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 (n1⁄4 6) and control skin (n1⁄4 6) fibroblasts was assessed by western blotting. Epidermis was also harvested from healthy (n1⁄45) and SSc (n1⁄49) 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.
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