymphocytes; Transforming Growth Factor beta; Young Adult

IL-35 is a member of the IL-12 family consisting of p35/IL-12a and EBI3/IL-27b subunits. IL-35 exerts immunomodulatory activities in experimental and human autoimmune inflammatory conditions. Our aim was to assess IL-35 expression in the skin and circulation of SSc patients and to characterize its potential association with SSc-related features.. Expression of IL-35 in skin and dermal fibroblasts was quantified by quantitative PCR, immunohistochemistry and immunofluorescence. Serum levels of IL-35 (by ELISA), CRP (by turbidimetry), ANA (by immunofluorescence) and autoantibodies of the ENA complex (by immunoblot) were measured in 40 SSc patients. Serum IL-35 was determined in 40 age- and sex-matched healthy controls.. IL-35 expression was increased in SSc skin and dermal fibroblasts in a TGF-β-dependent manner. IL-35 induced an activated phenotype in resting fibroblasts and enhanced the release of collagen. IL-35 serum levels were increased in patients with SSc compared with healthy controls [median 83.9 (interquartile range 45.1-146.1) vs 36.2 (interquartile range 17.2-49.4) pg/ml, P < 0.0001]. Serum IL-35 was negatively correlated with disease duration (r = -0.4339, P = 0.0052). In line with this finding, serum IL-35 was increased in patients with an early SSc pattern on capillaroscopy assessment compared with those with active and late SSc patterns.. The present study demonstrates overexpression of IL-35 in SSc skin, dermal fibroblasts and serum. TGF-β induces IL-35, which in turn activates resting fibroblasts and enhances the release of collagen, thereby contributing to aberrant TGF-β signalling in SSc. Increased serum IL-35 is associated with early, inflammatory stages of SSc.

Topics: Adult; Aged; Case-Control Studies; Cells, Cultured; Collagen; Female; Fibroblasts; Humans; Interleukins; Male; Middle Aged; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Up-Regulation

High endothelin-1 (ET-1) and transforming growth factor-β (TGF-β) levels may induce in healthy endothelial cells (EC) an endothelial-to-mesenchymal transition (EndMT). The same cytokines are associated with fibrosis development in systemic sclerosis (SSc). Although EndMT has not been definitively shown in SSc, this process, potentially induced by a stimulatory loop involving these 2 cytokines, overexpressed in this disease might contribute to fibroblast accumulation in affected tissues. Macitentan (MAC), an ET-1 receptor antagonist interfering with this loop, might prevent EndMT and fibroblast accumulation.. EC, isolated from healthy controls (HC) and patients with SSc, were treated with ET-1 and TGF-β and successively analyzed for gene and protein expressions of endothelial and mesenchymal markers, and for Sma- and Mad-related (SMAD) phosphorylation. Further, in the supernatants, we evaluated ET-1 and TGF-β production by ELISA assay. In each assay we evaluated the ability of MAC to inhibit both the TGF-β and ET-1 effects.. We showed that both TGF-β and ET-1 treatments induced an activation of the EndMT process in SSc-EC as reported in HC cells. The ELISA assays showed a mutual TGF-β and ET-1 induction in both SSc-EC and HC-EC. A statistically significant increase of SMAD phosphorylation after treatment was observed in SSc-EC. In each assay, MAC inhibited both TGF-β and ET-1 effects.. Our work is the first demonstration in literature that SSc-EC, under the synergistic effect of TGF-β and ET-1, may transdifferentiate toward myofibroblasts, thus contributing to fibroblast accumulation. MAC, interfering with this process in vitro, may offer a new potential therapeutic strategy against fibrosis.

Topics: Adult; Biomarkers; Case-Control Studies; Cell Transdifferentiation; Cells, Cultured; Endothelial Cells; Endothelin-1; Enzyme-Linked Immunosorbent Assay; Female; Humans; Italy; Male; Pyrimidines; Reference Values; Scleroderma, Systemic; Sensitivity and Specificity; Statistics, Nonparametric; Sulfonamides; Transforming Growth Factor beta; Young Adult

Persistent fibroblast activation initiated by transforming growth factor β (TGF-β) is a fundamental event in the pathogenesis of systemic sclerosis, and its pharmacological inhibition represents a potential therapeutic strategy. The nuclear receptor, peroxisome proliferator-activated receptor γ (PPAR-γ), exerts potent fibrotic activity. The synthetic oleanane triterpenoid, 2-cyano-3,12-dioxo-olean-1,9-dien-28-oic acid (CDDO), is a PPAR-γ agonist with potential effects on TGF-β signalling and dermal fibrosis.. To examine the modulation of fibrogenesis by CDDO in explanted fibroblasts, skin organ cultures and murine models of scleroderma.. The effects of CDDO on experimental fibrosis induced by bleomycin injection or by overexpression of constitutively active type I TGF-β receptor (TgfbR1ca) were evaluated. Modulation of fibrotic gene expression was examined in human skin organ cultures. To delineate the mechanisms underlying the antifibrotic effects of CDDO, explanted skin fibroblasts cultured in two-dimensional monolayers or in three-dimensional full-thickness human skin equivalents were studied.. CDDO significantly ameliorated dermal fibrosis in two complementary mouse models of scleroderma, as well as in human skin organ cultures and in three-dimensional human skin equivalents. In two-dimensional monolayer cultures of explanted normal fibroblasts, CDDO abrogated fibrogenic responses induced by TGF-β. These CDDO effects occurred via disruption of Smad-dependent transcription and were associated with inhibition of Akt activation. In scleroderma fibroblasts, CDDO attenuated the elevated synthesis of collagen. Remarkably, the in vitro antifibrotic effects of CDDO were independent of PPAR-γ.. The PPAR-γ agonist triterpenoid CDDO attenuates fibrogenesis by antagonistically targeting canonical TGF-β/Smad and Akt signalling in a PPAR-γ-independent manner. These findings identify this synthetic triterpenoid as a potential new therapy for the control of fibrosis.

Topics: Adipogenesis; Adult; Animals; Biopsy; Cells, Cultured; Collagen; Disease Models, Animal; Drug Evaluation, Preclinical; Female; Fibroblasts; Fibrosis; Humans; Infant, Newborn; Mice; Mice, Inbred C57BL; Oleanolic Acid; Organ Culture Techniques; PPAR gamma; Proto-Oncogene Proteins c-akt; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Targeted therapies for systemic sclerosis (SSc) and other fibrotic diseases are not yet available. We evaluated the efficacy of heat shock protein 90 (Hsp90) inhibition as a novel approach to inhibition of aberrant transforming growth factor (TGF)-β signalling and for the treatment of fibrosis in preclinical models of SSc.. Expression of Hsp90 was quantified by quantitative PCR, western blot and immunohistochemistry. The effects of Hsp90 inhibition were analysed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, in tight-skin (Tsk-1) mice and in mice overexpressing a constitutively active TGF-β receptor I (TβRI).. Expression of Hsp90β was increased in SSc skin and in murine models of SSc in a TGF-β-dependent manner. Inhibition of Hsp90 by 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) inhibited canonical TGF-β signalling and completely prevented the stimulatory effects of TGF-β on collagen synthesis and myofibroblast differentiation. Treatment with 17-DMAG decreased the activation of canonical TGF-β signalling in murine models of SSc and exerted potent antifibrotic effects in bleomycin-induced dermal fibrosis, in Tsk-1 mice and in mice overexpressing a constitutively active TβRI. Dermal thickness, number of myofibroblasts and hydroxyproline content were all significantly reduced on treatment with 17-DMAG. No toxic effects were observed with 17-DMAG at antifibrotic doses.. Hsp90 is upregulated in SSc and is critical for TGF-β signalling. Pharmacological inhibition of Hsp90 effectively blocks the profibrotic effects of TGF-β in cultured fibroblasts and in different preclinical models of SSc. These results have translational implications, as several Hsp90 inhibitors are in clinical trials for other indications.

Topics: Adult; Aged; Animals; Benzoquinones; Cells, Cultured; Disease Models, Animal; Female; Fibroblasts; Fibrosis; HSP90 Heat-Shock Proteins; Humans; Lactams, Macrocyclic; Male; Mice; Mice, Transgenic; Middle Aged; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc)-related interstitial lung disease (ILD) is one of the leading causes of mortality. We undertook this study to analyze the gene expression of lung tissue in a prospective cohort of patients with SSc-related ILD and to compare it with that in control lungs and with 2 prospective clinical parameters in order to understand the molecular pathways implicated in progressive lung disease.. Lung tissue was obtained by open lung biopsy in 28 consecutive patients with SSc-related ILD and in 4 controls. High-resolution computed tomography (HRCT) and pulmonary function testing (PFT) were performed at baseline and 2-3 years after treatment based on lung histologic classification. Microarray analysis was performed, and the results were correlated with changes in the HRCT score (FibMax) and PFT values. Quantitative polymerase chain reaction (qPCR) and immunohistochemistry were used to confirm differential levels of messenger RNA and protein.. Lung microarray data distinguished patients with SSc-related ILD from healthy controls. In the lungs of patients with SSc-related ILD who had nonspecific interstitial pneumonia (NSIP), expressed genes included macrophage markers, chemokines, collagen, and transforming growth factor β (TGFβ)- and interferon (IFN)-regulated genes. Expression of these genes correlated with progressive lung fibrosis defined by the change in FibMax. Immunohistochemistry confirmed increased markers of collagen (COL1A1), IFN (OAS1 and IFI44), and macrophages (CCL18 and CD163), and the positive correlation with the change in FibMax was confirmed by qPCR in a larger group of SSc patients with NSIP. Several genes correlated with both the change in FibMax (r > 0.4) and the change in % predicted forced vital capacity (r < -0.1), including IFN and macrophage markers, chemokines, and heat-shock proteins.. These results highlight major pathogenic pathways relevant to progressive pulmonary fibrosis in SSc-related ILD: macrophage emigration and activation, and up-regulated expression of TGFβ- and IFN-regulated genes.

Topics: 2',5'-Oligoadenylate Synthetase; Adult; Antigens; Antigens, CD; Antigens, Differentiation, Myelomonocytic; Chemokines, CC; Collagen Type I; Collagen Type I, alpha 1 Chain; Cytoskeletal Proteins; Disease Progression; Female; Humans; Lung; Macrophage Activation; Male; Middle Aged; Pulmonary Fibrosis; Receptors, Cell Surface; Respiratory Function Tests; Scleroderma, Systemic; Transforming Growth Factor beta

Interstitial lung disease (ILD) is the leading cause of death in patients with systemic sclerosis (SSc; scleroderma). Although SSc-related ILD is more common and severe in African Americans than in Caucasians, little is known about factors underlying this significant health disparity. The aim of this study was to examine the role that low expression of caveolin-1 might play in susceptibility to ILD among African Americans.. Assays of monocyte migration toward stromal cell-derived factor 1 (SDF-1) were performed using monocytes from Caucasian and African American healthy donors and patients with SSc. For fibrocyte differentiation studies, total peripheral blood mononuclear cells were incubated on fibronectin-coated plates. Protein expression was evaluated by immunohistochemistry and Western blotting.. Monocytes from healthy African American donors and those from patients with SSc had low caveolin-1 levels, enhanced migration toward the CXCR4 ligand SDF-1, and enhanced differentiation to fibrocytes. Enhanced migration and differentiation of monocytes from African Americans and patients with SSc appeared to be attributable to the lack of caveolin-1, because restoring caveolin-1 function using a caveolin-1 scaffolding domain peptide inhibited these processes. Although they differed from monocytes from Caucasians, monocytes from both African Americans and patients with SSc were not identical, because SSc monocytes showed major increases from baseline in ERK, JNK, p38, and Smad2/3 activation, while monocytes from African Americans showed only limited ERK activation and no activation of JNK, p38, or Smad2/3. In contrast, SDF-1 exposure caused no additional ERK activation in SSc monocytes but did cause significant additional activation in monocytes from African Americans.. African Americans may be predisposed to SSc-related ILD due to low baseline caveolin-1 levels in their monocytes, potentially affecting signaling, migration, and fibrocyte differentiation. The monocytes of African Americans may lack caveolin-1 due to high levels of transforming growth factor β in their blood.

Topics: Black or African American; Caveolin 1; Cell Differentiation; Cell Movement; Cytoskeleton; Fibroblasts; Humans; In Vitro Techniques; Lung Diseases, Interstitial; MAP Kinase Signaling System; Monocytes; Receptors, CXCR4; Risk Factors; Scleroderma, Systemic; Transforming Growth Factor beta; White People

Topics: Angiotensin II; Angiotensin II Type 1 Receptor Blockers; Animals; Awards and Prizes; Fibrillins; Humans; Loeys-Dietz Syndrome; Marfan Syndrome; Mice; Microfilament Proteins; Scleroderma, Systemic; Signal Transduction; Societies, Medical; Transforming Growth Factor beta; Translational Research, Biomedical; United States

Systemic sclerosis (SSc), or scleroderma, similar to many fibrotic disorders, lacks effective therapies. Current trials focus on anti-inflammatory drugs or targeted approaches aimed at one of the many receptor mechanisms initiating fibrosis. In light of evidence that a myocardin-related transcription factor (MRTF)-and serum response factor (SRF)-regulated gene transcriptional program induced by Rho GTPases is essential for myofibroblast activation, we explored the hypothesis that inhibitors of this pathway may represent novel antifibrotics. MRTF/SRF-regulated genes show spontaneously increased expression in primary dermal fibroblasts from patients with diffuse cutaneous SSc. A novel small-molecule inhibitor of MRTF/SRF-regulated transcription (CCG-203971) inhibits expression of connective tissue growth factor (CTGF), α-smooth muscle actin (α-SMA), and collagen 1 (COL1A2) in both SSc fibroblasts and in lysophosphatidic acid (LPA)-and transforming growth factor β (TGFβ)-stimulated fibroblasts. In vivo treatment with CCG-203971 also prevented bleomycin-induced skin thickening and collagen deposition. Thus, targeting the MRTF/SRF gene transcription pathway could provide an efficacious new approach to therapy for SSc and other fibrotic disorders.

Topics: Animals; Cell Culture Techniques; Cell Proliferation; Collagen Type I; Connective Tissue Growth Factor; Disease Models, Animal; DNA-Binding Proteins; Female; Humans; Lysophospholipids; Male; Mice; Mice, Inbred C57BL; Middle Aged; Myofibroblasts; NIH 3T3 Cells; Nipecotic Acids; Oncogene Proteins, Fusion; Scleroderma, Systemic; Serum Response Factor; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta

To determine serum and sputum Caveolin-1 (Cav-1) levels and their associations with transforming growth factor- ß (TGF-ß) and interstitial lung disease (ILD) in systemic sclerosis (SSc).. Serum and induced sputum samples from 55 patients with SSc, 25 asthma patients and 16 healthy volunteers (HC) were tested for Cav-1 and TGF-ß by the ELISA technique. As a possible downstream signaling regulator of TGF-ß, Endothelin-1 (ET-1), a potent profibrotic protein, was also measured in all serum and sputum samples and relations with Cav-1 and TGF-ß were sought. All scleroderma patients were evaluated for their clinical and laboratory parameters. Pulmonary function tests (PFT) and high resolution computerized tomography (HRCT) were performed for the diagnosis of ILD. The alveolitis-fibrosis index and the SSc disease severity scores were noted for each patient.. Serum Cav-1 levels were lower in SSc compared to HC (p<0.01). Cav-1 levels were significantly lower in the sputum of SSc patients compared to both control groups (p<0.001). It was also found significantly lower in SSc-ILD compared to those without ILD (0.19±0.04 vs 0.25±0.07, respectively, p<0.01). Although no difference was found in the serum TGF-ß levels among the groups, sputum TGF-ß levels correlated positively with the alveolitis index (r=0.34) and correlated inversely with FVC measurements (r=-0.44, p<0.05) among SSc patients. Serum ET-1 was significantly higher in SSc patients (p<0.01) but no association was found between ET-1 and Cav-1 or TGF-ß.. These results suggest that decreased sputum Cav-1 levels is associated with SSc related-ILD and may be used as a marker for the detection of SSc-ILD.

Topics: Adult; Aged; Case-Control Studies; Caveolin 1; Endothelin-1; Female; Humans; Lung Diseases, Interstitial; Male; Middle Aged; Prospective Studies; Scleroderma, Systemic; Sputum; Transforming Growth Factor beta

Topics: Animals; Collagen; Fibroblasts; Fibrosis; Humans; Mice; S100 Calcium-Binding Protein A4; S100 Proteins; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a chronic autoimmune disease clinically manifesting as progressive fibrosis of the skin and internal organs. Recent microarray studies demonstrated that cadherin 11 (Cad-11) expression is increased in the affected skin of patients with SSc. The purpose of this study was to examine our hypothesis that Cad-11 is a mediator of dermal fibrosis.. Biopsy samples of skin from SSc patients and healthy control subjects were used for real-time quantitative polymerase chain reaction analysis to assess Cad-11 expression and for immunohistochemistry to determine the expression pattern of Cad-11. To determine whether Cad-11 is a mediator of dermal fibrosis, Cad-11-deficient mice and anti-Cad-11 monoclonal antibodies (mAb) were used in the bleomycin-induced dermal fibrosis model. In vitro studies with dermal fibroblasts and bone marrow-derived macrophages were used to determine the mechanisms by which Cad-11 contributes to the development of tissue fibrosis.. Levels of messenger RNA for Cad-11 were increased in skin biopsy samples from patients with SSc and correlated with the modified Rodnan skin thickness scores. Cad-11 expression was localized to dermal fibroblasts and macrophages in SSc skin. Cad-11-knockout mice injected with bleomycin had markedly attenuated dermal fibrosis, as quantified by measurements of skin thickness, collagen levels, myofibroblast accumulation, and profibrotic gene expression, in lesional skin as compared to the skin of wild-type mice. In addition, anti-Cad-11 mAb decreased fibrosis at various time points in the bleomycin-induced dermal fibrosis model. In vitro studies demonstrated that Cad-11 regulated the production of transforming growth factor β (TGFβ) by macrophages and the migration of fibroblasts.. These data demonstrate that Cad-11 is a mediator of dermal fibrosis and TGFβ production and suggest that Cad-11 may be a therapeutic target in SSc.

Topics: Adult; Animals; Cadherins; Cell Movement; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Humans; Macrophages; Male; Mice; Mice, Knockout; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Excessive extracellular matrix deposition that occurs in many fibrotic skin disorders such as hypertrophic scarring and scleroderma is often associated with hypoxia. CD109 is a novel TGF-β co-receptor and TGF-β antagonist shown to inhibit TGF-β-induced extracellular matrix protein production in vitro. We examined whether CD109 is able to regulate extracellular matrix deposition under low oxygen tension in vivo using transgenic mice overexpressing CD109 in the epidermis. By creating dorsal bipedicle skin flaps with centrally located excisional wounds in these mice and their wild-type littermates, we generated a novel murine hypoxic wound model. Mice were sacrificed on 7 or 14 days post-wounding, and tissues were harvested for histological and biochemical analysis. Hypoxic wounds in both transgenic and wild-type mice showed increased levels of HIF-1α and delayed wound closure, validating this model in mice. Hypoxic wounds in CD109 transgenic mice demonstrated decreased collagen type 1 and fibronectin expression, and reduced dermal thickness on day 7 post-wounding as compared to those in wild-type mice and to non-hypoxic control wounds. These results suggest that CD109 decreases extracellular matrix production and fibrotic responses during hypoxic wound healing. Manipulating CD109 levels may have potential therapeutic value for the treatment of fibrotic skin disorders associated with poor oxygen delivery.

Topics: Animals; Antigens, CD; Cicatrix; Fibrosis; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Transgenic; Neoplasm Proteins; Oxygen; Scleroderma, Systemic; Transforming Growth Factor beta; Wound Healing

The crucial role of innate immunity in the pathogenesis of systemic sclerosis (SSc) is well established, and in the past few years the hypothesis that Toll-like receptor 4 (TLR4) activation induced by endogenous ligands is involved in fibrogenesis has been supported by several studies on skin, liver, and kidney fibrosis. These findings suggest that TLR4 activation can enhance transforming growth factor beta (TGF-β) signaling, providing a potential mechanism for TLR4/Myeloid differentiation factor 88 (MyD88)-dependent fibrosis.. The expression of TLR4, CD14 and MD2 genes was analyzed by real-time polymerase chain reaction from skin biopsies of 24 patients with diffuse cutaneous SSc. In order to investigate the effects of the chronic skin exposure to endotoxin (Lipopolysaccharide (LPS)) in vivo we examined the expression of inflammation, TGF-β signaling and cellular markers genes by nanostring. We also identified cellular subsets by immunohistochemistry and flow cytometry.. We found that TLR4 and its co-receptors, MD2 and CD14, are over-expressed in lesional skin from patients with diffuse cutaneous SSc, and correlate significantly with progressive or regressive skin disease as assessed by the Delta Modified Rodnan Skin Score. In vivo, a model of chronic dermal LPS exposure showed overexpression of proinflammatory chemokines, recruitment and activation of macrophages, and upregulation of TGF-β signature genes.. We delineated the role of MyD88 as necessary for the induction not only for the early phase of inflammation, but also for pro-fibrotic gene expression via activation of macrophages. Chronic LPS exposure might be a model of early stage of SSc when inflammation and macrophage activation are important pathological features of the disease, supporting a role for innate immune activation in SSc skin fibrosis.

Topics: Animals; Fibrosis; Flow Cytometry; Humans; Immunohistochemistry; Inflammation; Macrophage Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Differentiation Factor 88; Real-Time Polymerase Chain Reaction; Scleroderma, Systemic; Skin; Toll-Like Receptor 4; Transcriptome; Transforming Growth Factor beta

Nuclear receptors regulate cell growth, differentiation, and homeostasis. Selective nuclear receptors promote fibroblast activation, which leads to tissue fibrosis, the hallmark of systemic sclerosis (SSc). This study was undertaken to investigate the effects of constitutive androstane receptor (CAR)/NR1I3, an orphan nuclear receptor, on fibroblast activation and experimental dermal fibrosis.. CAR expression was quantified by quantitative polymerase chain reaction, Western blotting, immunohistochemistry, and immunofluorescence. CAR expression was modulated by small molecules, small interfering RNA, forced overexpression, and site-directed mutagenesis. The effects of CAR activation were analyzed in cultured fibroblasts, in bleomycin-induced dermal fibrosis, and in mice overexpressing a constitutively active transforming growth factor β (TGFβ) receptor type I (TβRI-CA).. Up-regulation of CAR was detected in the skin and in dermal fibroblasts in SSc patients. Stimulation of healthy fibroblasts with TGFβ induced the expression of CAR messenger RNA and protein in a Smad-dependent manner. Pharmacologic activation or overexpression of CAR in healthy fibroblasts significantly increased the stimulatory effects of TGFβ on collagen synthesis and myofibroblast differentiation, and amplified the stimulatory effects of TGFβ on COL1A2 transcription activity. Treatment with CAR agonist increased the activation of canonical TGFβ signaling in murine models of SSc and exacerbated bleomycin-induced and TβRI-CA-induced fibrosis with increased dermal thickening, myofibroblast counts, and collagen accumulation.. Our findings indicate that CAR is up-regulated in SSc and regulates TGFβ signaling. Activation of CAR increases the profibrotic effects of TGFβ in cultured fibroblasts and in different preclinical models of SSc. Thus, inactivation of CAR might be a novel approach to target aberrant TGFβ signaling in SSc and in other fibrotic diseases.

Topics: Adult; Aged; Animals; Bleomycin; Cells, Cultured; Collagen Type I; Constitutive Androstane Receptor; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Humans; Male; Mice; Mice, Inbred C57BL; Middle Aged; Receptors, Cytoplasmic and Nuclear; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Up-Regulation

The mammalian target of rapamycin (mTOR) regulates cellular activity in many diseases, but the complex interplay with PI3K/Akt pathway may hampers its function.. This study was undertaken to determine the activity of PI3K/Akt/mTOR signaling in the fibroblasts from systemic sclerosis (SSc) patients, and compare the effects of vertical inhibiting PI3K/Akt/mTOR by BEZ235 and inhibiting mTOR alone by rapamycin in fibroblast activation and in two complementary established mouse model of SSc.. Pharmaceutical specific inhibitors BEZ235 and rapamycin were used to vertical inhibit PI3K/Akt/mTOR signaling and mTOR signaling alone in cultured fibroblasts and in mice. SSc mouse model was established by daily injecting bleomycin subcutaneously or by overexpression of constitutively active type I TGF-β receptor (TβRI(ca)). To delineate the mechanisms underlying the antifibrotic effects of BEZ235 and rapamycin, activity of PI3K/Akt/mTOR signaling was analyzed by determining the expressions of phosphorylated Akt, GSK-3β, mTOR and S6 ribosomal protein (S6).. Primary dermal fibroblasts demonstrated hyperactivity of PI3K/Akt and mTOR signaling. mTOR inhibitor rapamycin failed to inhibit dermal fibrosis in an established SSc mouse model. However, administration of a dual inhibitor for PI3K/Akt and mTOR signaling BEZ235 attenuated dermal fibrosis by reversing increased dermal thickness and collagen deposition in two SSc mouse models. Furthermore, BEZ235 showed superior inhibitory effect on fibroblast activation relative to rapamycin in vitro. Also both BEZ235 and rapamycin could prevent the phosphorylation of mTOR and S6 completely. BEZ235 also blocked the activation of Akt and GSK-3β dramatically, whereas rapamycin has been shown to increase further upregulation of phosphorylated Akt on Ser473 both in vitro and in vivo.. These data show that blocking PI3K/Akt/mTOR with BEZ235 leads to superior inhibitory effect for dermal fibrosis, suggesting that vertical inhibition of PI3K/Akt/mTOR signaling may have therapeutic potential for SSc.

Topics: Animals; Cells, Cultured; Fibrosis; Humans; Imidazoles; Mice; Mice, Inbred C57BL; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Quinolines; Scleroderma, Systemic; Signal Transduction; Skin; TOR Serine-Threonine Kinases; Transforming Growth Factor beta

To evaluate the anti-fibrotic effects of Astragaloside IV in systemic sclerosis.. Treated or untreated systemic sclerosis (SSc) and normal fibroblast isolated from corresponding pairs were utilized to detect expression of collagen and fibronectin by western blot, quantitative real-time RT-PCR (RT-qPCR), immunofluorescence staining and histopathological examination. SSc mouse model induced by bleomycin was used to evaluate the effects of the drug in vivo.. Compared to normal fibroblast (NF), the expression of collagen and fibronectin in SSc (SScF) dramatically increased, and this could be reduced by Astragaloside IV (AST) in a dose- or time-dependent manner at both protein and mRNA levels. Administration of Astragaloside IV consistently decreased collagen formation and partially restored the structure, as well as suppressing collagen and fibronectin expression in the skin lesions of SSc-model mice. Mechanistically, Astragaloside IV-induced fibrosis reduction may be due to deregulation of Smad 3/Fli-1, the major mediators of the fibrotic response and key molecules for TGF-β signaling. Astragaloside IV also decreased the level of p-SMAD3 and completely blocked its relocation into the nuclei.. Astragaloside IV attenuates fibrosis by inhibiting the TGF-β-Smads3 axis in systemic sclerosis.

Topics: Animals; Disease Models, Animal; Fibroblasts; Fibrosis; Humans; Mice; RNA, Messenger; Saponins; Scleroderma, Systemic; Smad3 Protein; Transforming Growth Factor beta; Triterpenes

Vascular involvement is a key feature of Systemic sclerosis (SSc). Although the pericytes/endothelial cells (ECs) cross-talk regulates vessels formation, no evidences about the pericytes contribution to ineffective angiogenesis in SSc are available. Recent findings showed similarities between pericytes and Bone Marrow Mesenchymal Stem Cells (BM-MSCs). Due to difficulties in pericytes isolation, this work explores the possibility to use BM-MSCs as pericytes surrogate, clarifying their role in supporting neo-angiogenesis during SSc.. To demonstrate their potential to normally differentiate into pericytes, both SSc and healthy controls (HC) BM-MSCs were treated with TGF-β and PDGF-BB. The expression of pericytes specific markers (α-SMA, NG2, RGS5 and desmin) was assessed by qPCR, western blot, and immunofluorescence; chemioinvasion and capillary morphogenesis were also performed. Cell-sorting of BM-MSCs co-cultured with HC-ECs was used to identify a possible change in contractile proteins genes expression.. We showed that BM-MSCs isolated from SSc patients displayed an up-regulation of α-SMA and SM22α genes and a reduced proliferative activity. Moreover during SSc, both TGF-β and PDGF-BB can specifically modulate BM-MSCs toward pericytes. TGF-β was found interfering with the PDGF-BB effects. Using BM-MSCs/MVECs co-culture system we observed that SSc BM-MSCs improve ECs tube formation in stressed condition, and BM-MSCs, sorted after co-culture, showed a reduced α-SMA and SM22α gene expression.. BM-MSCs from SSc patients behave as pericytes. They display a more mature and myofibroblast-like phenotype, probably related to microenvironmental cues operating during the disease. After their co-culture with HC-MVECs, SSc BM-MSCs underwent to a phenotypic modulation which re-programs these cells toward a pro-angiogenic behaviour.

Topics: Actins; Becaplermin; Blotting, Western; Cell Differentiation; Cells, Cultured; DNA Primers; Dose-Response Relationship, Drug; Endothelium, Vascular; Flow Cytometry; Fluorescent Antibody Technique; Humans; Mesenchymal Stem Cells; Microfilament Proteins; Muscle Proteins; Neovascularization, Physiologic; Pericytes; Phenotype; Proto-Oncogene Proteins c-sis; Real-Time Polymerase Chain Reaction; Regenerative Medicine; Scleroderma, Systemic; Transforming Growth Factor beta

Transforming growth factor β (TGFβ) is a profibrotic cytokine, and its aberrant function is implicated in several types of fibrotic pathologies including scleroderma (systemic sclerosis [SSc]). Multiple lines of evidence show that increased TGFβ signaling contributes to progressive fibrosis in SSc by promoting fibroblast activation, excessive extracellular matrix (ECM) deposition, and dermal thickening. We have previously identified CD109 as a TGFβ coreceptor and have shown that it antagonizes TGFβ signaling and TGFβ-induced ECM expression in vitro in human keratinocytes and fibroblasts. The aim of the present study was to examine the ability of CD109 to prevent skin fibrosis in a mouse model of bleomycin-induced SSc.. Transgenic mice overexpressing CD109 in the epidermis and their wild-type (WT) littermates were injected with bleomycin in phosphate buffered saline (PBS) or with PBS alone every other day for 21 days or 28 days. Dermal thickness and collagen deposition were determined histologically using Masson's trichrome and picrosirius red staining. In addition, collagen and fibronectin content was analyzed using Western blotting, and activation of TGFβ signaling was examined by determining phospho-Smad2 and phospho-Smad3 levels using Western blotting and immunohistochemistry.. Transgenic mice overexpressing CD109 in the epidermis showed resistance to bleomycin-induced skin fibrosis, as evidenced by a significant decrease in dermal thickness, collagen crosslinking, collagen and fibronectin content, and phospho-Smad2/3 levels, as compared to their WT littermates.. Our findings suggest that CD109 inhibits TGFβ signaling and fibrotic responses in experimental murine scleroderma. They also suggest that CD109 regulates dermal-epidermal interactions to decrease extracellular matrix synthesis in the dermis. Thus, CD109 is a potential molecular target for therapeutic intervention in scleroderma.

Topics: Animals; Antibiotics, Antineoplastic; Antigens, CD; Bleomycin; Collagen; Cross-Linking Reagents; Dermis; Disease Models, Animal; Extracellular Matrix; Fibronectins; Fibrosis; GPI-Linked Proteins; Male; Mice; Mice, Transgenic; Neoplasm Proteins; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Aim of the study was to assess whether Iloprost treatment summer suspension modifies systemic cytokines levels, cutaneous thermal properties and functional response to a cold-induced stress in patients affected by systemic sclerosis (SSc).. Twenty-eight patients fulfilling the American College of Rheumatology (ACR) criteria for SSc were included in the study. Patients recorded number, duration and pain-severity of Raynaud phenomenon (RP). Pain-severity was determined by a visual analog scale. Cytokines expression and production in peripheral blood mononuclear cells and serum were evaluated by RT-PCR and ELISA assay. Basal finger temperature (Tb), distal-dorsal difference temperature (DTdd) and thermal recovery time (tr) from cold stress were measured by means of functional infrared imaging (fIR). Measurements were performed in late spring, during routine Iloprost therapy (1-3 days infusion of 0.5-2 ng/kg every month), and in late summer after a therapy-withdrawal period.. Deterioration of SSc patients' skin thermal properties was observed in the period of therapy withdrawal (Tb reduction and tr enhancement; no DTdd differences) despite the improvement in symptoms of RP. A reduction in IL-12/23p40 gene expression was recorded after therapy withdrawal and a direct correlation between IL-12/23p40 and IL-23p19 gene expression was observed, stronger after therapy suspension.. Our data suggest that Iloprost treatment summer suspension may induce the loss of the therapy beneficial effect on microcirculation despite the objective reduction of RP, thus favouring a continuous use of Iloprost in absence of severe side effects. Iloprost showed to modulate only IL-23 expression corroborating the idea that this cytokine is crucial for SSc development and progression.

Topics: Adjuvants, Immunologic; Aged; Biomarkers; Cold Temperature; Cytokines; Female; Humans; Iloprost; Interleukin-12; Interleukin-23; Male; Middle Aged; Pain Measurement; Raynaud Disease; Scleroderma, Systemic; Seasons; Time Factors; Transforming Growth Factor beta; Treatment Outcome; Tumor Necrosis Factor-alpha; Vasodilator Agents; Withholding Treatment

Systemic sclerosis (SSc) is a chronic disease, with early activation of the immune system. The aim of our work was to address how SSc-mesenchymal stem cells (MSCs), although senescent, might preserve specific immunomodulatory abilities during SSc. MSCs were obtained from 10 SSc patients and 10 healthy controls (HC). Senescence was evaluated by assessing cell cycle, β-galactosidase (β-Gal) activity, p21 and p53 expression; doxorubicin was used as acute senescence stimulus to evaluate their ability to react in stressed conditions. Immunomodulatory abilities were studied co-culturing MSCs with peripheral blood mononuclear cells (PBMCs) and CD4(+) cells, in order to establish both their ability to block proliferation in mixed lymphocyte reaction and in regulatory T cells (Tregs) induction. SSc-MSC showed an increase of senescence biomarkers. Eighty per cent of MSCs were in G0-G1 phase, without significant differences between SSc and HC. SSc-MSCs showed an increased positive β-Gal staining and higher p21 transcript level compared to HC cells. After doxorubicin, β-Gal staining increased significantly in SSc-MSCs. On the contrary, doxorubicin abolished p21 activation and elicited p53 induction both in SSc- and HC-MSCs. Interleukin (IL)-6 and transforming growth factor (TGF)-β-related transcripts and their protein levels were significantly higher in SSc-MSCs. The latter maintained their immunosuppressive effect on lymphocyte proliferation and induced a functionally regulatory phenotype on T cells, increasing surface expression of CD69 and restoring the regulatory function which is impaired in SSc. Increased activation of the IL-6 pathway observed in our cells might represent an adaptive mechanism to senescence, but preserving some specific cellular functions, including immunosuppression.

Topics: Antigens, CD; Antigens, Differentiation, T-Lymphocyte; beta-Galactosidase; Cell Proliferation; Cell- and Tissue-Based Therapy; Cells, Cultured; Cellular Senescence; Coculture Techniques; Cyclin-Dependent Kinase Inhibitor p21; Doxorubicin; Humans; Immunomodulation; Interleukin-6; Lectins, C-Type; Mesenchymal Stem Cells; Scleroderma, Systemic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Suppressor Protein p53

The antifibrotic effect of simvastatin has been demonstrated in human lung fibroblasts. This study aimed to measure the effects of simvastatin in the development of pulmonary and cutaneous fibrosis in a murine model of SSc and to explore the mechanisms of these effects.. Chronic oxidant stress SSc was induced in BALB/c mice by daily s.c. injections of HOCl for 6 weeks. Mice were randomized in three arms: treatment with HOCl, HOCl plus simvastatin or vehicle alone. Statin treatment was initiated 30 min after HOCl s.c. injection and continued daily for 6 weeks. Skin and lung fibrosis were evaluated by histological methods. Immunohistochemical staining for α-smooth muscle actin in cutaneous and pulmonary tissues was performed to evaluate myofibroblast differentiation. Lung and skin concentrations of VEGF, extracellular signal-related kinase (ERK), rat sarcoma protein (Ras), Ras homologue gene family (Rho) and TGF-β were analysed by western blot.. Injections of HOCl induced cutaneous and lung fibrosis in BALB/c mice. Simvastatin treatment prevented both skin thickness and pulmonary fibrosis. Myofibroblast differentiation was also inhibited by simvastatin in the skin and in the lung. Increased cutaneous and pulmonary expression of VEGF, ERK, Ras and Rho in mice treated with HOCl was significantly lower in mice treated with HOCl plus simvastatin.. Simvastatin reduces the development of pulmonary fibrosis, potentially modulating adverse lung remodelling, as shown by the reduced deposition of collagen in alveolar septae. Simvastatin also reduces skin thickness in this model.

Topics: Animals; Biopsy, Needle; Blotting, Western; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrosis; Immunohistochemistry; Mice; Mice, Inbred BALB C; Pulmonary Fibrosis; Random Allocation; Reactive Oxygen Species; Scleroderma, Systemic; Sensitivity and Specificity; Simvastatin; Skin Diseases; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

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

To delineate the constitutive pulmonary vascular phenotype of the TβRIIΔk-fib mouse model of scleroderma, and to selectively induce pulmonary endothelial cell injury using vascular endothelial growth factor (VEGF) inhibition to develop a model with features characteristic of pulmonary arterial hypertension (PAH).. The TβRIIΔk-fib mouse strain expresses a kinase-deficient transforming growth factor β (TGFβ) receptor type II driven by a fibroblast-specific promoter, leading to ligand-dependent up-regulation of TGFβ signaling, and replicates key fibrotic features of scleroderma. Structural, biochemical, and functional assessments of pulmonary vessels, including in vivo hemodynamic studies, were performed before and following VEGF inhibition, which induced pulmonary endothelial cell apoptosis. These assessments included biochemical analysis of the TGFβ and VEGF signaling axes in tissue sections and explanted smooth muscle cells.. In the TβRIIΔk-fib mouse strain, a constitutive pulmonary vasculopathy with medial thickening, a perivascular proliferating chronic inflammatory cell infiltrate, and mildly elevated pulmonary artery pressure resembled the well-described chronic hypoxia model of pulmonary hypertension. Following administration of SU5416, the pulmonary vascular phenotype was more florid, with pulmonary arteriolar luminal obliteration by apoptosis-resistant proliferating endothelial cells. These changes resulted in right ventricular hypertrophy, confirming hemodynamically significant PAH. Altered expression of TGFβ and VEGF ligand and receptor was consistent with a scleroderma phenotype.. In this study, we replicated key features of systemic sclerosis-related PAH in a mouse model. Our results suggest that pulmonary endothelial cell injury in a genetically susceptible mouse strain triggers this complication and support the underlying role of functional interplay between TGFβ and VEGF, which provides insight into the pathogenesis of this disease.

Topics: Angiogenesis Inhibitors; Animals; Disease Models, Animal; Endothelium, Vascular; Familial Primary Pulmonary Hypertension; Female; Hypertension, Pulmonary; Hypoxia; Indoles; Lac Operon; Male; Mice; Mice, Transgenic; Phenotype; Protein Serine-Threonine Kinases; Pulmonary Circulation; Pyrroles; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Receptors, Vascular Endothelial Growth Factor; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Although several miRNAs have been shown to regulate autoimmune pathogenesis by affecting lymphocyte function, the roles of miRNAs in the pathogenesis of SSc remain unclear. Therefore the purpose of this study was to identify miRNAs that play a role in the pathogenesis of SSc by quantitative PCR screening of serum miRNAs.. Ninety-five miRNAs that were predicted to target SSc-related genes [IL-4, TGF-β, CTGF, PDGF-B, PDGF receptor (PDGFR) α/β and COL1A2) by in silico analyses were selected. The expression of these miRNAs in sera of SSc patients and healthy controls was measured by quantitative PCR. Involvement of miR-30b, which was most strongly down-regulated in SSc patients, in the regulation of PDGFR-β expression was examined by transfection experiments and 3'-untranslated region (3'-UTR) target luciferase assays. The expression of miR-30b in skin was evaluated in a bleomycin-induced dermal fibrosis model in mice and in SSc patients.. Nineteen of 95 miRNAs were significantly decreased in the sera of SSc patients. Among them, miR-30b was most strongly down-regulated in SSc patients (P = 0.00006) and the levels of miR-30b were inversely correlated with modified Rodnan skin scores. Transfection of a miR-30b mimic repressed PDGFR-β expression in dermal fibroblasts and the activity of a luciferase reporter containing 3'-UTR of PDGFR-β. Moreover, the expression of miR-30b was down-regulated in bleomycin-treated sclerotic skin and in affected skin in SSc patients.. Down-regulation of miR-30b might be involved in the pathogenesis of SSc.

Topics: Adult; Aged; Animals; Bleomycin; Case-Control Studies; Cells, Cultured; Down-Regulation; Female; Fibroblasts; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred BALB C; MicroRNAs; Middle Aged; Receptor, Platelet-Derived Growth Factor beta; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Sclerosis; Skin; Transfection; Transforming Growth Factor beta

Members of the early growth response (Egr) gene family of transcription factors have nonredundant biological functions. Although Egr-3 is implicated primarily in neuromuscular development and immunity, its regulation and role in tissue repair and fibrosis has not been studied. We now show that in normal skin fibroblasts, Egr-3 was potently induced by transforming growth factor-β via canonical Smad3. Moreover, transient Egr-3 overexpression was sufficient to stimulate fibrotic gene expression, whereas deletion of Egr-3 resulted in substantially attenuated transforming growth factor-β responses. Genome-wide expression profiling in fibroblasts showed that genes associated with tissue remodeling and wound healing were prominently up-regulated by Egr-3. Notably, <5% of fibroblast genes regulated by Egr-1 or Egr-2 were found to be coregulated by Egr-3, revealing substantial functional divergence among these Egr family members. In a mouse model of scleroderma, development of dermal fibrosis was accompanied by accumulation of Egr-3-positive myofibroblasts in the lesional tissue. Moreover, skin biopsy samples from patients with scleroderma showed elevated Egr-3 levels in the dermis, and Egr-3 mRNA levels correlated with the extent of skin involvement. These results provide the first evidence that Egr-3, a functionally distinct member of the Egr family with potent effects on inflammation and immunity, is up-regulated in scleroderma and is necessary and sufficient for profibrotic responses, suggesting important and distinct roles in the pathogenesis of fibrosis.

