The fibrotic phenotype of systemic sclerosis fibroblasts varies with disease duration and severity of skin involvement: reconstitution of skin fibrosis development using a tissue engineering approach (original) (raw)
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The Journal of Pathology
We set out to examine the pathophysiological mechanisms of fibrosis in diffuse systemic sclerosis (SSc) using a tissue engineering approach. Skin fibroblasts were isolated from lesional skin of SSc patients with a disease duration of less than 1 year (early-stage SSc) or more than 10 years (late-stage SSc). Fibroblasts were also isolated from non-lesional skin and compared with normal fibroblasts isolated from healthy adults. Cells were cultured using a tissue engineering method to reconstruct a human dermis, and histologically observed. Dermal thickness was measured, as it reflects the global and intrinsic capacity of cells to reconstitute matrix. Collagen I, MMP-1, and MMP activity were evaluated. Cells were treated with TGFβ1 or CTGF during dermis formation to study their fibrogenic role. Clinical severity of skin involvement was measured by a modified Rodnan score. Thickness of the dermis generated with non-lesional early-stage SSc fibroblasts was similar to normal cells. In contrast, reconstructed dermis from lesional early-stage SSc fibroblasts and nonlesional late-stage SSc cells was thinner, while lesional late-stage SSc fibroblasts made a thicker dermis. Dermis was always thicker when produced with TGFβ1-treated cells, except when lesional late-stage SSc fibroblasts from patients with high Rodnan skin scores were used. CTGF did not affect dermal thickness. Measurements of collagen I and collagenases in the culture medium of the various reconstructed dermis could explain some of the changes observed. We conclude that the fibrotic phenotype of SSc fibroblasts varies with disease duration and with severity of skin involvement, and this is clearly visualized during in vitro dermis reconstruction.
Journal of Clinical Investigation, 1994
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-ft 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-,6 in the skin of patients with SSc.
Rheumatology, 2013
Background: SSc is an autoimmune disease characterized by progressive fibrosis of the skin and internal organs. Development of an activated population of fibroblasts and myofibroblasts in lesional tissue is likely to be central to pathogenesis. In this study, we identify stem cell factor (SCF) as a potential driving factor in this process and future therapeutic target. SCF is a cytokine which acts via c-Kit a tyrosine kinase receptor. In a phosphorylation array analysis of migrating fibroblasts c-Kit was found to be upregulated. Our aim was to examine the role of SCF in the migration and proliferation of fibroblasts in SSc and healthy controls. Methods: Primary skin fibroblast lines were cultured in vitro with varying concentrations of SCF, or neutralizing antibodies directed against its receptor c-Kit or SCF itself. Proliferation was assessed using the WST-1 assay and by direct cell counting. Migration was assessed using the scratch wound assay and area of wound invasion measured following treatment with either SCF or anti-Kit antibodies. The expression and quantity of SCF and c-Kit by SSc (n ¼ 6) and control skin (n ¼ 6) fibroblasts was assessed by western blotting. Epidermis was also harvested from healthy (n ¼ 5) and SSc (n ¼ 9) skin using the blister technique. SCF gene expression was assessed using quantitative PCR (qPCR). Results: SCF expression was enhanced in SSc epidermis samples by qPCR as compared with healthy controls (566 vs 336 copy numbers respectively). In the Western of fibroblasts, both c-KIT and SCF were strongly present in SSc skin derived fibroblasts and hardly detectable in healthy controls (normalized band intensity 2.14 vs 0.03 for c-KIT and 1.81 vs 0.07 in SSc vs normal skin respectively P < 0.002 in both). Human recombinant SCF (rSCF) promoted the migration of normal human fibroblasts. At both 24 and 48 h, cells treated with rSCF showed a much greater percentage wound coverage as compared treatment with media only-with greatest migration at the rSCF concentration of 2.5 ng/ml (wound invasion 86% compared with 52% at 48 h P < 0.002). Conversely, treatment with antibody to c-KIT resulted in a reduction of wound invasion (invasion 19% compared with 62% P < 0.04). Addition of rSCF to normal fibroblast cell induced proliferation as measured by the WST-1 assay (13% increase, not statistically significant). Conversely, adding anti-Kit or anti-SCF neutralizing antibodies to cell culture medium resulted in reduction of proliferation (30% reduction, P < 0.05 and 20% reduction P < 0.029 respectively). Conclusions: SCF is found at higher levels in the skin of SSc subjects compared with controls. We have demonstrated that in vitro, SCF promotes proliferation and migration of fibroblasts and that its blockade using specific antibodies results in reduction in both these processes. SCF appears to be a potential therapeutic target for future treatment in SSc. Disclosures: The authors have declared no conflicts of interest.
