Molecular subsets in the gene expression signatures of scleroderma skin

Background

Scleroderma is a clinically heterogeneous disease with a complex phenotype. The disease is characterized by vascular dysfunction, tissue fibrosis, internal organ dysfunction, and immune dysfunction resulting in autoantibody production.

Methodology and Findings

We analyzed the genome-wide patterns of gene expression with DNA microarrays in skin biopsies from distinct scleroderma subsets including 17 patients with systemic sclerosis (SSc) with diffuse scleroderma (dSSc), 7 patients with SSc with limited scleroderma (lSSc), 3 patients with morphea, and 6 healthy controls. 61 skin biopsies were analyzed in a total of 75 microarray hybridizations. Analysis by hierarchical clustering demonstrates nearly identical patterns of gene expression in 17 out of 22 of the forearm and back skin pairs of SSc patients. Using this property of the gene expression, we selected a set of ‘intrinsic’ genes and analyzed the inherent data-driven groupings. Distinct patterns of gene expression separate patients with dSSc from those with lSSc and both are easily distinguished from normal controls. Our data show three distinct patient groups among the patients with dSSc and two groups among patients with lSSc. Each group can be distinguished by unique gene expression signatures indicative of proliferating cells, immune infiltrates and a fibrotic program. The intrinsic groups are statistically significant (p<0.001) and each has been mapped to clinical covariates of modified Rodnan skin score, interstitial lung disease, gastrointestinal involvement, digital ulcers, Raynaud''s phenomenon and disease duration. We report a 177-gene signature that is associated with severity of skin disease in dSSc.

Conclusions and Significance

Genome-wide gene expression profiling of skin biopsies demonstrates that the heterogeneity in scleroderma can be measured quantitatively with DNA microarrays. The diversity in gene expression demonstrates multiple distinct gene expression programs in the skin of patients with scleroderma.     

Establishment and characterization of scleroderma fibroblast clonal cell lines by introduction of the hTERT gene

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‐βRI indicative of altered TGF‐β 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.     

Levels of adiponectin,a marker for PPAR-gamma activity,correlate with skin fibrosis in systemic sclerosis: potential utility as biomarker?

Introduction

Progressive fibrosis in systemic sclerosis (SSc) is linked to aberrant transforming growth factor beta (TGF-beta) signaling. Peroxisome proliferator-activated receptor gamma (PPAR-gamma) blocks fibrogenic TGF-beta responses in vitro and in vivo. Reduced expression and function of PPAR-gamma in patients with SSc may contribute to progression of fibrosis. Here we evaluated the levels of adiponectin, a sensitive and specific index of PPAR-gamma activity, as a potential fibrogenic biomarker in SSc.

Methods

Adiponectin levels were determined in the sera of 129 patients with SSc and 86 healthy controls, and serial determinations were performed in 27 patients. Levels of adiponectin mRNA in skin biopsies from SSc patients were assessed in an expression profiling microarray dataset. Regulation of adiponectin gene expression in explanted human subcutaneous preadipocytes and fibroblasts was examined by real-time quantitative PCR.

Results

Patients with diffuse cutaneous SSc had reduced serum adiponectin levels. A significant inverse correlation between adiponectin levels and the modified Rodnan skin score was observed. In longitudinal studies changes in serum adiponectin levels were inversely correlated with changes in skin fibrosis. Skin biopsies from a subset of SSc patients showed reduced adiponectin mRNA expression which was inversely correlated with the skin score. An agonist ligand of PPAR-gamma potently induced adiponectin expression in explanted mesenchymal cells in vitro.

Conclusions

Levels of adiponectin, reflecting PPAR-gamma activity, are correlated with skin fibrosis and might have potential utility as a biomarker in SSc.     

Chronic Toll-like receptor 4 stimulation in skin induces inflammation,macrophage activation,transforming growth factor beta signature gene expression,and fibrosis

Introduction

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.

Methods

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.

Results

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.

Conclusions

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.     

Cartilage oligomeric matrix protein is overexpressed by scleroderma dermal fibroblasts.

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.     

TGF-β-mediated downregulation of microRNA-196a contributes to the constitutive upregulated type I collagen expression in scleroderma dermal fibroblasts

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.     

Cutaneous Chronic Graft-Versus-Host Disease Does Not Have the Abnormal Endothelial Phenotype or Vascular Rarefaction Characteristic of Systemic Sclerosis

Background

The clinical and histologic appearance of fibrosis in cutaneous lesions in chronic graft-versus -host disease (c-GVHD) resembles the appearance of fibrosis in scleroderma (SSc). Recent studies identified distinctive structural changes in the superficial dermal microvasculature and matrix of SSc skin. We compared the dermal microvasculature in human c-GVHD to SSc to determine if c-GVHD is a suitable model for SSc.

Methodology/Principal Findings

We analyzed skin biopsies of normal controls (n = 24), patients with SSc (n = 30) and c-GVHD with dermal fibrosis (n = 133)). Immunostaining was employed to identify vessels, vascular smooth muscle, dermal matrix, and cell proliferation. C-GVHD and SSc had similar dermal matrix composition and vascular smooth muscle pathology, including intimal hyperplasia. SSc, however, differed significantly from c-GVHD in three ways. First, there were significantly fewer (p = 0.00001) average vessels in SSc biopsies (9.8) when compared with c-GVHD (16.5). Second, in SSc, endothelial markers were decreased significantly (19/19 and 12/14 for VE cadherin and vWF (p = <0.0001 and <0.05), respectively). In contrast, 0/13 c-GVHD biopsies showed loss of staining with canonical endothelial markers. Third, c-GVHD contained areas of microvascular endothelial proliferation not present in the SSc biopsies.

