Epithelial cell activation of fibroblasts in pulmonary fibrosis

肺纤维化中成纤维细胞的上皮细胞活化

• 批准号: 9164852
• 负责人: Katharine E. Black
• 金额: $ 17.35万
• 依托单位: MASSACHUSETTS GENERAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9164852
• 关键词: Acids; Actins; Advisory Committees; Affinity Chromatography; Alveolar; Animals; Apoptosis; Award; Basic Science; Bioinformatics; Biology; Bleomycin; Cell Culture Techniques; Cell Line; Cells; Cellular biology; Center for Translational Science Activities; Cessation of life; ChIP-seq; Chemicals; Chronic; Clinical; Clinical Sciences; Collaborations; Communication; Consultations; Critical Care; Data; Data Set; Deposition; Development; Disease; Disease Progression; Endoplasmic Reticulum; Environment; Epithelial; Epithelial Cells; Evaluation; Extracellular Matrix; Faculty; Fibroblasts; Fibrosis; Gases; Gene Expression; General Hospitals; Genetic Transcription; Goals; Grant; Hamman-Rich syndrome; Human; In Vitro; Inflammation; Inflammatory; Injury; Interstitial Lung Diseases; Knowledge; Laboratories; Link; Lung; Massachusetts; Mediator of activation protein; Medicine; Mentors; Mentorship; Messenger RNA; Modeling; Molecular; Molecular Chaperones; Mus; Pathogenesis; Pathway interactions; Patient Care; Patients; Physicians; Positioning Attribute; Production; Proteins; Public Health Schools; Pulmonary Fibrosis; Research; Research Training; Resources; Rho-associated kinase; Ribosomes; Sampling; Scientist; Sequence Analysis; Set protein; Signal Pathway; Small Interfering RNA; Source; Stimulus; Stress; Structure of parenchyma of lung; System; Techniques; Tissue Banking; Tissue Banks; Tissue Microarray; Training; Training Activity; Training Programs; Translating; Work; alveolar epithelium; base; career; catalyst; cell injury; cellular development; connective tissue growth factor; curative treatments; design; endoplasmic reticulum stress; human disease; human subject; human tissue; in vivo; indium-bleomycin; inhibitor/antagonist; injured; instructor; interest; knock-down; medical schools; member; mouse model; mutant; novel; polymerization; protein misfolding; repaired; research study; response; response to injury; targeted treatment; tenure track; transcription factor

Project Summary Candidate Katharine Black, MD is a faculty member of the Division of Pulmonary and Critical Care Medicine (DPCCM) at Massachusetts General Hospital (MGH), and an Instructor in Medicine on the tenure track at Harvard Medical School (HMS). She came to MGH with a strong interest and research background in interstitial lung disease, and is now working in the laboratory of Dr. Andrew Tager at the MGH Center for Inflammation and Inflammatory Diseases (CIID). Dr. Tager is a recognized leader in basic mechanisms underlying fibroblast activation, and in his laboratory she has established expertise in the evaluation of mediators of fibroblast activation. She is now focusing on alveolar epithelial cell (AEC) injury as a source of these fibroblast-activating mediators, and on profiling AEC responses to injury in animal and human cell culture models of fibrosis. Her long-term career goal is to determine the pathways that trigger and exacerbate pulmonary fibrosis, and to move the discoveries of basic biology closer to patient care, bringing the basic science and the clinical problem of interstitial lung disease together. The short-term goals of this grant are to delineate the pro-fibrotic AEC responses to endoplasmic reticulum (ER) stress pathways in vitro and in vivo. The experiments, training, and mentoring plans outlined in the proposal will position Dr. Black extremely well for her first R01 application, and for an independent career as a physician-scientist. Mentorship, Training Activities and Environment The training program described in this proposal is located primarily in the DPCCM and the CIID, both well- established environments for training high successful physician-scientists. Under the mentorship of Dr. Tager, Dr. Black has developed a research and training plan that will equip her with the necessary knowledge and experimental techniques required to move successfully from a mentored to an independent position. This K08 award will provide additional training in epithelial cell biology and in analysis of large sequencing data sets, and will expand the human subject work she began in establishing a tissue bank of lung explants from patients with pulmonary fibrosis. To accomplish her research and career goals, Dr. Black will make use of Dr. Tager’s laboratory’s expertise in modeling pulmonary fibrosis. She will additionally obtain training in epithelial cell biology and in more advanced sequencing analysis through collaboration and consultation with a carefully assembled team of experts, including those who form her Training Advisory Committee. Drs. Jay Rajagopal, Timothy Blackwell, and Benjamin Humphreys will share their expertise in lung epithelial cell development and repair, pathways of ER stress in pulmonary fibrosis and human tissue-based research, and the use of cell-specific translational profiling in analysis of fibrotic injury, respectively. In addition, she will receive formal training in epithelial cell development, cellular responses to injury, and bioinformatics through HMS, as well as Harvard School of Public Health, and the Harvard Clinical and Translational Science Center (Harvard Catalyst). The collaborative opportunities, intellectual environment, and resources available to Dr. Black consequently are exceptional. Research Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by epithelial cell injury and aberrant repair, with accumulation of fibroblasts and extracellular matrix leading to increasingly impaired gas exchange and ultimately death. While the current paradigm of IPF pathogenesis postulates that its initiation and progression begins with damage to the alveolar epithelium, little is known of the mechanisms linking alveolar epithelial injury to fibroblast activation. While ER stress of the alveolar epithelium has been well described in IPF, the direct links between ER stress of AEC and release of pro-fibrotic mediators are unknown. This K08 proposal aims to fill this knowledge gap. Specifically, we propose to: (1) Describe the mechanisms by which ER stress in AECs leads to release of known pro-fibrotic mediator Connective Tissue Growth factor (CTGF), and with ChiP-Seq, identify other pro-fibrotic mediators whose transcription is induced in stressed AECs by the same transcription factors that induce CTGF. (2) Analyze the AEC-specific translational response to fibrogenic injury in mouse models of pulmonary fibrosis, using a novel mouse system to perform translating ribosomal affinity purification (TRAP) to examine AEC translational profiles in mice with standard or with genetically exaggerated ER stress at baseline, in response to bleomycin injury, and with pharmacologic reduction of ER stress. (3) Evaluate the functional importance of AEC-derived mediators by evaluating the effects of siRNA knockdown of these mediators on AEC-induced fibroblast activation, and evaluate their relevance to the human disease by determining their expression in AECs in lung tissue microarrays constructed with lung samples from IPF patients and normal controls.

