Role of Heparan Sulfate 6-O-Endosulfatase 1 and 2 in Pulmonary Fibrosis

硫酸乙酰肝素 6-O-内切硫酸酯酶 1 和 2 在肺纤维化中的作用

• 批准号: 7897159
• 负责人: XINPING YUE
• 金额: $ 21.13万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-04-02 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7897159
• 关键词: A549; Address; Adenovirus Vector; Alveolar; Animal Model; Apoptosis; Area; Bleomycin; Cell Culture Techniques; Cell Line; Cell surface; Cells; Data; Development; Epithelial; Epithelial Cells; Extracellular Matrix; Fibroblast Growth Factor; Fibroblasts; Fibrosis; Gene Expression; Genetic; Growth; Hamman-Rich syndrome; Harvest; Heparitin Sulfate; Human; Hyperplasia; Immunohistochemistry; In Situ Hybridization; In Vitro; Inorganic Sulfates; Knock-out; Knockout Mice; Knowledge; Laboratories; Lead; Ligands; Literature; Lung; Mesenchymal; Messenger RNA; Modeling; Mus; Myofibroblast; Organ; Pathogenesis; Pathology; Patients; Play; Process; Production; Property; Proteins; Publishing; Pulmonary Fibrosis; Research; Resolution; Reverse Transcriptase Polymerase Chain Reaction; Role; Sampling; Sentinel; Signal Pathway; Signal Transduction; Site; Source; Structure of parenchyma of lung; Testing; Time; Transforming Growth Factors; United States; Unspecified or Sulfate Ion Sulfates; Viral; Viral Vector; Western Blotting; alveolar type II cell; base; cell type; fibrogenesis; in vivo; injury and repair; novel; novel therapeutic intervention; prevent; public health relevance; repaired; response; vector control

DESCRIPTION (provided by applicant): Transforming growth factor (TGF)-(1 plays a central role in the pathobiology of pulmonary fibrosis (PF), a debilitating and fatal illness that currently has no cure. TGF-(1 promotes PF by acting on both fibroblasts and epithelial cells, which are the two major cell types involved in the pathogenesis of PF. TGF-(1 initiates and sustains fibroblast activation and trans-differentiation into myofibroblasts, a hallmark of PF. In addition, TGF-(1 induces extracellular matrix (ECM) gene expression in myofibroblasts, the primary cellular source of ECM production in areas of active fibrogenesis. TGF-(1 induces growth arrest and apoptosis in alveolar type II (ATII) epithelial cells, thus preventing epithelial repair necessary for the resolution of PF. TGF-(1 also induces epithelial-mesenchymal transition in ATII cells, which has now been shown to occur in both human and animal models of PF. Our laboratory has made the sentinel observation that TGF-(1 induces Heparan sulfate (HS) 6- O-endosulfatase 1 and 2 (Sulf1 and Sulf2) in murine models of PF. Sulf1 and Sulf2 are the newly identified cell surface HS 6-O-endosulfatases, and have been shown to modulate HS-protein interactions by removing 6-O- sulfates from specific HS intra-chain sites. In vitro, TGF-(1 induces Sulf1 and Sulf2 in a cell-type specific manner, specifically Sulf1 in primary normal human lung fibroblasts and Sulf2 in A549 cells, an ATII cell line. Furthermore, blocking Sulf induction in lung fibroblasts and A549 cells altered their responses to TGF-(1. Finally, both Sulf1 and Sulf2 are up-regulated in human idiopathic pulmonary fibrosis. Based on our preliminary data, we hypothesize that heparan sulfate 6-O-endosulfatases, Sulf1 and Sulf2, modulate TGF-(1 function and the development of PF. In Aim 1, we will examine the role of Sulf1 in the development of PF. First, we will compare TGF-(1-induced PF in wild-type (WT) and Sulf1 knockout (KO) mice. Second, we will isolate primary lung fibroblasts from WT and Sulf1 KO mice and compare their fibrogenic properties in vitro. In Aim 2, we will examine the role of Sulf2 in the development of PF. First, we will compare TGF-(1-induced PF in WT and Sulf2 KO mice. Second, we will isolate primary ATII cells from WT and Sulf2 KO mice and compare their fibrogenic properties in vitro. In the in vivo studies, adenoviral vectors encoding active TGF-(1 or viral vector control will be used to induce PF in mice, and lung tissues will be harvested and examined at different time points following viral exposure for evidence of fibrosis and expression of fibrotic markers at both protein and mRNA levels using standard immunohistochemistry, in situ hybridization, Western Blotting and quantitative real-time RT-PCR. In the in vitro cell culture studies, cellular responses to TGF-(1 as well as other factors involved in PF will be examined including FGF and Wnt. To our knowledge, this is the first study to address the role of Sulf1 and Sulf2 in the development of PF. Our findings could lead to the development of new therapeutic interventions for PF as well as fibrogenic pathology in other organs. PUBLIC HEALTH RELEVANCE: There are currently over 200,000 patients in the United States and five million people worldwide suffering from pulmonary fibrosis (PF). Combining mouse genetics and in vitro cell culture studies, we will address in this study, for the first time, the role of Sulf1 and Sulf2 in the development of PF. Findings from this study may lead to the development of novel therapies for PF.

