Cellular crosstalk and molecular mechanisms in the initiation and progression of pulmonary fibrosis

总结 反复的肺泡损伤和肺泡细胞生态位中的信号失调已经被牵连在 肺纤维化的发病机制。虽然全基因组关联研究进一步支持了这一模型， 精确的细胞触发器和驱动肺泡成纤维细胞复制的分子机制， 转化为过量的细胞外基质产生肌成纤维细胞仍然是难以捉摸的。目前，我们缺乏一个 全面了解细胞的内在和外在稳态机制，通常控制 成纤维细胞复制并阻止其转化为病理性肌成纤维细胞。 我们最近的发现，无论是肺泡上皮1型（AT 1）细胞的条件性消融，还是Pdgfra的丢失， 特别是在体内表达PDGFRA的成纤维细胞（肺泡成纤维细胞）中，导致自发转化 转化为肌纤维母细胞和纤维化。单细胞转录组指导的配体-受体对 预测加上肺泡成纤维细胞培养在无血清条件下表明，AT 1细胞来源的 需要适量的PDGFA来维持肺泡成纤维细胞的身份（即，阻止他们的转变 成肌纤维细胞）并控制复制。探索下游机制，我们发现 转录因子RUNX 1在复制的肺泡成纤维细胞以及TGFβ- 诱导体内和离体肌纤维化。值得注意的是，我们的初步数据还表明， Runx 1的缺失消除了PDGFA或TGFβ诱导的肺泡成纤维细胞增殖或转化为 肌成纤维细胞。这些初步数据使我们假设AT 1细胞维持肺泡 通过PDGFA-PDGFRA信号轴和PDGFA的量的精细平衡， 是成纤维细胞静止和复制稳态和损伤修复所必需的。我们也 假设RUNX 1是PDGFRA和TGFβ信号传导重要调节因子， 再生和纤维化过程中肺泡成纤维细胞的复制和肌纤维化程序。 该建议的主要目标是定义AT 1细胞和肺泡上皮细胞之间的通信。 成纤维细胞和研究肌纤维形成的转录控制。在Aim 1中，我们将测试以下假设： AT 1细胞和肺泡成纤维细胞之间的PDGFA-PDGFRA信号传导对于肺泡内稳态是必不可少的。在 目的2，我们将确定RUNX 1在肺泡成纤维细胞复制中的特定功能， 肌纤维化我们将使用新的体内遗传小鼠模型和药理学功能丧失模型， 人肺成纤维细胞和精确切割的肺切片培养物，以及研究这些特定目的的分子测定。 我们先前在肺再生和细胞命运转录控制方面的专业知识将有助于我们研究拟议的 目标。拟议研究的结果将对肺再生医学产生更广泛的影响， 形成开发预防和逆转人肺纤维化的疗法的基础。

SUMMARY Recurrent alveolar injury and dysregulated signaling in alveolar cell niche have been implicated in the pathogenesis of pulmonary fibrosis. While genome-wide association studies further supported this model, the precise cellular triggers and the molecular mechanisms that drive alveolar fibroblasts replication and their conversion into excessive extracellular matrix producing myofibroblasts remain elusive. Currently we lack a comprehensive understanding of the cell intrinsic and extrinsic homeostatic mechanisms that normally control fibroblasts replication and prevent their conversion into pathological myofibroblasts. Our recent finding that either conditional ablation of alveolar epithelial type-1 (AT1) cells, or loss of Pdgfra specifically in PDGFRA-expressing fibroblasts (alveolar fibroblasts) in vivo, results in spontaneous conversion of alveolar fibroblasts into myofibroblasts and fibrosis. Single cell transcriptome guided ligand-receptor pair predictions coupled with alveolar fibroblast cultures in serum-free conditions suggest that AT1 cell-derived PDGFA is required in proper amounts to both maintain alveolar fibroblasts identity (i.e., prevent their conversion to myofibroblasts) and control replication. Exploring downstream mechanisms, we have uncovered an increase in the expression of the transcription factor RUNX1 in both replicating alveolar fibroblasts as well as TGFβ- induced myofibrogenesis both in vivo and ex vivo. Significantly, our preliminary data also indicate that genetic loss of Runx1 abrogates PDGFA or TGFβ-induced alveolar fibroblasts proliferation or conversion into myofibroblasts, respectively. These preliminary data lead us to hypothesize that AT1 cells maintain alveolar fibroblasts identity via PDGFA – PDGFRA signaling axis and that a fine balance in the amount of PDGFA is essential for fibroblast quiescence and replication at homeostasis and injury repair. We also hypothesize that RUNX1 is an essential regulator of both PDGFRA and TGFβ signaling to regulate replication and myofibrogenic programs in alveolar fibroblasts during regeneration and fibrosis. The major objectives of this proposal are to define the communication between AT1 cells and alveolar fibroblasts and to study transcriptional control of myofibrogenesis. In Aim1, we will test the hypothesis that PDGFA-PDGFRA signaling between AT1 cells and alveolar fibroblasts is essential for alveolar homeostasis. In Aim2, we will determine context specific functions of RUNX1 in alveolar fibroblasts replication and myofibrogenesis. We will use novel in vivo genetic mouse models and pharmacological loss-of-function models, human lung fibroblasts and precision cut lung slice cultures, and molecular assays to study these specific aims. Our prior expertise in lung regeneration and transcriptional control of cell fates will aid us in studying the proposed aims. The outcomes from the proposed studies will have broader impact on lung regenerative medicine and will form the basis for development of therapeutics to prevent and reverse human lung fibrosis.