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

摘要外泌体信号对急性呼吸道损伤肺泡再生的调节作用 急性呼吸窘迫综合征（ARDS）的研究还不完全。我们在支气管肺泡上皮细胞中发现了外泌体蛋白， 急性呼吸窘迫综合征（ARDS）患者肺泡灌洗液（BALF）的变化与肺损伤的严重程度密切相关。在ARDS中，临床 通过动脉氧分压（PaO 2，mmHg）与吸入氧分数（FiO 2）的比率对严重程度进行分级。 我们已经确定了干细胞相关的小生境蛋白，这些蛋白存在于轻度（200 kPa O2/FiO 2 <300 mmHg）至 中度ARDS（100 kPa PaO 2/FiO 2 <200 mmHg），但重度ARDS（PaO 2/FiO 2 <100 mmHg）降低。一个关键 ARDS的病理过程是肺泡上皮细胞的损伤，这已经在许多研究中得到证实。 与ARDS严重程度相关的临床前和临床研究。因此，我们假设外泌体 肺干细胞介导的再肺泡化信号是ARDS结果的决定因素。几 研究结果支持这一假设。1)外泌体是干细胞谱系的有效自分泌/旁分泌途径 受伤的器官。2)外泌体蛋白被保护免受发炎组织中的酶的催化。3)无偏 高通量蛋白质组学分析和先进的生物信息学平台已经成功地鉴定了新的 其他疾病的生物标志物。4)虽然ARDS的病因多种多样，但修复过程介导的 基于临床前研究，由小生境调节的上皮干/祖细胞的作用似乎是相似的。我们 目的是用来自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，并验证基因生物工程小鼠和类器官的结果 肺泡2型（AT 2）上皮细胞。我们将应用新的“外泌体”方法，尖端的生物信息学， 三维培养模型、强大的细胞来源跟踪和监督机器学习算法。那里 有三个具体目标：目标1旨在鉴定灌洗液中的外泌体信号通路和网络， 调节肺干细胞谱系以重新肺泡化。我们假设干细胞介导的再生- 在严重的ARDS患者中，由于关键的外泌体小生境的破坏，肺泡化受到抑制。我们 将优先考虑差异表达的外泌体蛋白和相关的信号通路和网络， 包装件.目的2旨在检测和优化损伤肺再肺泡化的再生预测因子。 我们假设灌洗液中显著差异的外泌体分子与临床相关性。 ARDS患者的数据。我们将执行无偏聚类和监督机器学习算法， 开发模型，以发现肺再生的关键信号。优先考虑的外泌体预测因子将是 与传统的全蛋白标记物相比，其准确性和适用性更高。目标3将验证选定的 用于AT 2介导的再肺泡化的外来体信号。我们将证实外泌体分子调节 在3D类器官和小鼠中的人类和小鼠AT 2细胞谱系。这些研究将确定干细胞特异性 外泌体分子作为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.