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

• 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
• 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

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）是一种慢性致命疾病，具有有限的治疗选择。尽管 IPF肺具有血管异常，包括毛细血管稀疏和血管泄漏，血管的影响 该疾病进展的内皮功能障碍尚未探索。该建议的设计 填补这一知识差距。来自年轻小鼠肺和老年小鼠肺的内皮细胞（EC）的多词分析 在我们的实验室中表演的是转录因子ERG作为肺血管的编排 修复和炎症，其稳态功能随着衰老而受损。遗传消融 年轻小鼠的内皮ERG导致炎症增加，血管稀疏性和永久性肺纤维化 在博来霉素挑战之后，反映了老化的肺表型。 ERG在体外LED中的人肺EC中沉默 增加了促进IPF衍生的肺成纤维细胞激活的纤维化介质的分泌。所有的 肺scrna-seq与FACS分析相结合，显示肺祖细胞EC数量减少，称为 与WT小鼠相比，ERG缺乏小鼠的一般毛细血管（GCAP）ECS；在 来自IPF患者的肺。增强子结合蛋白和表观遗传学的药理抑制 调节剂BRD4逆转了ERG脱毛的人肺EC中的炎症反应，并恢复了GCAP IPF肺外植体中的EC身份。此外，IPF和博来霉素诱导的小鼠肺纤维化是 与调节坏死性的基因过表达相关，这是一种编程炎症细胞的形式 死亡与衰老和纤维化有关。另外，用特异性抑制剂颗粒素抑制坏死性抑制 1 ERG沉默在人肺EC中引起的炎症。根据这些发现，我们 假设与衰老相关的表观遗传重塑损害受伤的肺GCAP EC中的ERG转录 导致炎症，毛细血管稀疏性和持续性肺纤维化失调。 目的1本提案 将表征ERG对肺GCAP EC再生和纤维化的影响。 AIM 2将定义 肺内皮ERG染色质相互作用，转录和血管衰老的表观遗传机制。目标3 将建立内皮坏死对肺血管异常和纤维化的贡献 具有受损的ERG功能。我们将使用体外，体内和体内模型，包括 人类IPF的器官模型。富含GCAP的EC和成纤维细胞将从人IPF肺中分离出来。 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 敲除和谱系跟踪方法将用于研究ERG在内皮体内平衡中的作用 和肺纤维化。将用分子评估纤维化，血管渗漏，毛细血管稀疏和缺氧将评估 生化和组织学方法。其他方法包括免疫组织化学，CHIP-seq分析， 和对选定靶标的药理抑制。该建议将定义至关重要的内皮改变 在肺纤维化朝着我们的长期目标的发展中，即确定新的IPF治疗目标。