Discovering Extracellular Modulators of Lung Fibrogenesis by Profiling Newly Synthesized Extracellular Matrix

项目摘要 间质性肺病（ILD）是引起肺组织进行性瘢痕形成（即纤维化）的破坏性疾病， 并由细胞异常和细胞外基质（ECM）失调的相互作用引起。 目前的ILD治疗主要针对细胞信号传导，仍然无法停止或逆转纤维化。 尽管ECM是ILD的关键病理标志，但很少直接靶向治疗 干预从根本上说，肺纤维化进展的细胞外机制仍然难以捉摸。 为了弥补这一差距，我们的目标是开发一种创新的方法， 合成ECM（newsECM）沿着肺纤维化。拟议中的技术将有选择地标记 newsECM生产在定义的短时间跨度通过纳入化学选择性叠氮基标签通过后- 翻译糖基化这将使newsECM从预先存在的ECM中解放出来，从而 能够以前所未有的每日时间对动态ECM合成进行灵敏的蛋白质组学检测， 解决方案，无论丰富的预先存在的ECM背景。我们提出的方法具有内在的 优选ECM蛋白，其中大多数是糖基化的。建议的newsECM分析将 解决了传统的非选择性质谱分析中的主要技术障碍， 这是因为在检测通常丰度低的动态新ECM沉积时灵敏度有限。的 ECM分析技术是通用的，将在三个肺纤维化模型中实施， 包括体内小鼠纤维化模型、离体供体肺灌注模型和体外合成纤维化模型。 模型我们打算实现以下具体目标。Aim 1将在体内追踪新闻ECM动态 肺纤维化的进展和解决，这有望揭示促纤维化和抗纤维化ECM因子。 目的2将使用小鼠肺作为实验材料，建立体内和体外newsECM谱之间的相关性。 模型，并将得到的优化的newsECM分析条件和算法应用于离体肺灌注 本发明提供了携带特发性肺纤维化（IPF）（ILD的最常见形式）的供体人肺的EVLP（EVLP）， 揭示了人类特异性的ECM致病机制。目的3建立人工合成肺纤维化模型 在脱细胞的天然细胞内结合成纤维细胞、上皮细胞、内皮细胞和巨噬细胞的组织工程 肺ECM支架，并将其用作实验上易于处理的系统以进一步解码纤维化肺细胞-ECM 互动此外，结合newsECM标记和天然ECM生物材料工程，我们将提供一个 用于有效功能评估候选纤维化调节ECM因子的化学选择性平台 一种仿生的ECM相关的方式。总之，这项研究将促进ILD的范式转变 通过促进ECM靶向治疗的发展和通过使联合治疗 针对细胞和细胞外靶点的干预措施，为受影响的患者带来新的希望。

Project Summary Interstitial lung diseases (ILD) are devastating disorders causing progressive scarring (i.e. fibrosis) of lung tissue, and result from reciprocal interactions of cellular abnormalities and extracellular matrix (ECM) dysregulation. Current ILD treatment primarily targets cellular signaling and remains unable to halt or reverse fibrogenesis. Despite being a key pathological hallmark of ILD, the ECM has rarely been directly targeted for therapeutic intervention. Fundamentally, the extracellular mechanism underlying lung fibrosis progression remains elusive. To bridge this gap, our objective is to develop an innovative approach for selective profiling of newly synthesized ECM (newsECM) along lung fibrogenesis. The proposed technology will selectively label newsECM produced over defined short time spans by incorporating chemoselective azido-tags via post- translational glycosylation. This will enable enrichment of newsECM free from the pre-existing ECM, and thereby enables sensitive proteomic detection of the dynamic ECM synthesis with unprecedented daily temporal resolution, irrespective of the abundant pre-existing ECM background. Our proposed approach has an inherent preference to ECM proteins, the majority of which are glycosylated. The proposed newsECM profiling will address a major technical barrier in conventional, non-selective mass spectrometry analysis, which suffers from limited sensitivity in detecting the dynamic new ECM deposition that is usually in low abundance. The proposed newsECM profiling technology is versatile and will be implemented in three lung fibrosis models, including in vivo mouse fibrosis models, an ex vivo donor lung perfusion model, and an in vitro synthetic fibrosis model. We intend to pursue the following specific aims. Aim 1 will track newsECM dynamics in vivo during the progression and resolution of lung fibrogenesis, which is expected to reveal pro- and anti-fibrotic ECM factors. Aim 2 will establish the correlation between the in vivo and ex vivo newsECM profiles using murine lungs as a model, and apply the resulting optimized newsECM profiling condition and algorithm to the ex vivo lung perfusion (EVLP) of donor human lungs bearing idiopathic pulmonary fibrosis (IPF), the most common form of ILD, to reveal human-specific pathogenic ECM mechanism. Finally, Aim 3 will establish a synthetic lung fibrosis model tissue-engineered combining fibroblast, epithelium, endothelium and macrophage within decellularized native lung ECM scaffold, and use it as an experimentally tractable system to further decode fibrogenic lung cell-ECM interaction. Furthermore, combining newsECM labeling and native ECM biomaterial engineering, we will offer a chemoselective platform for effective functional evaluation of candidate fibrosis-modulating ECM factors in a biomimetic, ECM-associated manner. In summary, this research will facilitate a paradigm shift in ILD treatment by promoting ECM-targeted therapeutic development and by enabling combined therapeutic interventions aiming at both cellular and extracellular targets to bring new hope for the impacted patients.