Lung Remodeling Mediated by Telomere Dysfunction in Alveolar Type II Cells

II 型肺泡细胞端粒功能障碍介导的肺重塑

• 批准号: 10660570
• 负责人: Paul j WOLTERS
• 金额: $ 79.93万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-05 至 2027-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10660570
• 关键词: Adenovirus Infections; Adult; Age; Alveolar; Alveolar fibrosis; Appearance; Automobile Driving; Biological Assay; Bleomycin; Cell Communication; Cell Reprogramming; Cell division; Cells; Complex; Cytoprotection; DNA Damage; Data; Development; Disease; Distal; Dose; Elements; Epithelial Cells; Epithelium; Event; Evolution; Fibrosis; Functional disorder; Gamma-H2AX; Gene Expression; Genes; Harvest; Health; Human; Injury; Knowledge; Length; Lung; Lung diseases; Measures; Mediating; Mediator; Modeling; Molecular; Mus; Organoids; Pathogenesis; Phase; Phenotype; Play; Pneumonectomy; Population; Prevalence; Production; Proliferating; Protein Array; Proteins; Pulmonary Fibrosis; Pulmonary Surfactant-Associated Protein C; Role; Serial Passage; Stains; Stimulus; Study models; System; TERF1 gene; TP53 gene; Tamoxifen; Telomerase; Testing; Time; Translating; aged; alveolar type II cell; beta-Galactosidase; comparative; comparison control; defined contribution; fibrotic lung; human disease; idiopathic pulmonary fibrosis; inhibitor; insight; lung regeneration; mouse model; novel; overexpression; senescence; single-cell RNA sequencing; stressor; telomere; transcription factor; transdifferentiation

Idiopathic pulmonary fibrosis (IPF) is a genetically mediated, progressive, lung disorder. Evidence points to dysfunctional epithelial telomeres as a core disease-initiating abnormality in IPF. There is limited knowledge of the relationship between alveolar type 2 (AT2) cell telomere dysfunction and the cellular and molecular elements of the remodeled fibrotic lung. Attempts to elucidate this connection in mouse models of telomere dysfunction have been limited by the inherently long telomeres of mice and the use additional fibrogenic stimuli such as low dose bleomycin, which may obscure the distinct input of dysfunctional telomeres to the pathobiology. This proposal seeks to overcome these limitations by applying a novel model of progressive lung fibrosis driven solely by telomere dysfunction isolated to AT2 cells (TRF1SC mice) to study mechanisms of lung fibrosis. The hypothesis is that events developing in TRF1SC mice mimic early epithelial-specific events in the pathogenesis of IPF and that this model will provide new insights into the origins of epithelial cell remodeling observed in IPF. To test this hypothesis, the following aims will be explored: Aim 1 proposes to use TRF1SC/p53-/- mice to establish whether p53 is a molecular mediator of progressive epithelial transdifferentiation and remodeling driven by AT2 cell telomere dysfunction in TRF1SC mice. Single cell RNAseq (scRNAseq) will be used to define the role of p53 on reprogramming of TRF1SC AT2 cells after telomere uncapping, and the appearance of other profibrotic epithelial cell subtypes. Aim 2 proposes to study TRF1SC/TZAP-/- mice to test the hypothesis that the latency of onset of fibrosis in TRF1SC mice requires telomere trimming. TRF1SC/TZAP-/- mice will be treated with tamoxifen for 9 months and measures of telomere attrition, epithelial remodeling, and fibrosis compared to control TRF1SC mice. scRNAseq will be used to define the contribution of telomere trimming to AT2 cell reprogramming in control, TRF1SC, and TRF1SC/TZAP-/- mice. Telomere trimming in normal AT2 cells will be investigated by studying changes in telomere lengths in aged TZAP-/- mice. Aim 3 proposes to culture IPF and control AT2 cells in the presence and absence of hTERT overexpression in an organoid system to establish whether augmenting telomere length restores epithelial cell fate and function of IPF lung epithelial cells. Comparative endpoints include: telomere length measures, DNA damage detected by co-staining for γH2AX and TRF2, senescence reprogramming by β-galactosidase and p16 staining. AT2 cell transdifferentiation into AT1 cells and by qPCR and immunostaining. The influence of hTERT overexpression on other IPF epithelial cell subtypes will be studied by comparing gene expression by scRNAseq of mixed populations of IPF epithelial cells cultured in the presence or absence of hTERT. If successful, these studies will provide an understanding into cell-cell interactions, mediated by telomere dysfunction, driving lung fibrosis in IPF.

特发性肺纤维化（IPF）是一种遗传介导的进行性肺部疾病。证据表明 上皮端粒功能障碍是IPF的核心疾病引发异常。我们的知识有限 肺泡2型（AT 2）细胞端粒功能障碍与细胞和分子 重塑纤维化肺的元素。试图在端粒的小鼠模型中阐明这种联系 功能障碍受到小鼠固有的长端粒和使用额外的纤维化刺激的限制 例如低剂量的博来霉素，这可能掩盖功能失调的端粒对病理生物学的独特输入。 该提议试图通过应用一种新的进行性肺纤维化驱动模型来克服这些局限性。 仅通过分离到AT 2细胞（TRF 1 SC小鼠）的端粒功能障碍来研究肺纤维化的机制。的 假设TRF 1 SC小鼠中发生的事件模拟发病机制中的早期上皮特异性事件 该模型将为IPF中观察到的上皮细胞重塑的起源提供新的见解。 为了检验这一假设，将探索以下目的：目的1提出使用TRF 1 SC/p53-/-小鼠来 确定p53是否是进行性上皮转分化和重塑驱动的分子介质 TRF 1 SC小鼠中AT 2细胞端粒功能障碍。将使用单细胞RNAseq（scRNAseq）来定义 p53在端粒脱帽后TRF 1 SC AT 2细胞重编程中的作用，以及其他蛋白的出现。 促纤维化上皮细胞亚型。目的2提出研究TRF 1 SC/TZAP-/-小鼠，以检验TRF 1 SC/TZAP-/-小鼠的免疫反应性的假设。 TRF 1 SC小鼠中纤维化发作的潜伏期需要端粒修剪。将对TRF 1 SC/TZAP-/-小鼠进行处理 他莫昔芬治疗9个月，并与 对照TRF 1 SC小鼠。scRNAseq将用于定义端粒修剪对AT 2细胞的贡献。 在对照、TRF 1 SC和TRF 1 SC/TZAP-/-小鼠中的重编程。正常AT 2细胞中的端粒修剪将是 通过研究老年TZAP-/-小鼠中端粒长度的变化来研究。目标3建议培养IPF， 在类器官系统中存在和不存在hTERT过表达的情况下对照AT 2细胞，以建立 增加端粒长度是否恢复IPF肺上皮细胞的上皮细胞命运和功能。 比较终点包括：端粒长度测量，通过γ H2 AX共染色检测的DNA损伤， TRF 2，β-半乳糖苷酶和p16染色的衰老重编程。AT 2细胞转分化为AT 1 细胞，并通过qPCR和免疫染色。hTERT过表达对其他IPF上皮细胞的影响 将通过比较IPF上皮细胞混合群体的scRNAseq基因表达来研究亚型 在存在或不存在hTERT的情况下培养。如果成功，这些研究将提供一个了解， 由端粒功能障碍介导的细胞-细胞相互作用，驱动IPF中的肺纤维化。