Nuclear Heparan Sulfate Mediates Fibroblast Activation in Indiopathic Pulmonary Fibrosis

项目摘要/摘要 特发性肺纤维化(IPF)是一种以肺纤维化为特征的持续进行性肺部疾病。 细支气管周围间质。越来越多的人认识到，IPF代表着慢性后果 对急性肺损伤的不适应反应。因此，已知的急性肺损伤的介质和 急性呼吸窘迫综合征(ARDS)可能与IPF的发病机制有直接关系。 我的导师埃里克·施密特博士(科罗拉多大学)的实验室之前已经证明 肝素酶在ARDS发病中的重要性。肝素酶特异性地降解硫酸乙酰肝素(HS)， 一种细胞表面的糖胺多糖，对体内平衡的肺功能至关重要。乙酰肝素酶介导的乳清蛋白降解 内皮HS导致ARDS；然而，HS的降解与肺成纤维细胞和慢性 肺损伤的研究相对较少。 在初步实验中，我们观察到肺外植体中肝素酶的表达增加 重度IPF患者。乙酰肝素酶与活化的肺成纤维细胞共定位并与 人IPF外植体分离的肺成纤维细胞表面HS丢失。这种细胞表面的损失 HS是促纤维化的，因为HS从正常的人肺成纤维细胞中实验性降解诱导表达 促纤维化基因。有趣的是，这种成纤维细胞的激活与HS的核定位有关 从降解的细胞表面释放的碎片。核HS易位足以诱导前- 类似诱导的正常小鼠肺成纤维细胞(MLF)核定位的纤维化基因表达 促纤维化基因转化生长因子β、胶原1A1和ɑ平滑肌肌动蛋白的表达。AS 核HS此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激活可能反映了表观遗传学 核HS易位的后果。 在这些初步研究的基础上，我假设损伤诱导的肺组织表达 乙酰肝素酶切割成纤维细胞表面的HS，导致HS片段的核内化。 核HS进而抑制组蛋白乙酰转移酶活性，诱导促纤维化基因 在IPF中的表达。这些研究代表了一项具有强大初步数据的新的、高影响力的调查， 为我提供了一个独特的、高收益的培训机会，可以在不同的领域进行培训，比如糖胺聚糖 生物学、肺损伤和修复，以及最新的分子生物学技术。此外，建议的 研究具有翻译相关性，代表了通过以下途径治疗肺纤维化的新机会 防止HS降解(和HS片段的释放)或增强组蛋白 乙酰化。

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.