Fibroblast heterogeneity in pulmonary fibrosis

肺纤维化中成纤维细胞的异质性

基本信息

批准号：
9979120
负责人：
Dean Sheppard
金额：
$ 64.1万
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：
2020
资助国家：
美国
起止时间：
2020-04-01 至 2024-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9979120
关键词：
Ablation Acute Address Adoptive Transfer Aftercare Alveolar Aspergillus Behavior Biology Bleomycin Cell Lineage Cell surface Cells Cessation of life Characteristics Chronic Chronic Disease Cicatrix Collagen Complex Development Diagnosis Diagnostic Diffuse Disease Dose Extracellular Matrix Extracellular Matrix Proteins Fibrillar Collagen Fibroblasts Fibrosis Functional disorder Gases Gel Gene Deletion Genes Geographic Distribution Geography Health Heterogeneity Histology Homeostasis Human Impairment In Situ Hybridization In Vitro Inflammation Lead Ligation Lung Modeling Molecular Molecular Profiling Mus Myofibroblast Nature Organ Pathologic Patients Pericytes Phase Play Population Prevalence Procedures Production Proteins Pulmonary Fibrosis Research Resolution Respiratory Failure Role Site Smooth Muscle Actin Staining Method Smooth Muscle Myocytes Structure of parenchyma of lung Therapeutic Intervention Time Tissues cell type design disability effective therapy fluorophore in vivo insight lung development migration mouse development novel prevent response single-cell RNA sequencing tool triple helix

项目摘要

ABSTRACT Pulmonary fibrosis, characterized by accumulation of extracellular matrix (ECM) proteins that impair normal function, is a major cause of disability and death. Although considerable research has been done to identify multiple potential lineages of cells that can give rise to the fibroblasts responsible for ECM production, it has been widely assumed that these cells are a relatively homogeneous population of cells called myofibroblasts, characterized by high expression of a smooth muscle actin (aSMA). However, limited information about the molecular characteristics of these cells in vivo has limited our understanding of the basic biology underlying fibrosis and hampered the development of effective therapies. To address this important gap, we have used single cell RNA sequencing (scRNAseq) of collagen producing cells to identify multiple distinct cell types that produce collagen in the normal and fibrotic murine and human lung. Using proximity ligation in situ hybridization (PLISH) we identified subsets of collagen-producing cells with distinct molecular signatures that were concentrated within the walls of conducting airways (peribronchial), surrounding bronchovascular bundles (adventitial) and diffusely distributed in gas exchanging regions (alveolar). After treatment with bleomycin, a distinct new subset emerged that expressed high levels of collagens and other ECM proteins and was uniquely marked by expression of collagen triple helix repeat containing protein 1 (cthrc1). scRNAseq of dissociated cells from normal and fibrotic human lungs also identified a population of cells marked by CTHRC1-expression that expressed the highest levels of collagens and other ECM proteins and was only seen in lungs from patients with pulmonary fibrosis. In the studies proposed here, we will first determine the geographic and temporal distribution and lineage of cthrc1+ cells in single and repeated dose bleomycin models using PLISH, adoptive transfer and novel ERcre lines we are developing to track cells derived from peribronchial, adventitial and alveolar fibroblasts. Next, we will evaluate the functional roles of cthrc1+ cells using adoptive transfer into normal or bleomycin-treated mice, in vitro studies of an array of behaviors associated with pathologic fibroblasts, and through deletion of these cells by crossing a novel cthrc1-ERcre line we have generated to mice expressing lox-stop-lox dta in the Rosa locus. Finally, we will examine the functional significance of each of the major collagen-producing cell populations we have identified in normal lungs at baseline and in models of alveolar and airway fibrosis, using a similar ablation strategy or through deletion of genes previously shown to contribute to tissue fibrosis. From these studies we hope to gain novel insights into the roles each of these unique fibroblast subsets plays in lung homeostasis and disease.

抽象的肺纤维化，其特征是细胞外基质 (ECM) 蛋白的积累，损害正常功能功能障碍，是导致残疾和死亡的主要原因。尽管已经进行了大量研究来确定多种潜在的细胞谱系可以产生负责 ECM 产生的成纤维细胞，它具有人们普遍认为这些细胞是相对同质的细胞群，称为肌成纤维细胞，其特征是平滑肌肌动蛋白 (aSMA) 高表达。然而，有关该项目的信息有限这些细胞在体内的分子特征限制了我们对基础生物学的理解纤维化并阻碍了有效疗法的开发。为了解决这个重要的差距，我们使用了对胶原蛋白生成细胞进行单细胞 RNA 测序 (scRNAseq)，以识别多种不同的细胞类型在正常和纤维化的小鼠和人肺中产生胶原蛋白。使用原位邻近连接通过杂交（PLISH），我们鉴定出具有不同分子特征的胶原蛋白生成细胞亚群集中在传导气道（支气管周围）壁内，周围支气管血管束（外膜）并广泛分布在气体交换区域（肺泡）。用博莱霉素治疗后，出现了独特的新亚群，表达高水平的胶原蛋白和其他 ECM 蛋白，并且是独一无二的以含有蛋白质 1 (cthrc1) 的胶原蛋白三螺旋重复序列的表达为标志。解离的 scRNAseq 来自正常和纤维化人肺的细胞还鉴定出一组以 CTHRC1 表达为标记的细胞表达最高水平的胶原蛋白和其他 ECM 蛋白，并且仅在肺部可见肺纤维化患者。在这里提出的研究中，我们将首先确定地理和使用 PLISH 在单剂量和重复剂量博莱霉素模型中 cthrc1+ 细胞的时间分布和谱系，我们正在开发过继转移和新型 ERcre 系，用于追踪来自支气管周围、外膜的细胞和肺泡成纤维细胞。接下来，我们将通过过继转移来评估 cthrc1+ 细胞的功能作用正常或博来霉素治疗的小鼠，对一系列与病理相关的行为进行体外研究成纤维细胞，并通过与我们生成的新型 cthrc1-ERcre 系杂交来删除这些细胞在 Rosa 基因座中表达 lox-stop-lox dta 的小鼠。最后，我们将研究每个的功能意义我们在基线和模型中在正常肺中鉴定出的主要胶原蛋白生成细胞群使用类似的消融策略或通过删除先前显示的基因来治疗肺泡和气道纤维化有助于组织纤维化。我们希望从这些研究中获得对每个角色的新见解独特的成纤维细胞亚群在肺稳态和疾病中发挥作用。