Novel exosomal niches for alveolar stem cell-based repair of ARDS

总结。急性呼吸损伤肺外信号对肺泡再生的调节作用 对窘迫综合征(ARDS)的研究还不够全面。我们在支气管肺泡中发现了胞外体蛋白 ARDS患者灌洗液(BALF)与肺损伤严重程度密切相关。在ARDS中，临床 病情严重程度由动脉血氧分压(PaO2，单位为毫米汞柱)与吸入氧分数(FiO2)之比进行分级。 我们已经确定了存在于轻度(200 GB PaO2/FiO2；300毫米汞柱)到 中度ARDS(100 GB PaO2/FiO2；200 mm Hg)，但重度ARDS(PaO2/FiO2；100 mm Hg)减少。一把钥匙 ARDS的病理过程是对肺泡上皮的损伤，在许多 与ARDS严重程度相关的临床前和临床研究。因此，我们假设外体 肺干细胞介导的再肺泡化信号是ARDS预后的决定因素。几个 研究结果支持这一假设。1)外切体是干细胞谱系的有效自分泌/旁分泌途径。 在受伤的器官里。2)外体蛋白在炎症组织中被酶保护而不被催化。3)不带偏见 高通量蛋白质组分析和先进的生物信息平台成功识别出新的 其他疾病的生物标志物。4)虽然ARDS的病因多种多样，但修复过程 在临床前研究中，受上皮干/祖细胞调控的干细胞和祖细胞似乎是相似的。我们的 目的是用NIH/NHLBI支持的BALF样本和患者的临床数据来验证这一假设 临床试验，优先考虑利基分子，并在基因生物工程小鼠和有机化合物中验证结果 肺泡2型(AT2)上皮细胞。我们将应用新的“外显体组学”方法，尖端生物信息学， 三维培养模型、稳健的细胞起源跟踪和有监督的机器学习算法。那里 有三个具体目标：Aim 1旨在识别灌洗液中的外体信号通路和网络 调节肺干细胞的谱系，以重新形成肺泡化。我们假设干细胞介导的重新- 严重ARDS患者的肺泡化受到抑制，原因是关键的胞外壁龛被破坏。我们 将使用R优先处理差异表达的胞外蛋白和相关信号通路和网络 包裹。AIM 2旨在检测和优化损伤肺重新肺泡化的再生预测指标。 我们推测，灌洗液中显著不同的外切体分子将与临床相关。 ARDS患者的临床资料。我们将执行无偏聚类和有监督的机器学习算法来 开发发现肺再生关键信号的模型。按优先顺序排列的胞外体预测因子将是 在准确性和适用性方面与传统的全蛋白标记进行了比较。AIM 3将验证所选内容 AT2介导的再牙槽化的外体信号。我们将证实外体分子调节 人和小鼠AT2细胞在3D有机物和小鼠中的谱系。这些研究将确定干细胞的特异性 胞外体分子作为ARDS的潜在药物靶点。

Summary. The regulation of alveolar regeneration of injured lungs by exosomal signals in acute respiratory distress syndrome (ARDS) are incompletely studied. We have identified exosomal proteins in bronchoalveolar lavage fluid (BALF) of ARDS patients that are closely association with the severity of lung injury. In ARDS, clinical severity is graded by the ratio of arterial oxygen tension (PaO2 in mmHg) to the fraction of inspired oxygen (FiO2). We have identified stem cell-related niche proteins that are present in mild (200£PaO2/FiO2<300 mmHg) to moderate ARDS (100£PaO2/FiO2<200 mmHg) but reduced in severe ARDS (PaO2/FiO2<100 mmHg). A key pathological process in ARDS is damage to the alveolar epithelium, which has been demonstrated in numerous preclinical and clinical studies to be associated with ARDS severity. Therefore, we hypothesize that exosomal signals for lung stem cell-mediated re-alveolarization are determinants of ARDS outcomes. Several findings support this hypothesis. 1) Exosomes are effective autocrine/paracrine pathways for stem cell lineage in injured organs. 2) Exosomal proteins are protected from catalysis by enzymes in inflamed tissues. 3) Unbiased high throughput proteomic analysis and advanced bioinformatic platforms have successfully identified novel biomarkers for other diseases. 4) Although the etiologies of ARDS are diverse, the repair processes mediated by epithelial stem/progenitor cells regulated by niches seem to be similar based on preclinical studies. Our objective is to test this hypothesis with BALF samples and patients' clinical data from NIH/NHLBI-supported clinical trials, prioritize niche molecules, and validate the results in genetically bioengineered mice and organoids of alveolar type 2 (AT2) epithelial cells. We will apply novel “exosomics” approaches, cutting-edge bioinformatics, three-dimensional culture models, robust cell origin tracking, and supervised machine learning algorithms. There are three specific aims: Aim 1 is designed to identify exosomal signaling pathways and networks in lavage that regulate the lineage of lung stem cells for re-alveolarization. We hypothesize that stem cell-mediated re- alveolarization has been suppressed in severe ARDS patients due to the disruption of key exosomal niches. We will prioritize differentially expressed exosomal proteins and related signaling pathways and networks using R packages. Aim 2 is designed to detect and optimize regenerative predictors for re-alveolarization of injured lungs. We hypothesize that the significantly differential exosomal molecules in lavage will be associated with clinical data in ARDS patients. We will perform unbiased clustering and supervised machine learning algorithms to develop models for discovering critical signals for lung regeneration. The prioritized exosomal predictors will be compared with traditional whole-protein markers for accuracy and applicability. Aim 3 will validate selected exosomal signals for AT2-mediated re-alveolarization. We will confirm that the exosomal molecules regulate the lineage of human and mouse AT2 cells in 3D organoids and mice. These studies will identify stem cell-specific exosomal molecules as potential pharmaceutic targets for ARDS.