Topics: Adult; Animals; Disease Models, Animal; Early Growth Response Protein 1; Early Growth Response Protein 2; Early Growth Response Protein 3; Female; Fibroblasts; Fibrosis; Gene Expression Profiling; Gene Expression Regulation; Humans; Intracellular Space; Male; Mice; Mice, Inbred BALB C; Middle Aged; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins; Transforming Growth Factor beta

In systemic sclerosis (SSc), a common and aetiologically mysterious form of scleroderma (defined as pathological fibrosis of the skin), previously healthy adults acquire fibrosis of the skin and viscera in association with autoantibodies. Familial recurrence is extremely rare and causal genes have not been identified. Although the onset of fibrosis in SSc typically correlates with the production of autoantibodies, whether they contribute to disease pathogenesis or simply serve as a marker of disease remains controversial and the mechanism for their induction is largely unknown. The study of SSc is hindered by a lack of animal models that recapitulate the aetiology of this complex disease. To gain a foothold in the pathogenesis of pathological skin fibrosis, we studied stiff skin syndrome (SSS), a rare but tractable Mendelian disorder leading to childhood onset of diffuse skin fibrosis with autosomal dominant inheritance and complete penetrance. We showed previously that SSS is caused by heterozygous missense mutations in the gene (FBN1) encoding fibrillin-1, the main constituent of extracellular microfibrils. SSS mutations all localize to the only domain in fibrillin-1 that harbours an Arg-Gly-Asp (RGD) motif needed to mediate cell-matrix interactions by binding to cell-surface integrins. Here we show that mouse lines harbouring analogous amino acid substitutions in fibrillin-1 recapitulate aggressive skin fibrosis that is prevented by integrin-modulating therapies and reversed by antagonism of the pro-fibrotic cytokine transforming growth factor β (TGF-β). Mutant mice show skin infiltration of pro-inflammatory immune cells including plasmacytoid dendritic cells, T helper cells and plasma cells, and also autoantibody production; these findings are normalized by integrin-modulating therapies or TGF-β antagonism. These results show that alterations in cell-matrix interactions are sufficient to initiate and sustain inflammatory and pro-fibrotic programmes and highlight new therapeutic strategies.

Topics: Amino Acid Motifs; Amino Acid Substitution; Animals; Antibodies, Antinuclear; Antibodies, Neutralizing; Autoimmunity; Contracture; Dendritic Cells; Female; Fibrillin-1; Fibrillins; Fibrosis; Integrins; Male; Mice; Microfilament Proteins; Mutation, Missense; Plasma Cells; Scleroderma, Systemic; Skin Diseases, Genetic; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta

Systemic sclerosis (SSc) is characterized by excess collagen deposition in the skin, due to intrinsic transforming growth factor-β (TGF-β) activation. We tried to determine the expression and the role of discoidin domain receptor 2 (DDR2) in SSc. The expression of DDR2 mRNA and protein was significantly decreased in SSc dermal fibroblasts, which was recovered by knocking down TGF-β. The knockdown of DDR2 in normal fibroblasts induced microRNA-196a expression, which led to type I collagen downregulation, indicating that DDR2 itself has a negative effect on microRNA-196a expression and inducible effect on collagen expression. In SSc fibroblasts, however, the DDR2 knockdown did not affect TGF-β signaling and microRNA-196a expression. The microRNA-196a levels were significantly decreased in normal fibroblasts treated with TGF-β and in SSc fibroblasts. Taken together our data indicate that, in SSc fibroblasts, intrinsic TGF-β stimulation induces type I collagen expression, and also downregulates DDR2 expression. This probably acts as a negative feedback mechanism against excess collagen expression, as a decreased DDR2 expression is supposed to stimulate the microRNA-196a expression and further change the collagen expression. However, in SSc fibroblasts the microRNA-196a expression was downregulated by TGF-β signaling. DDR2-microRNA-196a pathway may be a previously unreported negative feedback system, and its impairment may be involved in the pathogenesis of SSc.

Topics: Cells, Cultured; Collagen Type I; Dermis; Discoidin Domain Receptors; Feedback, Physiological; Fibroblasts; Gene Expression Regulation; Gene Knockdown Techniques; Humans; MicroRNAs; Receptor Protein-Tyrosine Kinases; Receptors, Mitogen; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Clinical evidence suggests that the vascular abnormalities of systemic sclerosis (SSc) precede the onset of fibrosis, but molecular cues accounting for a possible vascular connection of SSc fibrosis have been elusive, although they have been searched for intensively. Since we had previously shown that connective tissue growth factor (CCN2), endowed with fibroblast-oriented activities, was overexpressed by endothelial cells (ECs) from SSc patients, we undertook this study to investigate its role and mechanisms in fibroblast activation.. Normal fibroblasts were challenged with conditioned medium of normal microvascular ECs (MVECs) and MVECs obtained from SSc patients with the diffuse form of the disease. Fibroblast invasion was studied using the Boyden chamber Matrigel assay. Fibroblast activation was evaluated by Western blotting and immunofluorescence of α-smooth muscle actin (α-SMA), vimentin, and type I collagen. Matrix metalloproteinase (MMP) production was evaluated by zymography and reverse transcription-polymerase chain reaction. Signal transduction and activation of the small GTPases RhoA and Rac1 were studied by Western blotting. Inhibition of SSc MVEC conditioned medium-dependent fibroblast activation was obtained by anti-CCN2 antibodies and the transforming growth factor β (TGFβ) antagonist peptide p17.. SSc MVEC CCN2 stimulated fibroblast activation and invasion. Such activities depended on CCN2-induced overexpression of TGFβ and its type I, II, and III receptors combined with overproduction of MMP-2 and MMP-9 gelatinases. All of these effects were reversed by the TGFβ antagonist peptide p17. Motility increase required Rac1 activation and RhoA inhibition and was inhibited by an MMP inhibitor. These features connoted a mesenchymal style of cell invasion. Since fibroblast activation also fostered overexpression of α-SMA, vimentin, and type I collagen, the CCN2-dependent increase in fibroblast activities recapitulated the characteristics of a mesenchymal-to-mesenchymal transition.. SSc MVECs recruit and activate dermal fibroblasts by induction of a CCN2/TGFβ-dependent mesenchymal-to-mesenchymal transition.

Topics: Blotting, Western; Collagen; Connective Tissue Growth Factor; Drug Combinations; Endothelial Cells; Female; Fibroblasts; Fluorescent Antibody Technique; Humans; Laminin; Male; Mesoderm; Proteoglycans; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Systemic sclerosis (SSc) is characterized by fibrosis of the skin and visceral organs. Patients with SSc have enhanced plasma levels of the plasmin-α2-antiplasmin (α2AP) complex, and we recently implicated α2AP in the development of fibrosis through transforming growth factor β (TGFβ) production. This study was undertaken to clarify how α2AP induces TGFβ production and the development of fibrosis.. To clarify the detailed mechanism by which α2AP induces TGFβ production, we focused on adipose triglyceride lipase (ATGL)/calcium-independent phospholipase A(2) (iPLA(2)) and examined whether ATGL/ iPLA(2) is associated with α2AP-induced TGFβ production. The mouse model of bleomycin-induced SSc was used to evaluate the role of α2AP in the development of fibrosis. Dermal thickness and collagen content were determined in mouse skin treated with phosphate buffered saline or bleomycin. Moreover, we cultured SSc-like fibroblasts from the bleomycin-treated mouse skin and examined the production of TGFβ and prostaglandin F(2α) (PGF(2α)).. We found that α2AP binding to ATGL promoted PGF(2α) synthesis through iPLA(2) in fibroblasts, and the PGF(2α) synthesis that was promoted by α2AP induced TGFβ production in fibroblasts. In addition, the neutralization of α2AP attenuated the production of TGFβ and PGF(2α) in SSc-like fibroblasts from mice. The α2AP deficiency attenuated bleomycin-induced fibrosis and PGF(2α) synthesis, while the administration of PGF(2α) to α2AP-deficient mice facilitated α2AP deficiency-attenuated fibrosis.. These findings suggest that α2AP regulates the development of fibrosis by PGF(2α) synthesis through ATGL/iPLA(2). The inhibition of α2AP-initiated pathways might provide a novel therapeutic approach to fibrotic diseases.

Topics: alpha-2-Antiplasmin; Animals; Bleomycin; Cells, Cultured; Collagen; Dinoprost; Dinoprostone; Disease Models, Animal; Fibroblasts; Fibrosis; Lipase; Mice; Phospholipases A2, Calcium-Independent; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Because recent studies implicate Toll-like receptors (TLRs) in the pathogenesis of fibrosis, we sought to investigate the in vitro and in vivo role and mechanism of TLR4-mediated fibroblast responses in fibrogenesis. We found that TLR4 was constitutively expressed, and accumulation of endogenous TLR4 ligands significantly elevated, in lesional skin and lung tissues from patients with scleroderma. Activation of TLR4 signaling in explanted fibroblasts resulted in enhanced collagen synthesis and increased expression of multiple genes involved in tissue remodeling and extracellular matrix homeostasis. Moreover, TLR4 dramatically enhanced the sensitivity of fibroblasts to the stimulatory effect of transforming growth factor-β1. These profibrotic responses were abrogated by both genetic and pharmacological disruption of TLR4 signaling in vitro, and skin fibrosis induced by bleomycin in vivo was attenuated in mice harboring a mutated TLR4. Activation of TLR4 in fibroblasts augmented the intensity of canonical Smad signaling, and was accompanied by suppression of anti-fibrotic microRNA expression. Together, these results suggest a novel model to account for persistent fibrogenesis in scleroderma, in which activation of fibroblast TLR4 signaling, triggered by damage-associated endogenous TLR4 ligands, results in augmented transforming growth factor-β1 sensitivity with increased matrix production and progressive connective tissue remodeling. Under these conditions, fibroblast TLR4 serves as the switch for converting self-limited tissue repair into intractable fibrosis.

Topics: Adult; Aged; Animals; Biopsy; Bleomycin; Cells, Cultured; Collagen; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Ligands; Lung; Male; Mice; Mice, Inbred C3H; Mice, Mutant Strains; MicroRNAs; Middle Aged; Scleroderma, Systemic; Signal Transduction; Skin; Toll-Like Receptor 4; Transforming Growth Factor beta

To assess the antifibrotic effects of pregnane X receptors (PXRs) in experimental dermal fibrosis.. The antifibrotic effects of PXR activation by 5-pregnen-3β-ol-20-one-16α-carbonitrile (PCN) were studied in the bleomycin model for prevention of dermal fibrosis and the modified bleomycin model for the treatment of established bleomycin-induced dermal fibrosis. Activation of canonical transforming growth factor (TGF)β signalling was analysed by immunofluorescence staining for phosphorylated smads. The antifibrotic effects of PXR activation were further studied in murine fibroblasts and murine T cells under Th2 conditions. In the T cell experiments, synthesis of the profibrotic cytokines, interleukin (IL)-4 and IL-13, was assessed by quantitative PCR, and IL-13 levels in the murine skin were determined by multiplex bead array technology.. Activation of PXR effectively inhibited the development of bleomycin-induced dermal fibrosis and induced the regression of established dermal fibrosis as assessed by skin thickening, hydroxyproline content and myofibroblasts. Reduced levels of phosphorylated smad2 and smad3 suggested that the antifibrotic effects of PXRs were mediated by inhibition of canonical TGFβ signalling. While PXR activation appeared to have no direct effects on fibroblasts, it potently inhibited the release of the profibrotic cytokine, IL-13, from Th2 cells. Consistent with these findings, IL-13 levels were reduced in bleomycin-challenged murine skin upon PXR activation.. Our findings demonstrate a novel antifibrotic role for PXRs in inflammatory dermal fibrosis. The antifibrotic effects of PXRs appear to be indirect: PXR activation reduces the release of the Th2 cytokine, IL-13, from T cells resulting in decreased canonical TGFβ signalling.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; CD4-Positive T-Lymphocytes; Cells, Cultured; Dermis; Disease Models, Animal; Fibroblasts; Fibrosis; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Pregnane X Receptor; Pregnenolone Carbonitrile; Receptors, Steroid; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Fibrosis, the hallmark of systemic sclerosis (SSc), is characterized by persistent fibroblast activation triggered by transforming growth factor-β (TGF-β). As the acetyltransferase p300 has a key role in fibrosis and its availability governs the intensity of fibrotic responses, we investigated p300 expression in SSc and the molecular basis of its regulation. We found that expression of p300 was markedly elevated in SSc skin biopsies and was induced by TGF-β in explanted normal skin fibroblasts. Stimulation of p300 by TGF-β was independent of Smads and involved the early-immediate transcription factor Egr-1 (early growth response 1), a key regulator of profibrotic TGF-β signaling. Indeed, Egr-1 was both sufficient and necessary for p300 regulation in vitro and in vivo. Increased p300 accumulation in TGF-β-treated fibroblasts was associated with histone hyperacetylation, whereas p300 depletion, or selective pharmacological blockade of its acetyltransferase activity, attenuated TGF-β-induced responses. Moreover, TGF-β enhanced both p300 recruitment and in vivo histone H4 acetylation at the COL1A2 (collagen, type I, α2) locus. These findings implicate p300-mediated histone acetylation as a fundamental epigenetic mechanism in fibrogenesis and place Egr-1 upstream in TGF-β-driven stimulation of p300 gene expression. The results establish a firm link between fibrosis with aberrant p300 expression and epigenetic activity that, to our knowledge, is previously unreported. Targeted disruption of p300-mediated histone acetylation might therefore represent a viable antifibrotic strategy.

Topics: Acetylation; Adult; Animals; Biopsy; Cells, Cultured; Collagen Type I; Early Growth Response Protein 1; Epigenesis, Genetic; Fibroblasts; Fibrosis; Gene Expression Regulation; Histones; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; p300-CBP Transcription Factors; Scleroderma, Systemic; Skin; Smad3 Protein; Transforming Growth Factor beta

To determine the role of insulin-like growth factor binding protein 3 (IGFBP-3) in mediating the effects of transforming growth factor β (TGFβ) on tenascin-C (TN-C) production and to assess the levels of TN-C in vivo in patients with systemic sclerosis (SSc)-associated pulmonary fibrosis.. Human primary lung fibroblasts were stimulated with TGFβ or IGFBP-3 in the presence or absence of specific small interfering RNAs and chemical inhibitors of the signaling cascade. TN-C levels in lung tissue specimens obtained from patients with SSc-associated pulmonary fibrosis were assessed using immunohistochemical analysis and were compared with the levels in specimens obtained from normal donors. TN-C levels were quantified in sera from normal donors and patients with SSc with or without pulmonary fibrosis, using an enzyme-linked immunosorbent assay.. IGFBP-3 mediated the induction of TN-C by TGFβ. Direct induction of TN-C by IGFBP-3 occurred in a p38 MAP kinase-dependent manner. TN-C levels were abundant in lung tissues from patients with SSc and were localized to subepithelial layers of the distal airways. No TN-C was detectable around the proximal airways. Patients with SSc-associated pulmonary fibrosis had significantly higher levels of circulating TN-C compared with SSc patients without pulmonary fibrosis. Longitudinal samples obtained from patients with SSc before and after the onset of pulmonary fibrosis showed increased levels of TN-C after the onset of pulmonary fibrosis.. IGFBP-3, which is overexpressed in fibrotic lungs, induces production of TN-C by subepithelial fibroblasts. The increased lung tissue levels of TN-C parallel the levels detected in the sera of SSc patients with pulmonary fibrosis, suggesting that TN-C may be a useful biomarker for SSc-related pulmonary fibrosis.

Topics: Cells, Cultured; Culture Media, Conditioned; Fibroblasts; Humans; Insulin-Like Growth Factor Binding Protein 3; Lung; p38 Mitogen-Activated Protein Kinases; Pulmonary Fibrosis; Recombinant Proteins; RNA Interference; RNA, Small Interfering; Scleroderma, Systemic; Tenascin; Transforming Growth Factor beta

To investigate whether c-Jun and c-Fos contribute to the pathologic activation of fibroblasts in systemic sclerosis (SSc) and to evaluate the antifibrotic potential of selective activator protein 1 (AP-1) inhibition.. Expression of c-Jun and c-Fos was determined by real-time polymerase chain reaction (PCR) and immunohistochemical analysis. Fibroblasts were stimulated with transforming growth factor β (TGFβ) and incubated with T-5224, a small-molecule inhibitor of AP-1, or were transfected with small interfering RNA (siRNA) duplexes against c-Jun and c-Fos. Collagen synthesis was quantified by real-time PCR and hydroxyproline assay. Differentiation of resting fibroblasts into myofibroblasts was assessed by staining for α-smooth muscle actin and stress fibers. The antifibrotic potential of T-5224 was evaluated in mouse models of dermal fibrosis induced by bleomycin or by adenoviral overexpression of a constitutively active TGFβ receptor type I.. Up-regulation of c-Jun and c-Fos was detected in mouse models of SSc and in the skin and dermal fibroblasts of patients with SSc. Stimulation of healthy fibroblasts with TGFβ induced the expression of c-Jun and c-Fos. Treatment with T-5224 or nucleofection with siRNA directed against c-Jun and c-Fos abrogated the profibrotic effects of TGFβ. T-5224 decreased the release of collagen selectively in SSc fibroblasts. T-5224 was well tolerated and prevented dermal fibrosis induced by bleomycin or by adenoviral activation of TGFβ signaling.. AP-1 is up-regulated in a TGFβ-dependent manner in SSc. The selective AP-1 inhibitor T-5224 reduced collagen synthesis selectively in SSc fibroblasts and efficiently prevented the development of experimental dermal fibrosis. Thus, AP-1 might be a promising new molecular target for the treatment of SSc.

Topics: Animals; Benzophenones; Bleomycin; Cells, Cultured; Collagen; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Isoxazoles; JNK Mitogen-Activated Protein Kinases; Male; Mice; Proto-Oncogene Proteins c-fos; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Transcription Factor AP-1; Transfection; Transforming Growth Factor beta; Up-Regulation

The aim of the present study was to determine the expression and role of thrombospondin-2 (TSP-2) in systemic sclerosis (SSc). Both TSP-2 mRNA levels and protein synthesis in cell lysates were significantly lower in cultured SSc fibroblasts than in normal fibroblasts; however, the TSP-2 protein that accumulated in the conditioned medium of SSc fibroblasts was up-regulated, compared with that of normal fibroblasts, because of an increase in the half-life of the protein. In vivo serum TSP-2 levels were higher in SSc patients than in healthy control subjects, and SSc patients with elevated serum TSP-2 levels tended to have pitting scars and/or ulcers. TSP-2 knockdown resulted in the down-regulation of type I collagen expression and the up-regulation of miR-7, one of the miRNAs with an inhibitory effect on collagen expression. Expression levels of miR-7 were also up-regulated in SSc dermal fibroblasts both in vivo and in vitro. Taken together, these findings suggest that the increased extracellular TSP-2 deposition in SSc fibroblasts may contribute to tissue fibrosis by inducing collagen expression. Down-regulation of intracellular TSP-2 synthesis and the subsequent miR-7 up-regulation in SSc fibroblasts may be due to a negative feedback mechanism that prevents increased extracellular TSP-2 deposition and/or tissue fibrosis. Thus, TSP-2 may play an important role in the maintenance of fibrosis and angiopathy in patients with SSc.

Topics: Adult; Aged; Aged, 80 and over; Animals; Case-Control Studies; Cells, Cultured; Collagen Type I; Down-Regulation; Extracellular Matrix; Female; Fibroblasts; Humans; Male; Matrix Metalloproteinase 1; Matrix Metalloproteinase 2; Mice; Mice, Inbred C57BL; MicroRNAs; Middle Aged; RNA, Messenger; RNA, Small Interfering; Scleroderma, Systemic; Skin; Thrombospondins; Transforming Growth Factor beta; Up-Regulation; Young Adult

Idiopathic and inflammation-dependent fibrotic diseases such systemic sclerosis (SSc) impose a major burden on modern societies. Understanding endogenous mechanisms, which counteract fibrosis, may yield new therapeutic approaches. Lipoxins are highly potent lipid mediators, which have recently been found to be decreased in SSc.. To determine the potential role of 12/15-lipoxygenase (12/15-LO), the key enzyme for the synthesis of lipoxins, in fibrosis.. Two mouse models for experimental dermal fibrosis (bleomycin-induced dermal fibrosis and tight-skin 1 mouse model) together with bone marrow transfers were used in wildtype and 12/15-LO(-/-) mice to elucidate the role of this enzyme during dermal fibrosis. Primary dermal fibroblasts of wildtype and 12/15-LO(-/-) mice, and 12/15-LO-derived eicosanoids, were used to identify underlying molecular mechanisms. In both models, 12/15-LO(-/-) mice exhibited a significant exacerbation of the fibrotic tissue response. Bone marrow transfer experiments disclosed a predominant role of mesenchymal cell-derived 12/15-LO in these antifibrotic effects. Indeed, 12/15-LO(-/-) fibroblasts showed an enhanced activation of the mitogen-activated protein-kinase pathway and an increased col 1a2 mRNA expression in response to stimulation with transforming growth factor β (TGFβ), whereas 12/15-LO-derived eicosanoids blocked these TGFβ-induced effects.. These data indicate that 12/15-LO and its metabolites have a prominent antifibrotic role during dermal fibrosis. This opens new opportunities for therapeutic approaches in the treatment of fibrotic diseases.

Topics: Animals; Antibiotics, Antineoplastic; Arachidonate 12-Lipoxygenase; Arachidonate 15-Lipoxygenase; Bleomycin; Cells, Cultured; Dermis; Eicosanoids; Fibroblasts; Fibrosis; Lipoxins; Mesoderm; Mice; Mice, Inbred C57BL; Mice, Knockout; Phenotype; Protein Serine-Threonine Kinases; Scleroderma, Systemic; Transforming Growth Factor beta

Previous studies have shown that the transforming growth factor (TGF)β/Alk1/Smad1 signaling pathway is constitutively activated in a subset of systemic sclerosis (SSc) fibroblasts and this pathway is a critical regulator of CCN2 gene expression. Caveolin-1 (cav-1), an integral membrane protein and the main component of caveolae, has also been implicated in SSc pathogenesis. This study was undertaken to evaluate the role of caveolin-1 in Smad1 signaling and CCN2 expression in healthy and SSc dermal fibroblasts. We show that a significant subset of SSc dermal fibroblasts has up-regulated cav-1 expression in vitro, and that cav-1 up-regulation correlates with constitutive Smad1 phosphorylation. In addition, basal levels of phospho-Smad1 were down-regulated after inhibition of cav-1 in SSc dermal fibroblasts. Caveolin-1 formed a protein complex with Alk1 in dermal fibroblasts, and this association was enhanced by TGFβ. By using siRNA against cav-1 and adenoviral cav-1 overexpression we demonstrate that activation of Smad1 in response to TGFβ requires cav-1 and that cav-1 is sufficient for Smad-1 phosphorylation. We also show that cav-1 is a positive regulator of CCN2 gene expression, and that it is required for the basal and TGFβ-induced CCN2 levels. In conclusion, this study has revealed an important role of cav-1 in mediating TGFβ/Smad1 signaling and CCN2 gene expression in healthy and SSc dermal fibroblasts.

Topics: Activin Receptors, Type II; Blotting, Western; Caveolin 1; Cells, Cultured; Connective Tissue Growth Factor; Down-Regulation; Fibroblasts; Humans; Immunohistochemistry; Immunoprecipitation; Phosphorylation; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Skin; Smad1 Protein; Transfection; Transforming Growth Factor beta; Up-Regulation

Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA in paraffin section was performed by in situ hybridization. Several miRNAs were found to be downregulated in both TGF-β-stimulated normal fibroblasts and SSc fibroblasts compared with normal fibroblasts by PCR array. Among them, miR-196a expression was decreased in SSc both in vivo and in vitro by real-time PCR or in situ hybridization. In SSc fibroblasts, miR-196a expression was normalized by TGF-β small interfering RNA. miR-196a inhibitor leads to the overexpression of type I collagen in normal fibroblasts, whereas overexpression of the miRNA resulted in the downregulation of type I collagen in SSc fibroblasts. In addition, miR-196a was detectable and quantitative in the serum of SSc patients. Patients with lower serum miR-196a levels had significantly higher ratio of diffuse cutaneous SSc:limited cutaneous SSc, higher modified Rodnan total skin thickness score, and higher prevalence of pitting scars than those without. miR-196a may play some roles in the pathogenesis of SSc. Investigation of the regulatory mechanisms of type I collagen expression by miR-196a may lead to new treatments using miRNA.

Topics: 3' Untranslated Regions; Aged; Binding Sites; Cells, Cultured; Collagen Type I; Dermis; Female; Fibroblasts; Gene Expression Regulation; Humans; Male; MicroRNAs; Middle Aged; Phenotype; Scleroderma, Systemic; Transfection; Transforming Growth Factor beta

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1(+/-) and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1(+/-) mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc.

Topics: Animals; Case-Control Studies; Cathepsin B; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Female; Gene Silencing; Humans; Male; Mice; Microcirculation; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Vascular Diseases

Osteopontin (OPN) is a matricellular protein with proinflammatory and profibrotic properties. Previous reports demonstrate a role for OPN in wound healing and pulmonary fibrosis. Here, we determined whether OPN levels are increased in a large cohort of patients with systemic sclerosis (SSc) and whether OPN contributes to the development of dermal fibrosis. The plasma OPN levels were increased in SSc patients, including patients with limited and diffuse disease, compared with healthy controls. Immunohistology demonstrated OPN on fibroblast-like and inflammatory cells in SSc skin and lesional skin from mice in the bleomycin (bleo)-induced dermal fibrosis model. OPN-deficient (OPN(-/-)) mice developed less dermal fibrosis compared with wild-type (WT) mice in the bleo-induced dermal fibrosis model. Additional in vivo studies have demonstrated that lesional skin from OPN(-/-)mice had fewer Mac-3-positive cells, fewer myofibroblasts, decreased transforming growth factor (TGF)-β and genes in the TGF-β pathway, and decreased numbers of cells expressing phosphorylated SMAD2 (pSMAD) and extracellular signal-regulated kinase. In vitro, OPN(-/-) dermal fibroblasts had decreased migratory capacity but similar phosphorylation of SMAD2 by TGF-β. Finally, TGF-β production by OPN-deficient macrophages was reduced compared with WT. These data demonstrate an important role for OPN in the development of dermal fibrosis and suggest that it may be a new therapeutic target in SSc.

Topics: Adult; Animals; Antibiotics, Antineoplastic; Bleomycin; Cells, Cultured; Dermis; Disease Models, Animal; Female; Fibroblasts; Fibrosis; Humans; Macrophages; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Middle Aged; Osteopontin; Scleroderma, Systemic; Smad2 Protein; Transforming Growth Factor beta

Systemic infusion of bone marrow mesenchymal stem cells (BMMSCs) yields therapeutic benefit for a variety of autoimmune diseases, but the underlying mechanisms are poorly understood. Here we show that in mice systemic infusion of BMMSCs induced transient T cell apoptosis via the FAS ligand (FASL)-dependent FAS pathway and could ameliorate disease phenotypes in fibrillin-1 mutated systemic sclerosis (SS) and dextran-sulfate-sodium-induced experimental colitis. FASL(-/-) BMMSCs did not induce T cell apoptosis in recipients, and could not ameliorate SS and colitis. Mechanistic analysis revealed that FAS-regulated monocyte chemotactic protein 1 (MCP-1) secretion by BMMSCs recruited T cells for FASL-mediated apoptosis. The apoptotic T cells subsequently triggered macrophages to produce high levels of TGFβ, which in turn led to the upregulation of CD4(+)CD25(+)Foxp3(+) regulatory T cells and, ultimately, immune tolerance. These data therefore demonstrate a previously unrecognized mechanism underlying BMMSC-based immunotherapy involving coupling via FAS/FASL to induce T cell apoptosis.

Topics: Animals; Apoptosis; Bone Marrow Cells; Chemokine CCL2; Colitis; Dextran Sulfate; Fas Ligand Protein; fas Receptor; Fibrillin-1; Fibrillins; Humans; Immune Tolerance; Immunotherapy; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Knockout; Microfilament Proteins; Scleroderma, Systemic; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

To study the role of platelet-derived growth factor A (PDGF-A)/PDGF receptor-α (PDGFR-α) signaling pathway in the proliferation and transdifferentiation of fibroblasts (FB) into myofibroblasts (MFB) in the skin lesions of patients with systemic sclerosis (SSc).. The primary FBs isolated from the skin lesions of SSc patients and normal adult skin cultured in vitro were examined for α-smooth muscle actin (α-SMA) using immunocytochemistry. The FBs were incubated with different concentrations of PDGF-AA and the changes in their proliferative activity were quantified with MTT assay. RT-PCR was used to determine the effects of transforming growth factor-β(1) (TGF-β(1)) and PDGF-AA, either alone or in combination, on the expression levels of PDGFR-α and α-SMA mRNA in the FBs.. Although the FBs of the two groups were morphologically similar, only FBs from the skin lesion showed positive staining for α-SMA. Below the saturated concentration of PDGF, the FBs in the two groups both proliferated in a dose-dependent manner (P<0.05), but the FBs from the SSc lesions always showed a significantly higher proliferative activity (P<0.05). PDGF-AA and TGF-β(1), alone or in combination, up-regulated the expression level of PDGFR-α and α-SMA mRNA in the FBs from SSc lesions; similar results were obtained in the control FBs, except that TGF-β(1) alone did not influence PDGFR-α mRNA expression. PDGFR-α and α-SMA mRNA always showed higher expressions in FBs in SSc lesions than in the control FBs with the same treatments (P<0.05). The expression levels of PDGFR-α and α-SMA mRNA increased in the order of untreated, PDGF-AA, TGF-β(1) and PDGF-AA plus TGF-β(1) groups and showed a strong positive correlation between them (r=0.925, P<0.05).. The FBs from the skin lesions of SSc patients have a distinct feature of transdifferentiation into MFB. Over-expression of PDGFR-α on the surface of FBs from SSc lesions can bind more PDGF-AA ligands to increase cell proliferation and promote transdifferentiation to MFB, and TGF-β(1) further enhances this effect .

Topics: Actins; Adult; Cell Proliferation; Cell Transdifferentiation; Cells, Cultured; Female; Fibroblasts; Humans; Male; Middle Aged; Myofibroblasts; Platelet-Derived Growth Factor; Receptor, Platelet-Derived Growth Factor alpha; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

To investigate whether JAK-2 contributes to the pathologic activation of fibroblasts in patients with systemic sclerosis (SSc) and to evaluate the antifibrotic potential of JAK-2 inhibition for the treatment of SSc.. Activation of JAK-2 in human skin and in experimental fibrosis was determined by immunohistochemical analysis. JAK-2 signaling was inhibited by the selective JAK-2 inhibitor TG101209 or by small interfering RNA. Bleomycin-induced dermal fibrosis in mice and TSK-1 mice were used to evaluate the antifibrotic potential of specific JAK-2 inhibition in vivo.. Increased activation of JAK-2 was detected in the skin of patients with SSc, particularly in fibroblasts. The activation of JAK-2 was dependent on transforming growth factor β (TGFβ) and persisted in cultured SSc fibroblasts. Inhibition of JAK-2 reduced basal collagen synthesis selectively in SSc fibroblasts but not in resting healthy dermal fibroblasts. Moreover, inhibition of JAK-2 prevented the stimulatory effects of TGFβ on fibroblasts. Treatment with TG101209 not only prevented bleomycin-induced fibrosis but also effectively reduced skin fibrosis in TSK-1 mice.. We demonstrated that JAK-2 is activated in a TGFβ-dependent manner in SSc. Considering the potent antifibrotic effects of JAK-2 inhibition, our study might have direct translational implications, because inhibitors of JAK-2 are currently being evaluated in clinical trials for myeloproliferative disorders and would also be available for evaluation in patients with SSc.

Topics: Adult; Aged; Animals; Enzyme Inhibitors; Female; Fibrosis; Humans; Janus Kinase 2; Male; Mice; Middle Aged; Phosphorylation; Pyrimidines; Scleroderma, Systemic; Signal Transduction; Skin; Sulfonamides; Transforming Growth Factor beta

Scleroderma or systemic sclerosis (SSc) is a complex connective tissue disease characterized by fibrosis of skin and internal organs. Transforming growth factor beta (TGF-β) plays a key role in the pathogenesis of SSc fibrosis. We have previously identified CD109 as a novel TGF-β co-receptor that inhibits TGF-β signaling. The aim of the present study was to determine the role of CD109 in regulating extracellular matrix (ECM) production in human SSc skin fibroblasts.. CD109 expression was determined in skin tissue and cultured skin fibroblasts of SSc patients and normal healthy subjects, using immunofluorescence, western blot and RT-PCR. The effect of CD109 on ECM synthesis was determined by blocking CD109 expression using CD109-specific siRNA or addition of recombinant CD109 protein, and analyzing the expression of ECM components by western blot.. The expression of CD109 proteinis markedly increased in SSc skin tissue in vivo and in SSc skin fibroblasts in vitro as compared to their normal counterparts. Importantly, both SSc and normal skin fibroblasts transfected with CD109-specific siRNA display increased fibronectin, collagen type I and CCN2 protein levels and enhanced Smad2/3 phosphorylation compared with control siRNA transfectants. Furthermore, addition of recombinant CD109 protein decreases TGF-β1-induced fibronectin, collagen type I and CCN2 levels in SSc and normal fibroblasts.. The upregulation of CD109 protein in SSc may represent an adaptation or consequence of aberrant TGF-β signaling in SSc. Our finding that CD109 is able to decrease excessive ECM production in SSc fibroblasts suggest that this molecule has potential therapeutic value for the treatment of SSc.

Topics: Adult; Aged; Antigens, CD; Blotting, Western; Extracellular Matrix; Female; Fibroblasts; Fluorescent Antibody Technique; GPI-Linked Proteins; Humans; Immunohistochemistry; Male; Middle Aged; Neoplasm Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Up-Regulation

Topics: Animals; Disease Models, Animal; Fibrosis; Humans; Intercellular Signaling Peptides and Proteins; Mice; Mice, Transgenic; Scleroderma, Systemic; Transforming Growth Factor beta; Wnt Proteins; Wnt Signaling Pathway

Scleroderma (SSc) is characterized by excess production and deposition of extracellular matrix (ECM) proteins. Activated fibroblasts play a key role in fibrosis in SSc and are resistant to Fas-mediated apoptosis. Acid sphingomyelinase (ASMase), a major sphingolipid enzyme, plays an important role in the Fas-mediated apoptosis.. We investigated whether dysregulation of ASMase contributes to Fas-mediated apoptosis resistance in SSc fibroblasts.. Fibroblasts were isolated from SSc patients and healthy controls. Western blot was performed to analyze protein levels and quantitative real time RT-PCR was used to determine mRNA expression. Cells were transiently transfected with siRNA oligos against ASMase or transduced with adenoviruses overexpressing ASMase. Apoptosis was induced using anti-Fas antibody (1 μg/mL) and analyzed using caspase-3 antibody or Cell Death Detection ELISA.. SSc fibroblasts showed increased resistance to Fas-mediated apoptosis. ASMase expression was decreased in SSc fibroblasts and Transforming Growth Factor beta (TGFβ), the major fibrogenic cytokine involved in the pathogenesis of SSc, downregulated ASMase in normal fibroblasts. Forced expression of ASMase in SSc fibroblasts restored sensitivity of these cells to Fas-mediated apoptosis while blockade of ASMase was sufficient to induce partial resistance to Fas-induced apoptosis in normal fibroblasts. In addition, ASMase blockade decreased activity of protein phosphatase 2A (PP2A) through phosphorylation on Tyr(307) and resulted in activation of extracellular regulated kinase 1/2 (Erk1/2) and protein kinase B (Akt/PKB).. In conclusion, this study suggests that ASMase deficiency promotes apoptosis resistance and contributes to activation of profibrotic signaling in SSc fibroblasts.

Topics: Apoptosis; Cytokines; Extracellular Matrix; Extracellular Signal-Regulated MAP Kinases; fas Receptor; Fibroblasts; Fibrosis; Humans; Myofibroblasts; Niemann-Pick Disease, Type A; Oligonucleotides; Protein Phosphatase 2; RNA Interference; RNA, Small Interfering; Scleroderma, Systemic; Sphingomyelin Phosphodiesterase; Transforming Growth Factor beta; Wound Healing

Cutaneous fibrosis seen in systemic sclerosis (SSc) is caused by fibroblast activation and abnormal collagen accumulation due to 'autocrine transforming growth factor (TGF)-β/Smad signaling'. Hepatocyte growth factor (HGF) may have therapeutic value against SSc, because of its inducible effect on the expression of matrix metalloproteinase (MMP)-1. Previous studies indicated SSc dermal fibroblasts overexpress HGF receptor c-met, which suggest specific and effective induction of MMP-1 in SSc fibroblasts caused by HGF treatment. However, the exact mechanism of c-met overexpression in SSc cells was hardly investigated. We hypothesized that such c-met overexpression is also caused by autocrine TGF-β/Smad signaling. Expression of c-met protein in cultured SSc dermal fibroblasts was significantly up-regulated compared with that in normal fibroblasts. Ectopic TGF-β stimulation induced c-met synthesis in normal fibroblasts, while a TGF-β knockdown normalized the up-regulated c-met levels in SSc fibroblasts. Furthermore, we found the c-met promoter contains a putative binding site for Smads, and the binding activity of Smad2/3 to the c-met promoter was constitutively up-regulated in SSc fibroblasts as well as in normal fibroblasts treated with exogenous TGF-β1. Taken together, c-met may be overexpressed due to autocrine TGF-β/Smad signaling in SSc. Considering that HGF has an antifibrotic effect, such c-met overexpression in SSc fibroblasts may be a negative feedback against cutaneous fibrosis. Clarifying the mechanisms of c-met overexpression and controlling the HGF/c-met pathway may lead to a new therapeutic approach for this disease.

Topics: Autocrine Communication; Cells, Cultured; Fibroblasts; Fibrosis; Gene Expression Regulation; Gene Knockdown Techniques; Humans; Immunoblotting; Oligonucleotides; Proto-Oncogene Proteins c-met; Scleroderma, Systemic; Skin; Statistics, Nonparametric; Transforming Growth Factor beta

Fibrosis in scleroderma is associated with collagen deposition and myofibroblast accumulation. Peroxisome proliferator activated receptor gamma (PPAR-γ), a master regulator of adipogenesis, inhibits profibrotic responses induced by transforming growth factor-ß (TGF-β), and its expression is impaired in scleroderma. The roles of adiponectin, a PPAR-γ regulated pleiotropic adipokine, in regulating the response of fibroblasts and in mediating the effects of PPAR-γ are unknown.. Regulation of fibrotic gene expression and TGF-ß signaling by adiponectin and adenosine monophosphate protein-activated (AMP) kinase agonists were examined in normal fibroblasts in monolayer cultures and in three-dimensional skin equivalents. AdipoR1/2 expression on skin fibroblasts was determined by real-time quantitative PCR.. Adiponectin, an adipokine directly regulated by PPAR-γ, acts as a potent anti-fibrotic signal in normal and scleroderma fibroblasts that abrogates the stimulatory effects of diverse fibrotic stimuli and reduces elevated collagen gene expression in scleroderma fibroblasts. Adiponectin responses are mediated via AMP kinase, a fuel-sensing cellular enzyme that is necessary and sufficient for down-regulation of fibrotic genes by blocking canonical Smad signaling. Moreover, we demonstrate that endogenous adiponectin accounts, at least in part, for the anti-fibrotic effects exerted by ligands of PPAR-γ.. These findings reveal a novel link between cellular energy metabolism and extracellular matrix homeostasis converging on AMP kinase. Since the levels of adiponectin as well as its receptor are impaired in scleroderma patients with progressive fibrosis, the present results suggest a potential role for defective adiponectin expression or function in progressive fibrogenesis in scleroderma and other chronic fibrosing conditions. Restoring the adiponectin signaling axis in fibroblasts might, therefore, represent a novel pharmacological approach to controlling fibrosis.

Topics: Actins; Adenylate Kinase; Adiponectin; Adult; Cells, Cultured; Collagen; Female; Fibroblasts; Fibrosis; Humans; Male; Middle Aged; PPAR gamma; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Heightened production of collagen and other matrix proteins underlies the fibrotic phenotype of systemic sclerosis (SSc). Roscovitine is an inhibitor of cyclin-dependent kinases that promote cell cycling (CDK1, 2), neuronal development (CDK5) and control transcription (CDK7,9). In an in vivo glomerulonephritis model, roscovitine treatment decreased mesangial cell proliferation and matrix proteins [1]. We investigated whether roscovitine could regulate fibrotic protein production directly rather than through cell cycling. Our investigations revealed that roscovitine coordinately inhibited the expression of collagen, fibronectin, and connective tissue growth factor (CTGF) in normal and SSc fibroblasts. This effect occurred on a transcriptional basis and did not result from roscovitine-mediated cell cycle inhibition. Roscovitine-mediated suppression of matrix proteins could not be reversed by the exogenous profibrotic cytokines TGF-β or IL-6. To our knowledge, we are the first to report that roscovitine modulates matrix protein transcription. Roscovitine may thus be a viable treatment option for SSc and other fibrosing diseases.