描述（由申请人提供）：转化生长因子 (TGF)-(1 在肺纤维化 (PF) 的病理学中发挥核心作用，肺纤维化是一种目前无法治愈的使人衰弱的致命疾病。TGF-(1 通过作用于肺纤维化促进 PF成纤维细胞和上皮细胞是参与 PF 发病机制的两种主要细胞类型，TGF-(1 启动并维持成纤维细胞活化和转分化为肌成纤维细胞，此外，TGF-(1) 诱导肌成纤维细胞中的细胞外基质 (ECM) 基因表达，肌成纤维细胞是活跃纤维发生区域中 ECM 产生的主要细胞来源。TGF-(1 诱导 II 型肺泡生长停滞和细胞凋亡。 ATII) 上皮细胞，从而阻止 PF 消退所需的上皮修复，TGF-(1 还诱导 ATII 细胞中的上皮-间质转化，目前已证明这种转化发生在 ATII 细胞中。我们的实验室在 PF 的人类和动物模型中进行了前哨观察，发现 TGF-(1) 在 PF 小鼠模型中诱导硫酸乙酰肝素 (HS) 6-O-内切硫酸酯酶 1 和 2（Sulf1 和 Sulf2）。 Sulf1 和 Sulf2 是新鉴定的细胞表面 HS 6-O-内切硫酸酯酶，并已被证明可以通过从特定的 HS 链内位点去除 6-O- 硫酸盐来调节 HS-蛋白质相互作用。在体外，TGF-(1 以细胞类型特异性方式诱导 Sulf1 和 Sulf2，特别是原代正常人肺成纤维细胞中的 Sulf1 和 A549 细胞（一种 ATII 细胞系）中的 Sulf2。此外，阻断肺成纤维细胞和 A549 细胞中的 Sulf 诱导发生改变他们对 TGF-(1) 的反应。最后，Sulf1 和 Sulf2 在人类特发性肺纤维化中均上调。根据我们的初步数据，我们假设硫酸乙酰肝素 6-O-内切硫酸酯酶 Sulf1 和 Sulf2 调节 TGF-(1 功能和 PF 的发展。在目标 1 中，我们将研究 Sulf1 在 PF 发展中的作用。首先，我们将比较 TGF-(1) 的功能和 PF 的发展。 （1 在野生型 (WT) 和 Sulf1 敲除 (KO) 小鼠中诱导 PF。其次，我们将从 WT 和 Sulf1 KO 小鼠中分离原代肺成纤维细胞，并比较它们的体外纤维形成特性。在目标 2 中，我们将研究 Sulf2 在 PF 发展中的作用。首先，我们将比较 WT 和 Sulf2 KO 小鼠中 TGF-(1) 诱导的 PF。其次，我们将分离原发性 ATII。 WT 和 Sulf2 KO 小鼠的细胞并比较它们的体外纤维形成特性。在体内研究中，编码活性 TGF-(1) 的腺病毒载体或病毒载体对照将用于诱导小鼠 PF，并在病毒暴露后的不同时间点收获肺组织并进行检查，以获取纤维化和纤维化表达的证据。使用标准免疫组织化学、原位杂交、蛋白质印迹和定量实时 RT-PCR 在蛋白质和 mRNA 水平上标记在体外细胞培养研究中，细胞对 TGF-(1 以及其他因素) 的反应。据我们所知，这是第一个探讨 Sulf1 和 Sulf2 在 PF 发展中的作用的研究，我们的研究结果可能会导致 PF 以及新治疗干预措施的开发。其他器官的纤维化病理学。 公共卫生相关性：目前美国有超过 200,000 名患者，全球有 500 万人患有肺纤维化 (PF)。结合小鼠遗传学和体外细胞培养研究，我们将在本研究中首次探讨 Sulf1 和 Sulf2 在 PF 发展中的作用。这项研究的结果可能会促进肺纤维化新疗法的开发。