项目概要 候选人 Katharine Black 医学博士是肺科和重症监护医学科 (DPCCM) 的教员 马萨诸塞州总医院 (MGH) 和哈佛医学院终身教授医学讲师 她带着对间质性肺病的浓厚兴趣和研究背景来到麻省总医院。 现在在 MGH 炎症和疾病中心 Andrew Tager 博士的实验室工作 Tager 博士是炎症性疾病 (CIID) 领域公认的成纤维细胞基本机制领域的领导者。 激活，并在他的实验室中建立了评估成纤维细胞介质的专业知识 她现在专注于肺泡上皮细胞（AEC）损伤作为这些成纤维细胞激活的来源。 介质，以及分析动物和人类纤维化细胞培养模型中 AEC 对损伤的反应。 长期职业目标是确定引发和加剧肺纤维化的途径，并 让基础生物学的发现更贴近患者护理，将基础科学和临床问题结合起来 此项资助的短期目标是描绘促纤维化的 AEC。 体外和体内对内质网 (ER) 应激途径的反应 实验、训练和 提案中概述的指导计划将使 Black 博士在她的第一个 R01 申请中处于非常有利的位置，并且 作为一名医师科学家的独立职业。 指导、培训活动和环境 本提案中描述的培训计划主要位于 DPCCM 和 CIID，两者都很好 在泰格博士的指导下，建立了培养高度成功的医学科学家的环境。 布莱克博士制定了一项研究和培训计划，该计划将为她提供必要的知识和 这是 K08 成功从指导职位转变为独立职位所需的实验技术。 该奖项将提供上皮细胞生物学和大型测序数据集分析方面的额外培训，以及 将扩大她开始建立肺外植体组织库的人类受试者工作 肺纤维化。 为了实现她的研究和职业目标，布莱克博士将利用泰格博士实验室的专业知识 她还将获得上皮细胞生物学和更高级的培训。 通过与精心组建的专家团队合作和咨询进行测序分析， 包括她的培训咨询委员会的成员 Jay Rajagopal、Timothy Blackwell 和博士。 本杰明·汉弗莱斯 (Benjamin Humphreys) 将分享他们在肺上皮细胞发育和修复、ER 通路方面的专业知识 肺纤维化和基于人体组织的研究中的压力，以及细胞特异性转化的使用 此外，她还将接受上皮细胞方面的正式培训。 通过 HMS 和哈佛大学，研究发育、细胞对损伤的反应以及生物信息学 公共卫生和哈佛临床与转化科学中心（哈佛催化剂）。 因此，布莱克博士可获得的机会、智力环境和资源都是独一无二的。 研究 特发性肺纤维化（IPF）是一种破坏性疾病，其特征是上皮细胞损伤和异常。 修复，成纤维细胞和细胞外基质的积累导致气体交换日益受损 而目前 IPF 发病机制的范式假定其起始和最终死亡。 进展始于肺泡上皮的损伤，但对于与肺泡上皮细胞相关的机制知之甚少。 上皮损伤对成纤维细胞活化的影响，而肺泡上皮的 ER 应激已在 IPF、AEC 的 ER 应激与促纤维化介质释放之间的直接联系尚不清楚。 具体来说，我们建议： (1) 描述 AEC 中 ER 应激导致已知促纤维化介质释放的机制 结缔组织生长因子 (CTGF) 并通过 ChiP-Seq 识别其他促纤维化介质 其转录在应激的 AEC 中由诱导 CTGF 的相同转录因子诱导。 (2) 分析小鼠肺纤维化损伤模型中 AEC 特异性翻译反应 纤维化，使用新型小鼠系统进行核糖体亲和纯化（TRAP）翻译 检查具有标准 ER 应激或基因夸大 ER 应激的小鼠的 AEC 翻译谱 基线，响应博莱霉素损伤，并通过药物减少 ER 应激。 (3) 通过评估siRNA的效果来评估AEC衍生介质的功能重要性 敲低这些介质对 AEC 诱导的成纤维细胞活化的影响，并评估它们与 通过测定肺组织微阵列中 AEC 的表达来研究人类疾病 来自 IPF 患者和正常对照的肺部样本。