Fibroblast heterogeneity in pulmonary fibrosis

肺纤维化中成纤维细胞的异质性

• 批准号: 10167929
• 负责人: Dean Sheppard
• 金额: $ 18.8万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-04-01 至 2022-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10167929
• 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

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，其特征是损害正常的细胞外基质（ECM）蛋白的积累 功能是残疾和死亡的主要原因。尽管已经进行了大量研究以识别 细胞的多个潜在谱系可能引起负责ECM产生的成纤维细胞，它具有 被广泛认为这些细胞是一个相对均匀的细胞群，称为肌纤维细胞， 以平滑肌肌动蛋白（ASMA）的高表达为特征。但是，有关 这些细胞在体内的分子特征限制了我们对基本生物学的理解 纤维化并阻碍了有效疗法的发展。为了解决这一重要差距，我们已经使用了 胶原蛋白产生细胞的单细胞RNA测序（SCRNASEQ），以识别多种不同的细胞类型 在正常和纤维化的鼠和人类肺中产生胶原蛋白。使用原位接近连接 杂交（plish）我们鉴定出具有不同分子特征的产生胶原蛋白的细胞的子集 集中在导电道（周环）的壁上，周围是支气管血管束 （外囊）和扩散分布在气体交换区域（牙槽）。博来霉素治疗后 出现了独特的新子集，表达了高水平的胶原蛋白和其他ECM蛋白质，并且是独特的 由含有蛋白1的胶原蛋白三重螺旋重复的表达（CTHRC1）。解离的scrnaseq 来自正常和纤维化人肺的细胞也鉴定出以CTHRC1表达为标志的细胞群 这表达了最高水平的胶原蛋白和其他ECM蛋白，并且仅在肺中看到 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 CHRC1+细胞的时间分布和谱系在单个和重复剂量博来霉素模型中使用Plish， 我们正在开发采用转移和新型ERCRE线，以跟踪源自周围的源性细胞 和肺泡成纤维细胞。接下来，我们将使用收养转移到CTHRC1+细胞的功能作用 正常或博来霉素治疗的小鼠，在体外研究与病理相关的一系列行为 成纤维细胞，通过跨越这些细胞的删除，通过跨越新的CTHRC1- ercre系列，我们已经生成 在罗莎基因座中表达Lox-Stop-lox DTA的小鼠。最后，我们将检查每个功能意义 在基线和模型中，我们已经在正常肺中鉴定出的主要胶原蛋白细胞群体 使用类似的消融策略或通过先前显示的基因删除牙槽和气道纤维化 有助于组织纤维化。从这些研究中，我们希望能够对每一个的作用有新颖的见解 独特的成纤维细胞子集在肺稳态和疾病中发挥作用。