Topics: Animals; Collagen; Connective Tissue Growth Factor; Fibroblasts; Fibronectins; Fibrosis; Humans; Interleukin-6; Mice; NIH 3T3 Cells; Phosphorylation; Purines; Roscovitine; Scleroderma, Systemic; Signal Transduction; STAT3 Transcription Factor; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Systemic sclerosis (SSc) is a complex and incompletely understood disease associated with fibrosis in multiple organs. Recent findings identify transforming growth factor-ß (TGF-ß), Wnt ligands, toll-like receptor-mediated signaling, hypoxia, type I interferon, type 2 immune responses and mechanical stress as extracellular cues that modulate fibroblast function and differentiation, and as potential targets for therapy. Moreover, fibrillin-1 has a major role in storing and regulating the bioavailability of TGF-ß and other cytokines, and fibrillin-1 mutations are implicated in a congenital form of scleroderma called stiff skin syndrome. Fibrosis is due not only to the activation of tissue-resident fibroblasts and their transdifferentiation into myofibroblasts, but also the differentiation of bone marrow-derived fibrocytes, and transition of endothelial and epithelial cells, pericytes and adipocytes into activated mesenchymal cells. These responses are modulated by signaling mediators and microRNAs that amplify or inhibit TGF-ß and Wnt signaling. Gain-of-function and loss-of-function abnormalities of these mediators may account for the characteristic activated phenotype of SSc fibroblasts. The nuclear orphan receptor PPAR-γ plays a particularly important role in limiting the duration and intensity of fibroblast activation and differentiation, and impaired PPAR-γ expression or function in SSc may underlie the uncontrolled progression of fibrosis. Identifying the perturbations in signaling pathways, mediators and differentiation programs that are responsible for SSc tissue damage allows their selective targeting. This in turn opens the door for therapies utilizing novel compounds, or drug repurposing by innovative uses of already-approved drugs. In view of the heterogeneous clinical presentation and unpredictable course of SSc, as well as its complex pathogenesis, only robust clinical trials incorporating the judicious application of biomarkers will be able to clarify the clinical utility of these innovative approaches.

Topics: Adipocytes; Animals; beta Catenin; Biomarkers; Cell Transdifferentiation; Epithelial Cells; Fibrillin-1; Fibrillins; Fibroblasts; Fibrosis; Humans; Hypoxia; Mice; Microfilament Proteins; Molecular Targeted Therapy; Pericytes; PPAR gamma; Scleroderma, Systemic; Signal Transduction; Toll-Like Receptors; Transforming Growth Factor beta; Wnt Proteins

There is increasing evidence that the endocannabinoid system may be involved in pathological fibrosis, and that its modulation might limit fibrotic responses. The aim of this study was to examine the capacity of a synthetic cannabinoid receptor agonist to modify skin fibrosis in the bleomycin mouse model of scleroderma.. Skin fibrosis was induced by local injections of bleomycin in two groups of DBA/2J mice. One group was cotreated with the synthetic cannabinoid WIN55,212-2 at 1 mg/kg/day. Skin fibrosis was evaluated by histology and skin thickness and hydroxyproline content were quantified. Markers of fibroblast activation, including α smooth muscle actin and the profibrotic cytokines transforming growth factor (TGF)β, connective tissue growth factor (CTGF) and platelet-derived growth factor (PDGF)-BB, were examined. Levels of PSMAD2/3, which are crucial in extracellular matrix overproduction, were analysed.. Bleomycin treatment induced typical skin fibrosis. Upon WIN55,212-2 treatment dermal fibrosis was completely prevented. Subcutaneous inflammatory cell infiltration, dermal thickness and collagen content resulted similar to those of the control group. The synthetic cannabinoid prevented fibroblasts activation induced by bleomycin, paralleled by a strong inhibition of TGFβ, CTGF and PDGF-BB expression. Phosphorylation of SMAD2/3 was significantly downregulated after WIN55,212-2 exposure.. Taken together, the results indicate that the synthetic cannabinoid WIN55,212-2 is capable of preventing skin fibrosis in a mouse model of scleroderma.

Topics: Animals; Benzoxazines; Bleomycin; Dermatologic Agents; Disease Models, Animal; Drug Evaluation, Preclinical; Fibroblasts; Fibrosis; Mice; Mice, Inbred DBA; Morpholines; Naphthalenes; Platelet-Derived Growth Factor; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

We have previously demonstrated that in response to transforming growth factor β (TGFβ), Fli-1 activity is repressed through a series of sequential posttranslational modifications, consisting of protein kinase Cδ (PKCδ)-induced Thr312 phosphorylation, acetylation by p300/CREB binding protein-associated factor, and detachment from the collagen promoter. The purpose of this study was to further investigate the upstream events that lead to Fli-1 phosphorylation in response to TGFβ.. Dermal fibroblasts were isolated from systemic sclerosis (SSc) patients and healthy control subjects matched for age, sex, and ethnicity. Western blotting was used to analyze protein levels and real-time quantitative reverse transcription-polymerase chain reaction analysis was used to measure messenger RNA expression. Cells were transduced with constitutively active PKCδ adenovirus or were transiently transfected with a Bcr-Abl-overexpressing plasmid. Subcellular localization of PKCδ was examined by immunocytochemistry.. Western blot analysis of cell lysates demonstrated that the levels of phospho-Fli-1 (Thr312) were up-regulated in SSc fibroblasts, correlating with increased levels of type I collagen and c-Abl protein. Experiments using a constitutively activated form of c-Abl, small interfering RNA against c-Abl and the specific tyrosine kinase inhibitor imatinib, demonstrated the requirement of c-Abl for the TGFβ-induced phosphorylation of Fli-1. Additionally, we showed that c-Abl kinase activity was required for nuclear localization of PKCδ.. Our results demonstrate that in SSc fibroblasts, c-Abl is an upstream regulator of the profibrotic PKCδ/phospho-Fli-1 pathway, via induction of PKCδ nuclear localization. Additionally, the finding that Fli-1 is phosphorylated at higher levels in SSc fibroblasts supports the notion that the c-Abl/PKCδ/phospho-Fli-1 pathway is constitutively activated in these cells. Thus, blocking the TGFβ/c-Abl/PKCδ/phospho-Fli-1 pathway could be an attractive alternative approach to therapy for scleroderma.

Topics: Benzamides; Cell Nucleus; Cells, Cultured; Collagen Type I; Dermis; Female; Fibroblasts; Humans; Imatinib Mesylate; Male; Microfilament Proteins; Phosphorylation; Piperazines; Protein Kinase C-delta; Protein Kinase Inhibitors; Proto-Oncogene Proteins c-abl; Pyrimidines; Receptors, Cytoplasmic and Nuclear; RNA, Small Interfering; Scleroderma, Systemic; Trans-Activators; Transduction, Genetic; Transforming Growth Factor beta

Topics: Case-Control Studies; Flow Cytometry; Humans; Interleukin-10; Scleroderma, Systemic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a chronic disease of unknown etiology that is characterized by multiple tissue fibrosis. Transforming Growth Factor-beta (TGF-β) is thought to be the most important mediator that induces fibrosis. However, the molecular mechanisms by which fibrosis is induced have not been fully elucidated. In this study, the role of activin, a member of the TGF-β superfamily, was investigated in the pathogenesis of fibrosis in SSc. Serum activin A levels in patients with SSc were measured by ELISA, and the expression of the activin receptor type IB (ACVRIB/ALK4) and the activity of the signaling pathway via ACVRIB/ALK4 were investigated using western blotting. To evaluate a potential therapeutic strategy for SSc, we also attenuated the ACVRIB/ALK4 pathway using an inhibitor. Serum activin A levels were significantly higher in SSc patients than in normal controls. Activin A and ACVRIB/ALK4 expression were also higher in cultured SSc fibroblasts. Activin A stimulation induced phosphorylation of Smad2/3 and CTGF expression in SSc fibroblasts. Procollagen production and Col1α mRNA also increased upon stimulation by activin A. The basal level of Smad2/3 phosphorylation was higher in cultured SSc fibroblasts than in control cells, and treatment with the ALK4/5 inhibitor SB431542 prevented phosphorylation of Smad2/3 and CTGF expression. Furthermore, production of collagen was also induced by activin A. Activin A-ACVRIB/ALK4-Smad-dependent collagen production was augmented in SSc fibroblasts, suggesting the involvement of this signaling mechanism in SSc. Inhibition of the activin A-ACVRIB/ALK4-Smad pathway would be a new approach for the treatment of SSc.

Topics: Activin Receptors, Type I; Activins; Cells, Cultured; Collagen; Fibroblasts; Humans; Nitric Oxide; Scleroderma, Systemic; Signal Transduction; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Although the early growth response-2 (Egr-2, alias Krox20) protein shows structural and functional similarities to Egr-1, these two related early-immediate transcription factors are nonredundant. Egr-2 plays essential roles in peripheral nerve myelination, adipogenesis, and immune tolerance; however, its regulation and role in tissue repair and fibrosis remain poorly understood. We show herein that transforming growth factor (TGF)-β induced a Smad3-dependent sustained stimulation of Egr2 gene expression in normal fibroblasts. Overexpression of Egr-2 was sufficient to stimulate collagen gene expression and myofibroblast differentiation, whereas these profibrotic TGF-β responses were attenuated in Egr-2-depleted fibroblasts. Genomewide transcriptional profiling revealed that multiple genes associated with tissue remodeling and wound healing were up-regulated by Egr-2, but the Egr-2-regulated gene expression profile overlapped only partially with the Egr-1-regulated gene profile. Levels of Egr-2 were elevated in lesional tissue from mice with bleomycin-induced scleroderma. Moreover, elevated Egr-2 was noted in biopsy specimens of skin and lung from patients with systemic sclerosis. These results provide the first evidence that Egr-2 is a functionally distinct transcription factor that is both necessary and sufficient for TGF-β-induced profibrotic responses and is aberrantly expressed in lesional tissue in systemic sclerosis and in a murine model of scleroderma. Together, these findings suggest that Egr-2 plays an important nonredundant role in the pathogenesis of fibrosis. Targeting Egr-2 might represent a novel therapeutic strategy to control fibrosis.

Topics: Animals; Blotting, Western; Cells, Cultured; Early Growth Response Protein 2; Fibrosis; Humans; Immunohistochemistry; Mice; Microscopy, Confocal; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Transcription, Genetic; Transfection; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor β (TGFβ) has been identified as a key player in fibrotic diseases. However, the molecular mechanisms by which TGFβ activates fibroblasts are incompletely understood. Here, the role of JunD, a member of the activator protein 1 (AP-1) family of transcription factors, as a downstream mediator of TGFβ signalling in systemic sclerosis (SSc), was investigated.. The expression of JunD was analysed by real-time PCR, immunofluorescence, western blotting and immunohistochemistry. The canonical Smad pathway was specifically targeted by small interfering (si)RNA. The expression of extracellular matrix proteins in JunD deficient (JunD(-/-)) fibroblasts was analysed by real-time PCR and hydroxyproline assays. The mouse model of bleomycin-induced dermal fibrosis was used to assess the role of JunD in experimental fibrosis.. JunD was overexpressed in SSc skin and in cultured fibroblasts in a TGFβ dependent manner. The expression of JunD colocalised with pSmad 3 in fibrotic skin and silencing of Smad 3 or Smad 4 by siRNA prevented the induction of JunD by TGFβ. JunD(-/-) fibroblasts were less responsive to TGFβ and released less collagen upon stimulation with TGFβ. Moreover, JunD(-/-) mice were protected from bleomycin-induced fibrosis with reduced dermal thickening, decreased myofibroblast counts and lower collagen content of lesional skin.. These data demonstrate that JunD is overexpressed in SSc and that JunD is a mediator of the profibrotic effects of TGFβ. Considering that inhibitors of AP-1 signalling have recently been developed and are available for clinical trials in SSc, these findings may have translational implications.

Topics: Adult; Aged; Animals; Bleomycin; Cells, Cultured; Collagen; Female; Fibroblasts; Fibrosis; Humans; Male; Mice; Mice, Knockout; Middle Aged; Proto-Oncogene Proteins c-jun; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta; Up-Regulation

Vascular damage and platelet activation are associated with tissue remodeling in diseases such as systemic sclerosis, but the molecular mechanisms underlying this association have not been identified. In this study, we show that serotonin (5-hydroxytryptamine [5-HT]) stored in platelets strongly induces extracellular matrix synthesis in interstitial fibroblasts via activation of 5-HT(2B) receptors (5-HT(2B)) in a transforming growth factor β (TGF-β)-dependent manner. Dermal fibrosis was reduced in 5-HT(2B)(-/-) mice using both inducible and genetic models of fibrosis. Pharmacologic inactivation of 5-HT(2B) also effectively prevented the onset of experimental fibrosis and ameliorated established fibrosis. Moreover, inhibition of platelet activation prevented fibrosis in different models of skin fibrosis. Consistently, mice deficient for TPH1, the rate-limiting enzyme for 5-HT production outside the central nervous system, showed reduced experimental skin fibrosis. These findings suggest that 5-HT/5-HT(2B) signaling links vascular damage and platelet activation to tissue remodeling and identify 5-HT(2B) as a novel therapeutic target to treat fibrotic diseases.

Topics: Animals; Blood Platelets; Disease Models, Animal; Extracellular Matrix; Fibroblasts; Fibrosis; Humans; Mice; Mice, Knockout; Platelet Activation; Receptor, Serotonin, 5-HT2B; Scleroderma, Systemic; Serotonin; Signal Transduction; Transforming Growth Factor beta

Antinuclear antibodies (ANAs), usually detected by indirect immunofluorescence on HEp-2 cells, are identified in 90% of patients with systemic sclerosis (SSc). Thus, approximately 10% of SSc patients have no routinely detectable autoantibodies, and for 20% to 40% of those with detectable ANAs, the ANAs do not have identified specificity (unidentified ANAs). In this work, we aimed to identify new target autoantigens in SSc patients.. Using a proteomic approach combining two-dimensional electrophoresis and immunoblotting with HEp-2 cell total and enriched nuclear protein extracts as sources of autoantigens, we systematically analysed autoantibodies in SSc patients. Sera from 45 SSc patients were tested in 15 pools from groups of three patients with the same phenotype. A sera pool from 12 healthy individuals was used as a control. Proteins of interest were identified by mass spectrometry and analysed using Pathway Studio software.. We identified 974 and 832 protein spots in HEp-2 cell total and enriched nuclear protein extracts, respectively. Interestingly, α-enolase was recognised by immunoglobulin G (IgG) from all pools of patients in both extracts. Fourteen and four proteins were recognised by IgG from at least 75% of the 15 pools in total and enriched nuclear protein extracts, respectively, whereas 15 protein spots were specifically recognised by IgG from at least four of the ten pools from patients with unidentified ANAs. The IgG intensity for a number of antigens was higher in sera from patients than in sera from healthy controls. These antigens included triosephosphate isomerase, superoxide dismutase mitochondrial precursor, heterogeneous nuclear ribonucleoprotein L and lamin A/C. In addition, peroxiredoxin 2, cofilin 1 and calreticulin were specifically recognised by sera from phenotypic subsets of patients with unidentified ANAs. Interestingly, several identified target antigens were involved in the transforming growth factor β pathway.. We identified several new target antigens shared among patients with SSc or specific to a given phenotype. The specification of new autoantibodies could help in understanding the pathophysiology of SSc. Moreover, these autoantibodies could represent new diagnostic and/or prognostic markers for SSc.

Topics: Antibodies, Antinuclear; Antibody Specificity; Autoantigens; Biomarkers; Cell Line, Tumor; Electrophoresis, Gel, Two-Dimensional; Fluorescent Antibody Technique, Indirect; Humans; Immunoglobulin G; Immunophenotyping; Laryngeal Neoplasms; Nuclear Proteins; Prognosis; Proteome; Proteomics; Scleroderma, Systemic; Signal Transduction; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Transforming Growth Factor beta

The transforming growth factor-β (TGF-β) signaling pathway plays a key role in the fibrotic process in systemic scleroderma (SSc). Curcumin, a Turmeric root extract, has been demonstrated to exert antifibrotic activity. In the present study, we carefully investigated the effect of curcumin on TGF-β signaling and its potential mechanism in SSc fibroblasts. We demonstrated a potent inhibitory effect of curcumin on TGF-β signaling. Curcumin counteracted TGF-β-induced phosphorylation of Smad2 but not Smad3. Further study revealed curcumin induced upregulation of TGF-β-induced factor (TGIF), a negative regulator of TGF-β signaling. The TGIF silencing results evidenced the essential role of TGIF in curcumin-mediated TGF-β/Smad2 suppression. Moreover, our data indicated that the upregulation of TGIF by curcumin might result from decreased ubiquitination of TGIF, which blocks its proteasome-mediated degradation. Collectively, our data provide a novel mechanism of curcumin-mediated suppression of fibrotic process in scleroderma.

Topics: Antineoplastic Agents; Cells, Cultured; Curcumin; Female; Fibroblasts; Homeodomain Proteins; Humans; Middle Aged; Phosphorylation; Repressor Proteins; Scleroderma, Systemic; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor-ß (TGF-ß) signaling is implicated in the pathogenesis of fibrosis in scleroderma or systemic sclerosis (SSc), but the precise mechanisms are poorly understood. The immediate-early gene Egr-1 is an inducible transcription factor with key roles in mediating fibrotic TGF-ß responses. To elucidate Egr-1 function in SSc-associated fibrosis, we examined change in gene expression induced by Egr-1 in human fibroblasts at the genome-wide level. Using microarray expression analysis, we derived a fibroblast "Egr-1-responsive gene signature" comprising over 600 genes involved in cell proliferation, TGF-ß signaling, wound healing, extracellular matrix synthesis and vascular development. The experimentally derived "Egr-1-responsive gene signature" was then evaluated in an expression microarray dataset comprising skin biopsies from 27 patients with localized and systemic forms of scleroderma and six healthy controls. We found that the "Egr-1 responsive gene signature" was substantially enriched in the "diffuse-proliferation" subset comprising exclusively of patients with diffuse cutaneous SSc (dcSSc) of skin biopsies. A number of Egr-1-regulated genes was also associated with the "inflammatory" intrinsic subset. Only a minority of Egr-1-regulated genes was concordantly regulated by TGF-ß. These results indicate that Egr-1 induces a distinct profibrotic/wound healing gene expression program in fibroblasts that is associated with skin biopsies from SSc patients with diffuse cutaneous disease. These observations suggest that targeting Egr-1 expression or activity might be a novel therapeutic strategy to control fibrosis in specific SSc subsets.

Topics: Adenoviridae; Adult; Early Growth Response Protein 1; Fibroblasts; Fibrosis; Genomics; Humans; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Scleroderma, Systemic; Signal Transduction; Skin; Transcriptome; Transforming Growth Factor beta; Wound Healing

Lack of an adequate experimental model has hindered the ability to fully understand scleroderma (SSc) pathogenesis. Current SSc research is based on the study of cultured fibroblasts from skin biopsies. In depth characterization of the SSc fibroblast phenotype is hindered by the limited lifespan and heterogeneity of these cells. The goal of this study was to isolate high collagen-producing fibroblasts from SSc biopsies and extend their lifespan with hTERT immortalization to enable characterization of their phenotype. Fibroblasts from two pairs of closely matched normal and SSc biopsies were infected with an hTERT lentivirus. Infected colonies were isolated, cultured into clonal cell lines and analysed with respect to profibrotic gene expression. The mRNA levels of nine profibrotic genes were measured by quantitative real-time PCR. Protein levels were assessed by Western blot. The hTERT SSc clones were heterogeneous with regards to expression of the profibrotic genes measured. A subset of the SSc clones showed elevated expression levels of collagen I, connective tissue growth factor and thrombospondin 1 mRNA, while expression of other genes was not significantly changed. Elevated expression of collagen I protein and mRNA was correlative with elevated expression of connective tissue growth factor. Several hTERT clones expressed high levels of pSmad1, Smad1 and TGF-betaRI indicative of altered TGF-beta signalling. A portion of SSc clones expressed several profibrotic genes. This study demonstrates that select characteristics of the SSc phenotype are expressed in a subset of activated fibroblasts in culture. The clonal SSc cell lines may present a new and useful model to investigate the mechanisms involved in SSc fibrosis.

Topics: Cell Line; Cell Proliferation; Cell Shape; Cellular Senescence; Clone Cells; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Enzyme Assays; Female; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Humans; Kinetics; Middle Aged; RNA, Messenger; Scleroderma, Systemic; Telomerase; Transforming Growth Factor beta

Fra-2 belongs to the activator protein 1 family of transcription factors. Mice transgenic for Fra-2 develop a systemic fibrotic disease with vascular manifestations similar to those of systemic sclerosis (SSc). The aim of the present study was to investigate whether Fra-2 plays a role in the pathogenesis of SSc and to identify the molecular mechanisms by which Fra-2 induces fibrosis.. Dermal thickness and the number of myofibroblasts were determined in skin sections from Fra-2-transgenic and wild-type mice. The expression of Fra-2 in SSc patients and in animal models of SSc was analyzed by real-time polymerase chain reaction and immunohistochemistry. Fra-2, transforming growth factor beta (TGFbeta), and ERK signaling in SSc fibroblasts were inhibited using small interfering RNA, neutralizing antibodies, and small-molecule inhibitors.. Fra-2-transgenic mice developed a skin fibrosis with increases in dermal thickness and increased myofibroblast differentiation starting at age 12 weeks. The expression of Fra-2 was up-regulated in SSc patients and in different mouse models of SSc. Stimulation with TGFbeta and platelet-derived growth factor (PDGF) significantly increased the expression of Fra-2 in SSc fibroblasts and induced DNA binding of Fra-2 in an ERK-dependent manner. Knockdown of Fra-2 potently reduced the stimulatory effects of TGFbeta and PDGF and decreased the release of collagen from SSc fibroblasts.. We demonstrate that Fra-2 is overexpressed in SSc and acts as a novel downstream mediator of the profibrotic effects of TGFbeta and PDGF. Since transgenic overexpression of Fra-2 causes not only fibrosis but also vascular disease, Fra-2 might be an interesting novel candidate for molecular-targeted therapies for SSc.

Topics: Animals; Cells, Cultured; Dermis; Disease Models, Animal; Extracellular Matrix; Fibroblasts; Fibrosis; Fluorescent Antibody Technique, Indirect; Fos-Related Antigen-2; Humans; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Mice, Transgenic; Platelet-Derived Growth Factor; RNA Interference; RNA, Small Interfering; Scleroderma, Systemic; Transforming Growth Factor beta

The purpose of this study was to evaluate collagen deposition, mRNA collagen synthesis and TGF-beta expression in the lung tissue in an experimental model of scleroderma after collagen V-induced nasal tolerance.. Female New Zealand rabbits (N = 12) were immunized with 1 mg/ml of collagen V in Freund's adjuvant (IM). After 150 days, six immunized animals were tolerated by nasal administration of collagen V (25 microg/day) (IM-TOL) daily for 60 days. The collagen content was determined by morphometry, and mRNA expressions of types I, III and V collagen were determined by Real-time PCR. The TGF-beta expression was evaluated by immunostaining and quantified by point counting methods. To statistic analysis ANOVA with Bonferroni test were employed for multiple comparison when appropriate and the level of significance was determined to be p < 0.05.. IM-TOL, when compared to IM, showed significant reduction in total collagen content around the vessels (0.371 +/- 0.118 vs. 0.874 +/- 0.282, p < 0.001), bronchioles (0.294 +/- 0.139 vs. 0.646 +/- 0.172, p < 0.001) and in the septal interstitium (0.027 +/- 0.014 vs. 0.067 +/- 0.039, p = 0.026). The lung tissue of IM-TOL, when compared to IM, showed decreased immunostaining of types I, III and V collagen, reduced mRNA expression of types I (0.10 +/- 0.07 vs. 1.0 +/- 0.528, p = 0.002) and V (1.12 +/- 0.42 vs. 4.74 +/- 2.25, p = 0.009) collagen, in addition to decreased TGF-beta expression (p < 0.0001).. Collagen V-induced nasal tolerance in the experimental model of SSc regulated the pulmonary remodeling process, inhibiting collagen deposition and collagen I and V mRNA synthesis. Additionally, it decreased TGF-beta expression, suggesting a promising therapeutic option for scleroderma treatment.

Topics: Administration, Intranasal; Animals; Collagen Type V; Disease Models, Animal; Down-Regulation; Female; Gene Expression; Humans; Lung; Rabbits; RNA, Messenger; Scleroderma, Systemic; Transforming Growth Factor beta

To assess dermal expression and fibroblast production of fibrillin-1 (FBN-1) in SSc.. In vivo analysis of microfibrillar network was performed using EM from affected and unaffected skin biopsy specimens of dcSSc patients (n = 5) compared with healthy controls (n = 2). FBN-1 matrix deposition and organization by dermal fibroblast cultures from dcSSc (n = 6), healthy (n = 5) and Marfan (n = 4) controls was analysed in vitro by IF with or without TGF-beta activation. Finally, production of FBN-1 by cultured dermal fibroblasts was evaluated by western blot (WB) and real-time PCR.. We observed a striking decrease of tissue microfibrillar network in the dermis of SSc patients compared with healthy controls affecting both clinically involved and uninvolved skin. In cultures, SSc dermal fibroblasts displayed no apparent in vitro alteration of synthesis, secretion and organization of microfibril network. The WB and real-time PCR analyses showed similar FBN-1 amounts in matrix and FBN1 gene expression in SSc and healthy controls.. We observed a striking decrease of in vivo microfibrillar network in clinically affected and unaffected skin in early dcSSc patients. This does not relate to an inability of SSc dermal fibroblasts to produce, secrete and organize microfibrils in vitro. Therefore, the disturbances of microfibrils in SSc may be a secondary event to matrix remodelling that occurs in this disease.

Topics: Biopsy; Blotting, Western; Case-Control Studies; Dermis; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Fibroblasts; Humans; Microfilament Proteins; Polymerase Chain Reaction; Polymorphism, Genetic; Scleroderma, Systemic; Statistics as Topic; Transforming Growth Factor beta

The role and function of T regulatory (Treg) cells have not been fully investigated in patients with systemic sclerosis (SSc).. Ten patients with SSc donated 20ml of peripheral blood. Activity (Valentini) and severity (Medsger) scores for SSc were calculated for all patients. Healthy volunteers (controls) were matched to each patient by gender and age. CD4(+) cells were separated using the MACS system. The numbers of Treg cells were estimated by flow cytometry after staining for CD4, CD25, and FoxP3 and calculated as patient-to-control ratio separately for each experiment. Correlations with activity and severity indices of the disease were performed. Twenty-four-hour production of TGF-beta and IL-10 by activated CD4(+) cells was measured by ELISA in culture supernatants.. The numbers of Treg cells, expressed as patient-to-control ratio, correlated significantly with both activity and severity indices (r=0.71, p=0.034 and r=0.67, p=0.044, respectively). ELISA-measured production of TGF-beta and IL-10 by CD4(+) cells was similar in patients and controls.. Increased numbers of Treg cells are present in patients with SSc, correlating with activity and severity of the disease. This expansion of Treg cells was not accompanied, however, by heightened TGF-beta or IL-10 production. Further studies to elaborate the causes and functional significance of Treg cell expansion in SSc are needed.

Topics: Adult; CD4-Positive T-Lymphocytes; Disease Progression; Female; Forkhead Transcription Factors; Humans; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Male; Middle Aged; Scleroderma, Systemic; Severity of Illness Index; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

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

Systemic sclerosis (SSc) is classified among the complex genetic disorders and is characterized by massive extracellular matrix deposits. These may be due to overactivation of transforming growth factor ss that may be in part a result of abnormal remodeling of extracellular matrix and microfibrils. Metalloproteinases (MMP) are a family of proteolytic enzymes, and MMP 2, 9, and 14 contribute to the degradation of microfibrils. Our aim was to determine whether polymorphisms of the MMP2, MMP9, and MMP14 genes confer susceptibility to SSc in a large population.. A case-control study was performed in 659 SSc patients and 511 healthy matched controls from a European Caucasian population. Six Tag single-nucleotide polymorphisms (SNP) of the MMP2 gene and 2 SNP of MMP9 and MMP14 genes were genotyped.. All SNP were in Hardy-Weinberg equilibrium in the control population. There was no association between the MMP2, MMP9, and MMP14 variants we investigated and SSc for allelic and genotype frequencies. No association was observed for the different subphenotypes of SSc patients.. Our results in a large cohort of European Caucasian SSc patients do not support that MMP2, MMP9, and MMP14 genes are involved in the genetic background of SSc.

Topics: Aged; Case-Control Studies; Europe; Female; Gene Frequency; Genetic Predisposition to Disease; Genotype; Humans; Male; Matrix Metalloproteinase 14; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Microfibrils; Middle Aged; Polymorphism, Single Nucleotide; Scleroderma, Systemic; Transforming Growth Factor beta; White People

Improved outcome measures in systemic sclerosis (SSc) are critical to finding active therapeutics for this disease. The modified Rodnan skin thickness score (MRSS) is the current standard for evaluating skin disease in SSc, but it is not commonly used in the clinical setting, in part because it requires specialized training to perform accurately and consistently. The purpose of this study was to investigate whether skin gene expression might serve as a more objective surrogate outcome measure to supplement skin score evaluations.. Skin RNAs from a group of patients with diffuse cutaneous SSc were studied for expression levels of genes known to be regulated by transforming growth factor beta (TGFbeta) and interferon (IFN). These levels were correlated with the MRSS, using multiple regression analyses to obtain best-fit models.. Skin expression of the TGFbeta-regulated genes cartilage oligomeric matrix protein (COMP) and thrombospondin 1 (TSP-1) correlated moderately well with the MRSS, but the addition of other TGFbeta-regulated genes failed to significantly improve best-fit models. IFN-regulated genes were also found to correlate with the MRSS, and the addition of interferon-inducible 44 (IFI44) and sialoadhesin (Siglec-1) to COMP and TSP-1 in multiple regression analyses significantly improved best-fit models, achieving an R(2) value of 0.89. These results were validated using an independent group of skin biopsy samples. Longitudinal scores using this 4-gene biomarker indicated that it detects change over time that corresponds to changes in the MRSS.. We describe a 4-gene predictor of the MRSS and validate its performance. This objective measure of skin disease could provide a strong surrogate outcome measure for patient care and for clinical trials.

Topics: Antigens; Biopsy; Cartilage Oligomeric Matrix Protein; Cytoskeletal Proteins; Extracellular Matrix Proteins; Genetic Markers; Genetic Testing; Glycoproteins; Humans; Matrilin Proteins; Membrane Glycoproteins; Predictive Value of Tests; Receptors, Immunologic; Regression Analysis; Reproducibility of Results; Scleroderma, Systemic; Severity of Illness Index; Sialic Acid Binding Ig-like Lectin 1; Skin; Thrombospondin 1; Transforming Growth Factor beta

Abnormal fibrillinogenesis is associated with connective tissue disorders (CTDs), including Marfan syndrome (MFS), systemic sclerosis (SSc) and Tight-skin (Tsk) mice. We have previously shown that TGF-beta and Wnt stimulate fibrillin-1 assembly and that fibrillin-1 and the developmental regulator CCN3 are both highly increased in Tsk skin. We investigated the role of CCN3 in abnormal fibrillinogenesis in Tsk mice, MFS, and SSc. Smad3 deletion in Tsk mice decreased CCN3 overexpression, suggesting that TGF-beta mediates at least part of the effect of Tsk fibrillin on CCN3 which is consistent with a synergistic effect of TGF-beta and Wnt in vitro on CCN3 expression. Disruption of fibrillin-1 assembly by MFS fibrillin decreased CCN3 expression and skin from patients with early diffuse SSc showed a strong correlation between increased CCN3 and fibrillin-1 expression, suggesting that CCN3 regulation by fibrillin-1 extends to these CTDs. Diffuse SSc skin and sera also showed evidence of increased Wnt activity, implicating a Wnt stimulus behind this correlation. CCN3 overexpression markedly repressed fibrillin-1 assembly and also blocked other TGFbeta- and Wnt-regulated profibrotic gene expression. Together, these data indicate that CCN3 counter-regulates positive signals from TGF-beta and Wnt for fibrillin fibrillogenesis and profibrotic gene expression.

Topics: Animals; Biopsy; Case-Control Studies; CCN Intercellular Signaling Proteins; Cells, Cultured; Disease Models, Animal; Fibrillin-1; Fibrillins; Humans; Intracellular Signaling Peptides and Proteins; Marfan Syndrome; Mice; Mice, Mutant Strains; Microfilament Proteins; Nephroblastoma Overexpressed Protein; Proto-Oncogene Proteins; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Transforming Growth Factor beta; Wnt Proteins

The predisposition for scleroderma, defined as fibrosis and hardening of the skin, is poorly understood. We report that stiff skin syndrome (SSS), an autosomal dominant congenital form of scleroderma, is caused by mutations in the sole Arg-Gly-Asp sequence-encoding domain of fibrillin-1 that mediates integrin binding. Ordered polymers of fibrillin-1 (termed microfibrils) initiate elastic fiber assembly and bind to and regulate the activation of the profibrotic cytokine transforming growth factor-beta (TGFbeta). Altered cell-matrix interactions in SSS accompany excessive microfibrillar deposition, impaired elastogenesis, and increased TGFbeta concentration and signaling in the dermis. The observation of similar findings in systemic sclerosis, a more common acquired form of scleroderma, suggests broad pathogenic relevance.

Topics: Biopsy; Cell Adhesion; Cell Movement; Collagen; DNA Mutational Analysis; Elastin; Extracellular Matrix; Family; Female; Fibrillin-1; Fibrillins; Humans; Immunohistochemistry; Male; Mesoderm; Microfibrils; Microfilament Proteins; Mutation; Pedigree; Phenotype; Scleroderma, Systemic; Signal Transduction; Skin; Syndrome; Transforming Growth Factor beta

Vasculopathy, including altered vasoreactivity and abnormal large vessel biomechanics, is a hallmark of systemic sclerosis (SSc). However, the pathogenic link with other aspects of the disease is less clear. To assess the potential role of transforming growth factor beta (TGF-beta) overactivity in driving these cardiovascular abnormalities, we studied a novel transgenic mouse model characterized by ligand-dependent activation of TGF-beta signaling in fibroblasts.. The transgenic mouse strain Tbeta RIIDeltak-fib is characterized by balanced ligand-dependent upregulation of TGF-beta signaling. Aortic and cardiac tissues were examined with histologic, biochemical, and isolated organ bath studies. Vascular and perivascular architecture was examined by hematoxylin and eosin (H&E) and special stains including immunostaining for TGF-beta1 and phospho-Smad2/3 (pSmad2/3). Confirmatory aortic smooth muscle cell proliferation, phenotype, and functional assays, including signaling responses to exogenous TGF-beta and endothelin-1, were performed. Aortic ring contractile responses to direct and receptor-mediated stimulation were assessed.. Aortic ring contractility and relaxation were diminished compared with wild-type controls, and this was associated with aortic adventitial fibrosis confirmed histologically and with Sircol assay. TGF-beta1 and pSmad 2/3 expression was increased in the adventitia and smooth muscle layer of the aorta. Aortic smooth muscle cells from transgenic animals showed significant upregulation of TGF-beta- responsive genes important for cytoskeletal function, such as transgelin and smoothelin, which were then resistant to further stimulation with exogenous TGF-beta1. These cells promoted significantly more contraction of free floating type I collagen lattices when compared with the wild-type, but were again resistant to exogenous TGF-beta1 stimulation. Aortic ring responses to receptor-mediated contraction were reduced in the transgenic animals. Specifically, bosentan reduced endothelin-mediated contraction in wild-type animals, but had no effect in transgenic animals, and endothelin axis gene expression was altered in transgenic animals. Transgenic mice developed cardiac fibrosis.. The histologic, biochemical, and functional phenotype of this transgenic mouse model of scleroderma offers insight into the altered biomechanical properties previously reported for large elastic arteries in human SSc and suggests a role for perturbed TGF-beta and endothelin activity in this process.

Topics: Animals; Aorta, Thoracic; Cells, Cultured; Collagen; Disease Models, Animal; Endothelins; Female; Fibroblasts; Fibrosis; Male; Mice; Mice, Transgenic; Muscle Contraction; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Scleroderma, Systemic; Signal Transduction; Smad2 Protein; Systemic Vasculitis; Transforming Growth Factor beta

Nephrogenic systemic fibrosis (NSF) is an uncommon fibrotic disorder occurring after administration of linear gadolinium contrast agents in patients with severely decreased kidney function. The underlying pathogenetic mechanism of fibrosis remains to be elucidated. Transforming growth factor beta (TGF-beta), a key player in the pathogenesis of fibrotic disorders, has been found to be overexpressed in NSF skin lesions. The aim of this study is to analyze the TGF-beta-SMAD-connective tissue growth factor (CTGF) axis in NSF skin lesions compared with skin specimens from patients with systemic sclerosis, hemodialysis patients without NSF, and healthy controls. Additionally, expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) and antifibrotic tumor necrosis factor alpha (TNF-alpha) were examined.. Observational study.. Full-thickness skin biopsy specimens from fibrotic lesions or healthy skin were obtained from 10 patients with NSF, 16 patients with systemic sclerosis, 8 non-NSF hemodialysis patients, and 17 healthy participants.. Patient diagnosis of NSF, systemic sclerosis, non-NSF hemodialysis patients, and healthy participants, as defined using skin biopsy.. Dermal messenger RNA and protein expression of profibrotic TGF-beta, SMAD2, SMAD3, SMAD4, SMAD7, CTGF, TIMP-1, antifibrotic SMAD7, and TNF-alpha were analyzed using real-time reverse transcription-polymerase chain reaction and immunohistologic examination on formalin-embedded tissue.. Dermal expression of nearly all parameters differed in hemodialysis patients compared with healthy controls. In comparison to hemodialysis patients and healthy participants, we found increased messenger RNA levels for TGF-beta, the profibrotic receptor-activated SMAD2 and SMAD3, CTGF, and TIMP-1 in NSF and systemic sclerosis lesions. Few differences between NSF and non-NSF hemodialysis patients were observed for common SMAD4, inhibitory SMAD7, and TNF-alpha.. Small patient cohort.. Our results suggest a profibrotic imbalance in the TGF-beta-SMAD-CTGF axis in NSF skin lesions. Significantly increased dermal expression of TGF-beta and TIMP-1 in non-NSF hemodialysis patients in comparison to healthy participants emphasizes the need for a hemodialysis control group for future investigations and suggests a pre-existing profibrotic situation in the skin of hemodialysis patients.

Topics: Aged; Aged, 80 and over; Biopsy; Cohort Studies; Connective Tissue Growth Factor; Female; Humans; Kidney Diseases; Male; Middle Aged; Nephrogenic Fibrosing Dermopathy; Renal Dialysis; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad2 Protein; Smad3 Protein; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Systemic sclerosis (SSc) is a disorder of systemic and dermal fibrosis of uncertain etiology. Recently, we found that SSc epidermis is abnormal, taking on an activated phenotype observed during wound healing and tissue repair. As epithelial-fibroblast interactions are important during wound repair and in fibrosis in general, we investigated further the phenotype of the SSc epidermis, and tested whether the SSc epidermis provides a pro-fibrotic stimulus to fibroblasts. In this study we show that in SSc epidermis keratinocyte maturation is delayed, and wound-associated keratins 6 and 16 are induced, in both involved and clinically uninvolved skin. Phosphorylation array analysis revealed induction of stress-induced mitogen-activated protein kinase signaling and mesenchymal feedback through hepatocyte growth factor/c-Met in SSc epidermis. SSc epidermal cells maintained with normal fibroblasts in three-dimensional co-culture were found to stimulate fibroblasts, leading to contractility and connective tissue growth factor expression. These effects depend on elevation of IL-1alpha by the epidermal cells and induction of endothelin-1 and transforming growth factor-beta in fibroblasts. Antagonism of endogenous IL-1alpha using IL-1 receptor antagonist blocked gel contraction by SSc epidermis. We propose that in SSc, epidermal cells are in a persistently activated state and are able to promote dermal fibrosis. These findings are important because biologic therapies could target epithelial-fibroblast interactions in the disease.

Topics: Biopsy; Cell Communication; Cells, Cultured; Coculture Techniques; Connective Tissue Growth Factor; Endothelin-1; Epidermis; Epithelial Cells; Fibroblasts; Fibrosis; Humans; Interleukin 1 Receptor Antagonist Protein; Interleukin-1alpha; Keratin-16; Keratin-6; Phosphorylation; Proto-Oncogene Proteins c-met; Scleroderma, Systemic; Signal Transduction; Stress, Physiological; Transforming Growth Factor beta

Tissue fibrosis and vascular disease are hallmarks of systemic sclerosis (SSc). Transforming growth factor beta (TGFbeta) is a key-player in fibroblast activation and tissue fibrosis in SSc. In contrast to fibrosis, evidence for a role of TGFbeta in vascular disease of SSc is scarce. Using a transgenic mouse model with fibroblast-specific expression of a kinase-deficient TGFbeta receptor type II, Derrett-Smith and colleagues demonstrate that aberrant TGFbeta signaling in fibroblasts might result in activation of vascular smooth muscle cells and architectural changes of the vessel wall of the aorta.

Topics: Animals; Aorta; Disease Models, Animal; Fibroblasts; Mice; Mice, Transgenic; Muscle, Smooth, Vascular; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Vascular Diseases

Immune activation of fibrosis likely has a crucial role in the pathogenesis of systemic sclerosis (SSc). The aim of this study was to better understand the innate immune regulation and associated IFN- and transforming growth factor-β (TGFβ)-responsive gene expression in SSc skin and dermal fibroblasts, in particular the effect of different Toll-like receptor (TLR) ligands. To better understand the relationship between inflammation and fibrosis in vivo, we developed a murine model for chronic innate immune stimulation. We found that expression of both IFN- and TGFβ-responsive genes is increased in SSc skin and SSc fibroblasts when stimulated by TLR ligands. In contrast, cutaneous lupus skin showed much more highly upregulated IFN-responsive and much less highly upregulated TGFβ-responsive gene expression. Of the TLRs ligands tested, the TLR3 ligand, polyinosinic/polycytidylic acid (Poly(I:C)), most highly increased fibroblast expression of both IFN- and TGFβ-responsive genes as well as TLR3. Chronic subcutaneous immune stimulation by Poly(I:C) stimulated inflammation, and IFN- and TGFβ-responsive gene expression. However, in this model, type I IFNs had no apparent role in regulating TGFβ activity in the skin. These results suggest that TLR agonists may be important stimuli of dermal fibrosis, which is potentially mediated by TLR3 or other innate immune receptors.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Cells, Cultured; Dermatitis; Dermis; Disease Models, Animal; Fibroblasts; Gene Expression; GTP-Binding Proteins; Humans; Interferon Type I; Interferon-gamma; Lupus Erythematosus, Cutaneous; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Myxovirus Resistance Proteins; Poly I-C; Scleroderma, Systemic; Toll-Like Receptor 3; Transforming Growth Factor beta

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-beta 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-beta 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.

