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

摘要 复发性肺泡损伤和肺泡细胞巢信号异常与 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式，但 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难以捉摸。目前，我们缺少一个 全面了解细胞的内在和外在稳态机制，这些机制通常控制 成纤维细胞复制并防止其转化为病理性肌成纤维细胞。 我们最近的研究发现，无论是有条件地消融肺泡上皮1型(AT1)细胞，还是失去PDGFRA 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 肺泡成纤维细胞转化为肌成纤维细胞和纤维化。单细胞转录组引导的配体-受体对 与无血清条件下的肺泡成纤维细胞培养相结合的预测表明，AT1细胞来源于 需要适量的PDGFA来维持肺泡成纤维细胞的特性(即防止它们的转化 肌成纤维细胞)和控制复制。在探索下游机制时，我们发现了一个 转录因子RUNX1在复制的肺泡成纤维细胞和转化生长因子β中的表达 体内和体外诱导的肌纤维形成。值得注意的是，我们的初步数据还表明，基因 Runx1的缺失可阻断PDGFA或转化生长因子β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AT1细胞维持肺泡 成纤维细胞通过PDGFA-PDGFRA信号轴的识别以及PDGFA含量的微妙平衡 对于成纤维细胞的静止和复制在动态平衡和损伤修复中至关重要。我们也 假设RUNX1是PDGFRA和转化生长因子β信号调节的重要调节者 再生和纤维化过程中肺泡成纤维细胞的复制和肌纤维形成程序。 该方案的主要目的是定义AT1细胞和肺泡之间的通讯 成纤维细胞并研究肌纤维形成的转录调控。在Aim1中，我们将测试假设 AT1细胞和肺泡成纤维细胞之间的PDGFA-PDGFRA信号转导对肺泡内环境的稳定是必不可少的。在……里面 AIM2，我们将确定RUNX1在肺泡成纤维细胞复制和复制中的上下文特定功能 肌纤维形成。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密切片培养，以及分子分析来研究这些特定的目的。 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，并将 为开发预防和逆转人类肺纤维化的治疗方法奠定了基础。

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.