BackgroundScleroderma 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 FindingsWe 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 SignificanceGenome-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.

Systemic sclerosis is a complex disease with widespread skin fibrosis and variable visceral organ involvement. Since transforming growth factor-β (TGFβ) has been implicated in driving fibrosis in systemic sclerosis, a mechanism-derived gene expression signature was used to assay TGFβ-responsive gene expression in the skin of patients with systemic sclerosis (SSc). Primary dermal fibroblasts from patients with diffuse SSc (dSSc) and healthy controls were treated with TGFβ, and the genome-wide gene expression was measured on DNA microarrays over a time course of 24 hours. Eight hundred and ninety-four probes representing 674 uniquely annotated genes were identified as TGFβ responsive. Expression of the TGFβ-responsive signature was examined in skin biopsies from 17 dSSc, seven limited SSc (lSSc), three morphea patients, and six healthy controls. The TGFβ-responsive signature was expressed in 10 out of 17 dSSc skin biopsies, but was not found in lSSc, morphea, or healthy control biopsies. Expression of dSSC the TGFβ-responsive signature stratifies patients into two major groups, one of which corresponds to the “diffuse-proliferation” intrinsic subset that showed higher modified Rodnan skin score and a higher likelihood of scleroderma lung disease. The TGFβ-responsive signature is found in only a subset of dSSc patients who could be targeted by specific therapies.

Systemic sclerosis (SSc) is an autoimmune disease in which the tyrosine kinases platelet-derived growth factor receptor (PDGFR) and Abl are hypothesized to contribute to the fibrosis and vasculopathy of the skin and internal organs. Herein we describe 2 patients with early diffuse cutaneous SSc (dcSSc) who experienced reductions in cutaneous sclerosis in response to therapy with the tyrosine kinase inhibitor imatinib mesylate. Immunohistochemical analyses of skin biopsy specimens demonstrated reductions of phosphorylated PDGFR␤ and Abl with imatinib therapy. By gene expression profiling, an imatinib-responsive signature specific to dcSSc was identified (P < 10 ؊8 ). The response of these patients and the findings of the analyses suggest that PDGFR␤ and Abl play critical, synergistic roles in the pathogenesis of SSc, and that imatinib targets a gene expression program that is frequently dysregulated in dcSSc.

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-β 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-β mediates at least part of the effect of Tsk fibrillin on CCN3 and consistent with a synergistic effect of TGF-β 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 TGFβ- and Wnt-regulated profibrotic gene expression. Together these data indicate that CCN3 counter-regulates positive signals from TGF-β and Wnt for fibrillin fibrillogenesis and profibrotic gene expression.