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: 10444342
• 负责人: Giovanni Ligresti
• 金额: $ 71.71万
• 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/10444342
• 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; Disease; Disease Progression; Endothelial Cells; Endothelium; Enhancers; Epigenetic Process; Exhibits; Fibroblasts; Fibrosis; Foundations; Functional disorder; Gene Silencing; Genes; Genetic; Genetic Transcription; Goals; Grant; Histologic; Histones; Homeostasis; Human; Hypoxia; Immunohistochemistry; Impairment; In Vitro; Inflammation; Inflammatory; Inflammatory Response; Knowledge; Laboratories; Link; Lung; Mediating; Mediator of activation protein; Methods; Molecular; Molecular Abnormality; Mus; Natural Immunity; Pathway interactions; Patients; Pharmacology; Phase; Phenotype; Play; Production; Pulmonary Fibrosis; Reporter; Resolution; Role; STAT1 gene; Signal Transduction; System; Testing; Therapeutic; Transgenic Mice; Vascular Diseases; Vascular remodeling; aged; angiogenesis; base; 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; progenitor; repaired; response to injury; senescence; single-cell RNA sequencing; transcription factor; transcriptome sequencing; vascular abnormality; vascular endothelial dysfunction

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.

摘要 特发性肺纤维化(IPF)是一种慢性、致命性的老年疾病，治疗选择有限。尽管 IPF肺的特点是血管异常，包括毛细血管稀疏和血管漏，影响血管 血管内皮细胞功能障碍在本病进展中的作用尚不清楚。这份提案是专门设计的 来填补这一知识空白。幼龄和老年小鼠肺内皮细胞的多组学分析 在我们实验室进行的实验表明转录因子ERG是肺血管的协调者 修复和炎症，其体内平衡功能在随着年龄增长的纤维化过程中受到损害。遗传性消融 幼年小鼠内皮细胞ERG导致炎症增加、血管稀疏和持续性肺纤维化 博莱霉素激发后，反映了衰老的肺表型。ERG沉默体外培养的人肺内皮细胞 促进IPF来源的肺成纤维细胞活化的促纤维化介质的分泌增加。整体 肺scRNA-seq结合FACS分析显示肺祖细胞ECs数量减少，称为 与WT小鼠相比，ERG缺陷小鼠的一般毛细血管(GCap)内皮细胞也发生了这种变化 肺来源于IPF患者。增强子结合蛋白的药理抑制与表观遗传学 调节剂BRD4逆转ERG沉默的人肺内皮细胞的炎症反应并恢复GCap 体外培养的IPF肺组织中EC的鉴定。此外，IPF和博莱霉素诱导的小鼠肺纤维化 与调节坏死性下垂的基因过度表达有关，坏死性下垂是一种程序性炎症细胞 死亡与衰老和纤维化有关。此外，使用特异性抑制剂Necrostatin抑制坏死性下垂- 1 减轻ERG沉默所致的人肺内皮细胞炎症。基于这些发现，我们 衰老相关表观遗传学重塑损害受损肺GCap内皮细胞ERG转录的假说 导致炎症调节失调、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 肺内皮细胞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 将确定内皮细胞坏死性下垂在老年小鼠肺血管异常和纤维化中的作用 ERG功能受损。我们将使用体外、体外和体内模型来验证我们的假设，包括 人肺间质纤维化的器官模型。将从人IPF肺中分离出富含GCap的内皮细胞和成纤维细胞。 将在内皮-成纤维细胞共培养系统中评估内皮细胞的纤维化活性。有条件的 将使用基因敲除和谱系追踪的方法来研究ERG在内皮动态平衡中的作用 和肺纤维化。纤维化、血管渗漏、毛细血管疏松和缺氧将用分子生物学方法进行评估， 生化和组织学方法。其他方法包括免疫组织化学、芯片序列分析、 以及对选定靶点的药物抑制。这项提案将定义至关重要的内皮细胞改变 在肺纤维化的进展中，我们朝着确定治疗IPF的新靶点的长期目标前进。