Histologic and Transcriptional Profiling of Endothelial Cells During Progressive Pulmonary Fibrosis

进行性肺纤维化期间内皮细胞的组织学和转录谱

• 批准号: 10616601
• 负责人: Rachel S Knipe
• 金额: $ 8.33万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-01 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10616601
• 关键词: Abnormal Endothelial Cell; Actins; Apoptosis; Attenuated; Biological Assay; Bleomycin; Blood Vessels; Blood capillaries; Capillary Endothelial Cell; Cell Aging; Cell Communication; Cell physiology; Cells; Clinical; Clinical Trials; Cytoskeleton; DNA Sequence Alteration; Data; Deposition; Development; Diffuse; Disease; Disease Progression; Dose; Drug Targeting; Early Intervention; Endothelial Cells; Endothelium; Epithelial Cells; Epithelium; Equilibrium; Extracellular Matrix; Fibroblasts; Fibrosis; Functional disorder; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Genetic; Genetic Transcription; Goals; Histologic; Homeostasis; Human; Imaging Techniques; Immunofluorescence Immunologic; Injections; Injury; Knockout Mice; Knowledge; Lead; Link; Lung; Lung diseases; Lung fibrogenesis; Mediating; Mediator; Mesenchymal; Modeling; Mus; Myofibroblast; Myosin ATPase; Normal tissue morphology; Pathogenesis; Pathologic; Patients; Permeability; Phenotype; Play; Population; Process; Pulmonary Fibrosis; Research; Respiratory Failure; Rho-associated kinase; Role; Signal Transduction; Stains; Structure of parenchyma of lung; Therapeutic Intervention; Time; Vascular Permeabilities; angiogenesis; cell injury; density; effective therapy; endothelial dysfunction; experimental study; fibrogenesis; fibrotic lung; idiopathic pulmonary fibrosis; improved; insight; lung development; lung injury; migration; molecular imaging; mortality; mouse model; new therapeutic target; novel; prevent; programs; pulmonary function; pulmonary vascular permeability; repaired; senescence; sildenafil; single-cell RNA sequencing; tissue injury; tissue repair; tool; trafficking; transcriptome; wound healing

PROJECT SUMMARY Idiopathic Pulmonary Fibrosis (IPF) is a progressive scarring lung disease that very often leads to respiratory failure. There remains a large unmet need for effective therapies. IPF is thought to be driven by injury to a vulnerable epithelium, sensitive to dysfunction either by genetic mutations or by the dose or type of injury. Epithelial dysfunction and senescence lead to diffuse tissue injury and activation of myofibroblasts. Dysregulated wound repair mechanisms may play a role in amplification of the tissue injury, tipping the balance from repair to fibrosis. Data is emerging that there are abnormalities in the pulmonary endothelium of patients who develop pulmonary fibrosis, although the cause/effect of these changes on the process of fibrogenesis is not well understood. Increased vascular permeability has been shown in the lungs of IPF patients and predicts mortality. In addition, several recent single-cell RNA sequencing studies have demonstrated loss of pulmonary capillaries and an increase in a bronchial vessel population. The full mechanistic implications of these changes have not been defined. Rho-associated kinase (ROCK) signaling has been shown to play a major role in several endothelial cell functions, including cell contractility and subsequent alterations in vascular permeability. I have shown in preliminary data that loss of ROCK2 specifically in endothelial cells in a mouse model (EC ROCK2 KO) prevents the development of pulmonary fibrosis induced by the delivery of intratracheal bleomycin. In addition to less pulmonary fibrosis, these mice had less vascular permeability in the early time period after bleomycin, before fibrosis develops. This finding suggests a link between endothelial ROCK2, vascular function, and pulmonary fibrosis, and hints at the potential for an opportunity for early intervention, when fibrosis may be preventable or reversible. In this current proposal, I propose to explore the protective phenotype in EC ROCK2 KO mice further by using these mice in a repetitive bleomycin model of pulmonary fibrosis, which has been shown to produce progressive fibrosis and is more similar to IPF. I will characterize the endothelial changes in this repetitive bleomycin model through staining and permeability assays. I will also perform single-cell RNA sequencing of mouse lungs with or without ROCK2, in the repetitive bleomycin model of pulmonary fibrosis. I will look for mechanistic links between endothelial ROCK2 and angiogenesis, senescence, endothelial to mesenchymal transition and apoptosis, all in the context of fibrogenesis.

项目总结 特发性肺纤维化(IPF)是一种进行性疤痕肺疾病，通常导致 到呼吸衰竭。对有效疗法的需求仍然很大，尚未得到满足。IPF被认为是 是由脆弱的上皮损伤驱动的，对功能障碍敏感，要么是由于基因 突变或根据损伤的剂量或类型。上皮功能障碍和衰老导致弥漫性 肌成纤维细胞的组织损伤和激活。失调的伤口修复机制可能发挥作用 一种放大组织损伤的作用，扭转了从修复到纤维化的平衡。数据是 出现肺内皮细胞异常的患者 肺纤维化，尽管这些变化在纤维化形成过程中的因果关系是 不是很清楚。肺间质纤维化的肺血管通透性增加。 并预测死亡率。此外，最近的几项单细胞RNA测序研究 已显示肺毛细血管丢失和支气管血管增多 人口。这些变化的全部机械性影响还没有定义。 Rho相关激酶(ROCK)信号已被证明在几个 血管内皮细胞功能，包括细胞收缩和随后的血管改变 渗透性。我已经在初步数据中表明，ROCK2的丢失特别是在内皮细胞中 小鼠模型(EC ROCK2 KO)中的细胞可防止肺纤维化的发展 由气管内注射博莱霉素所致。除了肺纤维化较少外，这些 小鼠在博莱霉素治疗后的早期，即纤维化之前，血管通透性较低 发展起来。这一发现表明内皮细胞ROCK2、血管功能和 肺纤维化，并暗示有机会进行早期干预，当 纤维化可能是可预防的或可逆的。 在目前的建议中，我建议探索EC ROCK2 KO的保护性表型 进一步将这些小鼠用于重复的博莱霉素肺纤维化模型，该模型 已被证明会产生进行性纤维化，更类似于IPF。我将描述一下 通过染色和通透性观察博莱霉素重复模型中血管内皮细胞的变化 化验。我还将对有或没有ROCK2的小鼠肺进行单细胞RNA测序， 在重复的博莱霉素肺纤维化模型中。我将寻找机械上的联系 内皮细胞ROCK2与血管生成、衰老、内皮细胞向间充质转化和 细胞凋亡，ALL在纤维化形成的背景下。