Targeting vascular dysfunction to promote lung repair and fibrosis resolution

摘要 特发性肺纤维化（IPF）是一种慢性、致命的老年疾病，治疗选择有限。尽管 IPF肺以血管异常为特征，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仍未被探索。该提案旨在 来填补这一知识空白。青年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，其稳态功能在随着衰老的纤维化过程中受损。的基因切除 年轻小鼠的内皮ERG导致炎症增加、血管稀疏和永久性肺纤维化 在博来霉素激发后，反映了老化的肺表型。人肺内皮细胞ERG沉默的体外实验研究 促进IPF衍生的肺成纤维细胞活化的纤维化介质的分泌增加。整个 肺scRNA-seq结合FACS分析揭示了肺祖细胞EC数量的减少，称为 与WT小鼠相比，ERG缺陷小鼠中的一般毛细血管（gCap）EC; 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BRD 4逆转了ERG沉默的人肺EC的体外炎症反应并恢复了gCap 离体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是 与调节坏死性凋亡（一种程序性炎症细胞）的基因过表达有关 与衰老和纤维化有关的死亡此外，用特异性抑制剂Necrostatin- 1 减弱人肺EC中ERG沉默诱导的炎症。基于这些发现，我们 假设衰老相关的表观遗传重构损害受损的肺gCap EC中的ERG转录 导致失调的炎症、毛细血管稀疏和持续的肺纤维化。 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将确定以下方面的作用： 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作用 视网膜电图功能受损我们将使用体外、离体和体内模型测试我们的假设，包括 人IPF的器官型模型。将从人IPF肺中分离gCap富集的EC和成纤维细胞。 将在内皮-成纤维细胞共培养系统中评价内皮纤维化活性。条件 基因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用分子， 生物化学和组织学方法。另外的方法包括免疫组织化学、ChIP-seq分析、 和对所选靶点的药理学抑制。该提案将定义至关重要的内皮改变 在肺纤维化的进展中，我们的长期目标是确定治疗IPF的新靶点。

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.