Identification of Pathways that Regulate Fibrosis through Collagen Resorption

鉴定通过胶原吸收调节纤维化的途径

基本信息

批准号：
8250331
负责人：
KAMRAN ATABAI
金额：
$ 19.31万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-04-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8250331
关键词：
Aftercare Alveolar Alveolar Macrophages Animal Model Applications Grants Architecture Area Binding Biological Biological Assay Candidate Disease Gene Cell Line Cells Cicatrix Collagen Defect Deposition Diagnosis Digestion Disease Double-Stranded RNA Drosophila genome Drosophila genus Equilibrium Excision Experimental Models Extracellular Matrix Fibroblasts Fibrosis Flow Cytometry Future Gases Gene Proteins Gene Silencing Genes Glycoproteins Goals Grant Human Injury Libraries Lung Mediating Mediator of activation protein Metabolism Microscopy Mus Orthologous Gene Pathway interactions Patients Phagocytes Pharmacological Treatment Process Production Proteins Pulmonary Fibrosis RNA Interference RNA library Recombinant Proteins Research Role Severities Testing Tissues base cellular targeting design extracellular high throughput screening human subject in vivo innovation intervention effect lung injury macrophage new therapeutic target novel novel therapeutics prevent public health relevance research study response small hairpin RNA uptake

项目摘要

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a common disorder without effective pharmacological treatment. The metabolism of collagen, the main component of fibrotic tissue, is a dynamic process with the balance between collagen production and degradation determining tissue architecture. The pathways responsible for removal of collagen that has accumulated in areas of fibrosis are poorly understood. Whether therapies aimed at increasing collagen uptake will benefit patients with established fibrotic disease is unknown. We have recently described a novel pathway of collagen turnover that regulates the severity of tissue fibrosis. We have shown that the glycoprotein Mfge8 binds collagen that has accumulated in the extracellular matrix in areas of fibrosis. Mfge8-bound collagen is then taken up by macrophages for intracellular degradation. Mice deficient in Mfge8 develop an exaggerated fibrotic response after lung injury due to an in vivo defect in collagen degradation. We plan to build upon these findings by identifying other novel pathways by which collagen is taken up by cells. We have designed a Drosophila-based high-throughput screen of collagen uptake. In these studies we plan to identify novel mediators of collagen turnover through this screen and verify the function of human and orthologs of these genes. We will accomplish these goals through two specific aims. In the first aim, we will use a flow cytometry-based high-throughput screen of Drosophila S2 cell collagen uptake using a double stranded RNA library to identify candidate molecules involved in collagen turnover. The library created at UCSF covers Drosophila genes that have human and/or murine orthologs. Drosophila S2 cells are macrophage-like phagocytes that are highly susceptible to gene silencing with double-stranded RNA. We will subsequently rescreen candidate genes with both flow cytometry-based and fluorescent microscopy-based assays of collagen uptake. Candidate genes that remain after the secondary screen will be the focus of our second aim. In these studies we will confirm the function of human and murine orthologs of candidate molecules in collagen turnover. We will select the most compelling candidate genes identified through our Drosophila screen and evaluate their role in collagen turnover in both human and murine cell lines and primary cells. Our approach will involve both silencing candidate genes using shRNA and increasing candidate genes protein levels by adding exogenous recombinant protein. We will then test the effect of these interventions on collagen uptake and turnover by macrophages and fibroblasts. These studies have the potential to identify novel mediators of collagen turnover. The importance of a better understanding of the pathways responsible for collagen removal from the extracellular matrix is highlighted by the fact that no available therapies are efficacious in the treatment of patients with established fibrotic disease. PUBLIC HEALTH RELEVANCE: Pulmonary fibrosis is a common disorder without effective pharmacological treatment options. Research aimed at discovering pathways that remove fibrotic scar from the lung will provide new therapeutic options. The goals of this project are to identify and characterize the normal pathways that mediate scar remodeling with the goal of discovering new therapeutic targets for the treatment of pulmonary fibrosis.

描述（由申请人提供）：肺纤维化是一种常见疾病，没有有效的药理治疗。胶原蛋白的代谢是纤维化组织的主要成分，是一个动态过程，在胶原蛋白产生和降解之间的平衡确定了组织结构。对纤维化区域积累的胶原蛋白的去除的途径知之甚少。旨在增加胶原蛋白摄取的疗法是否会使既定的纤维化疾病患者受益。我们最近描述了一种胶原蛋白周转的新途径，该途径调节了组织纤维化的严重程度。我们已经表明，糖蛋白MFGE8结合了纤维化区域中在细胞外基质中积累的胶原蛋白。然后，通过巨噬细胞处理MFGE8结合的胶原蛋白，以进行细胞内降解。缺乏MFGE8的小鼠由于胶原蛋白降解的体内缺陷而导致肺损伤后产生夸张的纤维化反应。我们计划通过识别其他细胞吸收胶原蛋白的其他新型途径来建立这些发现。我们设计了一个基于果蝇的胶原蛋白摄取的高通量屏幕。在这些研究中，我们计划通过此屏幕确定胶原蛋白转移的新型介体，并验证这些基因的人类和直系同源物的功能。我们将通过两个具体目标来实现这些目标。在第一个目标中，我们将使用双链RNA库的果蝇S2细胞胶原蛋白摄取的基于流式细胞术的高通量屏幕来识别涉及胶原蛋白转换的候选分子。在UCSF创建的库涵盖了具有人类和/或鼠直系同源物的果蝇基因。果蝇S2细胞是巨噬细胞样的吞噬细胞，它非常容易受到双链RNA的基因沉默。随后，我们将与基于流式细胞术和基于荧光显微镜的胶原蛋白摄取的测定法重新分解候选基因。在次要屏幕之后保留的候选基因将成为我们第二个目标的重点。在这些研究中，我们将确认候选分子在胶原蛋白周转率中人类和鼠直系同源物的功能。我们将选择通过果蝇筛查确定的最引人注目的候选基因，并评估它们在人和鼠细胞系和原代细胞中胶原蛋白周转中的作用。我们的方法将既涉及使用shRNA的沉默候选基因，又通过添加外源重组蛋白来增加候选基因蛋白水平。然后，我们将通过巨噬细胞和成纤维细胞测试这些干预措施对胶原蛋白摄取和营业额的影响。这些研究有可能识别胶原蛋白周转的新型介体。更好地理解负责从细胞外基质中去除胶原蛋白的途径的重要性，这一事实强调了可用的疗法在治疗既定纤维化疾病的患者方面是有效的。公共卫生相关性：肺纤维化是一种常见疾病，没有有效的药理治疗选择。旨在发现从肺部去除纤维化疤痕的途径的研究将提供新的治疗选择。该项目的目标是识别和表征介导疤痕重塑的正常途径，目的是发现用于治疗肺纤维化的新治疗靶标。