Currently there remain very limited treatment options for patients with systemic sclerosis (SSc). Pharmacodynamic biomarker genes, such as THBS1 and COMP, developed in our previous CORT project, have proven valuable for assessing the efficacy of new therapies. Finding prognostic biomarkers is increasingly important for deciding which patients to treat with toxic therapies. In addition, genes/proteins associated with disease progression (prognostic biomarkers) likely drive disease. We have recently shown that COMP and SPP1, and CTGF and SERPINE1 are markers of progressive SSc associated interstitial lung disease (ILD)and skin disease, respectively. These genes are expressed by mesenchymal cells and are upregulated in vitro by TGFβ. Using single cell RNA-seq, in preliminary results we show transcriptome modules in healthy skin for mesenchymal cells, including subsets of pericytes and fibroblasts, and SSc skin identified a distinct transcriptome expressed uniquely on myofibroblasts. We propose that one or more of the mesenchymal cell types in normal skin are progenitors of myofibroblasts in SSc skin. Genes in this myofibroblast transcriptome include ADAM12, ITGA11, as well as biomarkers of SSc skin disease, such as THBS1, COMP, CTGF and SERPINE1. Thus, biomarkers of progressive SSc skin disease are expressed mainly by myofibroblasts. ADAM12 and ITGA11, expressed on cells identified as myofibroblasts, likely regulate the phenotype of these cells, as deletion of ITGA11 in mice markedly inhibits myofibroblast accumulation after wounding, and ADAM12 is a marker of pericyte progenitors that in mice differentiate into myofibroblasts. Our preliminary data support a singular hypothesis of this proposal that select biomarker genes drive differentiation of a mesenchymal cell type that is responsible for fibrosis in SSc skin and lungs. We will take advantage of unique resources developed in our laboratory to study thousands of single cell transcriptomes in skin, combining this with the exceptional access to patients with early diffuse cutaneous SSc provided by the UPMC Scleroderma Center. We propose three aims to evaluate this hypothesis. First, we will identify biomarkers of progressive fibrosis in SSc skin and SSc-ILD. We will identify and validate prognostic skin mRNA biomarkers of dcSSc skin disease and validate serum biomarkers identified using Somascan proteomic technology of progressive SSc-ILD. Second, we will investigate single cell transcriptomes and the relationship between dermal mesenchymal cells in normal and SSc skin. Further, we will compare quantitative and qualitative changes of the transcriptomes of mesenchymal cells from patients with dcSSc to healthy controls and identify profibrotic fibroblast and myofibroblast progenitors. Third, we will study the roles of TGFβ, ADAM12 and ITGA11 on regulating myofibroblast differentiation by testing the kinetics of TGFβ on inducing the myofibroblast transcriptome, by identifying myofibroblast progenitor cells, and by testing the effect of inhibiting ADAM12 and ITGA11 on myofibroblast differentiation and transcriptome.