Targeting vascular dysfunction to promote lung repair and fibrosis resolution

针对血管功能障碍促进肺修复和纤维化消退

基本信息

批准号：
10584589
负责人：
Giovanni Ligresti
金额：
$ 69.32万
依托单位：
BOSTON UNIVERSITY MEDICAL CAMPUS
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-05 至 2026-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10584589
关键词：
ATAC-seq Ablation Address Aging Attenuated Binding Biochemical Bleomycin Blood Vessels Blood capillaries Capillary Endothelial Cell Cell Death ChIP-seq Chromatin Chronic Coculture Techniques Collagen Coupled Data Deterioration Disease Disease Progression Endothelial Cells Endothelium Enhancers Epigenetic Process Exhibits Fibroblasts Fibrosis Foundations Functional disorder Gene Expression Gene Silencing Genes Genetic Genetic Transcription Goals Grant Histologic Histones Homeostasis Human Hypoxia Immunohistochemistry Impairment In Vitro Inflammation Inflammatory Inflammatory Response Knowledge Laboratories Link LoxP-flanked allele Lung Mediating Mediator Methods Molecular Molecular Abnormality Mus NF-kappa B Natural Immunity Pathway interactions Patients Phase Phenotype Play Production Pulmonary Fibrosis Reporter Resolution Role STAT1 gene Signal Transduction System Testing Therapeutic Transgenic Mice Vascular Diseases Vascular Permeabilities Vascular remodeling aged angiogenesis cell age cell regeneration conditional knockout deep sequencing defined contribution design endothelial dysfunction endothelial stem cell enhancer binding protein experimental study gene function gene repair genetic signature genome-wide human model idiopathic pulmonary fibrosis in vivo Model inhibitor injured lung injury lung repair multiple omics novel overexpression paracrine pharmacologic progenitor repaired response to injury senescence single-cell RNA sequencing transcription factor transcriptome sequencing vascular abnormality vascular endothelial dysfunction vascular inflammation

项目摘要

ABSTRACT Idiopathic Pulmonary Fibrosis (IPF) is a chronic, fatal disease of aging with limited therapeutic options. Despite IPF lungs feature vascular abnormalities, including capillary rarefaction and vascular leak, the impact of vascular endothelial dysfunction in the progression of this disease has remained unexplored. This proposal is designed to fill this knowledge gap. Multi-omics analysis of endothelial cells (ECs) from young and aged mouse lungs performed in our laboratory implicated the transcription factor ERG as an orchestrator of pulmonary vascular repair and inflammation, whose homeostatic function is impaired during fibrosis with aging. Genetic ablation of endothelial ERG in young mice led to increased inflammation, vascular rarefaction, and perpetuated lung fibrosis following bleomycin challenge mirroring the aged lung phenotype. ERG silencing in human lung ECs in vitro led to the increased secretion of fibrogenic mediators that promoted IPF-derived lung fibroblast activation. Whole lung scRNA-seq combined with FACS analysis revealed reduced number of lung progenitor ECs, known as general capillary (gCap) ECs, in ERG deficient mice compared to WT mice; this alteration was also observed in lungs derived from IPF patients. Pharmacologic inhibition of the enhancer-binding protein and epigenetic regulator BRD4 reversed inflammatory responses in ERG-silenced human lung ECs in vitro and restored gCap EC identity in IPF lung explants ex vivo. Moreover, IPF and bleomycin-induced mouse lung fibrosis were associated with overexpression of genes that regulate necroptosis, a form of programmed inflammatory cell death implicated in aging and fibrosis. In addition, inhibition of necroptosis with the specific inhibitor Necrostatin- 1 attenuated inflammation induced by ERG silencing in human lung ECs. Based on these findings we hypothesize that aging-associated epigenetic remodeling impairs ERG transcription in injured lung gCap ECs leading to dysregulated inflammation, capillary rarefaction, and persistent lung fibrosis. Aim 1 of this proposal will characterize the influence of ERG on lung gCap EC regeneration and fibrosis. Aim 2 will define the role of epigenetic mechanisms in lung endothelial ERG chromatin interaction, transcription, and vascular aging. Aim 3 will establish the contribution of endothelial necroptosis to lung vascular abnormalities and fibrosis in aged mice with compromised ERG functions. We will test our hypothesis using in vitro, ex vivo and in vivo models including an organotypic model of human IPF. gCap-enriched ECs and fibroblasts will be isolated from human IPF lungs. Endothelial fibrogenic activity will be evaluated in an endothelial-fibroblast co-culture systems. Conditional knockout and lineage tracing approaches will be used to investigate the role of ERG in endothelial homeostasis and lung fibrosis. Fibrosis, vascular leak, capillary rarefaction, and hypoxia will be evaluated with molecular, biochemical, and histological methods. Additional methods include immunohistochemistry, ChIP-seq analysis, and pharmacologic inhibition of selected targets. This proposal will define endothelial alterations that are critical in the progression of lung fibrosis toward our long-term goal of identifying novel targets for the treatment of IPF.