Skin involvement in scleroderma--where histological and clinical scores meet
Rheumatology, 2007
Objectives. A clinico-pathological study in diffuse systemic sclerosis (SSc) patients was performed to analyse whether the skin histological organization and the pro-fibrotic signals elicited by TGF-in fibroblasts vary according to the modified Rodnan skin score (mRSS). Methods. Twenty-seven SSc patients underwent 45 skin biopsies with simultaneous measure of mRSS before or after treatment by immunosuppressive drugs, with or without autologous peripheral haematopoietic stem cell transplantation (HSCT). Results. Double-blind optic microscopy analysis of the biopsies standard extracellular matrix stains allowed to define three histological subgroups: 6 with grade 1 weak fibrosis, 30 with grade 2 moderate fibrosis and 9 with grade 3 severe fibrosis. A significant correlation (P < 0.0001) was identified between the grades of fibrosis and the mRSS. In skin fibroblast cultures, Smad3 phosphorylation levels, as well as mRNA steady-state levels of two transforming growth factor (TGF)-/Smad3 targets, COL1A2 and PAI-1, increased in parallel with the mRSS. When compared with pre-transplant values the degree of fibrosis observed after HSCT in the papillary and in the reticular dermis decreased in parallel with the fall in mRSS (n ¼ 5 consecutive patients with repeated biopsies). Conclusions. The histological extent of skin fibrosis correlates closely with the mRSS. Both parameters appeared to regress after HSCT. The extent of TGF-signalling activation in SSc skin fibroblasts appears to parallel the severity of disease.
Biochemical and Biophysical Research Communications, 1992
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 (COLIA2/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 TGFgl. Analysis of the expression of COL1A2 promoter deletion constructs indicates that the TGFII 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 TGFI~ dependent pathway. , 1992 Acaaemic P ..... ~no. Scleroderma (systemic sclerosis; SSc) is a disease characterized by excessive deposition of connective tissue in the dermis, blood vessels and internal organs, in which lesional fibroblasts can be shown to produce increased quantities of extracellular matrix components on a per cell basis even after removal from the patient and propagation in vitro (1,2). Specifically, the increased synthesis of various collagen polypeptides is correlated with increased levels of corresponding mRNAs (3-5), at least in part due to increased transcription demonstrated in the fibroblasts of lesions of patients with localized scleroderma by run off assays for collagen c~2(I) (6). In addition to elevated synthesis of extracellular matrix components, SSc fibroblasts exhibit altered growth regulation, responding preferentially to a combination of TGFfl and PDGF AA due to a distinct upregulation of PDGFt~ receptors (7). Normal skin (NS) fibroblasts respond preferentially to bFGF (8). Though the mechanism of activation of SSc fibroblasts is unknown, it is currently believed that immune cells (possibly antigen-driven autoimmune T-cells) participate in tissue injury and Abbreviations used: SSc = systemic sclerosis (scleroderma); NS = normal skin; TGFi]l=one of several isoforms of transforming growth factor beta; COL1A2 = The gene expressing the alpha two chain of type I collagen; CAT = chloramphenicol acetyltransferase.