Topics: Case-Control Studies; Cell Proliferation; Cells, Cultured; Connective Tissue Growth Factor; Dermis; Extracellular Matrix; Fibroblasts; Gene Silencing; Humans; Matrix Metalloproteinase 1; RNA, Small Interfering; Scleroderma, Systemic; Time Factors; Transforming Growth Factor beta

The nuclear orphan receptor peroxisome proliferator-activated receptor-gamma (PPAR-γ) is expressed in multiple cell types in addition to adipocytes. Upon its activation by natural ligands such as fatty acids and eicosanoids, or by synthetic agonists such as rosiglitazone, PPAR-γ regulates adipogenesis, glucose uptake and inflammatory responses. Recent studies establish a novel role for PPAR-γ signaling as an endogenous mechanism for regulating transforming growth factor-ß (TGF-ß)-dependent fibrogenesis. Here, we sought to characterize PPAR-γ function in the prototypic fibrosing disorder systemic sclerosis (SSc), and delineate the factors governing PPAR-γ expression. We report that PPAR-γ levels were markedly diminished in skin and lung biopsies from patients with SSc, and in fibroblasts explanted from the lesional skin. In normal fibroblasts, treatment with TGF-ß resulted in a time- and dose-dependent down-regulation of PPAR-γ expression. Inhibition occurred at the transcriptional level and was mediated via canonical Smad signal transduction. Genome-wide expression profiling of SSc skin biopsies revealed a marked attenuation of PPAR-γ levels and transcriptional activity in a subset of patients with diffuse cutaneous SSc, which was correlated with the presence of a "TGF-ß responsive gene signature" in these biopsies. Together, these results demonstrate that the expression and function of PPAR-γ are impaired in SSc, and reveal the existence of a reciprocal inhibitory cross-talk between TGF-ß activation and PPAR-γ signaling in the context of fibrogenesis. In light of the potent anti-fibrotic effects attributed to PPAR-γ, these observations lead us to propose that excessive TGF-ß activity in SSc accounts for impaired PPAR-γ function, which in turn contributes to unchecked fibroblast activation and progressive fibrosis.

Topics: Adipogenesis; Adult; Animals; Animals, Newborn; Blotting, Western; Cells, Cultured; Down-Regulation; Female; Fibroblasts; Fibrosis; Gene Expression Profiling; Humans; Lung; Male; Mice; Mice, Knockout; Middle Aged; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Topics: Epithelium; Extracellular Matrix; Fibroblasts; Humans; Hyaluronic Acid; Hypertension, Pulmonary; Idiopathic Interstitial Pneumonias; Prognosis; Pulmonary Fibrosis; Randomized Controlled Trials as Topic; Scleroderma, Systemic; Transforming Growth Factor beta

We have recently shown a significant reduction in cytokine-induced transcription of type I collagen and fibronectin in systemic sclerosis (SSc) skin fibroblasts upon treatment with trichostatin A (TSA). Moreover, in a mouse model of fibrosis, TSA prevented the dermal accumulation of extracellular matrix. The purpose of this study was to analyze the silencing of histone deacetylase 7 (HDAC-7) as a possible mechanism by which TSA exerts its antifibrotic function.. Skin fibroblasts from patients with SSc were treated with TSA and/or transforming growth factor beta. Expression of HDACs 1-11, extracellular matrix proteins, connective tissue growth factor (CTGF), and intercellular adhesion molecule 1 (ICAM-1) was analyzed by real-time polymerase chain reaction, Western blotting, and the Sircol collagen assay. HDAC-7 was silenced using small interfering RNA.. SSc fibroblasts did not show a specific pattern of expression of HDACs. TSA significantly inhibited the expression of HDAC-7, whereas HDAC-3 was up-regulated. Silencing of HDAC-7 decreased the constitutive and cytokine-induced production of type I and type III collagen, but not fibronectin, as TSA had done. Most interestingly, TSA induced the expression of CTGF and ICAM-1, while silencing of HDAC-7 had no effect on their expression.. Silencing of HDAC-7 appears to be not only as effective as TSA, but also a more specific target for the treatment of SSc, because it does not up-regulate the expression of profibrotic molecules such as ICAM-1 and CTGF. This observation may lead to the development of more specific and less toxic targeted therapies for SSc.

Topics: Blotting, Western; Cells, Cultured; Collagen Type I; Collagen Type III; Connective Tissue Growth Factor; Enzyme Inhibitors; Extracellular Matrix Proteins; Fibroblasts; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Hydroxamic Acids; Intercellular Adhesion Molecule-1; Polymerase Chain Reaction; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Pulmonary diseases associated with fibrosis, including scleroderma lung disease, are characterized by the accumulation of T cells in the lungs. These cells are thought to facilitate lung fibrosis, but the exact mechanisms of their profibrotic action are not clear. Several alphaV-containing integrins, including alphaVbeta3 and alphaVbeta5, have been shown to directly activate transforming growth factor beta (TGFbeta) and promote collagen accumulation. The aim of this study was to investigate whether pulmonary T cells express profibrotic integrins and regulate collagen accumulation.. Expression of integrins was assessed by immunohistochemical analysis of lung tissue, by flow cytometry using bronchoalveolar lavage fluid from patients with systemic sclerosis (SSc), and in a CCL18 overexpression animal model of pulmonary T cell infiltration. Experiments in cell cultures were performed to determine whether integrin-expressing T cells are profibrotic in cocultures with pulmonary fibroblasts and, if so, through what possible mechanism.. Lymphocytes and integrin-positive cells were present in the lungs, and pulmonary T cells expressed integrins alphaVbeta3 and alphaVbeta5 in patients with SSc and in the animal model. Systemic administration of neutralizing anti-integrin alphaV antibody or a genetic deficiency of integrin beta3 in the CCL18 overexpression model significantly attenuated CCL18-driven pulmonary lymphocytic infiltration and collagen accumulation. Jurkat T cells overexpressing integrin alphaVbeta3 or integrin alphaVbeta5 in cocultures with primary pulmonary fibroblasts stimulated collagen accumulation and Smad2 nuclear translocation. Neutralizing anti-TGFbeta antibody attenuated the profibrotic effect of integrin-expressing T cells.. Pulmonary infiltrating T lymphocytes may express integrins alphaVbeta3 and alphaVbeta5 that are necessary for lymphocytic infiltration and T cell-associated TGFbeta activation and collagen accumulation.

Topics: Animals; Antibodies; Bronchoalveolar Lavage Fluid; Cells, Cultured; Collagen; Flow Cytometry; Humans; Immunohistochemistry; Integrin alphaV; Integrin alphaVbeta3; Jurkat Cells; Lung; Mice; Mice, Inbred C57BL; Pulmonary Fibrosis; Receptors, Vitronectin; Scleroderma, Systemic; T-Lymphocytes; Transforming Growth Factor beta

Systemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc.. Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFbeta and IFNgamma using flow cytometry. Levels of IL-17, IL-6, IL-1alpha and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNgamma and TGFbeta were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1alpha were increased in all or subsets of SSc patients.. The combination of IL-17, IFNgamma and TGFbeta levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.

Topics: Adult; Disease Progression; Female; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-17; Interleukin-23; Interleukin-6; Leukocyte Common Antigens; Male; Middle Aged; Phenotype; Scleroderma, Systemic; Transforming Growth Factor beta

Regulatory T cells (Tregs) are essential in the control of tolerance. Evidence implicates Tregs in human autoimmune conditions. Here we investigated their role in systemic sclerosis (SSc).. Patients were subdivided as having limited cutaneous SSc (lcSSc, n = 20) or diffuse cutaneous SSc (dcSSc, n = 48). Further subdivision was made between early dcSSc (n = 24) and late dcSSc (n = 24) based upon the duration of disease. 26 controls were studied for comparison. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, FoxP3, CD127, CD62L, GITR, CD69 using flow cytometry. T cell suppression assays were performed using sorted CD4CD25(high)CD127(-) and CD4CD25(low)CD127(high) and CD3(+) cells. Suppressive function was correlated with CD69 surface expression and TGFbeta secretion/expression. The frequency of CD4(+)CD25(+) and CD25(high)FoxP3(high)CD127(neg) T cells was highly increased in all SSc subgroups. Although the expression of CD25 and GITR was comparable between groups, expression of CD62L and CD69 was dramatically lower in SSc patients, which correlated with a diminished suppressive function. Co-incubation of Tregs from healthy donors with plasma from SSc patients fully abrogated suppressive activity. Activation of Tregs from healthy donors or SSc patients with PHA significantly up regulated CD69 expression that could be inhibited by SSc plasma.. These results indicate that soluble factors in SSc plasma inhibit Treg function specifically that is associated with altered Treg CD69 and TGFbeta expression. These data suggest that a defective Treg function may underlie the immune dysfunction in systemic sclerosis.

Topics: Adult; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Case-Control Studies; CD3 Complex; Female; Humans; Interleukin-2 Receptor alpha Subunit; Interleukin-7 Receptor alpha Subunit; Lectins, C-Type; Leukocytes, Mononuclear; Male; Middle Aged; Scleroderma, Systemic; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

Topics: Fibrosis; Humans; Scleroderma, Systemic; Transforming Growth Factor beta

Tissue fibrosis is a major cause of morbidity and mortality in systemic sclerosis (SSc), and an increasing number of promising molecular targets for antifibrotic therapies have been described recently. Transforming growth factor beta (TGFbeta) is well known to be the principal factor that leads to tissue fibrosis. The present study was undertaken to investigate the ability of HSc025, a novel small compound that antagonizes TGFbeta/Smad signaling through the activation of nuclear translocation of Y-box binding protein 1, to prevent tissue fibrosis in vitro or in mouse models of SSc.. Human dermal fibroblasts were exposed to HSc025 at various concentrations in the presence of TGFbeta, and levels of collagen or fibronectin expression were determined. HSc025 (15 mg/kg/day for 14 days) was administered orally to tight skin mice and to mice with bleomycin-induced pulmonary fibrosis. Improvement of tissue fibrosis was evaluated by histologic or biochemical examination in each model.. Pretreatment with HSc025 prevented Smad-dependent promoter activation, in a dose-dependent manner; however, HSc025 had no effect on TGFbeta-induced phosphorylation of Smad3. The inhibitory effects of HSc025 on TGFbeta-induced collagen or fibronectin expression were also confirmed in vitro. Orally administered HSc025 significantly reduced hypodermal thickness and hydroxyproline content in tight skin mice, and markedly decreased the histologic score and hydroxyproline content in the lungs of bleomycin-treated mice.. These results demonstrate that HSc025 is a novel inhibitor of TGFbeta/Smad signaling, resulting in the improvement of skin and pulmonary fibrosis. Orally available HSc025 might therefore be useful in the treatment of SSc.

Topics: Alkadienes; Animals; Bleomycin; Cells, Cultured; Collagen; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Fibroblasts; Fibronectins; Fibrosis; Humans; Mice; Mice, Inbred C57BL; Plant Extracts; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Skin; Smad Proteins; Transcriptional Activation; Transforming Growth Factor beta; Zanthoxylum

By stimulating collagen synthesis and myofibroblasts differentiation, transforming growth factor-beta (TGF- beta) plays a pivotal role in tissue repair and fibrosis. The early growth response-1 (Egr-1) transcription factor mediates profibrotic TGF-beta responses, and its expression is elevated in biopsies from patients with scleroderma. NGF1-A-binding protein 2 (Nab2) is a conserved transcriptional cofactor that directly binds to Egr-1 and positively or negatively modulates Egr-1 target gene transcription. Despite the recognized importance of Nab2 in governing the intensity of Egr-1-dependent responses, the regulation and function of Nab2 in the context of fibrotic TGF-beta signaling is unknown. Here we show that TGF-beta caused a time-dependent stimulation of Nab2 protein and mRNA in normal fibroblasts. Ectopic expression of Nab2 in these cells blocked Egr-1-dependent transcriptional responses, and abrogated TGF-beta-induced stimulation of collagen synthesis and myofibroblasts differentiation. These inhibitory effects of Nab2 involved recruitment of the NuRD chromatin remodeling complex to the COL1A2 promoter and were accompanied by reduced histone H4 acetylation. Mice with targeted deletion of Nab2 displayed increased collagen accumulation in the dermis, and genetic or siRNA-mediated loss of Nab2 in fibroblasts was associated with constitutively elevated collagen synthesis and accentuation of Egr-1-dependent TGF-beta responses in vitro. Expression of Nab2 was markedly up-regulated in skin biopsies from patients with scleroderma, and was localized primarily to epidermal keratinocytes. In contrast, little Nab2 could be detected in dermal fibroblasts. These results identify Nab2 as a novel endogenous negative regulator of Egr-1-dependent TGF-beta signaling responsible for setting the intensity of fibrotic responses. Defective Nab2 expression or function in dermal fibroblasts might play a role in persistent fibrotic responses in scleroderma.

Topics: Acetylation; Animals; Cell Line; Collagen; Collagen Type I; Fibroblasts; Gene Deletion; Gene Expression Regulation; Humans; Mice; Models, Genetic; NIH 3T3 Cells; Repressor Proteins; Scleroderma, Systemic; Transforming Growth Factor beta

Rho-associated kinases (Rock) are the major cellular mediators of Rho GTPases and play an important role in the organization of the actin cytoskeleton. Inhibitors of Rock are currently being evaluated for the treatment of pulmonary arterial hypertension. This study was undertaken to analyze the role of Rock in the activation of fibroblasts in systemic sclerosis (SSc).. Rock signaling was inhibited using chemical inhibitors and small interfering RNA. The expression of extracellular matrix (ECM) proteins and alpha-smooth muscle actin was analyzed by real-time polymerase chain reaction, Western blotting, and SirCol assay. Metabolic activity was quantified by MTT assay. Cell viability was assessed by staining with annexin V and propidium iodide. The role of MAP kinases was investigated using selective inhibitors and Western blotting.. Inhibition of Rock strongly reduced the synthesis of the major ECM proteins at the messenger RNA level as well as the protein level. Counterregulatory changes in the expression of tissue inhibitors of metalloproteinases and matrix metalloproteinases were not observed. Inhibition of Rock prevented myofibroblast differentiation. Transforming growth factor beta activated ERK in a Rock-dependent manner, and ERK mediated in part the stimulatory effects of Rock on myofibroblast differentiation. Toxic adverse effects of the inhibition of Rock were not observed.. Our findings demonstrate that Rock potently stimulates the differentiation of resting fibroblasts into myofibroblasts and the production of ECM at biologically relevant concentrations without cell toxicity. These findings, along with the beneficial effects of Rock inhibition on vascular disease, indicate that inhibition of Rock might be an interesting novel therapeutic approach for the treatment of SSc.

Topics: Adult; Aged; Amides; Case-Control Studies; Cell Differentiation; Cells, Cultured; Enzyme Inhibitors; Extracellular Matrix; Female; Fibroblasts; Humans; Male; Matrix Metalloproteinases; Middle Aged; Platelet-Derived Growth Factor; Pyridines; rho-Associated Kinases; Scleroderma, Systemic; Skin; Tissue Inhibitor of Metalloproteinases; Transforming Growth Factor beta

Activation of Smad1 signaling has recently been implicated in the development of fibrosis. The goal of the present study was to gain further insights into activation of the Smad1 pathway in fibrosis in systemic sclerosis (SSc) and to determine whether this pathway is targeted by the antifibrotic drug imatinib mesylate.. Levels of phosphorylated Smad1 and total Smad1 were examined in SSc and control skin biopsy samples by immunohistochemistry and in cultured fibroblasts by Western blotting. Activity of the CCN2 promoter was examined by a luciferase reporter gene assay. Interactions of Smad1 with the CCN2 promoter were examined by in vitro and in vivo DNA binding assays. Expression of the nonreceptor tyrosine kinase c-Abl and Smad1 was blocked using respective small interfering RNA.. Total and phosphorylated Smad1 levels were significantly elevated in SSc skin biopsy samples and in cultured SSc fibroblasts and correlated with elevated CCN2 protein and CCN2 promoter activity. DNA binding assays demonstrated that Smad1 was a direct activator of the CCN2 gene. Small interfering RNA-mediated depletion of Smad1 in SSc fibroblasts normalized the production of CCN2 and collagen. Imatinib mesylate blocked activation of the Smad1 pathway in transforming growth factor beta-stimulated control fibroblasts and reversed activation of this pathway in SSc fibroblasts. Likewise, blockade of c-Abl abrogated activation of the Smad1 pathway in SSc fibroblasts.. Our findings demonstrate that activation of Smad1 signaling occurs in a subset of SSc patients and contributes to persistent activation of SSc fibroblasts. Demonstration that the Smad1/CCN2 pathway is blocked by imatinib mesylate further clarifies the mechanism of the antifibrotic effects of this compound. This study suggests that SSc patients with activated Smad1 signaling may benefit from imatinib mesylate treatment.

Topics: Adult; Aged; Benzamides; Biopsy; Case-Control Studies; Cells, Cultured; Connective Tissue Growth Factor; Female; Fibroblasts; Humans; Imatinib Mesylate; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Piperazines; Protein Kinase Inhibitors; Pyrimidines; Scleroderma, Systemic; Signal Transduction; Skin; Smad1 Protein; Transforming Growth Factor beta

Topics: Extracellular Matrix; Fibrosis; Humans; Kidney Diseases; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Recent studies have implicated caveolin 1 in the regulation of transforming growth factor beta (TGFbeta) downstream signaling. Given the crucial role of TGFbeta in the pathogenesis of systemic sclerosis (SSc), we sought to determine whether caveolin 1 is also involved in the pathogenesis of tissue fibrosis in SSc. We analyzed the expression of CAV1 in affected SSc tissues, studied the effects of lack of expression of CAV1 in vitro and in vivo, and analyzed the effects of restoration of caveolin 1 function on the fibrotic phenotype of SSc fibroblasts in vitro.. CAV1 expression in tissues was analyzed by immunofluorescence and confocal microscopy. The extent of tissue fibrosis in Cav1-knockout mice was assessed by histologic/histochemical analyses and quantified by hydroxyproline assays. Cav1-null and SSc fibroblast phenotypes and protein production were analyzed by real-time polymerase chain reaction, immunofluorescence, Western blot, and multiplexed enzyme-linked immunosorbent assay techniques. The effects of restoration of caveolin 1 function in SSc fibroblasts in vitro were also examined using a cell-permeable recombinant CAV1 peptide.. CAV1 was markedly decreased in the affected lungs and skin of SSc patients. Cav1-knockout mice developed pulmonary and skin fibrosis. Down-regulation of caveolin 1 was maintained in cultured SSc fibroblasts, and restoration of caveolin 1 function in vitro normalized their phenotype and abrogated TGFbeta stimulation through inhibition of Smad3 activation.. Caveolin 1 appears to participate in the pathogenesis of tissue fibrosis in SSc. Restoration of caveolin 1 function by treatment with a cell-permeable peptide corresponding to the CAV1 scaffolding domain may be a novel therapeutic approach in SSc.

Topics: Animals; Blotting, Western; Caveolin 1; Cell Survival; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Fluorescent Antibody Technique; Humans; Lung; Mice; Mice, Knockout; Microscopy, Confocal; Pulmonary Fibrosis; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Transforming Growth Factor beta; Up-Regulation

Treatment of sclerodermatous chronic GVHD (cGVHD) remains disappointing. Imatinib mesylate enables selective, dual inhibition of the transforming growth factor beta (TGFbeta) and PDGF pathways. Recently, the drug's effects on fibroblasts have been reported in both in vitro and in vivo studies. The inhibition of fibroblast growth and decreased collagen production in dermal fibroblasts is thus a logical therapeutic approach. Two patients who developed refractory sclerodermatous cGVHD following allo-SCT received imatinib mesylate at the dose of 400 mg/day. In both patients, the scleroderma symptoms disappeared within 3 months of initiation of the treatment. At the time of this report, the two patients were both alive and had a very good skin response. This report shows that imatinib is effective in patients with refractory sclerodermatous cGVHD. Considering its well-documented clinical profile in other diseases, imatinib is a promising candidate for the treatment of sclerodermatous cGVHD.

Topics: Adult; Antineoplastic Agents; Benzamides; Bone Marrow Transplantation; Female; Fibroblasts; Graft vs Host Disease; Humans; Imatinib Mesylate; Male; Middle Aged; Piperazines; Pyrimidines; Scleroderma, Systemic; Transforming Growth Factor beta; Transplantation, Homologous; Treatment Outcome

Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

Topics: Adolescent; Adult; Aged; Animals; Bleomycin; Early Growth Response Protein 1; ets-Domain Protein Elk-1; Female; Fibroblasts; Fibrosis; Humans; Lung; Mice; Middle Aged; Mitogen-Activated Protein Kinases; NIH 3T3 Cells; Promoter Regions, Genetic; Scleroderma, Systemic; Skin; Smad3 Protein; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Scleroderma is a common autoimmune disorder with no effective therapy. Current concepts of scleroderma include the hypothesis that scleroderma results from excess conversion of endothelial cells to fibroblast like cells, called endothelial mesenchymal transformation. This process is thought to be mediated by cytokines including transforming growth factor beta (TGFb), which causes increased collagen synthesis, resulting in fibrosis, the hallmark of the disease. In vitro studies have hypothesized that rapamycin may be of benefit in scleroderma due to antagonism of collagen synthesis. Given that rapamycin has antiangiogenic activities, inhibits wound healing, and prevents the synthesis of collagen in vivo, we tried rapamycin in a patient with scleroderma. We observed rapid improvement in skin stiffness and mobility. Our results provide the rationale for larger clinical trials of rapamycin in scleroderma and other fibrotic disorders.

Topics: Adult; Angiogenesis Inhibitors; Collagen; Female; Fibrosis; Humans; Immunosuppressive Agents; Mesoderm; Pregnancy; Pregnancy Complications; Scleroderma, Systemic; Sirolimus; Transforming Growth Factor beta; Treatment Outcome

Topics: Bone Marrow Transplantation; Clinical Trials as Topic; Fibrosis; Genetic Predisposition to Disease; Humans; Hypertension, Pulmonary; Kidney Diseases; Oligonucleotide Array Sequence Analysis; Pulmonary Fibrosis; Scleroderma, Systemic; Skin; Stem Cell Transplantation; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta has a central role in driving many of the pathological processes that characterize pulmonary fibrosis. Inhibition of the integrin alpha(v)beta6, a key activator of TGF-beta in lung, is an attractive therapeutic strategy, as it may be possible to inhibit TGF-beta at sites of alpha(v)beta6 up-regulation without affecting other homeostatic roles of TGF-beta.. To analyze the expression of alpha(v)beta6 in human pulmonary fibrosis, and to functionally test the efficacy of therapeutic inhibition of alpha(v)beta6-mediated TGF-beta activation in murine bleomycin-induced pulmonary fibrosis.. Lung biopsies from patients with a diagnosis of systemic sclerosis or idiopathic pulmonary fibrosis were stained for alpha(v)beta6 expression. A range of concentrations of a monoclonal antibody that blocks alpha(v)beta6-mediated TGF-beta activation was evaluated in murine bleomycin-induced lung fibrosis.. Alpha(v)beta6 is overexpressed in human lung fibrosis within pneumocytes lining the alveolar ducts and alveoli. In the bleomycin model, alpha(v)beta6 antibody was effective in blocking pulmonary fibrosis. At high doses, there was increased expression of markers of inflammation and macrophage activation, consistent with the effects of TGF-beta inhibition in the lung. Low doses of antibody attenuated collagen expression without increasing alveolar inflammatory cell populations or macrophage activation markers.. Partial inhibition of TGF-beta using alpha(v)beta6 integrin antibodies is effective in blocking murine pulmonary fibrosis without exacerbating inflammation. In addition, the elevated expression of alpha(v)beta6, an activator of the fibrogenic cytokine, TGF-beta, in human pulmonary fibrosis suggests that alpha(v)beta6 monoclonal antibodies could represent a promising new therapeutic strategy for treating pulmonary fibrosis.

Topics: Animals; Antibodies, Monoclonal; Antigens, Neoplasm; Collagen; Disease Models, Animal; Dose-Response Relationship, Drug; Integrins; Mice; Mice, Inbred BALB C; Pulmonary Alveoli; Pulmonary Fibrosis; Scleroderma, Systemic; Transforming Growth Factor beta

Diseases are believed to arise from dysregulation of biological systems (pathways) perturbed by environmental triggers. Biological systems as a whole are not just the sum of their components, rather ever-changing, complex and dynamic systems over time in response to internal and external perturbation. In the past, biologists have mainly focused on studying either functions of isolated genes or steady-states of small biological pathways. However, it is systems dynamics that play an essential role in giving rise to cellular function/dysfunction which cause diseases, such as growth, differentiation, division and apoptosis. Biological phenomena of the entire organism are not only determined by steady-state characteristics of the biological systems, but also by intrinsic dynamic properties of biological systems, including stability, transient-response, and controllability, which determine how the systems maintain their functions and performance under a broad range of random internal and external perturbations. As a proof of principle, we examine signal transduction pathways and genetic regulatory pathways as biological systems. We employ widely used state-space equations in systems science to model biological systems, and use expectation-maximization (EM) algorithms and Kalman filter to estimate the parameters in the models. We apply the developed state-space models to human fibroblasts obtained from the autoimmune fibrosing disease, scleroderma, and then perform dynamic analysis of partial TGF-beta pathway in both normal and scleroderma fibroblasts stimulated by silica. We find that TGF-beta pathway under perturbation of silica shows significant differences in dynamic properties between normal and scleroderma fibroblasts. Our findings may open a new avenue in exploring the functions of cells and mechanism operative in disease development.

Topics: Algorithms; Cells, Cultured; Environment; Fibroblasts; Gene Regulatory Networks; Humans; Physical Stimulation; Scleroderma, Systemic; Signal Transduction; Silicon Dioxide; Systems Biology; Transforming Growth Factor beta

Animal models are useful tools to study various aspects of human diseases. Bleomycin (BLM)-induced scleroderma mouse has been widely investigated as an animal model of scleroderma. Repeated injections of BLM, either daily or every other day, for 3-4 weeks are required to induce scleroderma in mice. Poly(L-lactic acid) (PLA) is a biodegradable, biocompatible and bioabsorbable device that has been widely investigated for controlled drug release. In this study, we fabricated BLM-containing PLA microspheres and subcutaneously injected them into C3H mice for only one time.. Treated skins were harvested at days 7 and 21. Then, histological examination and collagen content measurement assay were performed. The mRNA expression of alpha1(I) collagen (COL1A1), monocyte chemoattractant protein-1 (MCP-1), TGF-beta(1) and connective tissue growth factor (CTGF) were quantified by real-time PCR.. Dermal fibrosis was histologically observed at day 7 after injection and remained present at day 21. Tissue responses against BLM-PLA microspheres alone were mild. Soluble collagen content and expression level of alpha1(I) collagen mRNA were significantly elevated at day 21. Expression levels of MCP-1 mRNA and TGF-beta(1) mRNA at day 7 and CTGF mRNA at day 21 were also elevated.. The present study demonstrated for the first time that one-time injection of BLM-PLA microspheres can induce dermal fibrosis in C3H mice. BLM-PLA microspheres thus offer a labour-saving, simple and powerful tool to establish an animal model of BLM-induced dermal fibrosis.

Topics: Animals; Bleomycin; Chemokine CCL2; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Connective Tissue Growth Factor; Delayed-Action Preparations; Disease Models, Animal; Female; Fibrosis; Gene Expression; Immediate-Early Proteins; Injections, Subcutaneous; Intercellular Signaling Peptides and Proteins; Lactic Acid; Mice; Mice, Inbred C3H; Microspheres; Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Acutely transforming retrovirus AKT8 in rodent T-cell lymphoma (Akt) is a serine/threonine kinase that plays important roles in survival, cell-cycle progression, and cell proliferation, and has recently been implicated in collagen regulation. The aim of this study was to determine the role of Akt in collagen deposition by normal dermal fibroblasts, and to determine the sensitivity of cultured systemic sclerosis (SSc) fibroblasts to Akt inhibition. We show that blockade of Akt using pharmacological inhibitors, small interfering RNA (siRNA), and a dominant-negative Akt mutant led to inhibition of the basal type I collagen production. Furthermore, inhibition of Akt upregulated basal matrix metalloproteinase 1 (MMP1) production and reversed the inhibitory effect of transforming growth factor-beta (TGF-beta) on MMP1 gene expression. In addition, SSc fibroblasts were more sensitive to Akt inhibition, with respect to collagen and MMP1 production. These findings suggest that in human dermal fibroblasts, Akt has dual profibrotic effects, increasing collagen synthesis and decreasing its degradation via downregulation of MMP1. Akt could directly contribute to elevated collagen in SSc fibroblasts and it may represent an attractive target for therapy of SSc fibrosis.

Topics: Actins; Biopsy; Case-Control Studies; Cells, Cultured; Collagen; Collagen Type I; Collagen Type I, alpha 1 Chain; Dose-Response Relationship, Drug; Fibroblasts; Gene Expression Regulation; Humans; Matrix Metalloproteinase 1; Oncogene Protein v-akt; RNA, Small Interfering; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

To explore increased susceptibility to fibrosis following experimental injury to alveolar epithelial cells (AECs) in a novel transgenic mouse model of scleroderma with fibroblast-specific perturbation of transforming growth factor beta (TGFbeta) signaling (TbetaRIIDeltak-fib mice).. Wild-type (WT) and transgenic mice were injured with intratracheally administered saline or bleomycin, and the lungs were harvested for biochemical, histologic, and electron microscopic analysis.. Electron microscopy revealed AEC abnormalities in the lungs of untreated transgenic mice and bleomycin-treated WT mice; the lungs of transgenic mice treated with bleomycin showed severe epithelial damage. Compared with lungs from bleomycin-treated WT mice, lungs from bleomycin-treated transgenic mice demonstrated increased fibroproliferation, myofibroblast persistence, and impaired hyperplasia and increased apoptosis of type II AECs. The lungs from saline-treated transgenic mice and those from bleomycin-treated WT mice had phenotypic similarities, suggesting enhanced susceptibility to minor epithelial injury in the transgenic strain. The level of collagen was increased in the lungs from transgenic mice compared with that in the lungs from WT mice after treatment with either bleomycin or saline. Persistent fibrosis in bleomycin-treated transgenic mice was independent of ongoing neutrophil inflammation but was associated with impaired alveolar epithelial repair.. These results suggest that in the context of fibroblast-specific perturbation of TGFbeta signaling, even minor epithelial injury induces significant fibrosis. The model supports a central role for TGFbeta in determining fibrosis and demonstrates that lung fibroblasts may regulate the response of AECs to injury. Our findings provide insight into likely pathogenic mechanisms in scleroderma-associated pulmonary fibrosis.

Topics: Animals; Bleomycin; Cells, Cultured; Disease Models, Animal; Epithelial Cells; Fibroblasts; Irritants; Mice; Mice, Transgenic; Pulmonary Alveoli; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Sodium Chloride; Transforming Growth Factor beta

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.

Topics: Animals; Bleomycin; Boronic Acids; Bortezomib; Cells, Cultured; Collagen Type I; Fibrosis; Hydroxyproline; Leupeptins; Lung; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Proteasome Inhibitors; Protein Serine-Threonine Kinases; Pulmonary Fibrosis; Pyrazines; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive accumulation of extracellular matrix (ECM) and are understood to develop under the influence of fibrogenic growth factors. To better understand the detailed mechanisms of persistent fibrosis in SSc, we have previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, transforming growth factor-beta (TGF-beta) transiently induced subcutaneous fibrosis and serial injections of connective tissue growth factor (CTGF) after TGF-beta caused persistent fibrosis. These results suggest that CTGF plays an important role in the development of persistent skin fibrosis and that CTGF may be a potential and specific therapeutic target in skin fibrosis. Therefore, the aim of the current study is to develop a neutralizing monoclonal antibody against human CTGF. We also investigated the neutralizing effect of the antibodies in our animal model. Firstly, by using the DNA immunization method, we developed a panel of anti-CTGF antibodies recognizing the native conformation of human CTGF. Next, to examine the anti-fibrosing effects of these antibodies, newborn B6 mice received subcutaneous injections of TGF-beta for 3 days with either anti-CTGF neutralizing antibodies or control purified immunoglobulin. Anti-CTGF antibodies significantly reduced skin fibrosis and collagen contents compared with the control group. These results suggest that our anti-CTGF antibodies are capable of blocking the development of skin fibrosis at least partially and these anti-CTGF neutralizing antibodies may be useful as the feasible strategy to treat skin fibrotic diseases as SSc.

Topics: Animals; Antibodies, Monoclonal; Collagen Type I; Connective Tissue Growth Factor; Female; Fibrosis; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Increased signaling by transforming growth factor beta (TGFbeta) has been implicated in systemic sclerosis (SSc; scleroderma), a complex disorder of connective tissues characterized by excessive accumulation of collagen and other extracellular matrix components in systemic organs. To directly assess the effect of sustained TGFbeta signaling in SSc, we established a novel mouse model in which the TGFbeta signaling pathway is activated in fibroblasts postnatally.. The mice we used (termed TBR1(CA); Cre-ER mice) harbor both the DNA for an inducible constitutively active TGFbeta receptor I (TGFbetaRI) mutation, which has been targeted to the ROSA locus, and a Cre-ER transgene that is driven by a fibroblast-specific promoter. Administration of 4-hydroxytamoxifen 2 weeks after birth activates the expression of constitutively active TGFbetaRI.. These mice recapitulated clinical, histologic, and biochemical features of human SSc, showing pronounced and generalized fibrosis of the dermis, thinner epidermis, loss of hair follicles, and fibrotic thickening of small blood vessel walls in the lung and kidney. Primary skin fibroblasts from these mice showed elevated expression of downstream TGFbeta targets, reproducing the hallmark biochemical phenotype of explanted SSc dermal fibroblasts. The mouse fibroblasts also showed elevated basal expression of the TGFbeta-regulated promoters plasminogen activator inhibitor 1 and 3TP, increased Smad2/3 phosphorylation, and enhanced myofibroblast differentiation.. Constitutive activation of TGFbeta signaling in fibroblastic cells of mice after birth caused a marked fibrotic phenotype characteristic of SSc. These mice should be excellent models with which to test therapies aimed at correcting excessive TGFbeta signaling in human scleroderma.

Topics: Activin Receptors, Type I; Animals; Cell Differentiation; Cells, Cultured; Disease Models, Animal; Female; Fibroblasts; Gene Targeting; Male; Mice; Mice, Inbred C57BL; Mice, Transgenic; Phosphorylation; Plasminogen Activator Inhibitor 1; Protein C Inhibitor; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin; Smad2 Protein; Smad3 Protein; Tamoxifen; Transforming Growth Factor beta

Systemic sclerosis (SSc) is a connective tissue disorder characterized by fibrosis of the skin and internal organs. Although the esophagus is the most frequently affected part of the gastrointestinal tract, all other segments can be involved. The present study was undertaken to evaluate the fibrotic process and the expression of fibrogenic cytokines in the gastric wall of SSc patients with gastroesophageal involvement.. Full-thickness surgical and endoscopic gastric biopsy samples were obtained from 14 SSc patients and 10 controls. Tissue sections were either stained with Masson's trichrome or by immunohistochemistry and analyzed for the expression of types I, III, and IV collagen, alpha-smooth muscle actin (alpha-SMA), transforming growth factor beta (TGFbeta), connective tissue growth factor (CTGF), and endothelin 1 (ET-1).. In the gastric wall of SSc patients, Masson's trichrome staining and immunohistochemistry for types I and III collagen revealed a high amount of collagen in the lamina propria that increased toward the muscularis mucosae. In addition, muscle layers showed features of atrophy, with wide areas of focal fibrosis surrounding smooth muscle cells. Type IV collagen was present around glands and small vessels, suggesting a thickening of the basal lamina. The expression of the fibrogenic cytokines TGFbeta and CTGF, ET-1, and the myofibroblast marker alpha-SMA was stronger in SSc patients than in controls.. A pronounced deposition of collagen, the presence of myofibroblasts, and increased expression of several profibrotic factors are important hallmarks in the stomach of patients with SSc. The fibrotic involvement of the gastric wall may account for muscle atrophy leading to stomach hypomotility in SSc.

Topics: Actins; Aged; Biopsy; Case-Control Studies; Collagen Type I; Collagen Type III; Collagen Type IV; Connective Tissue Growth Factor; Cytokines; Endothelin-1; Female; Fibrosis; Gastroscopy; Gene Expression; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Male; Middle Aged; Scleroderma, Systemic; Severity of Illness Index; Stomach; Transforming Growth Factor beta

Insufficient angiogenesis with tissue ischemia and accumulation of extracellular matrix are hallmarks of systemic sclerosis (SSc). Based on the severely decreased oxygen levels in the skin of patients with SSc, we aimed to investigate the role of hypoxia in the pathogenesis of SSc.. Subtractive hybridization was used to compare gene expression in dermal fibroblasts under hypoxic and normoxic conditions. Dermal fibroblasts were further characterized by exposure to different concentrations of oxygen and for different time periods as well as by interference with hypoxia-inducible factor 1alpha (HIF-1alpha). The systemic normobaric hypoxia model in mice was used for in vivo analyses.. Several extracellular matrix proteins and genes involved in extracellular matrix turnover, such as thrombospondin 1, proalpha2(I) collagen, fibronectin 1, insulin-like growth factor binding protein 3, and transforming growth factor beta-induced protein, were induced by hypoxia in SSc and healthy dermal fibroblasts. The induction of these genes was time- and dose-dependent. Experiments with HIF-1alpha-knockout mouse embryonic fibroblasts, deferoxamine/cobalt ions as chemical stabilizers of HIF-1alpha, and HIF-1alpha small interfering RNA consistently showed that extracellular matrix genes are induced in dermal fibroblasts by HIF-1alpha-dependent, as well as HIF-1alpha-independent, mechanisms. Using the systemic normobaric hypoxia mouse model, we demonstrated that dermal hypoxia leads to the induction of the identified extracellular matrix genes in vivo after both short exposure and prolonged exposure to hypoxia.. These data show that hypoxia contributes directly to the progression of fibrosis in patients with SSc by increasing the release of major extracellular matrix proteins. Targeting of hypoxia pathways might therefore be of therapeutic value in patients with SSc.

Topics: Animals; Biopsy; Cell Hypoxia; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Oxygen; RNA, Messenger; Scleroderma, Systemic; Skin; Time Factors; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

This is the first report that characterizes specific inhibitor of Smad3 (SIS3) as a potent and selective inhibitor of Smad3 function. In the reporter assay, the increased luciferase activity of p3TP-lux by the overexpression of constitutively active form of ALK-5 was abrogated by the treatment with SIS3 in a dose-dependent manner. Immunoprecipitation revealed that SIS3 attenuated the transforming growth factor (TGF)-beta1-induced phosphorylation of Smad3 and interaction of Smad3 with Smad4. On the other hand, this reagent did not affect the phosphorylation of Smad2. Thereafter, we evaluated the ability of SIS3 in the suppression of the TGF-beta1-induced type I procollagen up-regulation in human dermal fibroblasts. We found that the addition of SIS3 attenuated the effects of TGF-beta1 by reducing the transcriptional activity. SIS3 also inhibited the myofibroblast differentiation of fibroblasts by TGF-beta1. Moreover, we demonstrated that SIS3 completely diminished the constitutive phosphorylation of Smad3 as well as the up-regulated type I collagen expression in scleroderma fibroblasts. Together, our study suggested that SIS3 is a useful tool to evaluate the TGF-beta-regulated cellular mechanisms via selective inhibition of Smad3.

Topics: Cell Differentiation; Collagen Type I; Extracellular Matrix; Fibroblasts; Genes, Reporter; Humans; Isoquinolines; Luciferases; Phosphorylation; Promoter Regions, Genetic; Pyridines; Pyrroles; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

To elucidate the role of transforming growth factor-beta (TGF-beta)/Smad signalling in the increased expression of the collagen gene in systemic sclerosis (SSc) fibroblasts.. Dermal fibroblasts from seven patients with diffuse SSc of recent onset and from seven healthy individuals were studied. The expression levels of Smad2, Smad3 and Smad4 proteins were determined by immunoblotting. Smad3 phosphorylation and the interaction of Smad3 with Sp1 or p300 were analysed using immunoprecipitation. The effects of overexpression of Smad proteins or Sp1 on the human alpha2(I) collagen gene transcription were investigated with chloramphenicol acetyltransferase (CAT) assays using the -772 COL1A2/CAT construct.. Constitutive increased Smad3 phosphorylation was detected in SSc fibroblasts compared with normal fibroblasts. Increased interaction of Smad3 with Sp1 as well as p300 was also detected in SSc fibroblasts. The overexpression of Smad3 caused an increase of up to 5-fold in COL1A2 promoter activity in normal fibroblasts, while Smad3 caused a small increase in COL1A2 promoter activity in SSc fibroblasts. However, neither Smad2 nor Smad4 caused significant effects in COL1A2 promoter activity in normal fibroblasts or SSc fibroblasts. The overexpression of Sp1 caused further increase in COL1A2 promoter activity stimulated by TGF-beta in normal fibroblasts, but did not change COL1A2 promoter activity in the presence of TGF-beta in SSc fibroblasts. The combined overexpression of Smad3 and Sp1 significantly enhanced TGF-beta response in normal fibroblasts, but less markedly in SSc fibroblasts.. These results suggested that SSc fibroblasts are less sensitive to exogenous TGF-beta stimulation because they are already activated by the autocrine TGF-beta loop.

Topics: Cells, Cultured; Collagen; Collagen Type I; E1A-Associated p300 Protein; Fibroblasts; Gene Expression Regulation; Humans; Phosphorylation; Recombinant Proteins; Scleroderma, Systemic; Skin; Smad2 Protein; Smad3 Protein; Smad4 Protein; Sp1 Transcription Factor; Transcriptional Activation; Transforming Growth Factor beta

The role of fibroblasts in inflammatory processes and their cross-talk with T cells is increasingly being recognized. Our aim was to explore the capacity of dermal fibroblasts to produce inflammatory chemokines potentially involved in fibrosis occurring in response to contact with polarized human T cells. Our findings indicate that the program of chemokine production by fibroblasts is differentially regulated depending on the T-helper (Th) cell subset used to activate them. Thus, Th1 and Th2 cells preferentially induced production of IFN-gamma inducible protein (IP)-10 and IL-8, respectively, whereas monocyte chemoattractant protein (MCP)-1 was equally induced by both subsets at mRNA and protein levels. Neutralization experiments indicated that membrane-associated tumour necrosis factor-alpha and IL-1 played a major role in the induction of IL-8 and MCP-1 by Th1 and Th2 cells, whereas membrane-associated IFN-gamma (present only in Th1 cells) was responsible, at least in part, for the lower IL-8 and higher IP-10 production induced by Th1 cells. The contributions of tumour necrosis factor-alpha, IL-1 and IFN-alpha were confirmed when fibroblasts were cultured separated in a semipermeable membrane from living T cells activated by CD3 cross-linking. We observed further differences when we explored signal transduction pathway usage in fibroblasts. Pharmacological inhibition of c-Jun N-terminal kinase and nuclear factor-kappaB resulted in inhibition of IL-8 mRNA transcription induced by Th1 cells but not that by Th2 cells, whereas inhibition of MEK/ERK (mitogen-activated protein kinase of extracellular signal-regulated kinase/extracellular signal-regulated kinase) and nuclear factor-kappaB resulted in inhibition of MCP-1 mRNA induced by Th2 but not by Th1 cells. Finally, no distinct differences in chemokine production were observed when the responses to T cell contact or to prototypic Th1 and Th2 cytokines were examined in systemic sclerosis versus normal fibroblasts. These findings indicate that fibroblasts have the potential to participate in shaping the inflammatory response through the activation of flexible programs of chemokine production that depend on the Th subset eliciting their response.

