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

项目摘要 间质性肺疾病(ILD)是导致肺组织进行性瘢痕形成(即纤维化)的破坏性疾病， 这是细胞异常和细胞外基质(ECM)失调相互作用的结果。 目前的ILD治疗主要针对细胞信号转导，仍然无法阻止或逆转纤维化。 尽管ECM是ILD的主要病理标志，但很少直接作为治疗的靶点。 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。 为了弥合这一差距，我们的目标是开发一种创新的方法，用于选择性地对新的 合成ECM(新闻ECM)沿肺纤维化发展。拟议中的技术将选择性地标记 在定义的短时间跨度内，通过将化学选择性叠氮标签通过POST-TRAGE-TAG生成的新闻ECM 翻译糖基化。这将使新闻ECM能够在没有预先存在的ECM的情况下进行丰富，从而 实现对动态ECM合成的敏感蛋白质组检测，具有前所未有的每日时间 解决方案，而不考虑丰富的预先存在的ECM背景。我们提议的方法有一个固有的 偏爱ECM蛋白，其中大部分是糖基化的。拟议的新闻ECM分析将 解决常规、非选择性质谱分析中的一个主要技术障碍，这一障碍 从检测通常低丰度的动态新ECM沉积的有限灵敏度。这个 提议的NewECM简档技术是通用的，将在三个肺纤维化模型中实施， 包括体内小鼠纤维化模型、体外供体肺灌流模型和体外合成纤维化模型 模特。我们打算追求以下具体目标。AIM 1将在体内跟踪新闻ECM动态 肺纤维化的进展和消退，有望揭示促纤维化和抗纤维化的ECM因素。 目的2将以小鼠肺为研究对象，建立体内和体外新闻ECM图谱之间的相关性 模型，并将优化的NewECM轮廓条件和算法应用于体外肺灌流 携带特发性肺纤维化(IPF)的供体肺(EVLP)是ILD的最常见形式，以 揭示人类特有的致病ECM机制。最后，目标3将建立一种合成肺纤维化模型 脱细胞天然内成纤维细胞、上皮细胞、内皮细胞和巨噬细胞的组织工程化 肺ECM支架，并将其用作实验上易于处理的系统，进一步解码纤维化的肺细胞-ECM 互动。此外，结合新闻ECM标记和本土ECM生物材料工程，我们将提供 肝纤维化调节细胞外基质候选因子有效功能评价的化学选择平台 一种仿生的、ECM相关的方式。总之，这项研究将促进ILD范式的转变 通过促进ECM靶向治疗开发和实现联合治疗进行治疗 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.