Proliferation and collagen synthesis by human dermal fibroblasts cultured from different body sites
Journal of Dermatological Science, 1991
Distal areas in systemic sclerosis (scleroderma), such as the dorsal skin of the hand are more frequently involved and more indurated than proximal areas. On the contrary, the observation often made after surgical excision or trauma is that distal body areas heal more slowly than proximal areas. A possible explanation may be that dermal tibroblasts from distal body parts are more capable, when stimulated, to synthesize greater or lesser amounts of collagen and proliferate at different rates than dermal fibroblasts from more proximal skin. In this study, cultures of dermal fibroblasts from three different body sites (arm, forearm, and hand) of healthy volunteers were investigated for their proliferative activity and collagen synthesis after stimulation in 3% or 10% fetal bovine serum. No significant differences were observed in cell proliferation or in the relative or absolute collagen synthesis by fibroblasts cultured from the hand, forearm or upper arm. We conclude that other in vivo factors are responsible for the observed differences in fibrosis and healing at different body sites. Moreover, if clonal expansion of different fibroblast phenotypes occurs in these physiologic or disease states, it must be of a magnitude that overcomes the fundamental proliferative and biosynthetic baseline.
N-terminal connective tissue growth factor is a marker of the fibrotic phenotype in scleroderma
QJM, 2005
Background: Over-expression of connective tissue growth factor (CTGF) is a hallmark of fibrotic disease, including scleroderma. CTGF acts with the pro-fibrotic cytokine TGFb to promote sustained fibrotic responses in vivo. Elevated production of CTGF might be responsible for maintenance of the fibrotic phenotype in scleroderma. Assays of CTGF or of its fragments are potential non-invasive measures of the fibrotic response in scleroderma. Aim: To determine the utility of whole, N-terminal, and C-terminal CTGF as surrogate markers for fibrosis in scleroderma. Design: Cross-sectional controlled study. Methods: Plasma was collected prospectively from 47 scleroderma patients (26 diffuse scleroderma, 21 limited scleroderma) and 18 healthy controls. At the same time, dermal interstitial fluid was derived by a suction blister technique from the lesional skin of scleroderma patients, and from the forearm skin of healthy controls. Whole, N-terminal, and C-terminal CTGF were assayed by ELISA, using monoclonal antibodies specific for N-and C-terminal epitopes. Results: N-terminal cleavage products of CTGF were present at elevated levels in the plasma and dermal interstitial fluid of scleroderma patients, compared to healthy controls. N-terminal CTGF levels in plasma and dermal interstitial fluid correlated with severity of skin disease and (negatively) with disease duration. Whole and C-terminal CTGF levels were low in blister fluid and plasma levels were not elevated in disease. Discussion: These results support a role for CTGF in scleroderma-associated fibrosis and the utility of N-terminal CTGF as a marker of fibrosis.
Arthritis and Rheumatism, 2003
ObjectiveIn systemic sclerosis (SSc; scleroderma), T cells infiltrate organs undergoing fibrotic changes and may participate in dysregulated production of collagen by fibroblasts. The objective of this study was to functionally characterize T cells infiltrating skin lesions in early SSc and investigate their capacity to affect production of type I collagen and interstitial collagenase (matrix metalloproteinase 1 [MMP-1]) by dermal fibroblasts.In systemic sclerosis (SSc; scleroderma), T cells infiltrate organs undergoing fibrotic changes and may participate in dysregulated production of collagen by fibroblasts. The objective of this study was to functionally characterize T cells infiltrating skin lesions in early SSc and investigate their capacity to affect production of type I collagen and interstitial collagenase (matrix metalloproteinase 1 [MMP-1]) by dermal fibroblasts.MethodsFour-color cytometric analysis was used to characterize subset distribution and production of interferon-γ (IFNγ) and interleukin-4 (IL-4) in T cell lines generated from the skin of patients with SSc. T cell clones were generated, and their capacity to modulate collagen and MMP-1 production by fibroblasts derived from patients with SSc and from normal individuals was assessed. Neutralizing reagents were used to identify T cell mediators involved in fibroblast modulation.Four-color cytometric analysis was used to characterize subset distribution and production