Nuclear Heparan Sulfate Mediates Fibroblast Activation in Indiopathic Pulmonary Fibrosis

核硫酸乙酰肝素介导特发性肺纤维化中成纤维细胞活化

• 批准号: 10199011
• 负责人: Wells B LaRiviere
• 金额: $ 5.1万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-07-13 至 2022-07-12
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10199011
• 关键词: Acetyltransferase; Acute; Acute Lung Injury; Adult Respiratory Distress Syndrome; Attenuated; Beta-glucuronidase; Binding; Biological Assay; Biology; Bleomycin; Calorimetry; Cell Nucleus; Cell surface; Characteristics; Chronic; Cleaved cell; Clinical; Collagen; Colorado; Complement Factor B; Data; Deposition; Endothelium; Epigenetic Process; Fibroblasts; Fibrosis; Gene Expression; Genes; Glycosaminoglycans; Heparitin Sulfate; Histology; Histone Acetylation; Histone Deacetylase; Histone Deacetylase Inhibitor; Human; Influenza; Injury; Investigation; Laboratories; Lung; Lung Compliance; Lung diseases; Mass Spectrum Analysis; Measures; Mediating; Mediator of activation protein; Mentors; Molecular Biology Techniques; Mus; Nuclear; Nuclear Translocation; Oligosaccharides; Oncogenes; Pathogenesis; Pathologic; Patients; Pharmacology; Prevention; Pulmonary Edema; Pulmonary Fibrosis; Role; Sampling; Smooth Muscle Actin Staining Method; Structure; Sulfate; Testing; Transforming Growth Factor beta; Transforming Growth Factors; Transgenic Organisms; Universities; Western Blotting; analog; base; cell type; experimental study; gene induction; heparanase; heparinase III; histone acetyltransferase; idiopathic pulmonary fibrosis; in vivo; inhibitor/antagonist; injury and repair; lung injury; mouse model; novel; nuclear transfer; prevent; pulmonary function; response; therapeutic target; training opportunity

PROJECT SUMMARY/ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a relentlessly progressive lung disease characterized by fibrosis of the peribronchiolar interstitium. There is increasing appreciation that IPF represents the chronic consequence of a maladaptive response to acute lung injury. Accordingly, known mediators of acute lung injury and the acute respiratory distress syndrome (ARDS) may have direct relevance to IPF pathogenesis. The laboratory of my mentor, Dr. Eric Schmidt (University of Colorado), has previously demonstrated the importance of heparanase to the onset of ARDS. Heparanase specifically degrades heparan sulfate (HS), a cell-surface glycosaminoglycan essential for homeostatic lung function. Heparanase-mediated degradation of endothelial HS leads to ARDS; however, the relevance of HS degradation to pulmonary fibroblasts and chronic lung injury is relatively unexplored. In preliminary experiments, we observed increased expression of heparanase in lung explants from patients with severe IPF. Heparanase co-localized with activated pulmonary fibroblasts and was associated with loss of cell-surface HS in pulmonary fibroblasts isolated from human IPF explants. This loss of cell-surface HS is profibrotic, as experimental degradation of HS from normal human lung fibroblasts induced expression of pro-fibrotic genes. Intriguingly, this fibroblast activation was associated with nuclear localization of HS fragments released from the degraded cell surface. Nuclear HS translocation was sufficient to induce pro- fibrotic gene expression, as nucleofection of normal mouse lung fibroblasts (MLFs) similarly induced expression of profibrotic genes transforming growth factor β, collagen 1A1, and ɑ Smooth Muscle Actin. As nuclear HS has been previously implicated as an inhibitor of histone acetyltransferases, the observed profibrotic activation of pulmonary fibroblasts after degradation of cell-surface HS may reflect the epigenetic consequence of nuclear HS translocation. Based upon these preliminary studies, I hypothesize that injury-induced expression of pulmonary heparanase cleaves fibroblast cell-surface HS, leading to nuclear internalization of HS fragments. Nuclear HS in turn suppresses histone acetyltransferase activity, inducing pro-fibrotic gene expression in IPF. These studies represent a novel, high-impact investigation with robust preliminary data, providing me with a unique and high-yield opportunity for training in diverse field such as glycosaminoglycan biology, lung injury and repair, and state-of-the-art molecular biology techniques. Furthermore, the proposed studies have translational relevance, representing a novel opportunity to therapeutically target lung fibrosis via either the prevention of HS degradation (and release of HS fragments) or the augmentation of histone acetylation.

项目概要/摘要 特发性肺纤维化（IPF）是一种持续进行性肺部疾病，其特征是肺纤维化 细支气管周围间质。人们越来越认识到 IPF 是其长期后果 对急性肺损伤的适应不良反应。因此，已知的急性肺损伤介质和 急性呼吸窘迫综合征 (ARDS) 可能与 IPF 发病机制直接相关。 我的导师 Eric Sc​​hmidt 博士（科罗拉多大学）的实验室之前已经证明 乙酰肝素酶对 ARDS 发病的重要性。乙酰肝素酶特异性降解硫酸乙酰肝素 (HS)， 一种对肺稳态功能至关重要的细胞表面糖胺聚糖。乙酰肝素酶介导的降解 内皮 HS 导致 ARDS；然而，H2S 降解与肺成纤维细胞和慢性疾病的相关性 肺损伤相对尚未被探索。 在初步实验中，我们观察到肺外植体中乙酰肝素酶的表达增加 严重IPF患者。乙酰肝素酶与活化的肺成纤维细胞共定位并相关 从人 IPF 外植体中分离出的肺成纤维细胞的细胞表面 HS 丢失。这种细胞表面的损失 HS 是促纤维化的，因为正常人肺成纤维细胞中 HS 的实验降解诱导了 促纤维化基因。有趣的是，这种成纤维细胞激活与 HS 的核定位有关 从降解的细胞表面释放的碎片。核 HS 易位足以诱导亲 正常小鼠肺成纤维细胞 (MLF) 的核转染同样诱导纤维化基因表达 促纤维化基因转化生长因子 β、胶原蛋白 1A1 和 ɑ 平滑肌肌动蛋白的表达。作为 核 HS 先前被认为是组蛋白乙酰转移酶的抑制剂，观察到 细胞表面 HS 降解后肺成纤维细胞的促纤维化活化可能反映了表观遗传 核 HS 易位的结果。 基于这些初步研究，我假设损伤诱导的肺表达 乙酰肝素酶裂解成纤维细胞表面的 HS，导致 HS 片段的核内化。 核 HS 反过来抑制组蛋白乙酰转移酶活性，诱导促纤维化基因 IPF 中的表达。这些研究代表了一项新颖的、高影响力的调查，具有可靠的初步数据， 为我提供了在糖胺聚糖等不同领域进行培训的独特且高收益的机会 生物学、肺损伤和修复以及最先进的分子生物学技术。此外，拟议的 研究具有转化相关性，代表了通过治疗靶向肺纤维化的新机会 防止 HS 降解（和释放 HS 片段）或增强组蛋白 乙酰化。

项目成果

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining.

• DOI: 10.3791/62319
• 发表时间: 2021-02-25
• 期刊: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS
• 影响因子: 1.2
• 作者: LaRiviere, Wells B.;Han, Xiaorui;Oshima, Kaori;McMurtry, Sarah A.;Linhardt, Robert J.;Schmidt, Eric P.
• 通讯作者: Schmidt, Eric P.