Topics: Biopsy; Cell Membrane; Chemokines; Clone Cells; Coculture Techniques; Cytokines; Female; Fibroblasts; Humans; Inflammation; Male; Reference Values; RNA; Scleroderma, Systemic; Skin; T-Lymphocytes, Helper-Inducer; Transforming Growth Factor beta

Tissue fibrosis results when dysregulation of extracellular matrix (ECM) turnover favors deposition of collagen and other ECM proteins over degradation. Fibrosis may then lead to organ dysfunction and pathology as observed in systemic sclerosis (SSc). In the present study, we investigated the antifibrotic properties of proteasome blockade. A dose- and time-dependent reduction in type-I collagen and tissue inhibitor of metalloproteinase-1 (TIMP-1) production was observed in normal fibroblasts exposed to proteasome inhibitors (PI). In the same culture conditions, metalloproteinase-1 (MMP-1) protein and the collagenolytic activity on type I collagen was increased. The steady-state mRNA levels of COL1A1, TIMP-1, and MMP-1 paralleled protein levels. These effects were dominant over the profibrotic properties of TGF-beta and were observed with fibroblasts generated from normal and SSc skin. PI decreased type I collagen mRNA levels with kinetics similar to those observed with DRB, a specific RNA polymerase II inhibitor, thus indicating transcriptional inhibition. Of interest, PI induced c-Jun phosphorylation and c-Jun nuclear accumulation. The specific N-terminal Jun-kinase inhibitor SP-600125 selectively abrogated c-Jun phosphorylation and, in a dose-dependent fashion, the up-regulated synthesis of MMP-1 induced by PI. Finally, PI did not affect fibroblast viability. Thus, the coordinated down-regulation of collagen and TIMP-1 and up-regulation of MMP-1 renders proteasome blockade an attractive strategy for treating conditions as SSc, characterized by excessive fibrosis.

Topics: Acetylcysteine; Anthracenes; Boronic Acids; Bortezomib; Collagen Type I; Dose-Response Relationship, Drug; Down-Regulation; Extracellular Matrix; Fibroblasts; Fibrosis; Genes, jun; Humans; JNK Mitogen-Activated Protein Kinases; Leupeptins; Matrix Metalloproteinase 1; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-jun; Pyrazines; RNA Polymerase II; RNA, Messenger; Scleroderma, Systemic; Skin; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Up-Regulation

Activated fibroblasts, which have previously been obtained from bronchoalveolar lavage fluid (BALF), are proposed to be important cells in the fibrotic processes of asthma and scleroderma (SSc). We have studied the motility for BALF derived fibroblasts in patients with SSc that may explain the presence of these cells in the airway lumen. Furthermore, we have compared phenotypic alterations in activated fibroblasts from BALF and bronchial biopsies from patients with mild asthma and SSc that may account for the distinct fibrotic responses.. Fibroblasts were cultured from BALF and bronchial biopsies from patients with mild asthma and SSc. The motility was studied using a cell migration assay. Western Blotting was used to study the expression of alpha-smooth muscle actin (alpha-SMA), ED-A fibronectin, and serine arginine splicing factor 20 (SRp20). The protein expression pattern was analyzed to reveal potential biomarkers using two-dimensional electrophoresis (2-DE) and sequencing dual matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-TOF). The Mann-Whitney method was used to calculate statistical significance.. Increased migration and levels of ED-A fibronectin were observed in BALF fibroblasts from both groups of patients, supported by increased expression of RhoA, Rac1, and the splicing factor SRp20. However, these observations were exclusively accompanied by increased expression of alpha-SMA in patients with mild asthma. Compared to BALF fibroblasts in mild asthma, fibroblasts in SSc displayed a differential protein expression pattern of cytoskeletal- and scavenger proteins. These identified proteins facilitate cell migration, oxidative stress, and the excessive deposition of extracellular matrix observed in patients with SSc.. This study demonstrates a possible origin for fibroblasts in the airway lumen in patients with SSc and important differences between fibroblast phenotypes in mild asthma and SSc. The findings may explain the distinct fibrotic processes and highlight the motile BALF fibroblast as a potential target cell in these disorders.

Topics: Adult; Aged; Asthma; Biopsy; Bronchoalveolar Lavage Fluid; Cell Movement; Female; Fibroblasts; Fibronectins; GTP Phosphohydrolases; Humans; Male; Middle Aged; Myocytes, Smooth Muscle; Phenotype; Proteome; RNA-Binding Proteins; Scleroderma, Systemic; Serine-Arginine Splicing Factors; Transforming Growth Factor beta

The biological effect of cytokines is mainly determined by the cytokine-receptor interaction, which is modulated by the concentration and the activity of cytokines and/or their receptors. Because alphav-containing integrins can bind to and/or activate latent TGF-beta, these integrins have been thought to be involved in the pathogenesis of fibrotic disorders. Our recent observations that alphavbeta5 is up-regulated in scleroderma fibroblasts and that the transient overexpression of alphavbeta5 increases the human alpha2(I) collagen gene expression in normal fibroblasts suggest the involvement of alphavbeta5 in the self-activation system in scleroderma fibroblasts. In this study, we established stable transfectants with alphavbeta5 using normal dermal fibroblasts and demonstrated that such cells differentiated into myofibroblasts by the stimulation of autocrine TGF-beta. This observation is explained by 1) alphavbeta5 recruiting latent TGF-beta1 on the cell surface, 2) endogenous active TGF-beta localizing on the cell surface, and 3) alphavbeta5 interacting with TGF-beta receptors. Furthermore, blockade of alphavbeta5 reversed the myofibroblastic phenotype in scleroderma fibroblasts. These data identify a novel mechanism for the establishment of autocrine TGF-beta signaling in dermal fibroblasts by the up-regulation of alphavbeta5 and suggest the possibility of regulating fibrotic disorders, especially scleroderma, by targeting this integrin.

Topics: Activin Receptors, Type I; Autocrine Communication; Blotting, Northern; Cell Adhesion; Cell Differentiation; Cells, Cultured; Dermis; Fibroblasts; Humans; Integrins; Myocytes, Smooth Muscle; Phenotype; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type I; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Receptors, Vitronectin; Recombinant Proteins; Scleroderma, Systemic; Smad2 Protein; Smad3 Protein; Transforming Growth Factor beta

Skin sclerosis that progresses in the earlier disease phase in systemic sclerosis (SSc) spontaneously regresses thereafter. We investigated the relationship between changes of the serum cytokine profile and changes in skin fibrosis in patients with SSc.. Serum cytokine levels were examined by ELISA using 180 sera samples from 26 patients with early diffuse cutaneous SSc (dcSSc) with mean disease duration of 2.1 years. The mean followup period was 4.9 years (range 2-8). Cytokine mRNA expression in the affected skin was quantified by real-time reverse transcription-polymerase chain reaction.. Modified Rodnan total skin thickness score decreased after 2, 4, and 6 years compared to that at first visit. Serum levels of the Th2 cytokines interleukin 6 (IL-6) and IL-10 and monocyte chemotactic protein-1 (MCP-1) were higher at first evaluation compared to healthy controls, while IL-4 levels were normal. Levels of all Th2 cytokines generally decreased as skin sclerosis regressed. Conversely, levels of serum IL-12, a Th1-inducing cytokine, were lower at first visit relative to controls, but increased by roughly 15-fold after 6 years to significantly higher levels than controls. Surviving dcSSc patients exhibited elevated IL-12 levels compared to deceased patients. Serum levels of transforming growth factor-ss1 (TGF-ss1), a fibrogenic cytokine, increased throughout followup, with slightly decreased levels at later timepoints. IL-12 mRNA expression was upregulated in affected skin from patients with late-stage dcSSc, while TGF-ss1 and MCP-1 expression was downregulated.. These results suggest that a shift from Th2 to Th1 response correlates with improvement in skin fibrosis in SSc, and that IL-12 level is a serologically useful marker for disease activity and prognosis.

Topics: Adult; Chemokine CCL2; Female; Fibrosis; Gene Expression; Humans; Interleukin-10; Interleukin-12; Interleukin-6; Longitudinal Studies; Male; Middle Aged; Remission, Spontaneous; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Cartilage oligomeric matrix protein (COMP) is an extracellular glycoprotein that belongs to the thrombospondin gene family. It is found predominantly in cartilage, tendon, ligament, and bone. Mutations in the COMP gene have been linked to the development of pseudoachondroplasia and multiple epiphysial dysplasia. COMP influences the organization of collagen fibrils by interacting with collagens I, II and IX. Gene expression profiling of cultured skin fibroblasts suggested that COMP mRNA levels were elevated in scleroderma. We therefore examined COMP expression in SSc and normal skin biopsies. Immunohistochemistry confirmed that COMP protein accumulates in SSc but not normal skin, with SSc skin showing striking deposition in the papillary and deeper dermis. Significant staining was also seen in non-lesional skin from patients. Due to its involvement in the development of fibrosis, TGFbeta was examined for a possible role in regulating COMP expression. Cultured SSc fibroblasts demonstrated greater staining for COMP compared to normal controls prior to stimulation, and TGFbeta-1 induced a large increase in mRNA and protein. Murine fibroblasts engineered to overexpress human COMP demonstrated increased levels of fibronectin and collagen in the extracellular matrix. Taken together, these data demonstrate that COMP is overexpressed in SSc skin and cultured fibroblasts possibly due to autocrine TGFbeta stimulation, and COMP overexpression is sufficient to stimulate excess matrix deposition. By interactions with other matrix proteins and cells, COMP may play a role in pathogenic matrix deposition.

Topics: Animals; Cartilage Oligomeric Matrix Protein; Cells, Cultured; Collagen; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Gene Expression; Glycoproteins; Humans; Matrilin Proteins; Mice; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

To determine whether biopsy specimens obtained from systemic sclerosis (SSc) lesions show a distinctive gene profile, whether that gene profile is maintained in fibroblasts cultured from SSc skin biopsy specimens, and whether results from tissue obtained from multiple clinical centers can be combined to yield useful observations in this rare disease.. Biopsy samples and passaged fibroblasts were stored in RNAlater solution prior to processing for RNA. RNA from SSc and control skin biopsy specimens, as well as SSc and control explanted passage 4 fibroblasts, from 9 patients and 9 controls was hybridized to Affymetrix HG-U133A arrays. Data were analyzed using the BRB ArrayTools system. When appropriate, findings were followed up with immunohistochemical analysis or TaqMan studies.. Biopsy samples obtained from patients with SSc had a robust and distinctive gene profile, with approximately 1,800 qualifiers distinguishing normal skin from SSc skin at a significant level. The SSc phenotype was the major driver of sample clusters, independent of origin. Alterations in transforming growth factor beta and Wnt pathways, extracellular matrix proteins, and the CCN family were prominent. Explanted fibroblasts from SSc biopsy samples showed a far smaller subset of changes that were relatively variable between samples, suggesting that either nonfibroblast cell types or other aspects of the dermal milieu are required for full expression of the SSc phenotype.. SSc has a distinct gene profile that is not confounded by geographic location, indicating that extended multicenter studies may be worthwhile to identify distinct subsets of disease by transcript profiling. Explanted SSc fibroblasts show an incomplete reflection of the SSc phenotype.

Topics: Adult; Biopsy; Extracellular Matrix Proteins; Female; Fibroblasts; Gene Expression Profiling; Humans; Immunohistochemistry; Male; Middle Aged; Phenotype; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Wnt Proteins

Topics: Adult; Cells, Cultured; Collagen Type I; Down-Regulation; Fibroblasts; Gene Expression; Genetic Therapy; Humans; Oligonucleotides; Scleroderma, Systemic; Transcription Factor AP-1; Transforming Growth Factor beta; Transforming Growth Factor beta1

Allograft inflammatory factor 1 (AIF-1), a protein initially identified in chronically rejected rat cardiac allografts, is involved in the immune response and proliferative vasculopathy that occurs during allograft rejection. Three well-characterized isoforms of AIF-1 result from alternative messenger RNA (mRNA) splicing. We previously identified a strong association of systemic sclerosis (SSc) with a polymorphism in AIF-1 isoform 2. The purpose of this study was to investigate AIF-1 expression in affected tissues from patients with SSc and to examine the regulation of its isoforms by transforming growth factor beta (TGFbeta).. AIF-1 in the skin and lung tissues of patients with SSc was analyzed by immunochemistry. AIF-1 isoform expression in response to TGFbeta and interferon-gamma stimulation was examined by quantitative polymerase chain reaction (PCR).. AIF-1 protein was present in affected vessels of the lung and skin lesions of patients with SSc. Quantitative PCR showed an average of 14-fold higher mRNA levels in affected SSc skin than in normal skin. Double-label immunofluorescence staining demonstrated that T cells, macrophages, and endothelial cells in affected tissues expressed AIF-1. Stimulation of peripheral blood mononuclear cells with TGFbeta caused a specific and significant increase in the expression of AIF-1 isoform 2 transcripts (P < 0.005), which was due to stabilization of AIF-1 isoform 2 mRNA.. These data suggest that AIF-1 plays an important role in the pathogenesis of SSc owing to its increased expression in affected tissues and to the specific stimulation of AIF-1 isoform 2 by TGFbeta.

Topics: Calcium-Binding Proteins; Cells, Cultured; DNA-Binding Proteins; Drug Combinations; Endothelial Cells; Endothelium, Vascular; Fibroblasts; Fluorescent Antibody Technique, Indirect; Gene Expression; Humans; Interferon-gamma; Leukocytes, Mononuclear; Lung; Macrophages; Microfilament Proteins; Nuclear Proteins; Protein Isoforms; Reverse Transcriptase Polymerase Chain Reaction; RNA Processing, Post-Transcriptional; RNA, Messenger; Scleroderma, Systemic; Skin; T-Lymphocytes; Transforming Growth Factor beta

Connective tissue growth factor (CTGF) plays a role in the fibrotic process of systemic sclerosis (SSc). Because hypoxia is associated with fibrosis in several profibrogenic conditions, we investigated whether CTGF expression in SSc fibroblasts is regulated by hypoxia. Dermal fibroblasts from patients with SSc and healthy controls were cultured in the presence of hypoxia or cobalt chloride (CoCl(2)), a chemical inducer of hypoxia-inducible factor (HIF)-1alpha. Expression of CTGF was evaluated by Northern and Western blot analyses. Dermal fibroblasts exposed to hypoxia (1% O(2)) or CoCl(2) (1-100 microM) enhanced expression of CTGF mRNA. Skin fibroblasts transfected with HIF-1alpha showed the increased levels of CTGF protein and mRNA, as well as nuclear staining of HIF-1alpha, which was enhanced further by treatment of CoCl(2). Simultaneous treatment of CoCl(2) and transforming growth factor (TGF)-beta additively increased CTGF mRNA in dermal fibroblasts. Interferon-gamma inhibited the TGF-beta-induced CTGF mRNA expression dose-dependently in dermal fibroblasts, but they failed to hamper the CoCl(2)-induced CTGF mRNA expression. In addition, CoCl(2) treatment increased nuclear factor (NF)-kappaB binding activity for CTGF mRNA, while decreasing IkappaBalpha expression in dermal fibroblasts. Our data suggest that hypoxia, caused possibly by microvascular alterations, up-regulates CTGF expression through the activation of HIF-1alpha in dermal fibroblasts of SSc patients, and thereby contributes to the progression of skin fibrosis.

Topics: Cell Hypoxia; Cells, Cultured; Cobalt; Connective Tissue Growth Factor; Fibroblasts; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; NF-kappa B; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Transfection; Transforming Growth Factor beta; Up-Regulation

Systemic sclerosis (SSc) is characterized by an excessive production of extracellular matrix. It is widely accepted that fibrosis is induced by transforming growth factor (TGF)-beta in the early stage and is subsequently maintained by connective tissue growth factor (CTGF). CTGF is a cysteine-rich mitogenic peptide that has been involved in various fibrotic disorders and can be induced in fibroblasts by activation with TGF-beta.. To evaluate the effect of small interfering RNA (siRNA) targeting CTGF on the expression of CTGF and type I and type III collagen in SSc.. Skin fibroblasts from patients with SSc were cultured in vitro and later transfected using four CTGF-specific siRNAs and one nonspecific siRNA. The effect of CTGF-specific siRNAs on the expression of CTGF and type I and type III collagen was examined and quantified by real-time reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and immunocytochemistry.. Semiquantitative RT-PCR analysis showed that the four CTGF-specific siRNAs significantly reduced CTGF mRNA expression (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 73%. Three of the four siRNAs could also depress the transcriptional levels of type I and type III collagen mRNA (P < 0.001), of which siRNA742 showed the strongest inhibitory effect with an inhibitory rate of 37% and 29% for type I and type III collagen, respectively. Western blot analysis further demonstrated that three CTGF-specific siRNAs could significantly decrease CTGF protein level (P < 0.001). In addition, immunocytochemical analysis showed that the expression of type I collagen was significantly decreased in fibroblasts after transfection with siRNA742, whereas inhibition of expression of type III collagen was modest.. Our data for the first time showed that CTGF RNA interference could inhibit expression of CTGF and type I and III collagen in SSc fibroblasts and indicated that CTGF might be an upstream factor regulating type I and type III collagen synthesis, particularly type I collagen. Our findings suggest that silencing CTGF expression might facilitate a potential therapeutic approach for SSc.

Topics: Blotting, Western; Collagen Type I; Collagen Type III; Connective Tissue Growth Factor; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Small Interfering; Scleroderma, Systemic; Transfection; Transforming Growth Factor beta

Fibrosis, the pathological hallmark of scleroderma and related conditions, is due to sustained activation of tissue fibroblasts. Accumulating evidence implicates cytokine networks in initiating, and propagating or terminating fibroblast activation, and the specific cytokine phenotype dictates evolution of the fibrotic response toward either resolution or scarring. In particular, cytokines that promote fibroblast proliferation and myofibroblast differentiation and extracellular matrix (ECM) accumulation functionally define a type 2 (Th2) immune response, whereas interferon-gamma, which suppresses diverse fibroblast activities, defines a type 1 (Th1) immune response. It remains unclear what role the balance between Th1 and Th2 cytokines plays in the pathogenesis of fibrosis. Here we used bleomycin-induced skin fibrosis as a murine model for human scleroderma in order to study the fibrotic response in mice lacking T-bet, a transcription factor that is essential for initiating Th1 lineage development of CD4+ T lymphocytes. Spleen cells from T-bet null (T-bet(-/-)) mice exhibited a typical Th2 cytokine profile ex vivo, with elevated production of interleukin-4 (IL-4), IL-5 and IL-13, and diminished production of interferon-gamma. Bleomycin-induced early mast cells and eosinophil accumulation, and eosinophil degranulation, in the lesional tissue were greater in T-bet(-/-) mice than in wild-type control mice. At a later time point, T-bet(-/-) mice developed significantly more extensive dermal and especially hypodermal fibrosis. Elevated TGF-beta expression and intracellular Smad activation were prominent in lesional skin. Infiltrating eosinophils appeared to be an important cellular source of TGF-beta. These results demonstrate that in mice lacking T-bet bleomycin induced exaggerated skin fibrosis, suggesting that T-bet has an important physiologic role in regulation of tissue repair by promoting Th1 immune responses that prevent excessive ECM accumulation.

Topics: Animals; Antibiotics, Antineoplastic; Bleomycin; Eosinophils; Female; Fibrosis; Immunohistochemistry; Mice; Mice, Mutant Strains; Scleroderma, Systemic; T-Box Domain Proteins; Th1 Cells; Transforming Growth Factor beta

Skin fibrotic disorders such as systemic sclerosis (SSc) are characterized by an excessive production of extracellular matrix (ECM) and understood to develop under the influence of certain growth factors. Connective tissue growth factor (CTGF) is a cysteine-rich mitogenic peptide that is implicated in various fibrotic disorders and induced in fibroblasts after activation with transforming growth factor-beta (TGF-beta). To better understand the mechanisms of persistent fibrosis seen in SSc, we previously established an animal model of skin fibrosis induced by exogenous application of growth factors. In this model, TGF-beta transiently induced subcutaneous fibrosis and serial injections of CTGF after TGF-beta caused persistent fibrosis. To further define the mechanisms of skin fibrosis induced by TGF-beta and CTGF in vivo, we investigated in this study, the effects of growth factors on the promoter activity of the proalpha2 (I) collagen (COL1A2) gene in skin fibrosis. For this purpose, we utilized transgenic reporter mice harboring the -17 kb promoter sequence of the mouse COL1A2 linked to either a firefly luciferase gene or a bacterial beta-galactosidase gene. Serial injections of CTGF after TGF-beta resulted in a sustained elevation of COL1A2 mRNA expression and promoter activity compared with consecutive injection of TGF-beta alone on day 8. We also demonstrated that the number of fibroblasts with activated COL1A2 transcription was increased by serial injections of CTGF after TGF-beta in comparison with the injection of TGF-beta alone. Furthermore, the serial injections recruited mast cells and macrophages. The number of mast cells reached a maximum on day 4 and remained relatively high up to day 8. In contrast to the kinetics of mast cells, the number of macrophages was increased on day 4 and continued to rise during the subsequent consecutive CTGF injections until day 8. These results suggested that CTGF maintains TGF-beta-induced skin fibrosis by sustaining COL1A2 promoter activation and increasing the number of activated fibroblasts. The infiltrated mast cells and macrophages may also contribute to the maintenance of fibrosis.

Topics: Animals; Chemotaxis, Leukocyte; Collagen Type I; Connective Tissue Growth Factor; Disease Models, Animal; Extracellular Matrix; Fibroblasts; Fibrosis; Gene Expression Regulation; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Macrophages; Mast Cells; Mice; Mice, Transgenic; Procollagen; Promoter Regions, Genetic; RNA, Messenger; Scleroderma, Systemic; Skin Diseases; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta3; Up-Regulation

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of cancer, autoimmune disease, and various pathologic conditions characterized by excessive fibrosis. In this study, we investigated the expression of MMP-9 and its clinical significance in systemic sclerosis (SSc). The patients (n = 42) with SSc had higher concentrations of MMP-9 and of tissue inhibitor of metalloproteinase-1 (TIMP-1) and a higher ratio of MMP-9 to TIMP-1 in sera than healthy controls (n = 32). Serum MMP-9 concentrations were significantly higher in the diffuse type (n = 23) than the limited type of SSc (n = 19). Serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score and with serum concentrations of transforming growth factor beta. Moreover, dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls when they were stimulated with IL-1beta, tumor necrosis factor alpha, or transforming growth factor beta. Such an increase in MMP-9 production was partially blocked by treatment with cyclosporin A. In summary, the serum MMP-9 concentrations were elevated in SSc patients and correlated well with skin scores. The increased MMP-9 concentrations may be attributable to overproduction by dermal fibroblasts in SSc. These findings suggest that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc.

Topics: Adult; Aged; Biomarkers; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Interleukin-1; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Middle Aged; Scleroderma, Systemic; Severity of Illness Index; Skin; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

In this study the effect of collagen-polyvinylpyrrolidone (collagen-PVP) vs. triamcinolone acetonide (Triam) in scleroderma (SSc) skin lesions was evaluated. Ten SSc patients were treated weekly with subcutaneous injections of 0.2 mL Triam (8 mg/mL) or 0.2 mL collagen-PVP (1.66 mg collagen). Skin biopsies were obtained from lesions before and after treatment. Tissue sections were evaluated by histology and immunohistochemistry (ELAM-1, VCAM-1, IL-1beta, TNF-alpha, TGF-beta1 and PDGF). The corticoid-treated group showed abnormal tissue architecture while the biodrug-treatment restored cutaneous appendages and type I/III collagen proportion. Cytokine and adhesion molecule expression was almost inhibited with Triam, while collagen-PVP down-regulated it. Collagen-PVP improved the tissue architecture of SSc lesions and down-regulated some proinflammatory parameters, without the side effects induced by corticoids.

Topics: Adult; Cell Adhesion Molecules; Cytokines; Double-Blind Method; Down-Regulation; E-Selectin; Female; Glucocorticoids; Humans; Injections, Subcutaneous; Middle Aged; Povidone; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Triamcinolone Acetonide; Tumor Necrosis Factor-alpha

Recent studies suggest that, in addition to activation and hypersecretion of matrix components, fibroblasts from patients with systemic sclerosis (SSc) are relatively resistant to apoptosis. Transforming growth factor-beta (TGF)-beta is strongly implicated in the pathogenesis of SSc and we and others have shown that TGF-beta can activate Akt, a kinase with potent anti-apoptotic effects. To determine whether Akt was activated in SSc, we quantified phospho-Akt expression in skin fibroblasts in vitro by western blot analysis and a functional kinase assay. In addition, the relative proportion of fibroblasts containing activated Akt in was quantified by immunohistochemistry on skin sections insitu. Analysis of Akt phosphorylation of skin fibroblasts in vitro suggested increased phosphorylation of Akt, and evaluation of skin sections by immunohistochemistry revealed significantly higher percentages of fibroblasts that stained for phospho-Akt compared with controls (78% +/- 14.0% vs 13% +/- 9%, p < 0.001). In addition, co-incident staining of phospho-Akt and alpha-smooth muscle actin was observed in some fibroblasts. These findings indicate that Akt is activated insitu in skin fibroblasts from patients with SSc. Akt activation may contribute to resistance to apoptosis, selection of disease-inducing fibroblasts, and, possibly, myofibroblasts.

Topics: Apoptosis; Cells, Cultured; Female; Fibroblasts; Humans; Male; Middle Aged; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Scleroderma, Systemic; Signal Transduction; Skin; Transforming Growth Factor beta

1 Although the pathogenesis of scleroderma is not fully understood, activation of connective-tissue-type mast cells (CTMCs) has been implicated in various fibrotic diseases. 2 Our previous study showed that the number of CTMCs was markedly increased during fibrous proliferation in the skin of a scleroderma model, namely tight-skin (Tsk) mice. Because mast cells express numerous bioactive factors, such as cytokines, growth factors, proteases, and others, it is crucial to identify the primary factors that may be involved in the pathogenesis of scleroderma. Our previous study also showed that a CTMC-specific protease, chymase-4, was selectively upregulated in accordance with the development of skin fibrosis in Tsk mice. 3 To further elucidate the role of chymase secreted from CTMCs, we evaluated the therapeutic effects of a synthetic chymase-specific inhibitor, SUN-C8257, on the development of skin fibrosis in Tsk mice. SUN-C8257 (50 mg kg-1 day-1) was administered via intraperitoneal injection in 13-week-old Tsk mice for a period of 2 weeks. 4 Treatment with SUN-C8257 significantly reduced chymase activity by 43% and the chymase-4 mRNA level by 47%, and also decreased the thickness of the subcutaneous fibrous layer of Tsk mice by 42% compared with that of Tsk mice injected with vehicle. 5 Furthermore, immunohistochemical analysis revealed that transforming growth factor (TGF)-beta1 staining in the fibrous layer of Tsk skin was markedly reduced by the treatment with SUN-C8257. This chymase inhibitor may prevent the chymase-dependent pathway that activates the latent TGF-beta1 in fibrous tissue, and may exhibit beneficial effects that inhibit the development of fibrosis. 6 In conclusion, our results strongly support the assumption that CTMC-derived chymase may play a key role in the pathogenesis of scleroderma.

Topics: Animals; Cell Proliferation; Chymases; Fibrosis; Immunohistochemistry; Mast Cells; Mice; Mice, Inbred C57BL; Protease Inhibitors; Quinazolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Serine Endopeptidases; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

The tight skin (Tsk) mouse develops many pathological changes seen in human scleroderma, such as increased collagen content and mast cell density. Although associations between mast cell expansion and skin fibrosis have been reported, the mechanisms underlying mast cell accumulation remain unclear. In this study, we have measured the density of skin mast cells in Tsk mice and their normal littermates (pa/pa) of 4-36 weeks of age, and in the skin heterografted between Tsk and pa/pa mice. Cytokines related to mast cell differentiation, proliferation and migration were examined by using RNase protection assays. Skin mast cell density in Tsk mice was significantly increased from 12 weeks of age, compared to that in pa/pa mice. The expression of transforming growth factor-beta1 (TGF-beta1), and to a lesser extent, stem cell factor (SCF) and interleukin-15 (IL-15) mRNA was higher in Tsk mice, compared to that in control mice. Mast cell density was unchanged in Tsk skin grafted onto pa/pa hosts, but dramatically increased in pa/pa skin grafted onto Tsk hosts. This latter mast cell hyperplasia was associated with the increases in mRNA levels of TGF-beta1, SCF and IL-15, whereas little change in cytokine levels was seen in heterografted Tsk skin. These results suggest that locally produced cytokines in Tsk skin influence mast cell accumulation in this animal model of human scleroderma.

Topics: Animals; Cell Differentiation; Cell Movement; Cell Proliferation; Collagen; Cytokines; Disease Models, Animal; Fibrosis; Heterozygote; Immunohistochemistry; Mast Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Ribonucleases; RNA, Messenger; Scleroderma, Systemic; Skin; Skin Transplantation; Time Factors; Transforming Growth Factor beta

Transforming growth factor beta (TGFbeta) induces profibrotic responses in normal fibroblasts, and plays a fundamental role in the pathogenesis of fibrosis in scleroderma (systemic sclerosis [SSc]). The intensity of cellular responses elicited by cytokines is modulated by transcriptional coactivators such as the histone acetylase p300. The objective of these studies was to delineate the physiologic role of p300 in Smad-dependent profibrotic responses elicited by TGFbeta.. Ectopic p300 was transiently expressed in normal dermal fibroblasts. Cellular p300 levels were suppressed using p300-specific ribozymes. The regulation of gene expression was examined by transient transfection assays, Northern blotting, and immunoblot analysis. The expression of p300 in normal and scleroderma fibroblasts was evaluated by confocal microscopy and immunoblotting, and p300 levels in skin from mice with experimental scleroderma were assessed by immunohistochemistry.. In normal fibroblasts, TGFbeta induced an increase in the levels of p300. Forced expression of ectopic p300 in these cells dramatically enhanced the magnitude of TGFbeta responses, whereas selective depletion of p300 using ribozyme resulted in abrogation of TGFbeta-induced collagen synthesis and promoter activity. Furthermore, TGFbeta lost its ability to induce Smad-dependent transcription in p300-depleted fibroblasts. These responses could be fully rescued with ectopic p300. Abrogation of Smad-mediated TGFbeta signaling was not due to alterations in the levels or the ligand-dependent phosphorylation or intracellular trafficking of endogenous Smads. Immunohistochemical analysis demonstrated substantially increased p300 expression in lesional skin from mice with chronic graft-versus-host disease, an animal model of scleroderma. Furthermore, levels of p300 were 2-3-fold higher in cultured fibroblasts derived from SSc patients than in fibroblasts from matched normal controls.. These results establish, for the first time, that the coactivator histone acetylase p300, itself a target of TGFbeta regulation, is an essential component of the cellular TGFbeta signal transduction pathways mediating stimulation of collagen synthesis in fibroblasts. Since the cellular abundance of p300 appears to govern the intensity of profibrotic responses elicited by TGFbeta, elevated p300 expression in lesional tissue may contribute to the progression of skin fibrosis in scleroderma.

Topics: Acetyltransferases; Animals; Cell Cycle Proteins; Cells, Cultured; Disease Models, Animal; DNA-Binding Proteins; Female; Fibroblasts; Fibrosis; Gene Expression Regulation, Enzymologic; Graft vs Host Disease; Histone Acetyltransferases; Humans; Infant, Newborn; Male; Mice; Mice, Inbred BALB C; Middle Aged; p300-CBP Transcription Factors; RNA, Catalytic; Scleroderma, Systemic; Skin; Smad3 Protein; Trans-Activators; Transcription Factors; Transforming Growth Factor beta

To investigate the effects of FK-506 on the expression of the human alpha2(I) collagen gene and transforming growth factor beta (TGFbeta) signaling in normal and scleroderma fibroblasts.. The expression levels of type I procollagen protein and alpha2(I) collagen messenger RNA (mRNA) were analyzed by immunoblotting and Northern blotting, respectively. The promoter activities of alpha2(I) collagen gene and 3TP-Lux were determined by transient transfection assay. Interaction between TGFbeta receptor type I and FK-506 binding protein 12 (FKBP12) was evaluated by immunoprecipitation.. FK-506 did not affect the basal expression of type I procollagen protein or alpha2(I) collagen mRNA, but it significantly reduced the TGFbeta1-induced expression of type I procollagen protein and alpha2(I) collagen mRNA in normal fibroblasts. The effect of FK-506 was regulated posttranscriptionally, but not transcriptionally. In scleroderma fibroblasts, FK-506 significantly reduced the expression of type I procollagen protein and alpha2(I) collagen mRNA through posttranscriptional regulation, but not transcriptional regulation. FK-506 increased the basal activity of the 3TP-Lux promoter, but it did not affect the TGFbeta1-induced promoter activity in normal fibroblasts. In contrast, FK-506 did not affect the basal or the TGFbeta1-induced 3TP-Lux promoter activity in scleroderma fibroblasts. Furthermore, FKBP12, which protects TGFbeta receptor type I from ligand-independent activation by TGFbeta receptor type II, constitutively dissociated from TGFbeta receptor type I in scleroderma fibroblasts.. FK-506 inhibits alpha2(I) collagen gene expression by reducing the stability of mRNA without exhibiting its activation effect on TGFbeta signaling in scleroderma fibroblasts.

Topics: Cells, Cultured; Collagen Type I; Fibroblasts; Gene Expression; Humans; Immunosuppressive Agents; Procollagen; Scleroderma, Systemic; Signal Transduction; Tacrolimus; Transforming Growth Factor beta

Most of the cultured scleroderma fibroblasts have been reported to be myofibroblasts that have the ability to express alpha smooth muscle actin (alphaSMA). It is reported that, in human lung fibroblasts, alphaSMA is induced by transforming growth factor-beta (TGF-beta), which requires focal adhesion kinase (FAK) phosphorylation on its Tyr-397 site. In this study, we investigated how alphaSMA expression is upregulated in cultured scleroderma fibroblasts. 4-amino-5-(4-chlorophenyl)-7-(butyl)pyrazolo[3,4-d]pyrimidine, which is a pharmacologic inhibitor of FAK/Src, markedly diminished upregulated alphaSMA expression in scleroderma fibroblasts as well as in normal fibroblasts stimulated with TGF-beta. Likewise, alphaSMA expression was significantly reduced in sclerderma fibroblasts transfected with kinase-deficient FAK mutant. FAK phosphorylation levels on Tyr-397 in scleroderma fibroblasts were significantly higher than those in normal fibroblasts. Both alphaSMA expression and FAK phosphorylation levels in scleroderma fibroblasts were markedly diminished by the treatment with TGF-beta antisense oligonucleotide. These results indicate that the constitutive phosphorylation of FAK, which is possibly because of the autocrine TGF-beta signaling, may play an important role in alphaSMA expression in scleroderma fibroblasts.

Topics: Actins; Cell Differentiation; Cells, Cultured; Fibroblasts; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Oligonucleotides, Antisense; Oligopeptides; Phosphorylation; Protein-Tyrosine Kinases; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

The extracellular matrix (ECM) glycoprotein thrombospondin-1 (TSP-1) has been reported to activate the latent complex of transforming growth factor-beta (TGF-beta), the major effects of which in mesenchymal cells is stimulation of the synthesis of ECM. Previous reports suggested the involvement of an autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we examined whether TSP-1 plays a role in maintaining the autocrine TGF-beta loop in scleroderma. TSP-1 expression was increased in scleroderma patients compared with in healthy controls in vivo and in vitro. TGF-beta blocking antibody or TGF-beta1 antisense oligonucleotide markedly reduced the up-regulated TSP-1 expression in scleroderma fibroblasts but had little effect on normal fibroblasts. The expression of TSP-1 is up-regulated in scleroderma fibroblasts, possibly at the post-transcriptional level just like in normal fibroblasts stimulated with exogenous TGF-beta1. TSP-1 blocking peptide or antisense oligonucleotide had an inhibitory effect on the up-regulated alpha2I collagen and phosopho-Smad3 levels in scleroderma fibroblasts but had little effects on normal fibroblasts. The transient overexpression of TSP-1 up-regulated alpha2I collagen and phospho-Smad3 levels in normal fibroblasts but had no major effect on scleroderma fibroblasts. Furthermore, these effects of transiently overexpressed TSP-1, which possibly occurred via the activation of latent TGF-beta1, were abolished by the TGF-beta1 antisense oligonucleotide. These results indicate that the constitutive overexpression of TSP-1 may play an important role in autocrine TGF-beta signaling and accumulation of ECM in scleroderma fibroblasts.

Topics: Autocrine Communication; Case-Control Studies; Cells, Cultured; Collagen; Collagen Type I; DNA-Binding Proteins; Fibroblasts; Humans; Phosphorylation; Promoter Regions, Genetic; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin; Smad3 Protein; Thrombospondin 1; Trans-Activators; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

A procollagen alpha2(I) messenger RNA (mRNA) variant, with a 115-bp band and an expected band of 180 bp, was found to be increased during early, acute scleroderma-like disease in UCD-200 chickens. The present study investigated the influence of cytokines on the expression of these 2 proalpha2(I) mRNA variants.. Embryonic fibroblasts of UCD-200 chickens (UCD-200-CEF) and normal white leghorns (NWL-CEF) were grown in 3-dimensional collagen gels. Procollagen mRNA expression was analyzed by RNase protection assay, and proliferation was determined by (3)H-thymidine incorporation. Transforming growth factor beta1 (TGFbeta1) and TGFbeta2 were measured in culture supernatants by enzyme-linked immunosorbent assay.. Compared with NWL-CEF, UCD-200-CEF expressed 7.2 times more of the smaller profibrotic proalpha2(I) mRNA variant. TGFbeta1 stimulated the proliferation of UCD-200-CEF, but not NWL-CEF. The 115 bp:180 bp ratio was increased by TGFbeta1 in both NWL-CEF and UCD-CEF. TGFbeta2 and TGFbeta3 reduced the expression of the profibrotic proalpha2(I) mRNA in UCD-200-CEF to the same levels observed in healthy control NWL-CEF. In culture supernatants, NWL-CEF produced 4.1 times more TGFbeta2 than that produced by UCD-CEF. Inhibition of endogenous TGFbeta2 in NWL-CEF resulted in the same 115 bp:180 bp ratio as seen in untreated UCD-CEF.. TGFbeta2 reduces the expression of a profibrotic proalpha2(I) mRNA variant in UCD-200-CEF. The constitutive overproduction of this proalpha2(I) mRNA variant and the diminished synthesis of TGFbeta2 in untreated UCD-200-CEF suggest that TGFbeta2 can act as an antifibrotic cytokine and might be a key player during fibrosis onset. These results shed light on the contradictory observations regarding the role of TGFbeta2 in human systemic sclerosis.

Topics: Animals; Cells, Cultured; Chick Embryo; Collagen Type I; Fibroblasts; Models, Animal; RNA, Messenger; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

Transforming growth factor-beta (TGF-beta) stimulates the transcription of the alpha2(I) collagen gene. The dermal fibroblast activation in systemic sclerosis (SSc) may be a result of stimulation by autocrine TGF-beta. In this study, we investigated whether p38 mitogen-activated protein kinase (MAPK) is involved in TGF-beta-induced transcriptional activation of the human alpha2(I) collagen gene in normal dermal fibroblasts and in upregulated extracellular matrix (ECM) expression in SSc fibroblasts. Type I collagen expression induced by TGF-beta was suppressed by the specific p38 MAPK inhibitors SB203580 or SB202190 in normal fibroblasts. TGF-beta induced phosphorylation and activation of p38 MAPK in normal dermal fibroblasts. Transient transfection of dominant-negative mutant p38 MAPK into normal fibroblasts abolished TGF-beta-induced promoter activity of the human alpha2(I) collagen gene in normal fibroblasts. Moreover, constitutive phosphorylation and activation of p38 MAPK was demonstrated in SSc fibroblasts, and the inhibition of p38 MAPK using specific p38 MAPK inhibitors or dominant-negative mutant p38 MAPK abolished the upregulated expression of type I collagen or fibronectin in SSc fibroblasts. These results strongly suggest the contribution of p38 MAPK signaling to the TGF-beta-mediated regulation of the human alpha2(I) collagen gene in normal dermal fibroblasts and constitutive upregulated expression of type I collagen and fibronectin in SSc fibroblasts.

Topics: Adult; Cells, Cultured; Collagen; Collagen Type I; Dermis; Enzyme Inhibitors; Female; Fibroblasts; Fibronectins; Humans; Male; MAP Kinase Signaling System; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

The tight skin 1 (B6.CgFbn1(Tsk)+/+Pldn(pa)/J, henceforth referred to as Tsk1/+) mouse was first described as a spontaneously occurring mutant that resulted in hyperplasia of the subcutaneous loose connective tissue, and has subsequently been proposed to be a model of the human fibrotic disorder scleroderma. We have investigated the Tsk1/+ mouse as a model system for testing the efficacy of antifibrotic agents against skin fibrosis. We find that the tightness of the skin at the scruff of the neck leads to a measurably thicker skin pinch, but we suggest that this is due to hyperplasia of the subdermal loose connective tissue, which results in increased tethering of the skin to the underlying muscle layers. In contrast to previously published data, we do not find a significant difference in the dermal thickness or collagen content of the Tsk1/+ mouse skin compared with wild-type controls. In addition, expression profiling of Tsk1/+ mouse skin indicated that there are very few changes in gene expression, and that there is no evidence for upregulation of the transforming growth factor beta signaling axis. Therefore, we conclude that this model is not suitable for testing the effect of antifibrotic agents on the dermis, and that changes potentially related to scleroderma may be confined to subdermal connective tissue.

