Project 1: Systemic Sclerosis Skin Biomarkers & Therapeutics

目前，系统性硬化症（SSc）患者的治疗选择仍然非常有限。 药效学生物标志物基因，如THBS 1和COMP，在我们以前的CORT项目中开发， 被证明对评估新疗法的疗效很有价值。寻找预后生物标志物越来越多 这对于决定哪些患者应该接受毒性治疗非常重要。此外，与以下相关的基因/蛋白质 疾病进展（预后生物标志物）可能驱动疾病。我们最近表明，COMP和 SPP 1、CTGF和SERPINE 1是进行性SSc相关间质性肺病（ILD）的标志物， 皮肤病，分别。这些基因由间充质细胞表达，并在体外被 转化生长因子β使用单细胞RNA-seq，在初步结果中，我们显示了健康皮肤中的转录组模块， 间充质细胞，包括周细胞和成纤维细胞的子集，和SSc皮肤鉴定出不同的 在肌成纤维细胞上独特表达的转录组。我们认为一个或多个间充质细胞 在正常皮肤中的类型是SSc皮肤中肌成纤维细胞的祖细胞。肌成纤维细胞转录组中的基因 包括ADAM 12、ITGA 11以及SSc皮肤病生物标志物，如THBS 1、COMP、CTGF和 SERPINE 1.因此，进行性SSc皮肤病的生物标志物主要由肌成纤维细胞表达。Adam12 和ITGA 11，在被鉴定为肌成纤维细胞的细胞上表达，可能调节这些细胞的表型， 在小鼠中，ITGA 11的缺失显著抑制了创伤后肌成纤维细胞的积累，而ADAM 12是一种抑制肌成纤维细胞增殖的基因。 在小鼠中分化成肌成纤维细胞的周细胞祖细胞的标志物。我们的初步数据 支持该提议单一假设，即选择生物标记基因驱动分化 是一种间充质细胞类型，负责SSc皮肤和肺部的纤维化。我们将采取 在我们的实验室开发的独特资源的优势，研究数以千计的单细胞转录组， 皮肤，结合这一点与特殊的访问与早期弥漫性皮肤SSc患者提供的 UPMC硬皮病中心。我们提出了三个目标来评估这一假设。首先，我们将确定 SSc皮肤和SSc-ILD中进行性纤维化的生物标志物。我们将识别和验证预后皮肤 dcSSc皮肤病的mRNA生物标志物和使用Somascan蛋白质组学鉴定的验证血清生物标志物 渐进式SSc-ILD技术。第二，我们将研究单细胞转录组和 真皮间充质细胞之间的差异。此外，我们将比较定量和 dcSSc患者与健康对照间充质细胞转录组的质变化 并鉴定促纤维化成纤维细胞和肌成纤维细胞祖细胞。第三，我们将研究TGFβ的作用， 通过检测TGFβ诱导成肌纤维细胞分化的动力学研究ADAM 12和ITGA 11对成肌纤维细胞分化的调节作用 肌成纤维细胞转录组，通过鉴定肌成纤维细胞祖细胞，并通过测试 抑制ADAM 12和ITGA 11对肌成纤维细胞分化和转录组的影响。

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.