Epithelial cell activation of fibroblasts in pulmonary fibrosis

项目摘要 候选 凯瑟琳·布莱克，医学博士是肺和重症监护医学部（DPCCM）的教员， 马萨诸塞州总医院（MGH），以及哈佛医学院终身教职医学讲师 学校（HMS）。她带着对间质性肺病的浓厚兴趣和研究背景来到MGH， 现在在MGH炎症中心的Andrew Tager博士的实验室工作， 炎症性疾病（CIID）。Tager博士是成纤维细胞基本机制的公认领导者 激活，并在他的实验室，她已经建立了评估成纤维细胞介质的专业知识， activation.她现在专注于肺泡上皮细胞（AEC）损伤作为这些成纤维细胞活化的来源。 介导物，以及在纤维化的动物和人细胞培养模型中分析AEC对损伤的反应。她 长期职业目标是确定触发和加剧肺纤维化的途径， 使基础生物学的发现更接近病人护理， 间质性肺病的症状这项资助的短期目标是描绘促纤维化AEC 内质网（ER）应激途径的反应在体外和体内。实验、训练和 建议书中概述的指导计划将使布莱克博士在她的第一个R 01申请中处于非常有利的地位， 作为一名独立的物理学家和科学家。 指导、培训活动和环境 本建议书中所述的培训计划主要位于DPC CM和CIID，两者都很好- 为培养成功的医学科学家建立了环境。在泰格医生的指导下， 博士布莱克已经制定了一项研究和培训计划，这将使她具备必要的知识， 成功地从一个指导到一个独立的位置移动所需的实验技术。K08 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， 她开始建立一个肺组织库， 肺纤维化 为了实现她的研究和职业目标，布莱克博士将利用塔格博士实验室的专业知识， 模拟肺纤维化。她还将获得上皮细胞生物学和更先进的 通过与精心组建的专家团队合作和协商进行测序分析， 包括她的培训咨询委员会的成员。Jay Rajagopal博士，Timothy Blackwell博士， Benjamin Humphreys将分享他们在肺上皮细胞发育和修复，ER途径 应激在肺纤维化和人体组织中的基础研究，以及利用细胞特异性翻译 在纤维化损伤的分析中的分析。此外，她还将接受上皮细胞 通过HMS以及哈佛医学院， 公共卫生和哈佛临床和转化科学中心（哈佛催化剂）。合作 因此，布莱克博士所拥有的机会、智力环境和资源都是例外的。 研究 特发性肺纤维化（IPF）是一种以上皮细胞损伤和异常增殖为特征的破坏性疾病， 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 最终导致死亡虽然目前IPF发病机制的范例假定其起始和 进展开始于肺泡上皮的损伤，很少有人知道肺泡上皮细胞的机制， 上皮损伤致成纤维细胞活化。虽然肺泡上皮细胞的ER应激已经在 IPF，AEC的ER应激和促纤维化介质释放之间的直接联系尚不清楚。K08 该提案旨在填补这一知识空白。具体而言，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），并与芯片测序，确定其他促纤维化介质 其转录在应激的AEC中由诱导CTGF的相同转录因子诱导。 (2)分析肺纤维化损伤小鼠模型中AEC特异性翻译反应 纤维化，使用新的小鼠系统进行翻译核糖体亲和纯化（TRAP）， 检查在标准或遗传夸大ER应激的小鼠中的AEC翻译谱， 基线，对博来霉素损伤的反应，以及ER应激的药理学降低。 (3)通过评估siRNA的作用，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，并评估其相关性 通过测定它们在肺组织微阵列中AEC中的表达来研究人类疾病，所述肺组织微阵列由 来自IPF患者和正常对照的肺样品。

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.