Topics: Animals; Collagen; Dermis; Disease Models, Animal; Fibrillins; Fibrosis; Gene Expression Profiling; Gene Expression Regulation; Lung; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microfilament Proteins; Mutation; Phenotype; Protein Serine-Threonine Kinases; Scleroderma, Systemic; Sex Factors; Signal Transduction; Skin; Transforming Growth Factor beta

To confirm the involvement of alphavbeta5 in the self-activation system in systemic sclerosis (SSc) fibroblasts.. Levels of alphavbeta5 expression were analyzed by immunoprecipitation. The promoter activity of the human alpha2(I) collagen gene was determined by transient transfection assay. Phosphorylation levels and DNA binding ability of Smad3 were investigated by immunoprecipitation and DNA affinity precipitation, respectively. The localization of active transforming growth factor beta (TGFbeta) was determined by coculture assay using TMLC cells (mink lung epithelial reporter cells that stably express a portion of the plasminogen activator inhibitor 1 promoter). The morphologic features of cells were determined by immunofluorescence analysis.. Levels of alphavbeta5 expression were significantly elevated in SSc fibroblasts compared with normal fibroblasts. Treatment with anti-alphavbeta5 antibody or beta5 antisense oligonucleotide significantly reduced human alpha2(I) collagen gene promoter activity in SSc fibroblasts. In SSc fibroblasts pretreated with TGFbeta1 antisense oligonucleotide, the exogenous latent TGFbeta1 stimulation significantly increased human alpha2(I) collagen gene promoter activity; this effect was significantly reduced in the presence of anti-alphavbeta5 antibody. Phosphorylation levels and DNA binding ability of Smad3 in SSc fibroblasts were significantly reduced by treatment with beta5 antisense oligonucleotide. The luciferase activity of TMLC cells cocultured with SSc fibroblasts was significantly elevated compared with that of TMLC cells cocultured with normal fibroblasts and was significantly reduced in the presence of anti-alphavbeta5 antibody. Anti-alphavbeta5 antibody reversed the myofibroblastic features of SSc fibroblasts.. Up-regulated expression of alphavbeta5 contributes to the establishment of autocrine TGFbeta signaling in SSc fibroblasts through activation of endogenous latent TGFbeta1.

Topics: Antibodies, Blocking; Cell Count; Cell Differentiation; Cells, Cultured; Dermis; Fibroblasts; Humans; Integrins; Intracellular Signaling Peptides and Proteins; Latent TGF-beta Binding Proteins; Oligonucleotides, Antisense; Receptors, Vitronectin; Scleroderma, Systemic; Signal Transduction; Transfection; Transforming Growth Factor beta

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-beta1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-beta1 with anti-TGF-beta1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-beta1 from fibrillin-1-containing microfibrils in the ECM.

Topics: Active Transport, Cell Nucleus; Adult; Autoantibodies; Biomarkers; Cell Nucleus; Cells, Cultured; Extracellular Matrix Proteins; Fibrillin-1; Fibrillins; Fibroblasts; Humans; Microfilament Proteins; Middle Aged; Phosphorylation; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis (SSc) is characterized by excessive fibrosis and obliterative vascular lesions. Abnormal TGFbeta activation is implicated in the pathogenesis of SSc. Aberrant TGFbeta/Smad signaling can be controlled by stabilization of microtubules with paclitaxel.. SSc and healthy human skin biopsies were incubated in the presence or absence of paclitaxel followed by transplantation into severe combined immunodeficient mice. TGFbeta signaling, fibrosis, and neovessel formation were evaluated by quantitative RT-PCR and immunohistochemical staining. Paclitaxel markedly suppressed Smad2 and Smad3 phosphorylation and collagen deposition in SSc grafts. As a result, the autonomous maintenance/reconstitution of the SSc phenotype was prevented. Remarkably, SSc grafts showed a 2-fold increase in neovessel formation relative to normal grafts, regardless of paclitaxel treatment. Angiogenesis in SSc grafts was associated with a substantial increase in mouse PECAM-1 expression, indicating the mouse origin of the neovascular cells.. Low-dose paclitaxel can significantly suppress TGFbeta/Smad activity and lessen fibrosis in SCID mice. Transplantation of SSc skin into SCID mice elicits a strong angiogenesis-an effect not affected by paclitaxel. Although prolonged chemotherapy with paclitaxel at higher doses is associated with pro-fibrotic and anti-angiogenic changes, the findings described here indicate that low-dose paclitaxel may have therapeutic benefits for SSc via modulating TGFbeta signaling.

Topics: Adult; Animals; Antineoplastic Agents, Phytogenic; Biopsy; Dose-Response Relationship, Drug; Female; Fibrosis; Humans; Male; Mice; Mice, SCID; Middle Aged; Neovascularization, Pathologic; Paclitaxel; Phenotype; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transplantation, Heterologous

The constitutive secretion of latent TGF-beta by many cell types in culture suggests that extracellular mechanisms to control the activity of this potent cytokine are important in the pathogenesis of the diseases in which this cytokine may be involved, including fibrotic disorders. In this study, we focused on the alpha(v)beta3 integrin, which is recently demonstrated to function as an active receptor for latent TGF-beta1 through its interaction with latency-associated peptide-beta1, and investigated the involvement of this integrin in the pathogenesis of scleroderma. Scleroderma fibroblasts exhibited increased alpha(v)beta3 expression compared with normal fibroblasts in vivo and in vitro. In scleroderma fibroblasts, ERK pathway was constitutively activated and such abnormality induced the up-regulation of alpha(v)beta3. Transient overexpression of alpha(v)beta3 in normal fibroblasts induced the increase in the promoter activity of human alpha2(I) collagen gene and the decrease in that of human MMP-1 gene. These effects of alpha(v)beta3 were almost completely abolished by the treatment with anti-TGF-beta Ab or TGF-beta1 antisense oligonucleotide. Furthermore, the addition of anti-alpha(v)beta3) Ab reversed the expression of type I procollagen protein and MMP-1 protein, the promoter activity of human alpha2(I) collagen gene, and the myofibroblastic phenotype in scleroderma fibroblasts. These results suggest that the up-regulated expression of alpha(v)beta3 contributes to the establishment of autocrine TGF-beta loop in scleroderma fibroblasts, and this integrin is a potent target for the treatment of scleroderma.

Topics: Autocrine Communication; Blotting, Northern; Cells, Cultured; Collagen Type I; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Gene Expression; Humans; Immunohistochemistry; Immunoprecipitation; Integrin alphaVbeta3; Matrix Metalloproteinase 1; RNA, Messenger; Scleroderma, Systemic; Transfection; 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

The principal effect of TGF-beta1 on mesenchymal cells is its stimulation of ECM synthesis. Previous reports indicated the significance of the autocrine TGF-beta loop in the pathogenesis of scleroderma. In this study, we focused on Smad7 and Smurfs, principal molecules in the negative regulation of TGF-beta signaling, to further understand the autocrine TGF-beta loop in scleroderma. Scleroderma fibroblasts exhibited increased Smad7 levels compared with normal fibroblasts in vivo and in vitro. Smad7 constitutively formed a complex with the TGF-beta receptors, and the inhibitory effect of Smad7 on the promoter activity of human alpha2(I) collagen and 3TP-lux was completely impaired in scleroderma fibroblasts. Furthermore, the protein stability of TGF-beta receptor type I was significantly increased in scleroderma fibroblasts compared with normal fibroblasts. There was no significant difference in Smurf1 and Smurf2 levels between normal and scleroderma fibroblasts, and the transiently overexpressed Smurf1 and/or Smurf2 did not affect TGF-beta receptor type I protein levels in scleroderma fibroblasts. These results indicate that the impaired Smad7-Smurf-mediated inhibitory effect on TGF-beta signaling might contribute to maintaining the autocrine TGF-beta loop in scleroderma fibroblasts. To our knowledge, this is the first report of a disturbed negative regulation of TGF-beta signaling in fibrotic disorders.

Topics: Blotting, Northern; Blotting, Western; Cells, Cultured; Collagen; Collagen Type I; Densitometry; DNA-Binding Proteins; Fibroblasts; Gene Expression Regulation; Humans; Immunoblotting; Immunohistochemistry; Microscopy, Fluorescence; Oligonucleotides; Phosphorylation; Plasmids; Precipitin Tests; Receptors, Transforming Growth Factor beta; RNA; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Smad7 Protein; Time Factors; Trans-Activators; Transfection; Transforming Growth Factor beta; Transforming Growth Factor beta1; Ubiquitin-Protein Ligases

Tissue fibrosis in systemic sclerosis (SSc) is attributed to excessive deposition of extracellular matrix components produced by fibroblasts in skin lesions. Angiotensin II (Ang II), a vasoconstrictive peptide, is reported to have profibrotic activity as a result of induction of the extracellular matrix. The aim of the present study was to examine the expression of Ang II and its type 1 (AT(1)) and type 2 (AT(2)) receptors in affected skin and dermal fibroblasts from patients with SSc and to study the role of Ang II in collagen production by SSc dermal fibroblasts.. Levels of Ang II in sera from SSc patients and normal subjects were measured by a solid-phase immobilized-epitope immunoassay. Expression of angiotensinogen (Angt) in the skin was evaluated by immunohistochemistry. Expression of Angt, AT(1), and AT(2) in cultured dermal fibroblasts was analyzed by reverse transcription-polymerase chain reaction and immunohistochemistry. Levels of type I procollagen produced by cultured dermal fibroblasts were measured by enzyme-linked immunosorbent assay.. Serum Ang II levels in patients with diffuse cutaneous SSc were significantly higher than those in patients with limited cutaneous SSc and in healthy donors. Immunohistochemical and immunoblotting analyses showed that Angt was present in skin from SSc patients, but not in normal skin. Angt messenger RNA (mRNA) was expressed in fibroblasts from patients with diffuse cutaneous SSc who had high levels of serum Ang II, but not in normal fibroblasts. AT(1) mRNA expression was found in both SSc and normal fibroblasts, whereas AT(2) mRNA was found only in SSc fibroblasts. Exogenous Ang II augmented the production of type I procollagen and transforming growth factor beta1 by cultured fibroblasts via activation of AT(1).. Aberrant Ang II production may be involved in tissue fibrosis through excessive production of the extracellular matrix components in SSc dermal fibroblasts. This suggests that the use of AT(1) receptor antagonists may be a novel strategy for the treatment of tissue fibrosis in SSc patients.

Topics: Adult; Aged; Angiotensin II; Angiotensinogen; Autocrine Communication; Blotting, Western; Cathepsin D; Cells, Cultured; Extracellular Matrix; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Immunohistochemistry; Male; Middle Aged; Procollagen; Receptor, Angiotensin, Type 1; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

In fibroblasts, transforming growth factor beta (TGF beta) stimulates collagen synthesis and myofibroblast transdifferentiation through the Smad intracellular signal transduction pathway. TGF beta-mediated fibroblast activation is the hallmark of scleroderma and related fibrotic conditions, and disrupting the intracellular TGF beta/Smad signaling may provide a novel approach to controlling fibrosis. Because of its potential role in modulating inflammatory and fibrotic responses, we examined the expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) in normal skin fibroblasts and its effect on TGF beta-induced cellular responses.. The expression and activity of PPAR gamma in normal dermal fibroblasts were examined by Northern and Western blot analyses, immunocytochemistry, flow cytometry, and transient transfections with reporter constructs. The same approaches were used to evaluate the effects of PPAR gamma activation by naturally occurring and synthetic ligands on collagen synthesis and alpha-smooth muscle actin (alpha-SMA) expression. Modulation of Smad-mediated transcriptional responses was examined by transient transfection assays using wild-type and dominant-negative PPAR gamma expression constructs.. The PPAR gamma receptor was expressed and fully functional in quiescent normal skin fibroblasts. Whereas ligand activation of cellular PPAR gamma resulted in modest suppression of basal collagen gene expression, it abrogated TGF beta-induced stimulation in a concentration-dependent manner. This response was mimicked by overexpressing PPAR gamma in fibroblasts, and was blocked by a selective antagonist of PPAR gamma signaling or by transfection of fibroblasts with dominant-negative PPAR gamma constructs. Furthermore, PPAR gamma ligands abrogated TGF beta-induced expression of alpha-SMA, a marker of myofibroblasts. Stimulation of Smad-dependent transcriptional responses by TGF beta was suppressed by PPAR gamma despite the absence of a consensus PPAR gamma-response element in the targeted promoters. Ligand-induced activation of fibroblast PPAR gamma had no effect on protein expression of cellular Smad3 or Smad7.. By abrogating of TGF beta-induced stimulation of collagen gene expression, myofibroblast transdifferentiation, and Smad-dependent promoter activity in normal fibroblasts, PPAR gamma may play a physiologic role in the regulation of the profibrotic response. Furthermore, our results suggest that PPAR gamma activation by pharmacologic agonists may represent a novel approach to the control of fibrosis in scleroderma.

Topics: Actins; Cells, Cultured; Collagen; Collagen Type I; Dermis; DNA-Binding Proteins; Fibroblasts; Fibrosis; Gene Expression; Humans; Receptors, Cytoplasmic and Nuclear; Scleroderma, Systemic; Signal Transduction; Smad Proteins; Trans-Activators; Transcription Factors; Transfection; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) plays an important role in the pathogenesis of systemic sclerosis (SSc). To investigate the role of TGF-beta1 gene polymorphisms in SSc, we genotyped six biallelic polymorphic positions (position -988, -800, and -509; and codons 10, 25, and 263) in 61 Korean SSc patients and in 148 healthy controls, using polymerase chain reaction-sequence-specific primers. Genetic polymorphisms were found at position -509 and codon 10 in Koreans. The allele frequencies of C/T at position -509 were 0.59/0.41 in patients and 0.56/0.44 in controls. The allele frequencies of C/T at codon 10 were 0.40/0.60 in patients and 0.50/0.50 in controls. In conclusion, no skewed distribution of TGF-beta1 gene polymorphisms was found in Korean patients with SSc.

Topics: Adult; Female; Gene Frequency; Genotype; Humans; Korea; Male; Polymorphism, Genetic; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1

TGF-beta1 plays a major role in fibrotic diseases including scleroderma. Human fibroblasts from sclerotic lesions display an increased sensitivity to TGF-beta1. Similarly, fibroblasts from TSK mice which develop a scleroderma-like syndrome are hyperresponsive to TGF-beta. The aim of the present study was to investigate whether the TGF-beta hypersensitivity demonstrated by TSK/+ fibroblasts is associated with polymorphisms of the TGF-beta1 promoter. Sequence analysis revealed one polymorphism (a G --> T at -1133 bp) unique to the TSK/+ mouse. Transfection of fibroblasts with a 1.8 kb fragment of the TGF-beta1 promoter containing the -1133 polymorphism exhibited increased basal TGF-beta1 promoter activity which was enhanced upon incubation with TGF-beta1. This may be related to the loss of a negative regulatory site in the TSK/+ TGF-beta1 promoter.

Topics: Animals; Base Sequence; Mice; Molecular Sequence Data; Polymorphism, Genetic; Promoter Regions, Genetic; Scleroderma, Systemic; Sequence Analysis, DNA; Transforming Growth Factor beta; Transforming Growth Factor beta1

We sought to determine whether the spontaneous production of transforming growth factor-beta (TGF-beta) by peripheral blood mononuclear cells (PBMC) is increased in patients with systemic sclerosis (SSc). Culture supernatants of PBMC from SSc patients (n = 88) and healthy controls (n = 44) were analyzed by enzyme-linked immunosorbent assay. The production of active TGF-beta1 and total (active and latent) TGF-beta1 by PBMC from patients with limited cutaneous SSc (lSSc) and by PBMC from patients with diffuse cutaneous SSc (dSSc) was significantly elevated compared to the production by PBMC from normal controls. Production of active TGF-beta1 by dSSc PBMC was higher than that by lSSc PBMC, although not significantly. Patients with PBMC with increased active or total TGF-beta1 production showed significantly shorter disease duration than patients with PBMC with normal production levels. PBMC from patients without anticentromere antibody showed enhanced active TGF-beta1 production more frequently than those from patients with anticentromere antibody. PBMC from SSc patients more frequently showed enhanced total TGF-beta2 production than PBMC from normal controls. Among each leukocyte subset, spontaneous production of total TGF-beta1 was significantly higher in cultured peripheral monocytes/macrophages, but not in T cells, B cells, or NK cells, from patients than from normal controls. Thus, the enhanced production of TGF-beta by PBMC may contribute to the disease process in SSc

Topics: Adult; Case-Control Studies; Cells, Cultured; Female; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Prednisolone; Scleroderma, Diffuse; Scleroderma, Limited; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2

We previously reported a mouse model for scleroderma by repeated local injections of bleomycin. In this study, we investigated the level of transforming growth factor-beta1 (TGF-beta1) in various mice strains, in order to determine whether the expression of TGF-beta1 correlates with the susceptibility to bleomycin-induced scleroderma. Histological examination revealed prominent dermal sclerosis with increased collagen deposition in the bleomycin-treated skin in B10.A and C3H/HeJ strains as compared with BALB/c, C57BL/6J and DBA/2 strains. Collagen contents in the skin were also increased in B10.A and C3H/HeJ strains. Analysis of skin lesions from B10.A and C3H/HeJ exhibited the increased mRNA expression and protein synthesis of TGF-beta1. TGF-beta1 concentrations in culture supernatants of skin fibroblasts and spleen macrophages were significantly increased by bleomycin stimulation in B10.A and C3H/HeJ strains, and TGF-beta1 gene expression in fibroblasts derived from B10.A and C3H/HeJ strains was significantly increased by bleomycin stimulation. Thus we conclude that C3H/HeJ and B10.A mice are susceptible to bleomycin-induced scleroderma, which may be, in part, due to increased TGF-beta1 gene expression and protein production.

Topics: Animals; Bleomycin; Collagen; Disease Models, Animal; Disease Susceptibility; Female; Fibroblasts; Germ-Free Life; Macrophages; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Mice, Inbred Strains; RNA, Messenger; Scleroderma, Systemic; Skin; Spleen; Transforming Growth Factor beta; Transforming Growth Factor beta1

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

Transforming growth factor-beta (TGF-beta) is a potent stimulus of connective tissue accumulation, and is implicated in the pathogenesis of scleroderma and other fibrotic disorders. Smad3 functions as a key intracellular signal transducer for profibrotic TGF-beta responses in normal skin fibroblasts. The potential role of Smad3 in the pathogenesis of scleroderma was investigated in Smad3-null (Smad3(-/-)) mice using a model of skin fibrosis induced by subcutaneous injections of bleomycin. At early time points, bleomycin-induced macrophage infiltration in the dermis and local TGF-beta production were similar in Smad3(-/-) and wild-type mice. In contrast, at day 28, lesional skin from Smad3(-/-) mice showed attenuated fibrosis, lower synthesis and accumulation of collagen, and reduced collagen gene transcription in situ, compared to wild-type mice. Connective tissue growth factor and alpha-smooth muscle actin expression in lesional skin were also significantly attenuated. Electron microscopy revealed an absence of small diameter collagen fibrils in the dermis from bleomycin-treated Smad3(-/-) mice. Compared to fibroblasts derived from wild-type mice, Smad3(-/-) fibroblasts showed reduced in vitro proliferative and profibrotic responses elicited by TGF-beta. Together, these results indicate that ablation of Smad3 is associated with markedly altered fibroblast regulation in vivo and in vitro, and confers partial protection from bleomycin-induced scleroderma in mice. Reduced fibrosis is due to deregulated fibroblast function, as the inflammatory response induced by bleomycin was similar in wild-type and Smad3(-/-) mice.

Topics: Actins; Animals; Bleomycin; Blotting, Western; Cells, Cultured; Collagen; Connective Tissue Growth Factor; Disease Models, Animal; Fibroblasts; Fibronectins; Fibrosis; Flow Cytometry; Gene Expression Regulation; Gene Targeting; Hydroxyproline; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Intercellular Signaling Peptides and Proteins; Mice; Mice, Knockout; Scleroderma, Systemic; Signal Transduction; Skin; Trans-Activators; 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

Topics: Adenocarcinoma; Aged; Aged, 80 and over; Diagnosis, Differential; DNA Primers; Humans; Male; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Scleroderma, Systemic; Stomach Neoplasms; Transforming Growth Factor beta; Treatment Outcome

Transforming growth factor-beta is responsible for triggering a cascade of events leading to fibrosis in scleroderma. The Smads are intracellular signal transducers recently shown to mediate fibroblast activation and other profibrotic responses elicited by transforming growth factor-betain vitro. To understand better the involvement of Smads in the pathogenesis of fibrosis, we examined Smad expression and activation in situ in a murine model of scleroderma. Bleomycin injections induced striking dermal infiltration with macrophages by 3 d, and progressive fibrosis by 2 wk. Infiltrating macrophages and resident fibroblasts expressed Smad3, the positive mediator for transforming growth factor-beta responses. Importantly, in bleomycin-injected skin, fibroblasts showed predominantly nuclear localization of Smad3 and intense staining for phospho-Smad2/3. Furthermore, phosphorylated Smad2/3 in fibroblasts was detected even after the resolution of inflammation. Expression of Smad7, the endogenous inhibitor of transforming growth factor-beta/Smad signaling, was strongly induced in dermal cells by transforming growth factor-beta, but not by bleomycin injections. Collectively, these results indicate that bleomycin-induced murine scleroderma is associated with rapid and sustained induction of transforming growth factor-beta/Smad signaling in resident dermal fibroblasts. Despite apparent activation of the intracellular transforming growth factor-beta signaling pathway in the lesional dermis, the expression of transforming growth factor-beta-inducible Smad7 was not upregulated. In light of the critical function of Smad7 as an endogenous inhibitor of Smad signaling that restricts the duration and magnitude of transforming growth factor-beta responses, and as a mediator of apoptosis, relative Smad7 deficiency observed in the present studies may account for sustained activation of transforming growth factor-beta/Smad signaling in lesional tissues. These findings raise the possibility that Smads plays an important part in the pathogenesis of fibrosis, and may therefore represent targets for selective anti-fibrotic interventions.

Topics: Animals; Antibiotics, Antineoplastic; Antibodies; Bleomycin; Cells, Cultured; Dermatitis; Disease Models, Animal; DNA-Binding Proteins; Female; Fibroblasts; Mice; Mice, Inbred C3H; Phosphorylation; Scleroderma, Systemic; Signal Transduction; Smad2 Protein; Smad3 Protein; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta

Scleroderma is characterized by excessive synthesis and accumulation of matrix proteins in lesional tissues. Transforming growth factor beta (TGFbeta) plays a central role in the pathogenesis of fibrosis by inducing and sustaining activation of fibroblasts; however, the underlying mechanisms are poorly understood. We undertook this study to examine the expression and function of SMADs, recently characterized intracellular effectors of TGFbeta signaling, in scleroderma fibroblasts.. Primary dermal fibroblasts obtained from 14 patients with scleroderma and from 4 healthy adult volunteers were studied. Northern analysis was used to determine the expression of endogenous SMAD messenger RNA (mRNA), and Western analysis was used to determine SMAD protein expression. Intracellular compartmentalization of cellular SMAD proteins in the presence and absence of TGFbeta was studied by antibody-mediated immunofluorescence confocal microscopy. The effect of TGFbeta blockade on SMAD subcellular distribution was determined using anti-TGFbeta antibodies as well as a dominant-negative TGFbeta receptor type II (TGFbetaRII) vector to disrupt TGFbeta responses. SMAD-regulated luciferase reporter expression was examined to investigate the potential functional significance of activation and nuclear accumulation of endogenous SMADs in scleroderma fibroblasts.. Protein and mRNA levels of SMAD3, but not of SMAD4 or SMAD7, were variably elevated in scleroderma fibroblasts compared with those from healthy controls. In sharp contrast to control fibroblasts, which displayed predominantly cytoplasmic localization of SMAD3/4 in the absence of exogenous TGFbeta, in scleroderma fibroblasts SMAD3 and SMAD4 consistently showed elevated nuclear localization. Furthermore, phosphorylated SMAD2/3 levels were elevated and nuclear localization of phosphorylated SMAD2/3 was increased, suggesting activation of the SMAD pathway in scleroderma fibroblasts. Blockade of autocrine TGFbeta signaling with antibodies or by expression of dominant-negative TGFbetaRII failed to normalize SMAD subcellular distribution, suggesting that elevated nuclear SMAD import was due to alterations downstream of the TGFbeta receptors. The activity of a SMAD-responsive minimal promoter-reporter construct was enhanced in transiently transfected scleroderma fibroblasts.. This study is the first to demonstrate apparently ligand-independent constitutive activation of the intracellular TGFbeta/SMAD signaling axis in scleroderma fibroblasts. SMAD signaling may be a mechanism contributing to the characteristic phenotype of scleroderma fibroblasts and playing a role in the pathogenesis of fibrosis.

Topics: Adult; Cell Nucleus; Cells, Cultured; DNA-Binding Proteins; Female; Fibroblasts; Fibrosis; Gene Expression; Humans; Male; Middle Aged; RNA, Messenger; Scleroderma, Systemic; Skin; Smad2 Protein; Smad3 Protein; Smad4 Protein; Smad7 Protein; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta

The molecular and cellular mechanisms that maintain proper collagen homeostasis in healthy human skin and are responsible for the dysregulated collagen synthesis in scleroderma remain primarily unknown. This study demonstrates that Fli1 is a physiological negative regulator of collagen gene expression in dermal fibroblasts in vitro and in human skin in vivo. This conclusion is supported by the analyses of mouse embryonic fibroblasts from Fli1(-/-), Fli1(+/-), and Fli1(+/+) mice. In cultured human and mouse fibroblasts Fli1 expression levels are inversely correlated with the collagen type I expression levels. These in vitro observations were validated in vivo. In healthy human skin Fli1 protein is expressed in fibroblasts and endothelial cells. Significantly, absence of Fli1 expression in individual fibroblasts correlates with elevated collagen synthesis. In contrast to healthy skin, Fli1 protein is consistently absent from fibroblasts and significantly reduced in endothelial cells in clinically involved scleroderma skin, which correlates with enhanced collagen synthesis in systemic sclerosis skin. This study supports the role of Fli1 as a suppressor of collagen transcription in human skin in vivo. Persistent down-regulation of Fli1 in scleroderma fibroblasts in vivo may directly contribute to uncontrolled matrix deposition in scleroderma skin.

Topics: Adult; Aged; Animals; Cells, Cultured; Collagen; DNA-Binding Proteins; Down-Regulation; Female; Fibroblasts; Fibrosis; Gene Expression Regulation; Humans; Male; Mice; Mice, Knockout; Middle Aged; Procollagen-Proline Dioxygenase; Proto-Oncogene Protein c-fli-1; Proto-Oncogene Proteins; Scleroderma, Systemic; Skin; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Fibrotic diseases such as scleroderma (systemic sclerosis, SSc) are characterized by an excessive production of extracellular matrix and profibrotic proteins such as connective tissue growth factor (CTGF). In normal dermal fibroblasts, CTGF is not expressed unless induced by proteins such as tumor growth factor-beta (TGFbeta). Conversely, in fibroblasts cultured from fibrotic lesions CTGF mRNA and protein are constitutively expressed, even in the absence of exogenously added TGFbeta. Thus, studying the mechanism underlying CTGF overexpression in SSc fibroblasts is likely to yield valuable insights into the basis of the fibrotic phenotype of SSc and possibly other scarring disease. CTGF overexpression is mediated primarily by sequences in the CTGF promoter. In this report, we identify the minimal promoter element involved with the overexpression of CTGF in SSc fibroblasts. This element is distinct from the element necessary and sufficient for the induction of CTGF expression by TGFbeta in normal fibroblasts. Within this region is a functional Sp1 binding site. Blocking Sp1 activity reduces the elevated, constitutive levels of CTGF promoter activity and protein expression observed in SSc fibroblasts. Relative to those prepared from normal dermal fibroblasts, nuclear extracts prepared from SSc fibroblasts possess increased Sp1 binding activity. Removal of phosphate groups from nuclear extracts enhanced Sp1 binding activity, suggesting that phosphorylation of Sp1 normally reduces Sp1 binding to DNA. Thus, the constitutive overexpression of CTGF in SSc fibroblasts seems to be independent of TGFbeta signaling but dependent at least in part on Sp1.

Topics: Base Sequence; Binding Sites; Connective Tissue Growth Factor; Fibroblasts; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Phosphorylation; Promoter Regions, Genetic; Scleroderma, Systemic; Sequence Deletion; Sp1 Transcription Factor; Transfection; Transforming Growth Factor beta

Pulmonary fibrosis is a major cause of death in scleroderma patients. Previous studies have shown an increase in CD8+ T cells in the lungs of scleroderma patients. In the present study, we sought to determine whether activated CD8+ T cells contribute to pulmonary fibrosis in scleroderma patients through the production and activation of profibrotic mediators.. CD8+ cells were isolated from bronchoalveolar lavage fluid obtained from 19 scleroderma patients and 7 healthy subjects. The phenotype of these cells was determined using DNA array technology. Expression of selected genes was confirmed in real-time polymerase chain reaction and enzyme-linked immunosorbent assay experiments.. Hierarchical clustering of gene expression profiles revealed 2 groups of subjects. Group 1 consisted of 11 patients (8 with and 3 without lung inflammation). Group 2 consisted of 15 subjects (7 healthy controls and 2 patients with and 6 without lung inflammation). Gene expression in group 1 indicated T cell activation, a type 2 phenotype, production of profibrotic factors and matrix metalloproteinases, and reduced activation-induced cell death. Increased expression of beta6 integrin messenger RNA by CD8+ T cells in group 1 suggested the possibility that these T cells might induce cell-contact-dependent activation of latent transforming growth factor beta (TGFbeta).. A subset of scleroderma patients at higher risk of progressive lung disease have activated, long-lived CD8+ T cells in their lungs that could promote fibrosis directly, through production of profibrotic factors such as interleukin-4 and oncostatin M, as well as indirectly, through activation of TGFbeta.

Topics: Adult; Bronchoalveolar Lavage Fluid; CD8-Positive T-Lymphocytes; Cell Adhesion Molecules; Cell Survival; Cluster Analysis; Ephrins; Extracellular Matrix; Female; Gene Expression; Humans; Integrins; Lung; Lymphocyte Activation; Male; Middle Aged; Oligonucleotide Array Sequence Analysis; Phenotype; Pulmonary Fibrosis; Scleroderma, Systemic; Transforming Growth Factor beta

Transforming growth factor-beta stimulates the production of the extracellular matrix, whereas TNF-alpha has antifibrotic activity. Understanding the molecular mechanism underlying the antagonistic activities of TNF-alpha against TGF-beta is critical in the context of tissue repair and maintenance of tissue homeostasis. In the present study, we demonstrated a novel mechanism by which TNF-alpha blocks TGF-beta-induced gene and signaling pathways in human dermal fibroblasts. We showed that TNF-alpha prevents TGF-beta-induced gene trans activation, such as alpha2(I) collagen or tissue inhibitor of metalloproteinases 1, and TGF-beta signaling pathways, such as Smad3, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinases, without inducing levels of inhibitory Smad7 in human dermal fibroblasts. TNF-alpha down-regulates the expression of type II TGF-beta receptor (TbetaRII) proteins, but not type I TGF-beta receptor (TbetaRI), in human dermal fibroblasts. However, neither TbetaRII mRNA nor TbetaRII promoter activity was decreased by TNF-alpha. TNF-alpha-mediated decrease of TbetaRII protein expression was not inhibited by the treatment of fibroblasts with either a selective inhibitor of I-kappaB-alpha phosphorylation, BAY 11-7082, or a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, PD98059. Calpain inhibitor I (ALLN), a protease inhibitor, inhibits TNF-alpha-mediated down-regulation of TbetaRII. We found that TNF-alpha triggered down-regulation of TbetaRII, leading to desensitization of human dermal fibroblasts toward TGF-beta. Furthermore, these events seemed to cause a dramatic down-regulation of alpha2(I) collagen and tissue inhibitor of metalloproteinases 1 in systemic sclerosis fibroblasts. These results indicated that TNF-alpha impaired the response of the cells to TGF-beta by regulating the turnover of TbetaRII.

Topics: Cells, Cultured; DNA-Binding Proteins; Down-Regulation; Extracellular Matrix; Fibroblasts; Gene Expression Regulation; Humans; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Skin Physiological Phenomena; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Murine sclerodermatous graft-versus-host disease (Scl GVHD), produced by transplanting B10.D2 bone marrow and spleen cells to lethally irradiated BALB/cJ mice, is a model for human scleroderma. Mice with Scl GVHD have skin thickening, lung fibrosis, cutaneous mononuclear cell infiltration, and upregulation of cutaneous transforming growth factor beta1 (TGF-beta1) and type I collagen mRNAs by day 21 after bone marrow transplantation. Elevated TGF-beta1 appears to be the critical cytokine driving fibrosis in Scl GVHD, which can be prevented with antibodies to TGF-beta administered early in disease. Here we demonstrate that we can also prevent skin thickening in mice with Scl GVHD with a naturally occurring antagonist to TGF-beta1, human latency-associated peptide (LAP). By quantitative real-time PCR analysis and immunostaining, LAP treatment also abrogates the upregulation of cutaneous TGF-beta1 and connective tissue growth factor mRNAs and type I collagen synthesis in Scl GVHD. In contrast to anti-TGF-beta antibodies, LAP at 4 ng total per mouse has no significant suppressive effect on cutaneous influx of T cells and monocytes or immune cell activation. LAP may be a potential new therapy in scleroderma and other TGF-beta-driven fibrosing disease that targets TGF-beta more specifically, without affecting systemic critical roles of TGF-beta on immune cell function.

Topics: Animals; Biomarkers; Collagen Type I; Connective Tissue Growth Factor; Disease Models, Animal; Female; Fibrosis; Gene Expression; Graft vs Host Disease; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; Mice, Inbred BALB C; Peptide Fragments; Protein Precursors; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation

Topics: Animals; Collagen; Fibrosis; Immunoglobulins, Intravenous; Interferon-gamma; Interleukin-4; Mice; Mice, Mutant Strains; Scleroderma, Systemic; Skin; Spleen; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Humans; Polymorphism, Genetic; Risk Factors; Scleroderma, Systemic; Transforming Growth Factor beta

To determine the distribution of transforming growth factor beta1 (TGFbeta1) genotypes at codon 10 (+869 polymorphism) and codon 25 (+915 polymorphism) in patients with scleroderma (SSc). Differences between diffuse and limited SSc (dSSc and lSSc) were also investigated.. Patients with lSSc (n=89) and dSSc (n=63) were compared with 147 controls. DNA was isolated from peripheral blood and polymorphisms at codons 10 (C/T) and 25 (G/C) of the TGFbeta1 gene analysed by polymerase chain reaction and sequence specific oligonucleotide probing.. Significantly more patients with SSc than controls carried allele C at codon 10 (controls v SSc, 38% v 48%, chi(2)=8.2, 1df, p=0.004), OR=1.95 (95% CI 1.16 to 3.27). The difference remained when patients with SSc were split into those with limited or diffuse disease, (controls v dSSc, chi(2)=5, 1df, p=0.02 and controls v lSSc, chi(2)=6, 1df, p=0.013). The patients with SSc had significantly more subjects heterozygous at codon 10 (controls v SSc, chi(2)=45, 1df, p<0.0001). Possession of allele C at codon 10 gave an OR=4.8 (95% CI 2.8 to 8.4). No difference in allele frequency was seen between patients with SSc and controls at codon 25. More patients with SSc than controls carried the GG genotype (controls v SSc, 80% v 88%, chi(2)=7, 2df, p=0.027). Possession of allele G gave an OR=1.7 (95% CI 0.5 to 5.9). There was no difference between diffuse and limited disease at either codon.. These results suggest that patients with SSc are genetically predisposed to high TGFbeta1 production. These polymorphisms do not, however, explain the difference in the clinical phenotypes of limited and diffuse SSc.

Topics: Codon; Gene Frequency; Genotype; Humans; Oligonucleotide Probes; Polymerase Chain Reaction; Polymorphism, Genetic; Scleroderma, Systemic; Transforming Growth Factor beta

As an initial approach to understanding the basis of the systemic sclerosis (SSc; scleroderma) phenotype, we sought to identify genes in the transforming growth factor beta (TGF beta) signaling pathway that are up-regulated in lesional SSc fibroblasts relative to their normal counterparts.. We used gene chip, differential display, fluorescence-activated cell sorter, and overexpression analyses to assess the potential role of TGF beta signaling components in fibrosis. Fibroblasts were obtained by punch biopsy from patients with diffuse cutaneous SSc of 2-14 months' duration (mean 8 months) and from age- and sex-matched healthy control subjects.. Unexpectedly, we found that fibroblasts from SSc patients showed elevated expression of the endothelial cell-enriched TGF beta receptor endoglin. Endoglin is a member of the nonsignaling high-affinity TGF beta receptor type III family. The expression of endoglin increased with progression of disease. Transfection of endoglin in fibroblasts suppressed the TGF beta-mediated induction of connective tissue growth factor promoter activity.. SSc is characterized by overproduction of matrix; that is, genes that are targets of TGF beta signaling in normal fibroblasts. Our findings suggest that lesional SSc fibroblasts may overexpress endoglin as a negative feedback mechanism in an attempt to block further induction of profibrotic genes by TGF beta.

Topics: Antigens, CD; Endoglin; Female; Fibroblasts; Humans; In Vitro Techniques; Receptors, Cell Surface; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta; Up-Regulation; Vascular Cell Adhesion Molecule-1

Smad7 is a major inhibitory regulator of transforming growth factor (TGF)-beta signaling. Smad7 expression is induced by TGF-beta itself and other signaling pathways, indicating a key role for Smad7 in feedback or cross-talk control of TGF-beta signaling. Recent reports have implicated Smad7 as a crucial regulator of TGF-beta activity in human disease; aberrant expression of Smad7 is involved in inflammatory bowel disease and scleroderma. Thus, modulation of Smad7 expression could provide a novel therapeutic basis for TGF-beta-associated disorders.

Topics: Animals; DNA-Binding Proteins; Gene Expression Regulation; Humans; Inflammatory Bowel Diseases; Models, Biological; Scleroderma, Systemic; Signal Transduction; Smad7 Protein; Trans-Activators; Transforming Growth Factor beta

Transforming growth factor (TGF)-beta has been shown to be a potent stimulator of collagen production by fibroblasts, and could play a role in the pathogenesis of systemic sclerosis (SSc).. To study the possible involvement of TGF-beta1 gene polymorphism in Japanese patients with SSc.. Fifty-nine patients with SSc and 110 normal subjects were studied. Genomic DNA was extracted from skin tissues, and was amplified in a thermal cycler, generating a TGF-beta1 gene fragment with a size of 294 bp. The T to C transition at T869C (Leu10Pro) and the G to C transition at G915C (Arg25Pro) were identified by digestion with MspA1I and BglI, respectively.. At T869C (Leu10Pro), the frequency of the C allele in SSc (65.3%) was significantly higher than in normal controls (50.5%) (P < 0.01). SSc showed C/C allele 42.4%, C/T 45.8% and T/T 11.2%. Normal controls showed C/C allele 26.4%, C/T 48.2% and T/T 25.5%. The frequency of the C/C allele in SSc was significantly higher than in normal controls, in comparison with the T/T allele (P < 0.02), but no significant difference was found between the frequency of the C/C allele vs. the C/T allele. The frequency of the C/C allele showed no significant difference between diffuse and limited SSc. At G915C (Arg25Pro), all the normal controls and SSc patients showed only the G/G allele. These results are different from a previous study in which the frequency of the T/T allele was high in SSc at T869C (Leu10Pro).. This discrepancy may indicate that Japanese patients with SSc show a different genetic predisposition to TGF-beta1.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Alleles; Amino Acid Substitution; Female; Genetic Predisposition to Disease; Humans; Male; Middle Aged; Polymorphism, Genetic; Scleroderma, Systemic; Transforming Growth Factor beta; Transforming Growth Factor beta1

Systemic sclerosis (SSc) is a connective tissue disorder that is characterized by excessive collagen synthesis by fibroblasts and by vascular hyperreactivity and obliteration phenomena. Excessive collagen production is the consequence of abnormal interactions between endothelial cells, fibroblasts and mononuclear cells. Immunological abnormalities are present very early in the development of SSc. Mononuclear cells, particularily macrophages and T lymphocytes play a prominent role in fibroblast activation and collagen synthesis through the cytokines they produce. Thus, lymphocytic infiltrates in the skin and in the lung are preferentially composed of CD8+ T lymphocytes, that produce important amounts of interleukin 4 (IL-4). The effects of IL-4 are added to these of transforming growth factor B (TGF-B) and connective tissue growth factor (CTGF) that stimulate collagen synthesis by fibroblasts. T lymphocytes produce important amounts of gamma interferon (INF-gamma) that is the best inhibitor of collagen synthesis by fibroblasts. However, the inhibitory effect of INF-gamma on collagen synthesis is diminished in SSc patients. Numerous autoantibodies can be evidenced in the serum of SSc patients. Three of them are specific for SSc and mutually exclusive: anti-centromere antibodies (Ab) in limited SSc, anti-Scl70 Ab in diffuse SSc and anti-RNA polymerase III Ab in diffuse SSc with renal involvement. These autoantibodies are good prognosis markers but their pathogenic role remains uncertain.

Topics: Autoantibodies; Collagen; Cytokines; Fibroblasts; Growth Substances; Humans; Immunity, Cellular; Interleukin-4; Scleroderma, Systemic; T-Lymphocytes; Transforming Growth Factor beta

The naturally occurring compound halofuginone has been shown to antagonize collagen synthesis by fibroblasts both in vitro and in vivo. We previously demonstrated that this inhibitory property was related to the ability of halofuginone to disrupt transforming growth factor beta signal transduction. The present study further analyzed the ability of halofuginone to affect transcription factors that can regulate type I collagen gene expression by examining its effect on c-Jun, the negative regulator of collagen gene transcription.. The phosphorylation state of c-Jun in the presence of halofuginone was examined via direct Western blotting, and the transcriptional activity of the activator protein 1 (AP-1) binding element via electrophoretic mobility shift assay and luciferase reporter assay. We determined whether the effect of halofuginone on collagen synthesis was dependent on the presence of c-Jun by ectopic expression of a wild-type or dominant-negative c-Jun construct in the presence of halofuginone and assaying alpha2(I) collagen promoter strength via luciferase reporter assay. The effect of halofuginone on alpha2(I) collagen message levels in fibroblasts when wild-type or dominant-negative c-Jun was overexpressed was determined. We also determined whether halofuginone had an effect on the phosphorylation state of c-Jun in the skin of TSK/+ mice via immunohistochemistry.. Treatment of fibroblasts with 10(-8)M halofuginone enhanced basal and mitogen-mediated phosphorylation of c-Jun in culture. This elevated phosphorylation of c-Jun correlated with enhanced DNA binding and transcriptional activation of an AP-1 complex consisting of c-Jun and Fos but lacking the c-Jun antagonist JunB. Overexpression of c-Jun enhanced in a dose-dependent manner the ability of halofuginone to inhibit the activity of a luciferase reporter construct under control of the -3200-bp to +54-bp COL1A2 promoter, whereas the expression of a dominant-negative c-Jun construct abolished this effect. Northern blotting showed that overexpression of c-Jun enhanced the ability of halofuginone to reduce collagen alpha2(I) messenger RNA levels in fibroblasts, whereas expression of the dominant-negative c-Jun abolished this effect. Topical administration of a halofuginone-containing cream for 20 days to TSK mice, which spontaneously develop dermal fibrosis, greatly increased the phosphorylated form of c-Jun in the skin; this was followed by a decrease in skin thickness and type I collagen messenger RNA expression.. Our findings illustrate the powerful down-regulatory property of c-Jun toward type I collagen and establish that halofuginone exerts its effect on collagen synthesis in a c-Jun-dependent manner.