Conclusions/Significance

The sclerosis associated with c-GVHD appears to resemble wound healing. Focal capillary proliferation occurs in early c-GVHD. In contrast, loss of canonical endothelial markers and dermal capillaries is seen in SSc, but not in c-GVHD. The loss of VE cadherin in SSc, in particular, may be related to microvascular rarefaction because VE cadherin is necessary for angiogenesis. C-GVHD is a suitable model for studying dermal fibrosis but may not be applicable for studying the microvascular alterations characteristic of SSc.     

mPGES-1 null mice are resistant to bleomycin-induced skin fibrosis

Introduction

Microsomal prostaglandin E2 synthase-1 (mPGES-1) is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H₂ to PGE₂. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrogenesis is largely unknown. Herein, we examine the role of mPGES-1 in a mouse model of skin scleroderma using mice deficient in mPGES-1.

Methods

Wild type (WT) and mPGES-1 null mice were subjected to the bleomycin model of cutaneous skin scleroderma. mPGES-1 expressions in scleroderma fibroblasts and in fibroblasts derived from bleomycin-exposed mice were assessed by Western blot analysis. Degree of fibrosis, dermal thickness, inflammation, collagen content and the number of α-smooth muscle actin (α-SMA)-positive cells were determined by histological analyses. The quantity of the collagen-specific amino acid hydroxyproline was also measured.

Results

Compared to normal skin fibroblasts, mPGES-1 protein expression was elevated in systemic sclerosis (SSc) fibroblasts and in bleomycin-exposed mice. Compared to WT mice, mPGES-1-null mice were resistant to bleomycin-induced inflammation, cutaneous thickening, collagen production and myofibroblast formation.

Conclusions

mPGES-1 expression is required for bleomycin-induced skin fibrogenesis. Inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control the onset of fibrogenesis.     

Estradiol promotes the development of a fibrotic phenotype and is increased in the serum of patients with systemic sclerosis

Introduction

Systemic sclerosis (SSc) is more prevalent in women. Our goal is to determine the effects of 17β-estradiol (E2) on the development of fibrosis and to compare circulating levels of estrogens in SSc patients and healthy controls.

Methods

Using primary human dermal fibroblasts, we evaluated the effect of E2 on fibronectin (FN) expression with and without the estrogen receptor (ER) antagonist ICI 182,780, inhibitors of signaling, propyl-pyrazole-triol, an ERα specific ligand, and genistein, an ERβ selective ligand, to identify the signaling pathways mediating E2''s effect. We confirmed the fibrotic effect of E2 in human skin using an ex vivo organ culture model. Lastly, we measured levels of E2 and estrone in serum samples from SSc patients with diffuse cutaneous involvement and healthy controls using mass spectrometry.

Results

E2 increased expression of FN in dermal fibroblasts. ICI 182,780, inositol-1,4,5-triphosphate inhibitor, and p38 mitogen-activated protein kinase inhibitor blocked the effects of E2 on FN. Propyl-pyrazole-triol, but not genistein, significantly increased FN expression. Ex vivo, E2 induced fibrosis of human skin. The effects of E2 were abrogated by ICI 182,780. Circulating levels of E2 and estrone were significantly increased in sera of patients with diffuse cutaneous SSc.

Conclusion

Our findings implicate estrogens in the fibrotic process and may explain the preponderance of SSc in women. ICI 182,780 or other ER signaling antagonists may be effective agents for the treatment of fibrosis.     

Tumor necrosis factor alpha suppresses the induction of connective tissue growth factor by transforming growth factor-beta in normal and scleroderma fibroblasts

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.     

Impaired IL-17 signaling pathway contributes to the increased collagen expression in scleroderma fibroblasts

Among IL-17 families, IL-17A and IL-17F share amino acid sequence similarity and bind to IL-17R type A. IL-17 signaling is implicated in the pathogenesis of various autoimmune diseases, but its role in the regulatory mechanism of extracellular matrix expression and its contribution to the phenotype of systemic sclerosis (SSc) both remain to be elucidated. This study revealed that IL-17A expression was significantly increased in the involved skin and sera of SSc patients, whereas the IL-17F levels did not increase. In contrast, the expression of IL-17R type A in SSc fibroblasts significantly decreased in comparison with that in normal fibroblasts, due to the intrinsic TGF-β1 activation in these cell types. Moreover, IL-17A, not IL-17F, reduced the protein expression of α1(I) collagen and connective tissue growth factor. miR-129-5p, one of the downregulated microRNAs in SSc fibroblasts, increased due to IL-17A and mediated the α1(I) collagen reduction. These results suggest that IL-17A signaling, not IL-17F, has an antifibrogenic effect via the upregulation of miR-129-5p and the downregulation of connective tissue growth factor and α1(I) collagen. IL-17A signaling is suppressed due to the downregulation of the receptor by the intrinsic activation of TGF-β1 in SSc fibroblasts, which may amplify the increased collagen accumulation and fibrosis characteristic of SSc. Increased IL-17A levels in the sera and involved skin of SSc may be due to negative feedback. Clarifying the novel regulatory mechanisms of fibrosis by the cytokine network consisting of TGF-β and IL-17A may lead to a new therapeutic approach for this disease.