Epithelial cell activation of fibroblasts in pulmonary fibrosis

项目摘要 侯选人 凯瑟琳·布莱克，医学博士，肺和重症监护医学部(DPCCM)教员，位于 马萨诸塞州综合医院(MGH)，哈佛医学院终身医学讲师 学校(HMS)。她带着对间质性肺病的浓厚兴趣和研究背景来到麻省理工学院， 目前在麻省理工学院炎症和治疗中心安德鲁·泰格博士的实验室工作 炎症性疾病(CIID)泰格博士在成纤维细胞的基本机制方面是公认的领导者 在他的实验室里，她已经在成纤维细胞介体的评估方面建立了专业知识。 激活。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 中介物，以及在动物和人类细胞培养的纤维化模型中对损伤的血管内皮细胞反应。她 长期的职业目标是确定触发和加剧肺纤维化的途径，并 使基础生物学的发现更贴近患者护理，带来基础科学和临床问题 间质性肺病的共同特征。这笔赠款的短期目标是描绘促肝纤维化的血管内皮细胞。 体内和体外对内质网应激途径的反应。实验、培训和 提案中概述的指导计划将使布莱克博士为她的第一个R01申请做好准备，并且 作为一名内科科学家的独立职业生涯。 师徒关系、培训活动和环境 本建议中描述的培训计划主要位于DPCCM和CIID，这两个地方都很好- 为培养高成就的内科科学家建立了良好的环境。在泰格博士的指导下， 布莱克博士已经制定了一项研究和培训计划，将使她具备必要的知识和 成功地从被指导的岗位转移到独立岗位所需的实验技术。这个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，以及 将扩展她开始的人类主题工作，建立一个肺移植的组织库，这些组织来自于 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在 建立肺纤维化模型。此外，她还将接受上皮细胞生物学和更高级的 通过与精心组建的专家团队合作和协商进行测序分析， 包括那些组成她的培训咨询委员会的人。Jay Rajagopal博士、Timothy Blackwell博士和 本杰明·汉弗莱斯将分享他们在肺上皮细胞发育和修复、ER途径方面的专业知识 肺纤维化和基于人体组织的研究中的应激以及细胞特异性翻译的使用 在纤维损伤分析中的侧写。此外，她还将接受有关上皮细胞的正式培训 发展，细胞对伤害的反应，和生物信息学通过HMS以及哈佛大学学院 公共卫生和哈佛临床与转化科学中心(哈佛大学促进会)。协作 因此，布莱克博士可以获得的机会、智力环境和资源都是非同寻常的。 研究 特发性肺纤维化(IPF)是一种以上皮细胞损伤和异常为特征的破坏性疾病 修复，成纤维细胞和细胞外基质堆积导致气体交换日益受损 最终导致死亡。而目前IPF发病机制的范式假设它的启动和 进展始于肺泡上皮的损伤，对肺泡连接的机制知之甚少。 上皮损伤导致成纤维细胞活化。而肺泡上皮细胞的内质网应激在 IPF，AEC的内质网应激与促纤维化介质释放之间的直接联系尚不清楚。这个K08 该提案旨在填补这一知识空白。具体来说，我们建议： (1)描述血管内皮细胞内质网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)，并用CHIP-SEQ鉴定其他促纤维化介质 其转录是由诱导CTGF的相同转录因子在应激的血管内皮细胞中诱导的。 (2)分析肺纤维化模型小鼠肺血管内皮细胞特异性的翻译反应。 纤维化，使用一种新的小鼠系统执行翻译核糖体亲和纯化(TRAP)到 检测具有标准内质网应激或遗传夸大内质网应激的小鼠的AEC翻译图谱 基线，对博莱霉素损伤的反应，以及药物减少内质网应激。 (3)通过评估siRNA的作用来评估AEC衍生的介体的功能重要性 这些介质对AEC诱导的成纤维细胞激活的抑制作用，并评估它们与 在构建的肺组织微阵列中通过检测它们在AEC中的表达来预防人类疾病 IPF患者和正常对照组的肺标本。

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.