抽象的特发性肺纤维化（IPF）是一种慢性致命的衰老疾病，治疗选择有限。尽管 IPF肺部的特点是血管异常，包括毛细血管稀疏和血管渗漏，血管的影响这种疾病进展中的内皮功能障碍尚未被探索。本提案设计来填补这一知识空白。对年轻和老年小鼠肺内皮细胞 (EC) 进行多组学分析我们实验室进行的研究表明转录因子 ERG 作为肺血管的协调者修复和炎症，其稳态功能在衰老纤维化过程中受损。基因消融年轻小鼠的内皮 ERG 导致炎症增加、血管稀疏和持久的肺纤维化博莱霉素激发后反映了老年肺表型。体外导致人肺内皮细胞 ERG 沉默纤维生成介质的分泌增加，促进 IPF 衍生的肺成纤维细胞活化。所有的肺 scRNA-seq 结合 FACS 分析显示肺祖细胞 EC 数量减少，称为与 WT 小鼠相比，ERG 缺陷小鼠中的一般毛细血管 (gCap) EC；这种变化也被观察到来自IPF患者的肺。增强子结合蛋白的药理学抑制和表观遗传调节剂 BRD4 在体外逆转了 ERG 沉默的人肺 EC 的炎症反应并恢复了 gCap IPF 肺外植体中的 EC 身份。此外，IPF 和博莱霉素诱导的小鼠肺纤维化与调节坏死性凋亡（一种程序性炎症细胞形式）的基因过度表达有关死亡与衰老和纤维化有关。此外，用特异性抑制剂 Necrostatin 抑制坏死性凋亡 1 减轻人肺 EC 中 ERG 沉默诱导的炎症。根据这些发现我们假设衰老相关的表观遗传重塑会损害受损肺 gCap EC 中的 ERG 转录导致炎症失调、毛细血管稀疏和持续性肺纤维化。本提案的目标 1 将表征 ERG 对肺 gCap EC 再生和纤维化的影响。目标 2 将定义以下角色：肺内皮 ERG 染色质相互作用、转录和血管老化的表观遗传机制。目标 3 将确定内皮坏死性凋亡对老年小鼠肺血管异常和纤维化的影响 ERG 功能受损。我们将使用体外、离体和体内模型来检验我们的假设，包括人类 IPF 的器官模型。将从人 IPF 肺部分离出富含 gCap 的 EC 和成纤维细胞。内皮纤维化活性将在内皮-成纤维细胞共培养系统中进行评估。有条件的敲除和谱系追踪方法将用于研究 ERG 在内皮稳态中的作用和肺纤维化。纤维化、血管渗漏、毛细血管稀疏和缺氧将通过分子、生物化学和组织学方法。其他方法包括免疫组织化学、ChIP-seq 分析、以及对选定靶标的药理学抑制。该提案将定义至关重要的内皮改变我们的长期目标是确定治疗 IPF 的新靶点，从而促进肺纤维化的进展。