Proteolysis in the Pathogenesis of ARDS

项目总结/摘要 急性呼吸窘迫综合征（ARDS）是急性肺损伤（ALI）的最常见形式， 其特征在于损伤肺泡毛细血管单位和损害肺泡上皮完整性，导致 高渗透性肺水肿和嗜酸性肺泡炎。无法修复损坏的 肺泡膜导致显著的死亡率。包括我们自己在内的几个实验室的研究表明， 发现动物模型中ALI的诱导与基质表达增加有关 金属蛋白酶-3（MMP-3），一种已显示直接靶向细胞-细胞连接并 降解基底膜，尽管还不清楚这一过程在ALI病理学的哪个阶段发生 最关键的其他实验室和我们的实验室也发现，缺乏MMP-3的转基因小鼠对 多种刺激诱导的肺损伤，表明MMP-3抑制剂具有作为治疗剂的潜力 对于ARDS，尽管这样的治疗策略需要高度选择性，因为其他MMPs已经 在肺损伤的修复中起关键作用。在这个翻译应用中，我们提出了实验， 将弥合这些关键差距。在目标1中，我们将使用生理相关的细胞培养和动物模型， ALI/ARDS包括新型转基因小鼠，以确定MMP-3如何影响ALI/ARDS中上皮损伤的消退。 对酸吸入或流感感染的反应。在目标2中，我们将使用定向分子进化 该平台用于产生金属蛋白酶组织抑制剂-1（TIMP-1）的变体，其对 MMP-3和减少结合有益的MMP，我们将确定这些TIMP的治疗效用 在ALI/ARDS小鼠模型中的变异。在目标3中，我们将询问ARDS的临床样本，以确定 人类疾病进展的精确阶段，在该阶段进行治疗干预将是最有益的。我们 一项研究提案将定义一种新发现的机制，MMP-3通过这种机制驱动ARDS病理， 一项综合研究计划，将从ARDS分离的细胞群的单细胞RNA测序联系起来， 患者组织、细胞培养物和ARDS动物模型，以及相关ARDS生物标志物和离体分析 培养精确切割的人肺切片，并将开发一种新的选择性抑制剂， 治疗潜力我们的多学科研究团队非常适合解决这些问题。我们 工作将解决ALI/ARDS的关键机制，将使用这些知识来开发个性化的 这些策略将识别出发生ALI/ARDS风险最高的患者， 基于MMP-3在导致ALI/ARDS的上皮损伤发病机制中的关键作用的方法。

PROJECT SUMMARY/ABSTRACT The Acute respiratory distress syndrome (ARDS) is the severest form of acute lung injury (ALI), and is characterized by injury to the alveolar-capillary unit and compromised alveolar epithelial integrity, leading to high permeability pulmonary edema and neutrophilic alveolar inflammation. Failure to repair the damaged alveolar membrane leads to significant mortality. Studies from several laboratories, including our own, have found that induction of ALI in animal models is associated with increased expression of matrix metalloproteinase-3 (MMP-3), a protease that has been shown to directly target cell-cell junctions and to degrade basement membranes, although it was unknown at what stages of ALI pathology this process was most critical. Other laboratories and ours have also found that transgenic mice lacking MMP-3 are resistant to lung injury induced by multiple stimuli, indicating that inhibitors of MMP-3 have potential as therapeutic agents for ARDS, although such a therapeutic strategy would need to be highly selective, as other MMPs have been shown to play key roles in repair of lung injury. In this translational application, we propose experiments which will bridge these critical gaps. In Aim 1, we will use physiologically relevant cell culture and animal models of ALI/ARDS including novel transgenic mice to define how MMP-3 affects the resolution of epithelial injury in response to acid aspiration or influenza infection. In Aim 2, we will use our directed molecular evolution platform to create variants of Tissue Inhibitor of Metalloproteinase-1 (TIMP-1) with greatly increased affinity for MMP-3 and decreased binding to beneficial MMPs, and we will define the therapeutic utility of these TIMP variants in mouse models of ALI/ARDS. In Aim 3, we will interrogate clinical samples of ARDS to determine the precise stages of human disease progression at which therapeutic intervention would be most beneficial. Our research proposal will define a newly-discovered mechanism by which MMP-3 drives ARDS pathology through an integrated research plan that links single cell RNA sequencing of cell populations isolated from ARDS patient tissue, cell culture and animal models of ARDS, and analysis of relevant ARDS biomarkers and ex vivo culture of precision cut human lung slices, and will develop a novel selective inhibitor with immediate therapeutic potential. Our multidisciplinary research team is uniquely suited to address these questions. Our work will address critical mechanisms underlying ALI/ARDS, will use this knowledge to develop personalized strategies that will identify patients at highest risk of developing ALI/ARDS, and will create therapeutic approaches based on the pivotal role of MMP-3 in the pathogenesis of epithelial injury leading to ALI/ARDS.