Topics: Administration, Topical; Animals; Cells, Cultured; Collagen; Collagen Type I; Drug Synergism; Fibroblasts; Gene Expression; Male; Mice; Mice, Mutant Strains; Mitogens; Phosphorylation; Piperidines; Promoter Regions, Genetic; Protein Synthesis Inhibitors; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-jun; Quinazolines; Quinazolinones; RNA, Messenger; Scleroderma, Systemic; Transcription Factor AP-1; Transcriptional Activation; Transforming Growth Factor beta; Transforming Growth Factor beta1

Fibrillin, an extracellular matrix protein implicated in dermal fibrosis, is increased in the reticular dermis of systemic sclerosis (SSc) skin. We undertook this study to investigate the hypothesis that transforming growth factor beta (TGFbeta) or other cytokines regulate fibrillin matrix formation by normal and SSc fibroblasts. We further investigated the mechanism of TGFbeta-induced fibrillin fibrillogenesis and its relationship to myofibroblasts.. Fibrillin and fibronectin matrix deposition and alpha-smooth muscle actin expression by fibroblast cultures from normal and SSc skin treated with TGFbeta or other cytokines were analyzed by immunofluorescence. Supernatant and extracellular matrix from normal and SSc fibroblasts treated with or without TGFbeta were evaluated by Western blot and Northern blot for fibrillin protein and messenger RNA (mRNA) expression, respectively.. Immunofluorescence demonstrated increased fibrillin matrix formation by normal and scleroderma fibroblasts after TGFbeta treatment. Other cytokines, including tumor necrosis factor alpha, interleukin-1beta (IL-1beta), IL-4, granulocyte-macrophage colony-stimulating factor, and platelet-derived growth factor, did not affect fibrillin fibrillogenesis. Fibrillin matrix formed in proximity to myofibroblasts and independently of up-regulation of fibronectin matrix or cell number. Western blot analysis of extracellular matrix confirmed increased fibrillin after TGFbeta stimulation of normal or scleroderma fibroblasts. However, TGFbeta did not alter the expression of either soluble fibrillin protein or fibrillin mRNA.. Our data show that TGFbeta induces fibrillin protein incorporation into the extracellular matrix without affecting fibrillin gene expression or protein synthesis, suggesting that fibrillin matrix assembly is regulated extracellularly. TGFbeta might increase fibrillin matrix by activating myofibroblasts. Such TGFbeta-mediated effects could account for the increased fibrillin matrix observed in SSc skin.

Topics: Cells, Cultured; Cytokines; Fibrillin-1; Fibrillins; Fibroblasts; Fluorescent Antibody Technique; Humans; Microfilament Proteins; RNA, Messenger; Scleroderma, Systemic; 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

Murine sclerodermatous graft-vs-host disease (Scl GVHD) models human scleroderma, with prominent skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA. Fibrosis in Scl GVHD may be driven by infiltrating TGF-beta1-producing mononuclear cells. Here we characterize the origin and types of those cutaneous effector cells, the cytokine and chemokine environments, and the effects of anti-TGF-beta Ab on skin fibrosis, immune cell activation markers, and collagen and cytokine synthesis. Donor cells infiltrating skin in Scl GVHD increase significantly at early time points post-transplantation and are detectable by PCR analysis of Y-chromosome sequences when female mice are transplanted with male cells. Cutaneous monocyte/macrophages and T cells are the most numerous cells in Scl GVHD compared with syngeneic controls. These immune cells up-regulate activation markers (MHC class II I-A(d) molecules and class A scavenger receptors), suggesting Ag presentation by cutaneous macrophages in early fibrosing disease. Early elevated cutaneous mRNA expression of TGF-beta1, but not TGF-beta2 or TGF-beta3, and elevated C-C chemokines macrophage chemoattractant protein-1, macrophage inflammatory protein-1alpha, and RANTES precede subsequent skin and lung fibrosis. Therefore, TGF-beta1-producing donor mononuclear cells may be critical effector cells, and C-C chemokines may play important roles in the initiation of Scl GVHD. Abs to TGF-beta prevent Scl GVHD by effectively blocking the influx of monocyte/macrophages and T cells into skin and by abrogating up-regulation of TGF-beta1, thereby preventing new collagen synthesis. The Scl GVHD model is valuable for testing new interventions in early fibrosing diseases, and chemokines may be new potential targets in scleroderma.

Topics: Animals; Bone Marrow Transplantation; Cell Migration Inhibition; Cell Movement; Chemokine CCL2; Chemokine CCL4; Chemokine CCL5; Chemokines; Collagen Type I; Cytokines; Disease Models, Animal; Female; Graft vs Host Disease; Histocompatibility Antigens Class II; Humans; Immune Sera; Leukocyte Common Antigens; Lymphocyte Activation; Macrophage Activation; Macrophage Inflammatory Proteins; Macrophage-1 Antigen; Macrophages; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Monocytes; Receptors, Immunologic; Receptors, Lipoprotein; Receptors, Scavenger; RNA, Messenger; Scavenger Receptors, Class A; Scavenger Receptors, Class B; Scleroderma, Systemic; Skin; Spleen; T-Lymphocytes; Transforming Growth Factor beta; Transforming Growth Factor beta1; Transforming Growth Factor beta2; Transforming Growth Factor beta3; Up-Regulation

Scleroderma is a chronic systemic disease that leads to fibrosis of affected organs. Transforming growth factor (TGF) beta has been implicated in the pathogenesis of scleroderma. Smad proteins are signaling transducers downstream from TGF-beta receptors. Three families of Smads have been identified: (i) receptor-regulated Smad2 and -3 (R-Smads); (ii) common partner Smad4 (Co-Smad); and (iii) inhibitory Smad6 and -7 (I-Smads, part of a negative feedback loop). We have investigated the signaling components for the TGF-beta pathway and TGF-beta activity in scleroderma lesions in vivo and in scleroderma fibroblasts in vitro. Basal level and TGF-beta-inducible expression of Smad7 are selectively decreased, whereas Smad3 expression is increased both in scleroderma skin and in explanted scleroderma fibroblasts in culture. TGF-beta signaling events, including phosphorylation of Smad2 and -3, and transcription of the PAI-1 gene are increased in scleroderma fibroblasts, relative to normal fibroblasts. In vitro adenoviral gene transfer with Smad7 restores normal TGF-beta signaling in scleroderma fibroblasts. These results suggest that alterations in the Smad pathway, including marked Smad7 deficiency and Smad3 up-regulation, may be responsible for TGF-beta hyperresponsiveness observed in scleroderma.

Topics: Adult; Animals; Blotting, Western; DNA-Binding Proteins; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Female; Fibroblasts; Humans; Immunohistochemistry; Male; Mice; Mice, SCID; Middle Aged; Phosphorylation; Plasminogen Activator Inhibitor 1; RNA, Messenger; Scleroderma, Systemic; Signal Transduction; Skin Transplantation; Smad3 Protein; Smad7 Protein; Trans-Activators; Transcription, Genetic; Transforming Growth Factor beta; Up-Regulation

Transforming growth factor beta has been implicated as a mediator of excessive extracellular matrix deposition in scar tissue and fibrosis, including systemic sclerosis. To further characterize the mechanism of collagen gene expression in systemic sclerosis and healthy skin fibroblasts, we examined the role of p38 MAPK signaling in collagen gene regulation by transforming growth factor beta. Treatment of dermal fibroblasts with transforming growth factor beta resulted in a prolonged activation of p38 MAPK. Furthermore, a specific inhibitor of p38 suppressed transforming growth factor beta stimulation of collagen type I mRNA and the alpha2(I) collagen promoter activity. To further probe the role of p38 in collagen regulation by transforming growth factor beta, we utilized an expression vector containing p38alpha cDNA. Ectopic expression of p38alpha enhanced COL1A2 promoter activity and potentiated transforming growth factor beta stimulation of this promoter. The p38 response element in the COL1A2 promoter overlapped with the previously characterized transforming growth factor beta response element. Consistent with these observations, collagen type I mRNA and protein levels were increased in transforming-growth-factor-beta-stimulated fibroblasts transduced with an adenoviral vector expressing p38alpha. To determine the possible role of p38 in abnormal collagen production by systemic sclerosis fibroblasts, p38 protein levels were compared in systemic sclerosis and healthy skin fibroblasts. Both cell types exhibited similar total levels of p38 MAPK and similar kinetics of p38 activation in response to transforming growth factor beta. In conclusion, this study demonstrates a costimulatory role for p38 MAPK in transforming growth factor beta induction of the collagen type I gene. Expression levels and activation status of p38 are not consistently elevated in systemic sclerosis fibroblasts suggesting that the p38 MAPK pathway is not dysregulated in systemic sclerosis fibroblasts.

Topics: Adult; Cells, Cultured; Collagen; Collagen Type I; Dermis; Enzyme Inhibitors; Extracellular Matrix; Female; Fibroblasts; Gene Expression; Humans; Imidazoles; Male; Middle Aged; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Pyridines; RNA, Messenger; Scleroderma, Systemic; Transforming Growth Factor beta

In normal adult fibroblasts, transforming growth factor-beta (TGFbeta) induces the expression of connective tissue growth factor (CTGF). CTGF independently promotes fibroblast proliferation and matrix deposition, and in acute models of fibrosis promotes cell proliferation and collagen deposition acting synergistically with TGFbeta. In contrast to normal fibroblasts, fibroblasts cultured from fibrotic tissues express high basal levels of CTGF, even in the absence of added TGFbeta. Induction of transcription by TGFbeta requires the action of SMAD proteins. In this report we have investigated the role of SMADs in the TGFbeta-induction of CTGF in normal fibroblasts and in the elevated levels of CTGF expression found in dermal fibroblasts cultured from lesional areas of patients with scleroderma, a progressive fibrotic disorder that can affect all organs of the body. We have identified a functional SMAD binding site in the CTGF promoter. TGFbeta-induction of CTGF is dependent on SMAD3 and SMAD4 but not SMAD2 and is p300-independent. However, mutation of the SMAD binding site does not reduce the high level of CTGF promoter activity observed in dermal fibroblasts cultured from lesional areas of scleroderma patients. Conversely, the previously termed TGFbetaRE in the CTGF promoter is required for basal CTGF promoter activity in normal fibroblasts and for the elevated level of CTGF promoter activity in scleroderma fibroblasts. Thus, the maintenance of the fibrotic phenotype in scleroderma fibroblasts, as visualized by excess CTGF expression, appears to be independent of SMAD-dependent TGFbeta signaling. Furthermore, given CTGF's activities, the high level of CTGF expression observed in scleroderma lesions may contribute to the excessive scarring observed in this disorder.

Topics: 3T3 Cells; Animals; Cell Differentiation; Connective Tissue Growth Factor; DNA-Binding Proteins; Fibroblasts; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mice; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Trans-Activators; Transforming Growth Factor beta

Scleroderma is a disorder characterized by fibrosis of the skin and internal organs and autoimmunity. Whereas the cause is unknown, interleukin-4 and transforming growth factor-beta have been postulated to play a major part in the fibrosis. To investigate the part played by these cytokines, we prepared TSK/+ mice with a targeted mutation in the interleukin-4R alpha or transforming growth factor-beta genes. The breeding failed to produce TSK/+ transforming growth factor-beta -/- mice so analysis of the role of transforming growth factor-beta was limited to TSK/+ transforming growth factor-beta +/- mice. We observed that TSK/+ interleukin-4R alpha -/- did not develop dermal thickening, and deletion of one allele of the transforming growth factor-beta gene resulted in diminished dermal thickness compared with TSK/+ mice; however, the deletion of interleukin-4R alpha or transforming growth factor-beta had no effect on lung emphysema, which is another characteristic of TSK syndrome. Electron microscopic analysis of skin showed that the collagen fibrils in TSK/+ interleukin-4R alpha -/- mice exhibit normal periodicity but have a smaller diameter than the fibers found in C57BL/6 mice. Analysis of skin and serum samples showed that the deletion of interleukin-4R alpha or one allele of transforming growth factor-beta prevented the increase of skin thickness paralleled with a decrease in the dermal hydroxyproline content and development of autoantibodies associated with TSK syndrome. These results demonstrate the importance of interleukin-4 and transforming growth factor-beta for the development of cutaneous fibrosis in vivo and suggest an important part for these cytokines in wound healing and connective tissue maintenance in general.

Topics: Animals; Collagen; Crosses, Genetic; Fibroblasts; Fibrosis; Interleukin-4; Lung; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Electron; Receptors, Interleukin-4; Scleroderma, Systemic; Skin; Skin Abnormalities; Transforming Growth Factor beta; Transforming Growth Factors

To elucidate the role of transforming growth factor beta (TGFbeta) in the increased expression of the collagen gene in scleroderma fibroblasts.. Dermal fibroblasts from 10 patients with diffuse systemic sclerosis (SSc) of recent onset and from 10 healthy individuals were studied. The production of active and total (active + latent) TGFbeta1 levels from cultured dermal fibroblasts was measured using a TGFbeta1 enzyme-linked immunosorbent assay system. Expression of the TGFbeta type I and type II receptor proteins in dermal fibroblasts was determined by immunoblotting, and the level of expression of human alpha2(I) collagen messenger RNA (mRNA) was evaluated by Northern blot analysis. The transcriptional activity of the human alpha2(I) collagen gene was examined with chloramphenicol acetyltransferase (CAT) assays using the -772 COL1A2/CAT construct.. SSc fibroblasts expressed increased levels of TGFbeta type I and type II receptors but secreted amounts of TGFbeta similar to those secreted by normal fibroblasts. The blockade of TGFbeta signaling with anti-TGFbeta antibodies or a TGFbeta1 antisense oligonucleotide abolished the increased mRNA expression, as well as the up-regulated transcriptional activity of the human alpha2(I) collagen gene in SSc fibroblasts.. These results suggest that TGFbeta plays a crucial role in the pathogenesis of SSc and raise the possibility of a therapeutic approach with anti-TGFbeta antibodies or a TGF11 antisense oligonucleotide.

Topics: Antibodies; Collagen; Fibroblasts; Gene Expression; Humans; Oligonucleotides, Antisense; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Topics: Animals; Disease Models, Animal; Endomyocardial Fibrosis; Endothelium, Vascular; Fibroblasts; Gene Targeting; Humans; Oxidative Stress; Pulmonary Fibrosis; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

Although the role of transforming growth factor beta (TGFbeta) in initiating fibrosis is well established, the role that TGFbeta plays in maintaining fibrosis is unclear. The gene encoding connective tissue growth factor (ccn2; ctgf), which promotes fibrosis, is not normally expressed in dermal fibroblasts unless TGFbeta is present. However, in dermal fibroblasts cultured from lesional areas of scleroderma, ccn2 (ctgf) is expressed constitutively. The contribution of several elements in the ccn2 (ctgf) promoter to basal and TGFbeta induced ccn2 (ctgf) expression in normal and scleroderma fibroblasts has been investigated. A functional SMAD binding site in the ccn2 (ctgf) promoter that is necessary for the TGFbeta mediated induction of this gene has been identified. The previously termed TGFbeta responsive enhancer (TGFbetaRE) in the ccn2 (ctgf) promoter has been found to be necessary for basal promoter activity in normal fibroblasts. The SMAD element is not necessary for the high ccn2 (ctgf) promoter activity seen in scleroderma fibroblasts. However, mutation of the previously termed TGFbetaRE reduces ccn2 (ctgf) promoter activity in scleroderma fibroblasts to that seen in normal fibroblasts. Thus, the maintenance of the scleroderma phenotype, as assessed by a high degree of ccn2 (ctgf) promoter activity, appears to be relatively independent of SMAD action and seems to reflect increased basal promoter activity.

Topics: Case-Control Studies; Connective Tissue Growth Factor; DNA-Binding Proteins; Fibroblasts; Gene Expression; Growth Substances; Humans; Immediate-Early Proteins; Intercellular Signaling Peptides and Proteins; Mutation; Phenotype; Promoter Regions, Genetic; Scleroderma, Systemic; Signal Transduction; Transforming Growth Factor beta

To evaluate the relative roles of endothelium and platelets in the pathogenesis of primary RP and RP secondary to SSc.. Endothelial derived ET-1, t-PA, PAI-1, and platelet derived beta-TG, PDGF, TGF-beta were measured in 36 patients with primary RP, 14 patients with RP secondary to SSc and 30 age and sex matched controls.. A significative increase of ET-1, t-PA, PAI-1, TGF-beta, and beta-TG were the most relevant changes in patients with RP secondary to SSc with respect to the controls. Less relevant increases of t-PA, PAI-1, PDGF, and beta-TG levels were observed in patients with primary RP vs controls.. These data seem to confirm the involvement of endothelial cells and platelets in the pathogenesis of RP, with mild changes in primary RP and more relevant changes in RP secondary to SSc.

Topics: Adult; beta-Thromboglobulin; Biomarkers; Endothelin-1; Endothelium; Female; Humans; Male; Middle Aged; Plasminogen Activator Inhibitor 1; Platelet Activation; Platelet-Derived Growth Factor; Raynaud Disease; Scleroderma, Systemic; Tissue Plasminogen Activator; Transforming Growth Factor beta

To determine whether there was altered elaboration of non-collagenous extracellular matrix proteins or expression of TGFbeta isoforms and their receptors in salivary glands of patients with systemic sclerosis (SSc) and Raynaud's phenomenon (RP). Because of the possible role of mast cells in the early stages of SSc their presence was also investigated.. Sections of normal labial salivary glands (n=10) and glands from patients with SSc (n = 13) and RP (n = 5) were stained immunohistochemically and using acid toluidine blue.. SSc glands contained more mast cells than control tissues (p<0.005) and similar numbers to those found in RP specimens. There were no differences in the pattern or amount of non-collagenous matrix proteins detected. Tenascin and elastin were predominantly found surrounding ducts whereas fibronectin had a more general distribution. TGFbeta isoforms and receptors were expressed by glandular epithelium, fibroblasts, vascular endothelium and inflammatory cells. Cell counts showed no differences in expression of TGFbeta1 or TGFbeta receptors between groups. However, the percentage of TGFbeta2 positive fibroblasts was significantly higher in SSc glands compared with controls (p<0.004). RP glands showed an intermediate level of expression. By contrast, a lower percentage of RP fibrolasts expressed TGFbeta3 compared with controls with SSc glands showing an intermediate level of expression.. These results show that (a) there are no changes in glandular expression of tenascin, elastin and fibronectin in SSc and RP and (b) both conditions are associated with an increased salivary gland mast cell population and changes in expression of TGFbeta2 and beta3 isoforms by glandular fibroblasts.

Topics: Adult; Aged; Aged, 80 and over; Dendritic Cells; Extracellular Matrix Proteins; Female; Humans; Immunoenzyme Techniques; Male; Mast Cells; Middle Aged; Protein Isoforms; Raynaud Disease; Receptors, Transforming Growth Factor beta; Salivary Glands; Scleroderma, Systemic; Transforming Growth Factor beta; Vimentin

Connective tissue growth factor (CTGF) is overexpressed in a variety of fibrotic disorders, presumably secondary to the activation and production of transforming growth factor-beta (TGF-beta), a key inducer of fibroblast proliferation and matrix synthesis. The CTGF gene promoter has a TGF-beta response element that regulates its expression in fibroblasts but not epithelial cells or lymphocytes. Recent studies have shown that the macrophage-produced cytokine tumor necrosis factor alpha (TNFalpha) is necessary to promote inflammation and to induce genes, such as matrix metalloproteinases, involved with the early stages of wound healing. In this study, we examined the ability of TNFalpha to modulate CTGF gene expression. TNFalpha was found to suppress the TGF-beta-induced expression of CTGF protein in cultured normal fibroblasts. The activity of TNFalpha was blocked by NF-kappaB inhibitors. We showed that sequences between -244 and -166 of the CTGF promoter were necessary for both TGF-beta and TNFalpha to modulate CTGF expression. There was a constitutive expression of CTGF by scleroderma fibroblasts that was increased by TGF-beta treatment. Although TNFalpha was able to repress TGF-beta-induced CTGF and collagen synthesis both in normal and scleroderma skin fibroblasts, fibroblasts cultured from scleroderma patients were more resistant to TNFalpha as TNFalpha was unable to suppress the basal level of CTGF expression in scleroderma fibroblasts. Thus, we suspect that the high level of constitutive CTGF expression in scleroderma fibroblasts and its inability to respond to negative regulatory cytokines may contribute to the excessive scarring of skin and internal organs in patients with scleroderma.

Topics: Cell Line; Cells, Cultured; Collagen; Connective Tissue Growth Factor; Fibroblasts; Growth Substances; Humans; Immediate-Early Proteins; Infant, Newborn; Intercellular Signaling Peptides and Proteins; Luciferases; Male; Mitogens; NF-kappa B; Promoter Regions, Genetic; Reference Values; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Abnormalities of transforming growth factor beta (TGFbeta) and platelet-derived growth factor (PDGF) alpha and beta and/or their receptors have been demonstrated in systemic sclerosis (SSc). This study aimed to determine whether genetic polymorphisms in or near the TGFbeta and PDGF gene families were associated with susceptibility to SSc in a Native American population with a high disease prevalence.. Genotyping of 5 intragenic polymorphisms within the TGFbeta1 gene and mapping of 35 microsatellites near the genes for TGFbeta1, latent TGFbeta1 binding protein (LTBP1), TGFbeta receptors I and II, PDGFalpha, PDGFbeta, PDGF receptor alpha, and PDGF receptor beta was performed in 19 SSc patients, 76 controls, and 42 family members. Allele distributions and frequencies were examined between SSc patients and controls, and marker haplotypes were examined in families when allele frequencies appeared to be different between patients and controls.. Although 1 polymorphism within the TGFbeta1 gene (TGFbeta1) was modestly increased in the SSc patients, this did not maintain statistical significance after correction. Similarly, 1 microsatellite (D9S120) near the TGFbeta receptor I gene (TGFBR1) showed a significant disturbance of allele frequencies between patients and controls; however, it did not form a disease-associated haplotype with other nearby markers. Weak disturbances of markers near PDGFalpha (PDGFA) and PDGFbeta, (PDGFB) also failed to maintain significance after correction. Both PDGF receptor genes (PDGFRA and PDGFRB) also showed no disease associations.. The results of these preliminary analyses suggest that genetic anomalies of the TGFbeta1 and PDGF gene families are not likely to explain the dysregulation seen in SSc or to account for the susceptibility to SSc in this population.

Topics: Alleles; Genetic Predisposition to Disease; Homozygote; Humans; Indians, North American; Microsatellite Repeats; Platelet-Derived Growth Factor; Polymorphism, Genetic; Receptors, Platelet-Derived Growth Factor; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Transforming Growth Factor beta

We have used representational difference analysis (RDA) to identify up-regulated genes in skin fibroblasts from fibrotic lesions obtained from patients with systemic sclerosis (scleroderma). RDA of cDNA libraries derived from fibroblasts from involved and uninvolved skin detected several differentially expressed genes. One such gene consistently up-regulated in scleroderma cells coded for human connective tissue growth factor (CTGF). Other studies described here show that the CTGF protein is readily detected in cultures of systemic sclerosis fibroblasts but was not detected in comparable normal cells. High levels of CTGF are also evident in biological fluids from patients with systemic sclerosis. TGFbeta stimulates CTGF production in both normal and systemic sclerosis fibroblasts with the latter found to be higher producers. Moreover, an analysis of constitutive and TGFbeta-induced CTGF gene activation showed altered and elevated transcriptional responses in systemic sclerosis cells compared with controls. CTGF stimulated a two- to threefold increase in proalpha1(I) collagen and fibronectin synthesis by both dermal and lung fibroblasts in culture and promoted significant matrix remodeling of fibroblast-populated three-dimensional collagen lattices. A direct relation between the overexpression of CTGF and elevated collagen synthesis was suggested by the observation that transfection of a CMV-CTGF cDNA construct and protein expression in fibroblasts increased the transcription of a Col 1alpha2 promoter-reporter construct to levels seen in systemic sclerosis fibroblasts. Using Col 1alpha2 promoter deletion constructs the CTGF responsive element was localized to the first 379 bp upstream of the transcriptional start site. These data indicate that there is an overexpression of CTGF in the systemic sclerosis cells, probably due to increased gene transcription, and suggest that the dysregulation of CTGF production is an important factor in fibroblast activation and the excessive deposition of collagen in systemic sclerosis.

Topics: 3T3 Cells; Animals; Autocrine Communication; Base Sequence; Bronchoalveolar Lavage Fluid; Connective Tissue Growth Factor; Extracellular Matrix Proteins; Fibrosis; Gene Expression Regulation; Gene Transfer Techniques; Growth Substances; Humans; Immediate-Early Proteins; In Vitro Techniques; Intercellular Signaling Peptides and Proteins; Mice; Molecular Biology; Molecular Sequence Data; Phenotype; Promoter Regions, Genetic; Respiratory Mucosa; RNA, Messenger; Scleroderma, Systemic; Transcriptional Activation; Transforming Growth Factor beta

We hypothesized that pathophysiologic events during the development of systemic sclerosis (SSc) may lead to selection and propagation of certain apoptosis-resistant fibroblast subpopulations. The aim of this study was to examine a possible role for apoptosis in fibroblast selection in SSc and the role of transforming growth factor beta1 (TGFbeta1).. We compared SSc and normal fibroblasts for their susceptibility to anti-Fas-induced apoptosis and analyzed 2 models that might lead to fibroblast resistance to apoptosis in this process: long-term exposure to either anti-Fas or TGFbeta1.. SSc-derived fibroblasts were resistant to anti-Fas-induced apoptosis, showing 5.5 +/- 17.2% (mean +/- SD) apoptosis, compared with 32.1 +/- 14.0% among normal fibroblasts (P < 0.05). Anti-Fas-selected normal fibroblasts showed 9.0 +/- 3.7% apoptosis, compared with 21.6 +/- 5.9% for sham-treated cells, which is consistent with the elimination of apoptosis-susceptible subpopulations. Normal fibroblasts subjected to 6 weeks of TGFbeta1 treatment showed not only resistance to apoptosis, but also proliferation (118.5 +/- 35.4%), after anti-Fas treatment, compared with sham-treated cells (35.1 +/- 11.1% apoptotic cell death). TGFbeta1 treatment also increased the proportion of myofibroblasts (47% versus 28% in controls). Cultured SSc fibroblasts had a greater proportion of myofibroblasts (32-83%) than did normal fibroblasts (4-25%). We also examined the relationship between collagen gene expression and the myofibroblast phenotype in normal and SSc skin sections. Only 2 of 7 normal sections had alpha-smooth muscle actin (a-SMA)-positive cells (mean +/- SD score 0.29 +/- 0.49 on a scale of 0-3), but all SSc sections were positive for alpha-SMA, with a mean score of 1.90 +/- 0.88 for lesional and 1.50 +/- 0.71 for nonlesional sections. Scores for alpha1(I) procollagen messenger RNA (mRNA) in lesional skin (mean +/- SD 3.30 +/- 0.82 on a scale of 1-4) were significantly higher than in normal (1.43 +/- 0.79) or nonlesional (1.40 +/- 0.52) skin, but scores varied, and there was no correlation between collagen mRNA and alpha-SMA levels.. Our results show that resistance to apoptosis is an important part of the SSc phenotype. TGFbeta1 may play a role by inducing apoptosis-resistant fibroblast populations, and also by inducing myofibroblasts and by enhancing extracellular matrix synthesis.

Topics: Actins; Adult; Aged; Antibodies, Monoclonal; Antibodies, Monoclonal, Murine-Derived; Apoptosis; Collagen; Female; Fibroblasts; Humans; Male; Middle Aged; Muscle, Smooth; Phenotype; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Transforming Growth Factor beta1

Topics: Calcinosis; CREST Syndrome; Female; Humans; Middle Aged; Scleroderma, Systemic; Skin Diseases; Transforming Growth Factor beta; Transforming Growth Factor beta3

To investigate whether six microsatellite markers known to map closely to genes involved in fibrosis are associated with systemic sclerosis (SSc).. Markers mapping to TGFbeta1, TGFbeta2, TGFbeta3, PDGFB, TIMP1 and COL5A2 were genotyped and allele frequency distributions compared in 191 patients and 196 controls. As TIMP1 maps to the X chromosome, male and females were analysed separately. Markers associated with SSc were further investigated according to whether patients had limited (lcSSc) or diffuse (dcSSc) cutaneous fibrosis.. Associations were found between SSc and markers for TGFbeta3 (chi(2)=17.3, df=8, P=0.02), TGFbeta2 (chi(2)=25.2, df=13, P=0.02) and TIMP1 (with male SSc, chi(2)=11.9, df=5, P=0.03), between lcSSc and the TGFss2 marker (chi(2)=25.6, df=13, P=0.02), and between dcSSc and TGFbeta3 marker (chi(2)=27.1, df=8, P=0.001). Between lcSSc and dcSSc patients, the allele frequency distribution differed only for the TGFbeta3 marker (chi(2)=16.5, df=6, P=0.01).. These associations indicate a possible role for TGFbeta3, TGFbeta2 and TIMP1 in genetic susceptibility to SSc and for TGFbeta3 in determining the degree of cutaneous fibrosis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Female; Fibrosis; Gene Expression Regulation; Humans; Male; Microsatellite Repeats; Middle Aged; Scleroderma, Systemic; Tissue Inhibitor of Metalloproteinase-1; Transforming Growth Factor beta

C-myb and B-myb belong to the myb family of transcription factors. We have shown previously that c-myb is deregulated in fibroblasts from systemic sclerosis (scleroderma) patients relative to normal fibroblasts. Scleroderma fibroblasts are known to express elevated levels of collagen genes and transforming growth factor beta is known to be a pro-fibrotic cytokine and to induce transcription of type I collagen genes. We have therefore investigated the role of c-myb and B-myb in the regulation of type I collagen genes in response to transforming growth factor beta in normal human fibroblasts. We show that, in these cells, transforming growth factor beta treatment induces c-myb as well as collagen alpha1(I) and alpha2(I) gene expression, but not B-myb. Furthermore we demonstrate by cotransfection assays that c-myb can upregulate alpha1(I) and alpha2(I) collagen promoters by 6-10-fold whereas B-myb is inactive. The activity of c-myb on both type I collagen promoters requires a functional c-myb DNA binding domain suggesting a direct interaction between c-myb and these promoters. Indeed c-myb is active also on a 500 bp fragment of the alpha2(I) collagen promoter and can bind to this fragment in electrophoretic mobility shift assays. Finally, we show that anti-c-myb anti-sense treatment reduces alpha1(I) and to a lesser extent alpha2(I) collagen gene expression. These data strongly suggest that c-myb, but not B-myb, plays a direct role in the upregulation of type I collagen gene expression in response to transforming growth factor beta.

Topics: Cell Cycle Proteins; Collagen; DNA-Binding Proteins; Down-Regulation; Fibroblasts; Gene Expression; Humans; Oligonucleotides, Antisense; Promoter Regions, Genetic; Protein Structure, Tertiary; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-myb; Scleroderma, Systemic; Trans-Activators; Transcriptional Activation; Transforming Growth Factor beta; Up-Regulation

We have established a mouse model for scleroderma induced by repeated local injections of bleomycin (BLM). Daily injection of BLM at a dose of >10 microg per ml for 4 wk induced histologic changes of dermal sclerosis, but not fibrosis, with thickened and homogenous collagen bundles and cellular infiltrates in BALB/C mice, whereas clinical signs of scleroderma were not apparent. In addition, lung fibrosis was also induced preceding the cutaneous changes. Sclerotic changes were not found in other sites of the skin distant from the injection site. Dermal sclerosis could also be induced by injecting BLM only every other day. The sclerotic changes of the dermis were sustained after ceasing BLM applications for at least 6 wk. Mast cells gradually increased in number as the sclerotic changes developed. Marked degranulation of mast cells was observed with elevated histamine release. The amount of hydroxyproline in skin was significantly increased at 4 wk of BLM treatment as compared with that in untreated or phosphate-buffered saline-treated mice. Anti-nuclear antibody was detected in serum of BLM-treated mice. Transforming growth factor-beta1 mRNA was detected at an early phase, while transforming growth factor-beta2 mRNA was strongly expressed at 4 wk when the sclerotic features were prominent. These results suggest that dermal sclerosis induced by BLM closely resembles systemic sclerosis both histologically and biochemically. Our mouse model can provide a powerful tool of inducing dermal sclerosis to examine the pathogenesis and the therapeutic approach of scleroderma.

Topics: Animals; Autoantibodies; Bleomycin; Collagen; Disease Models, Animal; Female; Mice; Mice, Inbred BALB C; Mice, Inbred C3H; Scleroderma, Systemic; Transforming Growth Factor beta

Dermatopontin is a recently discovered extracellular matrix protein with proteoglycan and cell-binding properties and is assumed to play important roles in cell-matrix interactions and matrix assembly. In this study we examined the expression of dermatopontin mRNA and protein in skin fibroblast cultures from patients with hypertrophic scar and patients with systemic sclerosis. Dermatopontin mRNA and protein levels were reduced in fibroblast cultures from hypertrophic scar lesional skin compared with fibroblasts from normal skin of the same hypertrophic scar patient. Fibroblast cultures from systemic sclerosis patient involved skin also showed significantly reduced expression of dermatopontin compared with normal skin fibroblasts from healthy individuals. We also investigated the effects of cytokines and matrix collagen on dermatopontin expression in normal cultured fibroblasts. Transforming growth factor-beta1 increased dermatopontin mRNA and protein levels, while interleukin-4 reduced dermatopontin expression. Substrate coated with type I collagen reduced dermatopontin mRNA levels, the reduction being more prominent in three-dimensional collagen matrices. Our results suggest that the decreased expression of dermatopontin is associated with the pathogenesis of fibrosis in hypertrophic scar and systemic sclerosis, and that the effect of the cytokines and matrix collagen on dermatopontin may have important implications for skin fibrosis.

Topics: Adolescent; Adult; Blotting, Northern; Carrier Proteins; Cell Adhesion Molecules; Cells, Cultured; Chondroitin Sulfate Proteoglycans; Cicatrix, Hypertrophic; Collagen; Dose-Response Relationship, Drug; Down-Regulation; Extracellular Matrix Proteins; Fibroblasts; Humans; Interleukin-4; Middle Aged; RNA, Messenger; Scleroderma, Systemic; Skin; Time Factors; Transforming Growth Factor beta

Recent studies have demonstrated the evidence of the crucial role of transforming growth factor-beta (TGF-beta) in the pathogenesis of tissue fibrosis; however, its precise role has not been fully elucidated. Administration of anti-TGF-beta antibody is shown to be effective for inhibiting lung fibrosis induced by bleomycin in an experimental animal model. We have recently established a mouse model for scleroderma by repeated injections of bleomycin. In this study, we examined whether the suppression of TGF-beta leads to the improvement of dermal sclerotic lesion by using this model. We induced dermal sclerosis in C3H mice by subcutaneous injections of bleomycin (100 microg/ml) for 3 weeks, and separate groups of mice were also injected with bleomycin with either anti-TGF-beta antibody (10 microg/ml) or control normal rabbit serum for 3 weeks. Thus treated skins were harvested and analyzed for histological sclerosis, serum cytokine, and influx of mast cells and eosinophils, both of which are known to release fibrogenic cytokines or several mediators responsible for tissue fibrosis. The result showed that anti-TGF-beta antibody caused a significant reduction in cutaneous sclerosis characterized by histological features and hydroxyproline contents. Examination of tissue sections also revealed a significant suppression of influx of mast cells and eosinophils. Serum interleukin-4 (IL-4) and IL-6 levels determined by enzyme-linked immunosorbent assay exhibited a significant reduction after anti-TGF-beta antibody treatment. Our results suggest that administration of an antibody against TGF-beta is useful in preventing experimental dermal sclerosis induced by bleomycin and raises a possibility of the therapeutic approach of anti-TGF-beta antibody in scleroderma.

Topics: Animals; Antibodies; Basophils; Bleomycin; Cell Count; Disease Models, Animal; Eosinophils; Female; Interleukin-4; Interleukin-6; Leukocyte Count; Mast Cells; Mice; Mice, Inbred C3H; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Scleroderma is a generalized or localized disorder which leads to fibrosis of the affected organs. TGF-beta has been implicated as a causal agent in its pathogenesis. In mammals, TGF-beta comprises a family of three members, beta 1, beta 2 and beta 3. Since cutaneous wound healing is thought to result either in formation of a scar or in scar-free tissue regeneration, depending on the relative amounts of the beta 3 isoform, the expression of all three isoforms was studied in skin biopsies of patients with either localized or systemic scleroderma. mRNA for all three isoforms was detected in inflammatory skin areas of both disease forms, but never in sclerotic or healthy skin. Immunohistochemical analysis confirmed expression of beta1 and beta 2 proteins in inflammatory skin of patients, whereas beta 3 protein appeared to be present in the subepidermal area and also found throughout the dermis of patients and healthy dermis as well.

Topics: Adult; Aged; Female; Humans; Immunohistochemistry; In Situ Hybridization; Male; Middle Aged; Protein Isoforms; RNA, Messenger; Scleroderma, Systemic; Transforming Growth Factor beta; Up-Regulation

Scleroderma, a debilitating acquired connective tissue disease, is characterized by fibrosis, particularly of the skin and lungs. Monocyte-produced TGF-beta1, a potent stimulus for collagen synthesis, is thought to drive the fibrosis. Here, we thoroughly characterize a murine sclerodermatous graft-vs-host disease (Scl GVHD) model for scleroderma that reproduces important features of scleroderma including skin thickening, lung fibrosis, and up-regulation of cutaneous collagen mRNA, which is preceded by monocyte infiltration and the up-regulation of cutaneous TGF-beta1 mRNA. Most importantly, we can prevent fibrosis in both the skin and lungs of mice with Scl GVHD by inhibiting TGF-beta with neutralizing Abs. The murine Scl GVHD model provides the unique opportunity to study basic immunologic mechanisms that drive fibrosing diseases and GVHD itself and will be useful for testing new therapies for these diseases.

Topics: Animals; Bone Marrow Transplantation; Cell Movement; Collagen; Disease Models, Animal; Female; Fibrosis; Graft vs Host Disease; Immune Sera; Injections, Intravenous; Leukocytes, Mononuclear; Macrophage-1 Antigen; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Pulmonary Fibrosis; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Up-Regulation

Scleroderma fibroblasts exhibit numerous phenotypic differences when compared with healthy skin fibroblasts. Some of these differences, in particular overexpression of collagen type I and other extracellular matrix proteins, parallel the effect of transforming growth factor-beta (TGF-beta) on dermal fibroblasts, suggesting that the scleroderma fibroblast phenotype may result from activation of autocrine TGF-beta signaling. To test this hypothesis we examined the role of TGF-beta Type I and Type II receptors in regulating collagen type I transcription. We have shown that overexpression of either Type I or Type II receptors significantly (3-4-fold) increases alpha2 (I) collagen promoter activity in transient transfection assays in dermal fibroblasts. Addition of anti-TGF-beta antibody abolished, whereas addition of plasmin enhanced, the stimulatory effect of receptor overexpression on collagen promoter activity, suggesting that this effect depends on autocrine TGF-beta. Moreover, these cotransfection experiments indicated that expression levels of TGF-beta receptors is a limiting factor in the autocrine regulation of collagen type I transcription by TGF-beta. Comparison of the TGF-beta receptor Type I and Type II mRA expression levels in scleroderma and normal fibroblasts have indicated elevated expression (2-fold) of both receptor types in scleroderma cells, which correlated with increased binding of TGF-beta. Significantly, elevated TGF-beta receptor levels correlated with elevated alpha2 (I) collagen mRNA levels. These results suggest that the elevated production of collagen type I by scleroderma fibroblasts results from overexpression of TGF-beta receptors.

Topics: Collagen; Fibroblasts; Humans; Phenotype; Receptors, Transforming Growth Factor beta; Scleroderma, Systemic; Signal Transduction; Transcription, Genetic; Transforming Growth Factor beta

A 19-year-old woman with acute lymphoblastic leukemia received an allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling during the second remission, on September 28, 1993. The conditioning regimen consisted of total body irradiation and cyclophosphamide. Short term methotrexate and cyclosporin A were given for prophylaxis of graft-versus-host disease (GVHD). On day 771 after BMT, she complained of bilateral forearm pain, and developed sclerotic lesions on the skin of the abdominal wall, forearms and legs. The diagnosis of sclerodermatous GVHD was established by skin biopsy on day 834. The values of CRP and IgG were elevated, and both antinuclear antibody and anti-DNA antibody became positive. Flow cytometric analysis showed a significant increase in the number of CD57+ cells after appearance of sclerotic change. In addition, 65% of CD8+ cells were positive for CD57. Circulating level of transforming growth factor (TGF)-beta 1 was high. These results suggest that overproduction of CD8+ CD57+ T cells and high level of circulating TGF-beta are related to the development of sclerodermatous GVHD.