of interferon-γ (IFNγ) and interleukin-4 (IL-4) in T cell lines generated from the skin of patients with SSc. T cell clones were generated, and their capacity to modulate collagen and MMP-1 production by fibroblasts derived from patients with SSc and from normal individuals was assessed. Neutralizing reagents were used to identify T cell mediators involved in fibroblast modulation.ResultsThe skin of individuals with early-stage SSc contained T cells preferentially producing high levels of IL-4. Cloned CD4+ Th2-like cells inhibited collagen production by normal fibroblasts. Th2 cell-dependent inhibition was, at least in part, contact-dependent, was essentially mediated by tumor necrosis factor α (TNFα), and was dominant over the enhancement induced by profibrotic IL-4 and transforming growth factor β cytokines. The simultaneous induction of MMP-1 production confirmed the specificity of these observations. To be inhibitory, Th2 cells required activation by CD3 ligation. Th2 cells were less potent than were Th1 cells in inhibiting collagen production by normal fibroblasts via cell-to-cell interaction, and SSc fibroblasts were resistant to inhibition.The skin of individuals with early-stage SSc contained T cells preferentially producing high levels of IL-4. Cloned CD4+ Th2-like cells inhibited collagen production by normal fibroblasts. Th2 cell-dependent inhibition was, at least in part, contact-dependent, was essentially mediated by tumor necrosis factor α (TNFα), and was dominant over the enhancement induced by profibrotic IL-4 and transforming growth factor β cytokines. The simultaneous induction of MMP-1 production confirmed the specificity of these observations. To be inhibitory, Th2 cells required activation by CD3 ligation. Th2 cells were less potent than were Th1 cells in inhibiting collagen production by normal fibroblasts via cell-to-cell interaction, and SSc fibroblasts were resistant to inhibition.ConclusionThese findings indicate that, despite their production of IL-4, Th2 cells reduce type I collagen synthesis by dermal fibroblasts because of the dominant effect of TNFα, and suggest that strategies based on TNFα blockade aimed at controlling fibrosis in SSc may be unwise.These findings indicate that, despite their production of IL-4, Th2 cells reduce type I collagen synthesis by dermal fibroblasts because of the dominant effect of TNFα, and suggest that strategies based on TNFα blockade aimed at controlling fibrosis in SSc may be unwise.
Annals of the New York Academy of Sciences, 1990
Systemic sclerosis (scleroderma) is characterized by vascular damage and fibrosis of skin and internal organs. Its pathogenesis remains unknown. Morphologic findings in the initial phases of the disease are endothelial cell damage and lymphocytic infiltrates in the dermis and subcutaneous tissue. Over the course of months or years, the dermis and subcutaneous tissues are replaced by a relatively avascular band of sclerosis, consisting mainly of collagen but also of other matrix proteins. Especially before the excessive accumulation of collagen, one can observe deposition of glycosaminoglycans (GAG) in the dermis and in and around blood vessels. Most patients have Raynaud's phenomenon, which refers to the pain and skin color changes in the hands and feet as a result of vascular spasm and intimal thickening. In the full expression of the disease, the clinical picture is one of hand contractures, extremely indurated skin predominantly over the hands, forearms, and face, and varying degrees of internal organ fibrosis, affecting the function of the gastrointestinal tract, the lung, the heart, and the kidney.' A unifying hypothesis for the fibrosis would favor soluble factors from plasma or platelets that, by stimulating fibroblasts, would lead to excessive deposition of connective tissue. However, no definite conclusions that prove or disprove this hypothesis have been reached? Cytokines derived from mononuclear cells have shown both stimulatory and inhibitory effects on macromolecular synthesis of fibroblasts cultured from scleroderma skin. One study showed scleroderma fibroblasts to be insensitive to optimal mitogenic doses of platelet-derived growth factor, but not to epidermal growth factor, fibroblast growth factor, or nerve growth factor? These findings led the authors to suggest that the relative insensitivity of scleroderma fibroblasts to platelet-derived growth factor was the result of their in viva exposure and already maximal response to this peptide. We have shown that E F-p l causes a selectively greater increase in GAG synthesis ' Supported by a grant from the Scleroderma Research Foundation.