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Cellular and molecular delineation of pathologic fibroblasts in pulmonary fibrosis

肺纤维化中病理性成纤维细胞的细胞和分子描绘

基本信息

批准号：
10462784
负责人：
Tatsuya Tsukui
金额：
$ 15.77万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-08-05 至 2023-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10462784
关键词：
Ablation Adoptive Transfer Alveolar Apoptosis Award Binding Biological Assay Bleomycin Candidate Disease Gene Cell Count Cell Cycle Kinetics Cell physiology Cells ChIP-seq Chromatin Chronic Cicatrix Collagen Collagen Gene Computer Analysis Data Deposition Deterioration Development Disease Effector Cell Epigenetic Process Extracellular Matrix Fibrillar Collagen Fibroblasts Fibrosis Gases Gene Expression Gene-Modified Genes Genetic Genetic Transcription Genome Genomic approach Goals Human Impairment In Situ Hybridization Inflammation Injury Knowledge Label Lead Light Lung Malignant neoplasm of lung Malignant neoplasm of pancreas Methodology Modeling Molecular Morphology Mouse Strains Mus Normal tissue morphology Pathologic Patients Phase Phenotype Play Population Process Proteins Protocols documentation Pulmonary Fibrosis RNA Regulator Genes Reporter Resolution Respiratory physiology Role Site Survival Rate Therapeutic Time Transcriptional Regulation Transposase Work XCL1 gene conditional knockout design epigenetic regulation experimental study fibrogenesis fibrotic lung functional genomics genetic approach idiopathic pulmonary fibrosis in vitro Assay in vivo indium-bleomycin injured innovation knock-down lung development migration mouse development novel overexpression programs single-cell RNA sequencing skills success therapeutic development therapeutic target tool training opportunity transcription factor transcriptome sequencing triple helix wound healing

项目摘要

PROJECT SUMMARY Pulmonary fibrosis is a chronic and intractable disease with a 5-year survival rate comparable to pancreatic or lung cancers. Deterioration of respiratory function in pulmonary fibrosis is caused by progressive replacement of normal tissue for gas exchange to dense fibrotic scar with fibrillar collagens. Pathologic fibroblasts accumulate at the sites of fibrogenesis and work as effector cells for excessive collagen deposition. Development of therapeutic strategies for targeting pathologic fibroblasts is hindered by the lack of understanding to cellular lineage and molecular detail of pathologic fibroblasts. In our previous study, we performed single-cell RNA-sequencing of normal and fibrotic lungs of mouse and human with a specialized protocol to identify all collagen-producing cells. We identified several fibroblast subsets that localize in different compartments of the lung. One of the fibroblast subsets emerge in fibrotic lungs of both mouse and human and show the highest levels of collagen gene expression and enhanced migratory capacity. These fibroblasts are characterized by specific expression of Cthrc1 (collagen triple helix repeat containing 1) and localized within fibroblastic foci of idiopathic pulmonary fibrosis, suggesting their pathologic role in pulmonary fibrosis. We recently generated and validated a novel mouse strain, Cthrc1-CreER, which allows us to specifically manipulate the pathologic fibroblast population in pulmonary fibrosis. The goal of this K99/R00 proposal is to elucidate the role and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis by using our innovative murine tools. Aim 1 (K99 phase) will reveal the role and fate of pathologic fibroblasts by ablating Cthrc1+ cells or lineage-tracing Cthrc1-CreER-labeled cells over the course of bleomycin-induced pulmonary fibrosis. Aim 2 (K99 phase) will reveal the transcriptional and epigenetic landscape of pathologic fibroblasts by performing RNA-seq, ChIP-seq, and ATAC-seq of purified Cthrc1-CreER-labeled cells at multiple time points of pulmonary fibrosis to seek master regulators for activation and deactivation. We will also seek the transcriptional regulations of CTHRC1+ cells in human pulmonary fibrosis. Aim 3 (R00 phase) will demonstrate the role of genes regulating pathologic fibroblasts by using intratracheal adoptive transfer of fibroblasts with lentiviral gene modifications and by conditionally knocking out candidate genes in fibroblasts in pulmonary fibrosis. These studies using the murine genetic tool highly specific for pathologic fibroblasts will shed light on cellular function and transcriptional regulations of pathologic fibroblasts in pulmonary fibrosis. This proposal is also designed to provide the candidate with training opportunity to obtain skill sets for murine genetic approach in search of therapeutic targets and functional genomics approach integrating RNA-seq, ChIP-seq, and ATAC- seq. The success of this project will enable the candidate to establish his expertise in the field of pulmonary fibrosis and lead to the candidate’s transition to scientific independence over the course of award period.

项目摘要肺纤维化是一种慢性难治性疾病，其5年生存率与胰腺或肺癌肺纤维化呼吸功能恶化是由进行性替代引起的正常组织的气体交换到致密的纤维化瘢痕与纤维状胶原。病理性成纤维细胞在纤维发生的部位积聚，并作为过度胶原沉积的效应细胞。靶向病理性成纤维细胞的治疗策略的开发受到缺乏免疫抑制剂的阻碍。了解病理性成纤维细胞的细胞谱系和分子细节。在之前的研究中，我们用专门的方法对小鼠和人类的正常和纤维化肺进行单细胞RNA测序，鉴定所有胶原蛋白产生细胞的方案。我们确定了几个成纤维细胞亚群，肺的腔室。成纤维细胞亚群之一出现在小鼠和人类的纤维化肺中，显示出最高水平的胶原基因表达和增强的迁移能力。这些成纤维细胞是其特征在于Cthrc 1（含1的胶原三螺旋重复序列）的特异性表达，并定位于特发性肺纤维化的成纤维细胞病灶，提示其在肺纤维化中的病理作用。我们最近产生并验证了一种新的小鼠品系Cthrc 1-CreER，它使我们能够特异性地操纵肺纤维化中的病理性成纤维细胞群体。本K99/R 00提案的目标是阐明病理性成纤维细胞在肺纤维化中的作用和转录调控，创新的鼠工具。目的1（K99期）通过消融病理性成纤维细胞，博来霉素诱导的肺动脉高压过程中Cthrc 1+细胞或谱系追踪的Cthrc 1-CreER标记的细胞纤维化目的2（K99期）将揭示病理性成纤维细胞的转录和表观遗传景观，在多个时间点对纯化的Cthrc 1-CreER标记的细胞进行RNA-seq、ChIP-seq和ATAC-seq，肺纤维化，以寻求激活和失活的主调节器。我们还将寻求 CTHRC 1+细胞在人肺纤维化中的转录调节。目标3（R 00阶段）将证明通过使用成纤维细胞的体内过继转移来调节病理性成纤维细胞的基因的作用慢病毒基因修饰和有条件地敲除肺成纤维细胞中的候选基因纤维化这些研究使用了对病理性成纤维细胞高度特异性的鼠遗传工具，肺纤维化中病理性成纤维细胞的细胞功能和转录调控。这项建议是还旨在为候选人提供培训机会，以获得鼠遗传方法的技能在寻找治疗靶点和整合RNA-seq、ChIP-seq和ATAC功能基因组学方法中， seq.该项目的成功将使候选人能够建立他在肺领域的专业知识纤维化，并导致候选人在获奖期间过渡到科学独立。