Topics: Adult; Bone Marrow Transplantation; CD57 Antigens; CD8-Positive T-Lymphocytes; Cell Division; Female; Graft vs Host Disease; Humans; Precursor Cell Lymphoblastic Leukemia-Lymphoma; Scleroderma, Systemic; Transforming Growth Factor beta; Transplantation, Homologous

Heat-shock protein 47 (HSP47) is a collagen-binding stress protein that is thought to act as a collagen-specific molecular chaperon during the biosynthesis and secretion of procollagen. In this study we examined the expression of HSP47 mRNA and protein in systemic sclerosis (SSc) skin fibroblasts. HSP47 mRNA and protein levels were significantly higher in fibroblast cultures from SSc patient-involved skin samples than in fibroblasts from normal skin from healthy individuals, as assessed by northern blot and immunoblot analyses, respectively. SSc cultured fibroblasts with increased levels of HSP47 mRNA and protein showed high expression of type I procollagen. By in situ hybridization, SSc skin had a higher number of fibroblasts with high HSP47 and procollagen alpha1(I) mRNA levels than normal skin, and the distribution of HSP47 mRNA was similar to that of procollagen alpha1(I) mRNA. We also investigated the effects of cytokines on the expression of HSP47 in normal cultured fibroblasts. Transforming growth factor-beta1 and interleukin-4 increased HSP47 mRNA and protein levels, whereas interferon-gamma reduced HSP47 expression. The same pattern of cytokine-regulated expression was observed for type I procollagen levels. These results indicate that HSP47 expression is closely associated with that of type I procollagen in skin fibroblasts, and that increased expression of HSP47 may be involved in the abundant production of type I procollagen by SSc fibroblasts.

Topics: Cells, Cultured; Fibroblasts; Gene Expression Regulation; Heat-Shock Proteins; HSP47 Heat-Shock Proteins; Humans; Immunoblotting; In Situ Hybridization; Interferon-gamma; Interleukin-4; Procollagen; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Pregnancy after the onset of scleroderma is uncommon; therefore, placental findings and perinatal outcome have rarely been correlated. The histopathologic features of placentas from 13 pregnancies in eight women with scleroderma were recorded and correlated with the clinical features of the mother and fetus. Adverse perinatal outcome included intrauterine fetal demise in five, and previable or preterm delivery in four. A decidual vasculopathy was seen in 5 of the 13 placentas, four of which were associated with intrauterine fetal demise. Decidual blood vessels in the scleroderma patients were evaluated immunohistochemically for platelet-derived growth factor (PDGF), transforming growth factor beta1 (TGF-beta1), T-helper and T-suppressor lymphocytes, macrophages, immunoglobulin (Ig) M, and IgG, and compared with those from hypertensive and uncomplicated third-trimester pregnancies. The atherotic blood vessels in scleroderma were characterized by mural macrophages and IgM and IgG deposition and were similar to those seen in placentas from hypertensive pregnancies. CD8-positive T cells predominated in normal and hypertensive decidua compared with scleroderma, in which CD4-positive T cells were more frequent. No difference in PDGF or TGF-beta1 staining was found between scleroderma and control groups. In conclusion, decidual vasculopathy is common in scleroderma, is similar to that seen in hypertension, and is associated with poor perinatal outcome. A trend toward a reversed ratio of decidual CD4 to CD8-positive T cells is seen in scleroderma compared with hypertension and uncomplicated pregnancies. PDGF and TGF-beta1 do not appear to be involved in the pathogenesis of decidual vasculopathy in scleroderma.

Topics: Abortion, Spontaneous; Adolescent; Adult; Antigens, CD; Arteriosclerosis; Blood Vessels; CD4-CD8 Ratio; Decidua; Female; Fetal Death; Gestational Age; Humans; Hypertension; Immunoglobulins; Infant, Newborn; Male; Obstetric Labor, Premature; Platelet-Derived Growth Factor; Pregnancy; Pregnancy Complications; Pregnancy Outcome; Scleroderma, Systemic; Transforming Growth Factor beta

Studies have shown that scleroderma (systemic sclerosis, SSc) and normal fibroblasts respond differently to basic fibroblast growth factor (bFGF), SSc fibroblasts being less responsive than normal fibroblasts in mitogenic assays in vitro, bFGF also stimulates the expression of platelet derived growth factor-alpha (PDGF-alpha) receptors in normal fibroblasts, but not in SSc fibroblasts. Conversely, transforming growth factor-beta (TGF-beta) stimulates PDGF-alpha receptor expression in SSc fibroblasts, but not in normal fibroblasts. Since bFGF has been shown to inhibit collagen gene expression in several cell types, we examined responses of SSc and normal fibroblasts to bFGF alone and in combination with TGF-beta with regard to collagen alpha 2(I) (COL1A2) expression.. Fibroblasts were obtained by skin biopsy from affected areas of patients with diffuse cutaneous SSc and from healthy donors and propagated in vitro. The effects of bFGF and TGF-beta on the COL1A2 mRNA expression levels in SSc and healthy fibroblasts were analyzed by Northern blot. The effects of bFGF on the COL1A2 promoter activities in both cell types were analyzed by transient transfection assays. The effects of bFGF and TGF-beta on collagen protein synthesis were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and fluorography.. While bFGF diminished COL1A2 mRNA in both SSc and normal cells, COL1A2 mRNA quantities in the SSc fibroblasts were not depressed to the levels expressed by normal controls. As anticipated, TGF-beta strongly induced COL1A2 mRNA levels in normal fibroblasts, and to a lesser degree in SSc fibroblasts. When cells were incubated with both TGF-beta and bFGF, the stimulatory effect of TGF-beta was completely suppressed in both cell types. bFGF decreased COL1A2 promoter activity in both cell types, suggesting that COL1A2 inhibition by bFGF occurs at least partially at the transcriptional level. The effects of bFGF and TGF-beta on the collagen protein synthesis correlated well with mRNA data, in that TGF-beta stimulated, while bFGF strongly inhibited, collagen synthesis.. bFGF is a potent inhibitor of basal and TGF-beta stimulated collagen expression in human fibroblasts, and this effect is not different between SSc and healthy fibroblasts.

Topics: Collagen; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Fibroblast Growth Factor 2; Fibroblasts; Fibronectins; Gene Expression; Humans; Promoter Regions, Genetic; RNA, Messenger; Scleroderma, Systemic; Sodium Dodecyl Sulfate; Time Factors; Transforming Growth Factor beta

To assess the ability of peripheral blood mononuclear cells (PBMC) of patients with systemic sclerosis (SSc) to produce interleukin 6 (IL-6), transforming growth factor beta 1 (TGF-beta 1), to identify the IL-6 producer cells in the in vitro model, and to correlate these data with the clinical evidence of our patients.. We used a sandwich ELISA to quantitate IL-6 and TGF-beta 1 levels in sera, plasma, and supernatants, and an imunofluorescence technique to evaluate IL-6 producing cells in our patients.. IL-6 was detected in sera from 8 of 20 patients and no controls (p < 0.05). A significant increase of IL-6 production was observed in both spontaneous and phytohemagglutinin (PHA) induced cultures of PBMC from patients with SSc vs controls. No differences in TGF-beta 1 production were observed, either in sera or supernatants, between patients and controls. A significant increase of IL-6 synthesizing cells was observed after 3 h of PHA stimulation in patients vs controls (p < 0.05).. Spontaneous IL-6 production and the higher number of IL-6 producing cells in patients with SSc suggest that these cells have been already primed in vivo. The absence of PBMC primed for TGF-beta 1 production supports the hypothesis that cells other than lymphocytes produce and secrete this cytokine in the skin of patients. Higher serum levels of IL-6 observed in a subset of patients did not correlate with either severity or duration of disease.

Topics: Adult; Aged; Female; Humans; Interleukin-6; Middle Aged; Monocytes; Scleroderma, Systemic; Transforming Growth Factor beta

We investigated the responses of normal and scleroderma fibroblasts to various growth factors, especially transforming growth factor-beta 1 (TGF-beta 1). The effects of various growth factors on [3H]thymidine incorporation in normal and scleroderma fibroblasts were examined. [125I]-labeled platelet-derived growth factor (PDGF)-BB binding in scleroderma and normal fibroblasts was examined both in the presence and absence of TGF-beta 1 (1 ng/ml). Cytoplasmic protein was isolated and analyzed by Western blotting. Total RNA from fibroblasts was also isolated and analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) using specific primer sets. Mitogenic responses to TGF-beta 1 (0.33-1 ng/ml) in seven scleroderma fibroblast strains were significantly greater than those in normal controls. [125I]-PDGF-BB binding to scleroderma fibroblasts was increased after TGF-beta 1 stimulation. The increased response to TGF-beta 1 was shown to be mediated through PDGF-like protein induction; TGF-beta 1-treated scleroderma fibroblasts produced greater amounts of 36-kD PDGF-like protein, which was reported previously as connective tissue growth factor (CTGF), than did TGF-beta 1-treated normal fibroblasts. TGF-beta 1 treatment also upregulated PDGF-alpha receptor expression in scleroderma fibroblasts but not in normal dermal fibroblasts. mRNA expression of CTGF and PDGF-alpha receptor was correlated with the above protein expression. These observations suggest that the increased growth response to TGF-beta 1 in scleroderma fibroblasts is mediated through the induction of CTGF and PDGF-alpha receptor.

Topics: Cell Division; Cells, Cultured; Female; Fibroblasts; Humans; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

Systemic sclerosis (SSc) is characterized by systemic fibrosis and microvascular lesions. As TGF-beta is suggested to be related to skin fibrosis, we examined the production of TGF-beta from peripheral mononuclear cells (MNC) of SSc patients. Since anti-TGF-beta neutralizing antibody improved the defective proliferative response in autologous mixed lymphocyte reaction (AMLR) of SSc patients, TGF-beta was thought to participate in the decreased AMLR of SSc patients. Greater amounts of TGF-beta in the active as well as in the latent forms were produced during AMLR of SSc patients than that of normal subjects. It was suggested that TGF-beta excessively produced from the MNC of SSc patients might play a major role in the fibrosis of the patients during AMLR-like in vivo responses.

Topics: Adult; Aged; Female; Humans; Immunity, Cellular; In Vitro Techniques; Leukocytes, Mononuclear; Lymphocyte Culture Test, Mixed; Male; Middle Aged; Scleroderma, Systemic; Transforming Growth Factor beta

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

Previous studies have demonstrated that platelet-derived growth factor (PDGF) alpha receptor expression is up-regulated by transforming growth factor-beta 1 (TGF-beta 1) in scleroderma dermal fibroblasts, but not in healthy control fibroblasts. We asked whether this selective effect in scleroderma cells was TGF-beta 1-specific or a general response by studying responses to other growth factors. In this study, we compared the expression of alpha and beta PDGF receptor subunits (mRNA and protein levels) in these two cell types in response to basic fibroblast growth factor (bFGF) and TGF-beta 1. bFGF coordinately stimulated mRNA levels of alpha and beta receptor subunits in healthy fibroblasts, but did not change PDGF receptor expression in scleroderma fibroblasts. Conversely, and in agreement with previous observations, TGF-beta 1 induced PDGF alpha receptor expression in scleroderma fibroblasts, but not in healthy fibroblasts. PDGF beta receptor mRNA levels were induced to similar degrees by TGF-beta 1 in both cell types. PDGF alpha receptor protein levels correlated directly with mRNA levels, induced by bFGF only in healthy fibroblasts and by TGF-beta 1 only in scleroderma fibroblasts. However, PDGF beta receptor protein levels were not altered by either growth factor in either cell type. Thus, the activated state of scleroderma fibroblasts does not include receptor-signaling pathways to bFGF. This distinct pattern of expression of PDGF alpha receptors in scleroderma fibroblasts suggests a possible role for the coordinately expressed PDGF AA ligand/alpha receptor system in the development of fibrosis.

Topics: Blotting, Western; Fibroblast Growth Factor 2; Fibroblasts; Humans; Proteins; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Scleroderma, Systemic; Time Factors; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

The distribution and amount of integrin alpha2 were studied in cultured fibroblasts from normal subjects and scleroderma patients by immunofluorescence and immunoblotting using a monoclonal antibody against the human integrin alpha2 subunit. Integrin alpha2 was concentrated at the perinuclear regions in a dot-like pattern in normal fibroblasts on the glass coverslips until the 14th day after planting, and the staining pattern of integrin alpha2 was gradually changed to a dispersed dot-like pattern by the 19th day as examined by immunofluorescence microscopy by using the anti-integrin alpha2 antibody. No difference was observed in the distribution patterns between normal and scleroderma fibroblasts. By immunoblotting study, the amount of integrin alpha2 in scleroderma fibroblasts (n = 10) was less than that of normal fibroblasts (n = 10) (P < 0.01) in both cytosol and cytoskeleton-associated fractions. Furthermore, transforming growth factor beta (TGF-beta) increased the amount of integrin alpha2 in both normal fibroblasts and scleroderma cells by 33%. The total amount of integrin alpha2 in TGF-beta-stimulated scleroderma fibroblasts was less than that in TGF-beta-stimulated normal fibroblasts. These findings suggest that the amount of integrin alpha2, a collagen receptor, is reduced in scleroderma fibroblasts, but the integrin alpha2 production by TGF-beta stimulation is not impaired in scleroderma fibroblasts.

Topics: Adult; Aged; Antigens, CD; Blotting, Western; Case-Control Studies; Cell Adhesion; Cells, Cultured; Endoplasmic Reticulum, Rough; Female; Fibroblasts; Golgi Apparatus; Humans; Integrin alpha2; Male; Microscopy, Fluorescence; Middle Aged; Scleroderma, Systemic; Transforming Growth Factor beta

Quantification of gene expression is of increasing interest in many medical sciences. Methods based on reverse transcription-polymerase chain reactions (RT-PCRs) are timesaving and require only very small amounts of RNA. A limiting factor, however, is the significant fluctuation in the efficacy of reverse transcription as well as in the polymerase chain reactions. Various external and internal standards have been suggested for correcting these fluctuations. We describe a novel way of creating an internal standard for assessing the expression of type VII collagen in human cells. The total RNA of a patient with hereditary epidermolysis bullosa dystrophica associated with a homozygous T to A point mutation in type VII collagen gene was reverse transcribed and a 382bp fragment of type VII collagen cDNA containing the mutation was amplified. The mutated cDNA, unlike normal type VII collagen cDNA could be cleaved by EarI endonuclease into 244bp and 138bp fragments. Semiquantitative PCR was performed with the mutated cDNA as internal standard and the studied cDNA sample in the same tube in the presence of alpha 32P-labeled dCTP. The reaction was followed by EarI digestion, electrophoresis on a polyacrylamide gel and exposure to a X-ray film. In conclusion, we describe a timesaving method for creating internal standards for semiquantitative RT-PCR.

Topics: Base Sequence; Collagen; DNA; Fibroblasts; Humans; Molecular Sequence Data; Mutation; Polymerase Chain Reaction; Reference Standards; Reference Values; RNA, Messenger; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

Systemic sclerosis (scleroderma, SSc) frequently affects the lungs, and interstitial pulmonary fibrosis is one of its major complications. The pathophysiology of SSc lung disease s poorly understood, but recent studies document an inflammatory process resembling that of idiopathic pulmonary fibrosis with increased numbers of activated alveolar macrophages and granulocytes in bronchoalveolar lavage (BAL) fluid). We determined levels of 2 potentially important mediators of fibroproliferative repair in BAL fluid from patients with SSc.. Using Western blot and ELISA techniques we measured levels of platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta in BAL fluid from patients with SSc and healthy controls. The mitogenic effect of these cytokines on SSc lung myofibroblasts was determined by [3H]thymidine incorporation.. SSc BAL fluid contains significantly elevated levels of PDGF-AA and PDGF-BB. Where TGF-beta 1 was significantly elevated in SSc lavage fluid, the amount of TGF-beta 2 was significantly less than that observed in normal lavage fluid. Myofibroblasts cultured from SSc lavage fluid exhibited enhanced [3H]thymidine incorporation upon exposure to the growth factors present in SSc BAL fluid: PDGF and TGF-beta 1. SSc lung myofibroblasts pretreated with TGF-beta 1 exhibited an enhanced mitogenic effect upon stimulation by PDGF, due in part to the induction of the PDGF alpha receptor.. Our studies support a role for PDGF and TGF-beta 1 in the pathogenesis of SSc lung disease.

Topics: Bronchoalveolar Lavage Fluid; Cell Division; Fibroblasts; Humans; Isomerism; Lung; Muscle, Smooth; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Reference Values; 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

A hallmark of systemic sclerosis (SSc) is the development of tissue fibrosis. Excessive production of several connective tissue components normally present in the dermis, including type I, III, V, and VI collagens as well as fibronectin and proteoglycans, is a consistent finding in the skin of SSc patients. Type VII collagen is a major constituent of anchoring fibrils, present in the skin at the dermal-epidermal basement membrane zone. TGF-beta has been shown to upregulate the expression of the type VII collagen gene. In this study, we assessed the expression of type VII collagen and TGF-beta in the skin of patients with SSc. Indirect immunofluorescence showed an abundance of type VII collagen in the patients' skin, including the dermis. Ultrastructural analysis of SSc skin revealed an abundance of fibrillar material, possibly representing type VII collagen. The increased expression of type VII collagen epitopes was accompanied by the elevated expression of immunodetectable TGF-beta 1 and TGF-beta 2. Dermal fibroblasts cultured from the affected individuals showed a statistically significant (P < 0.02) increase in the expression of type VII collagen at the mRNA level, as detected by reverse transcription-PCR with a mutated cDNA as an internal standard, and increased deposition of the protein as assessed by indirect immunofluorescence. Thus, type VII collagen is abundantly present in SSc patients' dermis, a location not characteristic of its normal distribution, and its aberrant expression may relate to the presence of TGF-beta in the same topographic distribution. The presence of type VII collagen in the dermis may contribute to the tightly bound and indurated appearance of the affected skin in SSc patients.

Topics: Adult; Aged; Base Sequence; Cells, Cultured; Collagen; Epitopes; Female; Fluorescent Antibody Technique; Humans; Male; Microscopy, Electron; Middle Aged; Molecular Sequence Data; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

To determine whether the active 25 kDa form of the fibrogenic cytokine transforming growth factor beta (TGF beta) can be detected in plasma from patients with systemic sclerosis and to examine the relationship between plasma TGF beta and clinical markers of disease severity and serum concentrations of the aminoterminal peptide of type III procollagen (PIIINP) (a laboratory marker of the fibrotic process).. A cross sectional study was made of 39 patients with systemic sclerosis (11 diffuse and 28 limited), nine patients with primary Raynaud's disease and 60 healthy controls. TGF beta 1 and TGF beta 2 were measured by enzyme linked immunosorbent assay (ELISA) (sensitivity 100 pg/ml) and PIIINP by radioimmunoassay.. TGF beta 1 was detected in plasma from six of 39 patients with systemic sclerosis but not in any patient with primary Raynaud's disease or healthy controls. TGF beta 2 was not detected in plasma from patients or controls. No clear relationship was demonstrated between TGF beta 1, clinical features or PIIINP concentrations.. The 25 kDa form of TGF beta 1 can be detected in the plasma of some patients with systemic sclerosis. This provides limited support for the hypothesis that this cytokine plays a role in the pathogenesis of this disease. However, longitudinal studies, particularly in early diffuse disease, are required to clarify the relationship between circulating TGF beta 1 and disease activity.

Topics: Adult; Cross-Sectional Studies; Disease Progression; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Middle Aged; Peptide Fragments; Procollagen; Raynaud Disease; Scleroderma, Systemic; Transforming Growth Factor beta

To study the role of transforming growth factor-beta 1 (TGF-beta 1) in the pathogenesis of pulmonary fibrosis we have examined lung biopsies from nine patients with systemic sclerosis and interstitial lung disease, eight with 'lone' cryptogenic fibrosing alveolitis, two with cystic fibrosis, two with extrinsic allergic alveolitis, two with Langerhans' cell histiocytosis, one with lymphangioleiomyomatosis, one with giant cell interstitial pneumonia, and one adenocarcinoma of the lung. In cryptogenic fibrosing alveolitis, both 'lone' and associated with systemic sclerosis alveolar macrophages, bronchial epithelium and hyperplastic type II pneumonocytes expressed intracellular TGF-beta 1. Extracellular TGF-beta 1 was found in the fibrous tissue immediately beneath the bronchial and hyperplastic alveolar epithelium. In normal lung, however, the alveolar epithelium and alveolar interstitium were negative for both forms of TGF-beta 1. There was strong expression of TGF-beta 1 in hyperplastic mesothelium and its underlying connective tissue and in Langerhans' cells in the two cases of histiocytosis. In the organizing pneumonia in cystic fibrosis, the intraalveolar buds of granulation tissue reacted strongly for the extracellular form of TGF-beta 1 and the overlying hyperplastic epithelium expressed the intracellular form. In lymphangioleiomyomatosis, the aberrant smooth muscle cells strongly expressed intracellular TGF-beta 1 and the extracellular form was expressed in the adjacent connective tissue. In giant cell interstitial pneumonia, the numerous alveolar macrophage including the multinucleate forms, expressed intracellular TGF-beta 1, as did the hyperplastic alveolar epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Humans; Immunohistochemistry; Lung Diseases, Interstitial; Pulmonary Fibrosis; Scleroderma, Systemic; Transforming Growth Factor beta

Tight skin (TSK/+) mice develop a cutaneous hyperplasia associated with the occurrence of autoantibodies characteristic for scleroderma. In order to study the role of autoimmunity in the production of skin fibrosis, we conducted adoptive transfer experiments in which bone marrow cells of TSK/pa mice were infused into pa/pa mice littermates. (C57BL/6 pa/pa mice are used to produce heterozygous TSK/pa mice). Our results showed that after a prodromal period of several months, the transfer of bone marrow cells led to skin fibrosis, the presence of autoantibodies, and increased transcription of (alpha 1) collagen I and TGF beta genes. Infusion of enriched B or T cells alone did not cause skin fibrosis but of B cells alone increased autoantibody production. By contrast, transfer of T and B lymphocytes led to earlier mild fibrosis, cellular infiltration and autoantibody production as well as increased transcription of the (alpha 1) collagen gene. Our results strongly demonstrate, for the first time, that immunocompetent cells can play a role in the activation of collagen synthesis leading to skin fibrosis.

Topics: Animals; Autoantibodies; Autoimmune Diseases; Bone Marrow Transplantation; Collagen; Connective Tissue Diseases; Disease Models, Animal; Fibrosis; Gene Expression Regulation; Hyperplasia; Immunocompetence; Immunoglobulin G; Immunoglobulin M; Immunotherapy, Adoptive; Lymphocyte Cooperation; Lymphocyte Subsets; Mice; Mice, Inbred C57BL; Mice, Mutant Strains; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

We documented the effect of the retinoid (4-hydroxyphenyl)retinamide on collagen expression in a tissue culture and in an animal model of scleroderma.. We used RNA analysis, chloramphenicol acetyltransferase assays, organ culture, and histologic evaluation.. We showed that (4-hydroxyphenyl)retinamide decreases alpha 1(I) collagen messenger RNA and transcription in cultured cells, and decreases collagen levels in the dermis of tight-skin mice.. These results provide a basis for further experiments to address the efficacy of (4-hydroxyphenyl)retinamide in the treatment of scleroderma.

Topics: Animals; Cell Division; Collagen; Disease Models, Animal; Fenretinide; Fibroblasts; Gene Expression; Humans; Male; Mice; Mice, Mutant Strains; RNA, Messenger; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

We have investigated the distribution of TGF beta using antibodies specific for its intracellular and extracellular forms in full-thickness biopsies of patients with SSc, primary Raynaud's phenomenon (PRP), systemic lupus erythematosus (SLE), and from normal subjects. Nine of 11 SSc biopsies demonstrated intracellular TGF beta in endothelial cells while only 6 exhibited extracellular TGF beta. Endothelial cells in skin biopsies of all PRP patients displayed both intracellular and extracellular TGF beta. All other control biopsies were negative. In patients with PRP, some positively staining fibroblasts were found scattered throughout the dermis. Lastly, extracellular TGF beta was localized in the papillary dermis of PRP and SSc biopsies and in all the dermal layers of SLE patients. No significant staining of TGF beta was observed in the endothelial cells, fibroblasts, or in the extracellular matrix of the majority of biopsies from normal subjects. These data suggest that TGF beta may be one of the cytokines involved in the early stages of pathogenesis of SSc, and that endothelial cells in SSc and PRP may be a source and/or a target of TGF beta.

Topics: Aged; Extracellular Space; Female; Histocytochemistry; Humans; Middle Aged; Raynaud Disease; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

We investigated the expression of transforming growth factor-beta (TGF-beta) isoforms in involved and uninvolved areas of skin from patients with systemic sclerosis (SSc) and normal controls. Paraffin-embedded skin specimens were stained for TGF-beta 1, TGF-beta 2, and TGF-beta 3 using the avidin-biotin-peroxidase technique. TGF-beta 2 was expressed intensely in the extracellular matrix of the skin biopsies obtained from involved areas of patients with SSc, in contrast to the uninvolved areas and normal individuals. TGF-beta 2 was deposited throughout the entire dermis and also in a linear fashion along the dermoepidermal junction and in the perivascular areas of SSc-involved skin. Although infiltrating mononuclear cells were not present in great numbers, they did not stain for TGF-beta 2. TGF-beta 1 and TGF-beta 3 were not expressed in the extracellular space in either patients or normal controls, except in rare cases. The cellular staining which was observed for all three isoforms did not differ between involved and uninvolved skin and normal controls. The finding of increased deposition of TGF-beta 2 in the involved skin of patients with SSc implies that it may be involved in the pathologic fibrotic process.

Topics: Adult; Epidermis; Extracellular Matrix; Female; Humans; Immunohistochemistry; Male; Middle Aged; Muscle, Smooth, Vascular; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Differences in the responses to growth factors of normal fibroblasts and scleroderma fibroblasts have been demonstrated previously. Because human dermal fibroblasts are heterogeneous populations, whether known differences between papillary and reticular dermal fibroblasts could account for the noted differences between normal and scleroderma fibroblasts was investigated. Papillary dermal fibroblasts were grown from a dermatome section of normal skin from an adult donor. Reticular dermal fibroblasts were cultured from punch biopsy specimens taken from the same location. In vitro, papillary dermal fibroblasts proliferated more rapidly, had a higher mitotic index and reached greater density at confluence, confirming previous observations. The reticular dermal fibroblasts were more dendritic. Reticular dermal fibroblasts had higher rates of tritiated thymidine uptake and larger increases in mitotic index in response to isoforms of platelet-derived growth factor (PDGF). The characteristic response of scleroderma fibroblasts, potentiation of the mitogenicity of PDGF AA by transforming growth factor-beta (TGF-beta), was not observed in either cell type. Therefore, the phenotypic characteristics of scleroderma fibroblasts cannot be explained by an unusual admixture of papillary and reticular fibroblasts.

Topics: Adult; Cell Count; Cells, Cultured; Fibroblasts; Humans; Platelet-Derived Growth Factor; Receptors, Platelet-Derived Growth Factor; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Topics: Adult; Cells, Cultured; DNA; Fibroblast Growth Factor 2; Fibroblasts; Humans; Infant, Newborn; Mitosis; Radioligand Assay; Receptors, Fibroblast Growth Factor; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta), a multifunctional cytokine, is an indirect mitogen for human fibroblasts through platelet-derived growth factor (PDGF), particularly the A ligand-alpha receptor arm of that system. TGF-beta effects on PDGF alpha receptor expression were studied in vitro using ligand binding techniques in three human dermal fibroblast strains: newborn foreskin, adult skin, and scleroderma (systemic sclerosis, SSc). Each cell strain responded differently to TGF-beta. In newborn foreskin fibroblasts, PDGF alpha receptor number decreased in a dose-dependent manner after exposure to low concentrations of TGF-beta (0.1-1 ng/ml). Responses of normal skin fibroblasts were varied, and mean net receptor number was unchanged. Increases in PDGF alpha receptor number by TGF-beta occurred consistently with SSc fibroblasts and low concentrations of TGF-beta (0.1-1 ng/ml) were particularly stimulatory. Increased surface expression of alpha receptor subunit by TGF-beta in SSc fibroblasts correlated with increased new PDGF alpha receptor synthesis as demonstrated by radioimmunoprecipitation analysis of metabolically labeled cells and with increased steady-state levels of corresponding mRNAs. In normal adult skin fibroblasts, TGF-beta had no effect on either synthesis or mRNA expression of alpha receptor subunits. Proliferative responses to PDGF-AA after pretreatment with TGF-beta correlated positively with effects of TGF-beta on expression of alpha receptor subunit. Decreased mitogenic responses to PDGF-AA were observed in foreskin fibroblasts, small changes in responses in adult fibroblasts, and significant increases in SSc fibroblasts. Thus, costimulation with PDGF-AA and TGF-beta selectively enhanced proliferation of fibroblasts with the SSc phenotype. Immunohistochemical examination of SSc and control skin biopsies revealed the presence of PDGF-AA in SSc skin. Data obtained by ligand binding, immunoprecipitation, mRNA, and mitogenic techniques are consistent with the hypothesis that activation of the PDGF-AA ligand/alpha receptor pathway is a characteristic of the SSc fibroblast and may contribute to the expansion of fibroblasts in SSc.

Topics: Adult; Blotting, Northern; Female; Fibroblasts; Humans; Infant, Newborn; Male; Radioimmunoprecipitation Assay; Receptors, Cell Surface; Receptors, Platelet-Derived Growth Factor; RNA, Messenger; Scleroderma, Systemic; Transforming Growth Factor beta; Up-Regulation

Effects of transforming growth factor beta (TGF-beta) on proliferative responses to basic fibroblast growth factor (bFGF) were studied in human diploid fibroblasts cell strains derived from three different sources: adult skin, scleroderma, and newborn foreskin. All three types of cell strains were similarly responsive to TGF-beta, whereas adult skin fibroblasts were significantly more responsive to bFGF. Incubation of cells with TGF-beta prior to bFGF addition substantially increased responsiveness of adult skin fibroblasts to this latter cytokine, slightly increased that of scleroderma fibroblasts, and decreased that of foreskin fibroblasts. Modulation of bFGF receptors by TGF-beta correlated positively with these mitogenic effects. Adult skin fibroblasts showed increases of both high- and low-affinity receptors and scleroderma fibroblasts showed small increases of high-affinity receptors only, whereas foreskin fibroblasts showed no changes. Heparitinase treatment of adult skin fibroblasts during TGF-beta pre-incubation resulted in reduced bFGF binding to low-affinity receptors and reduced mitogenic response to bFGF, suggesting that the TGF-beta-stimulated increase of low-affinity receptors in these cells contributes to the observed enhanced mitogenic effects of bFGF. Abnormal responses of scleroderma fibroblasts to TGF-beta/bFGF stimulation, particularly failure to synthesize low-affinity receptors in response to TGF-beta, adds a new characteristic to the fibrotic phenotype of scleroderma fibroblasts.

Topics: Adult; Fibroblast Growth Factor 2; Fibroblasts; Humans; Infant, Newborn; Male; Mitogens; Receptors, Cell Surface; Receptors, Fibroblast Growth Factor; Scleroderma, Systemic; Skin; Transforming Growth Factor beta

Prolyl 4-hydroxylase (PH) is an enzyme that catalyzes an essential step in procollagen synthesis. PH production, as measured by the new ELISA method, was significantly higher in fibroblast cultures from patients with systemic sclerosis (SSc; N = 7) than in cultures from healthy controls (NC; N = 5) (p less than 0.01). Transforming growth factor-beta and tumor necrosis factor-alpha significantly increased PH production, but recombinant interferon-gamma had a significant negative effect on PH production. Since the production of PH is relevant to collagen synthesis by fibroblasts of patients with SSc, regulation of PH production may have therapeutic value.

Topics: Cells, Cultured; Collagen; Cytokines; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Fibrosis; Humans; Immunoblotting; Interferon-gamma; Procollagen; Procollagen-Proline Dioxygenase; Radioimmunoassay; Recombinant Proteins; Scleroderma, Systemic; Skin; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha

Since transforming growth factor beta (TGF beta) has been implicated as an important mediator of pulmonary fibrosis, we measured TGF beta protein and gene expression in alveolar epithelial lining fluid (ELF) of fibrotic scleroderma lungs sampled by bronchoalveolar lavage (BAL). TGF beta protein was qualitatively examined by Western blot analysis, and quantitatively by radioreceptor assays. Gene expression was evaluated in BAL mononuclear cells by Northern blot analysis with quantification of relative gene expression by densitometric analysis of the autoradiograms.. Normal and scleroderma subjects had a 24-kd protein that comigrated with defined human TGF beta 1 and immunoreacted with anti-TGF beta antibody. The normal population had a significantly higher average TGF beta concentration (705 pM) compared with the scleroderma subjects (177 pM). The TGF beta 1 gene was expressed in amounts that did not significantly differ between the scleroderma and normal groups. On an individual subject basis, the TGF beta concentration variability did not correlate with variations in BAL cellularity or TGF beta 1 gene expression within the recovered mononuclear cells.. It is concluded that both normal and fibrotic lungs have TGF beta 1 present at the alveolar epithelial surface. However, in the fibrotic scleroderma lungs, TGF beta protein content and gene expression were not increased at the alveolar epithelial surface. The simultaneous analysis of TGF beta protein content, gene expression, and cellular constituents within individual ELF specimens showed that the cellular components of the ELF do not appear to be major determinants of TGF beta protein concentration at the alveolar epithelial surface.

Topics: Adult; Aged; Alabama; Autoradiography; Blotting, Northern; Blotting, Western; Bronchoalveolar Lavage Fluid; Evaluation Studies as Topic; Female; Gene Expression; Hospitals, University; Humans; Male; Middle Aged; Outpatient Clinics, Hospital; Pulmonary Fibrosis; Radioligand Assay; Scleroderma, Systemic; Sensitivity and Specificity; Transforming Growth Factor beta

Transforming growth factor beta (TGF-beta) 1 and 2 have both become increasingly important in cutaneous biology, but their expression and distribution in human skin are not entirely clear. In this report, normal forearm skin from four volunteers was investigated for TGF-beta 1 and beta 2 immunostaining with antibodies that detect preferentially either cell- or matrix-associated forms of these peptides. Marked cell-associated TGF-beta 1 was found in the dermis, particularly around blood vessels and ducts; cellular TGF-beta 2 immunostaining was less prominent, and was predominantly around blood vessels. Neither TGF-beta 1 nor -beta 2 could be detected in the epidermis or epithelial structures, and the dermal matrix contained minimally detectable amounts of the two isoforms. In all cases, dermal matrix and cells contained greater amounts of TGF-beta 1 than TGF-beta 2. Previous studies have shown that both TGF-beta 1 and -beta 2 can induce dramatic increases in extracellular matrix, and both peptides have been implicated in the pathogenesis of fibrosis. We therefore investigated TGF-beta 1 and -beta 2 immunostaining in involved forearm skin of four patients with systemic sclerosis. Compared to normal skin, fibrotic specimens showed increased amounts of matrix and epidermal TGF-beta 1, but not TGF-beta 2. We conclude that TGF-beta 1 and -beta 2 expression in human skin is differentially regulated, and that their distribution is varied and complex.

Topics: Adult; Extracellular Matrix; Gene Expression Regulation; Humans; Immunohistochemistry; Scleroderma, Systemic; Skin; Tissue Distribution; Transforming Growth Factor beta

Lesional fibroblasts propagated from the skin of patients with scleroderma, when compared to normal fibroblasts, show increased synthesis of several collagens and increased levels of their corresponding mRNAs. Using constructs (COL1A2/CAT) containing the promoter for the alpha 2 (I) collagen gene in transient transfection assays with matched pairs of scleroderma and normal skin fibroblasts, we observed higher transcriptional activity of the COL1A2 gene in scleroderma fibroblasts and, in contrast to normal fibroblasts, no further expression was observed in the presence of TGF beta 1. Analysis of the expression of COL1A2 promoter deletion constructs indicates that the TGF beta responsive element functional in normal fibroblasts and the sequence involved in intrinsic upregulation of COL1A2 gene expression in scleroderma fibroblasts are both located between bp-376 (Bgl II) and bp-108 (Sma I) sites. These data may indicate that intrinsic upregulation of extracellular matrix genes in scleroderma fibroblasts utilizes a TGF beta dependent pathway.

Topics: Base Sequence; Cells, Cultured; Collagen; Fibroblasts; Gene Expression Regulation; Humans; Molecular Sequence Data; Promoter Regions, Genetic; RNA, Messenger; Scleroderma, Systemic; Transcription, Genetic; Transfection; Transforming Growth Factor beta

The spontaneous increase in the transcription of the transforming growth factor beta (TGF beta) gene in bronchoalveolar mononuclear cells of patients with autoimmune diseases affecting the lung has been shown by northern blot assay and a nuclear run on transcription assay. Transcription of the TGF beta gene in bronchoalveolar mononuclear cells of patients with autoimmune diseases affecting the lung was increased 10 times compared with normal healthy subjects or patients with bronchial asthma as controls. This observation, confirmed by protein data, suggests that TGF beta, a potent mitogen for fibroblasts, may be produced in bronchoalveolar mononuclear cells during an active immune response in such patients and may be involved in autoimmune related changes of the pathophysiology of cytokine networks when the lung is affected, such as in lung fibrosis.

Topics: Blotting, Northern; Bronchoalveolar Lavage Fluid; Female; Fibrosis; Genetic Techniques; Humans; Lung; Lung Diseases; Lupus Erythematosus, Systemic; Male; Monocytes; RNA, Messenger; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

A characteristic feature of systemic scleroderma is fibrosis of the skin and eventually of internal organs resulting from an overproduction of collagen and other connective tissue components by the resident fibroblasts. The balance between the cells and the amount of the surrounding extracellular matrix is then altered. Because cellular metabolism depends to a large extent on cellular contacts and communications with connective tissue molecules, we have therefore investigated the interactions with extracellular matrix components of fibroblasts obtained from skin of patients affected with scleroderma. In comparison to fibroblasts from healthy skin, all fibroblasts from scleroderma patients had an increased adhesion capacity to collagens I, IV, VI, fibronectin, and laminin. In addition, whereas adhesion of control fibroblasts was stimulated by a pre-treatment with transforming growth factor-beta, adhesion patterns of scleroderma fibroblasts remained unchanged. However, pre-incubation of the cells with interferon-gamma decreased the adhesion of both scleroderma and control fibroblasts.

Topics: Cell Adhesion; Collagen; Connective Tissue; Female; Fibroblasts; Fibronectins; Fibrosis; Humans; In Vitro Techniques; Interferon-gamma; Male; Middle Aged; Scleroderma, Systemic; Transforming Growth Factor beta

Abnormal growth regulation in lesional skin fibroblasts may be related to scleroderma pathogenesis. We report on the abnormal response of cultured fibroblasts derived from sclerotic lesions to various growth factors. We investigated the responses of skin fibroblasts (10 strains) and normal fibroblasts (9 strains) to the growth factors as PDGF, TGF-beta 1, EGF and basic FGF. Experiments were conducted during the proliferation and confluent stages. PDGF, EGF and basic FGF stimulated fibroblast growth during the proliferation and confluent stages, but the response of scleroderma fibroblasts was significantly lower than that of normal fibroblasts. TGF-beta 1 slightly stimulated confluent fibroblast growth and inhibited proliferating fibroblasts, and the response of scleroderma fibroblasts exceeded that of normal fibroblasts. The decreased response to growth-stimulating factors observed in scleroderma fibroblasts suggests that cultured fibroblasts derived from scleroderma lesions were already senescent because they have been activated by growth-stimulating factors and repeatedly divided in vivo. Thus, abnormal growth regulation of skin fibroblasts may be partially related to the pathogenesis of scleroderma.

Topics: Adolescent; Adult; Aged; Cell Division; Cells, Cultured; Child; Epidermal Growth Factor; Female; Fibroblast Growth Factor 2; Fibroblasts; Growth Substances; Humans; Male; Middle Aged; Platelet-Derived Growth Factor; Scleroderma, Systemic; Transforming Growth Factor beta

The spontaneous elevation of the transcription of the transforming growth factor-beta (TGF-beta) gene in broncho-alveolar mononuclear cells (BMC) of individuals with autoimmune diseases with lung involvement, by nuclear run-on transcription assay, is shown in this study. In quantification analysis of TGF-beta gene transcription, we found more than 10 times the enhanced transcription of the TGF-beta gene in BMC of individuals with autoimmune diseases with lung involvement, in comparison to normal healthy subjects or patients with bronchial asthma used as controls. Our observation suggests that TGF-beta, a potent mitogen for fibroblasts, may be produced in BMC during an active immune response in individuals with systemic autoimmune diseases with lung involvement, and may be involved in autoimmune-related pathophysiological changes of cytokine networks in lung involvement such as lung fibrosis.

Topics: Autoimmune Diseases; Bronchoalveolar Lavage Fluid; Female; Humans; Leukocytes, Mononuclear; Lupus Erythematosus, Systemic; Male; Pulmonary Fibrosis; Scleroderma, Systemic; Transcription, Genetic; Transforming Growth Factor beta

Topics: Cell Line; Collagen; Humans; Lung; Scleroderma, Systemic; Transforming Growth Factor beta

To determine whether enhanced matrix synthesis by systemic sclerosis (SSc) fibroblasts in vitro is due to increased responsiveness to transforming growth factor-beta (TGF-beta), fibronectin release by SSc and normal fibroblasts (7 pairs) was measured at various concentrations of TGF-beta. In the absence of TGF-beta, SSc fibroblasts released 30 +/- 22% more fibronectin than normal fibroblasts. While both SSc and normal fibroblasts increased fibronectin release at all concentrations of TGF-beta tested, the percentage increases were not statistically greater for the SSc fibroblasts even though 4 of the SSc fibroblasts strains were selectively sensitive to low concentrations of TGF-beta. TGF-beta increased cell numbers of both SSc and normal strains equally. Our data confirm abnormal regulation of fibronectin gene expression in SSc fibroblasts and suggest increased sensitivity to TGF-beta by some SSc fibroblast strains.

Topics: Blotting, Western; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Extracellular Matrix; Fibroblasts; Fibronectins; Gene Expression Regulation; Humans; Scleroderma, Systemic; Skin; 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

The effects of sera and of platelet-poor plasma from patients with scleroderma on endothelial cell survival in vitro were studied. The survival ratio of rat heart endothelial cells was studied both in 10% test serum and in 10% platelet-poor plasma. Sera from patients with scleroderma decreased the survival ratio significantly when compared with sera from normal controls. In contrast, there was no significant difference between platelet-poor plasma from patients with scleroderma and that from normal controls. Our data indicate that platelets in the patients with scleroderma may cause vascular damage by affecting endothelial cell survival.

Topics: Adult; Aged; Animals; Antibodies, Antinuclear; Cell Survival; Cells, Cultured; Centromere; Endothelium, Vascular; Humans; Middle Aged; Neutralization Tests; Rats; Scleroderma, Systemic; Transforming Growth Factor beta