Proteolysis in the Pathogenesis of ARDS

项目摘要/摘要 急性呼吸窘迫综合征(ARDS)是急性肺损伤(ALI)的最严重形式， 以肺泡毛细血管单位受损和肺泡上皮完整性受损为特征，导致 高渗透性肺水肿和中性粒细胞肺泡炎。未能修复损坏的 肺泡膜可导致显著的死亡率。来自几个实验室的研究，包括我们自己的，已经 发现动物模型中ALI的诱导与基质表达的增加有关 金属蛋白酶-3(MMP3)，一种已被证明直接靶向细胞-细胞连接和 降解基底膜，尽管这一过程在ALI病理的哪个阶段尚不清楚 最关键的。其他实验室和我们的实验室也发现，缺乏基质金属蛋白酶-3的转基因小鼠对 多种刺激诱导的肺损伤，提示基质金属蛋白酶-3抑制剂具有潜在的治疗作用 对于ARDS，尽管这样的治疗策略需要像其他MMP一样具有高度选择性 显示在肺损伤的修复中起关键作用。在这个翻译应用中，我们提出了一些实验， 将弥合这些关键差距。在目标1中，我们将使用与生理相关的细胞培养和动物模型 ALI/ARDS包括新的转基因小鼠以确定基质金属蛋白酶-3如何影响急性肺损伤上皮细胞损伤的解决 对酸吸入或流感感染的反应。在目标2中，我们将使用我们的定向分子进化 创造金属蛋白酶组织抑制因子-1(TIMP-1)变异体的平台，其亲和力大大提高 MMP3和减少与有益MMPs的结合，我们将确定这些TIMP的治疗作用 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 在人类疾病发展的准确阶段，治疗干预将是最有益的。我们的 研究提案将定义一种新发现的机制，即基质金属蛋白酶-3通过其驱动ARDS病理 一项将ARDS分离细胞群体的单细胞RNA测序联系起来的综合研究计划 ARDS的患者组织、细胞培养和动物模型，以及相关ARDS生物标志物和体外分析 培养精密切割的人肺切片，并将开发一种新的选择性抑制剂，立即 治疗潜力。我们的多学科研究团队非常适合解决这些问题。我们的 工作将解决ALI/ARDS背后的关键机制，将使用这些知识来开发个性化 将确定ALI/ARDS风险最高的患者的策略，并将创建治疗性 基于基质金属蛋白酶-3在导致ALI/ARDS的上皮损伤发病机制中的关键作用的研究进展。

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.