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Objective The goal of this study was to define a pharmacodynamic biomarker based on gene expression in skin that would provide a biological measure of disease extent in patients with diffuse cutaneous systemic sclerosis (dcSSc) and that could be used to monitor skin disease longitudinally. Methods Skin biopsies taken from a cohort of dcSSc patients that included longitudinal samples were analyzed by microarray. Expression of genes correlating with the modified Rodnan skin score (MRSS) were examined by nanostring for change over time, and a generalized estimating equation used to define and validate longitudinal, pharmacodynamic biomarkers composed of multiple genes. Results Microarray analysis of genes parsed to include only genes correlating with the MRSS revealed prominent clusters of profibrotic/TGFβ-regulated, IFN-regulated/proteasome, macrophage and vascular marker genes. Using genes changing longitudinally with the MRSS, two multigene, pharmacodynamic biomarkers were defined. The first was defined mathematically, applying a generalized estimating equation to longitudinal samples. This modeling method selected cross-sectional THBS1 and longitudinal THBS1 and MS4A4A genes. The second model was based on a weighted selection of genes, including additional genes with statistically significant change over time: CTGF, CD163, CCL2 and WIF1. Biomarker levels calculated using both models correlated highly with the MRSS in an independent validation dataset. Conclusion Skin gene expression can be used effectively to monitor SSc skin disease change over time. We have implemented these relatively simple models on a nanostring platform permitting highly reproducible assays that can be applied directly to samples from patients or collected as part of clinical trials.

Glucocorticoid (GC) receptors (GRs) have profound anti-survival effects on human small cell lung cancer (SCLC). To explore the basis of these effects, protein partners for GRs were sought using a yeast two-hybrid screen. We discovered a novel gene, FAM33A, subsequently identified as a SKA1 partner and involved in mitosis, and so renamed Ska2. We produced an anti-peptide antibody that specifically recognized full-length human SKA2 to measure expression in human cell lines and tissues. There was a wide variation in expression across multiple cell lines, but none was detected in the liver cell line HepG2. A xenograft model of human SCLC had intense staining and archival tissue revealed SKA2 in several human lung and breast tumours. SKA2 was found in the cytoplasm, where it co-localized with GR, but nuclear expression of SKA2 was seen in breast tumours. SKA2 overexpression increased GC transactivation in HepG2 cells while SKA2 knockdown in A549 human lung epithelial cells decreased transactivation and prevented dexamethasone inhibition of proliferation. GC treatment decreased SKA2 protein levels in A549 cells, as did Staurosporine, phorbol ester and trichostatin A; all agents that inhibit cell proliferation. Overexpression of SKA2 potentiated the proliferative response to IGF-I exposure, and knockdown with shRNA caused cells to arrest in mitosis. SKA2 has recently been identified in HeLa S3 cells as part of a complex, which is critical for spindle checkpoint silencing and exit from mitosis. Our new data show involvement in cell proliferation and GC signalling, with implications for understanding how GCs impact on cell fate.

Tissue injury triggers the activation and differentiation of multiple cell types to minimize damage and initiate repair processes. In systemic sclerosis, these repair processes appear to run unchecked, leading to aberrant remodeling and fibrosis of the skin and multiple internal organs, yet the fundamental pathological defect remains unknown. We describe herein a transition wherein the abundant CD34 þ dermal fibroblasts present in healthy human skin disappear in the skin of systemic sclerosis patients, and CD34 À , podoplanin þ , and CD90 þ fibroblasts appear. This transition is limited to the upper dermis in several inflammatory skin diseases, yet in systemic sclerosis, it can occur in all regions of the dermis. In vitro, primary dermal fibroblasts readily express podoplanin in response to the inflammatory stimuli tumor necrosis factor and IL-1b. Furthermore, we show that on acute skin injury in both human and murine settings, this transition occurs quickly, consistent with a response to inflammatory signaling. Transitioned fibroblasts partially resemble the cells that form the reticular networks in organized lymphoid tissues, potentially linking two areas of fibroblast research. These results allow for the visualization and quantification of a basic stage of fibroblast differentiation in inflammatory and fibrotic diseases in the skin. (Am J Pathol 2016, 186: 2650e2664; http://dx

Objective: The mechanisms that lead to endothelial cell (EC) injury and propagate the vasculopathy in Systemic Sclerosis (SSc) are not well understood. Using single cell RNA sequencing (scRNA-seq), our goal was to identify EC markers and signature pathways associated with vascular injury in SSc skin.Methods: We implemented single cell sorting and subsequent RNA sequencing of cells isolated from SSc and healthy control skin. We used t-distributed stochastic neighbor embedding (t-SNE) to identify the various cell types. We performed pathway analysis using Gene Set Enrichment Analysis (GSEA) and Ingenuity Pathway Analysis (IPA). Finally, we independently verified distinct markers using immunohistochemistry on skin biopsies and qPCR in primary ECs from SSc and healthy skin.Results: By combining the t-SNE analysis with the expression of known EC markers, we positively identified ECs among the sorted cells. Subsequently, we examined the differential expression profile between the ECs from healthy and SSc skin. Using GSEA and IPA analysis, we demonstrated that the SSc endothelial cell expression profile is enriched in processes associated with extracellular matrix generation, negative regulation of angiogenesis and epithelial-to-mesenchymal transition. Two of the top differentially expressed genes, HSPG2 and APLNR, were independently verified using immunohistochemistry staining and real-time qPCR analysis.Conclusion: ScRNA-seq, differential gene expression and pathway analysis revealed that ECs from SSc patients show a discrete pattern of gene expression associated with vascular injury and activation, extracellular matrix generation and negative regulation of angiogenesis. HSPG2 and APLNR were identified as two of the top markers of EC injury in SSc.