The role of Pdgfra+ fibroblasts in lung fibrosis and alveolar homeostasis

描述（由申请人提供）：该项目的目标是提供支持和持续的指导，因为我发展成为一个独立的医生，科学家研究肺纤维化和上皮间质相互作用在疾病的发病机制中的作用。该课程是专门设计来加强我在肺纤维化，遗传和表型分析，以及人类肺细胞生物学的小鼠模型的理论和实践知识基础。这将通过实验室实践经验、教学培训、数据和结果介绍、与相关领域专家的互动以及专门咨询委员会的指导来实现。如本提案所述，我将至少有75%的时间用于研究。在此期间，我将能够获得一些实用的专业知识， 新技术，包括流式细胞术，荧光激活细胞分选（FACS），小鼠呼吸力学的评估，以及人肺细胞的分离和培养。我还将在基因组学和基因表达分析的相关领域建立我的知识基础。鉴于我经验丰富的导师和丰富而充满活力的研究环境，我有能力成功完成提案中概述的工作。研究项目：特发性肺纤维化（IPF）是一种以严重和进行性肺泡纤维化为特征的疾病，最常发生在老年人中，除了肺移植外没有有效的治疗方法。基于人类遗传学研究揭示了2型肺泡上皮细胞（AEC 2）特异性基因突变与家族性肺纤维化之间的联系，公认肺泡上皮异常与疾病发展相关。然而，我们缺乏知识，这些上皮异常如何触发旺盛的反应，从底层间充质，导致显着的和渐进的疤痕形成。我们也不知道是哪些细胞引起的病理性肌成纤维细胞是疾病的特征。我在肺间质中发现了一个独特的成纤维细胞群，可能是肌成纤维细胞的来源。这些表达PDGF受体α（Pdgfra）的细胞与AEC 2非常接近，并且在博来霉素肺损伤后增殖，上调肌成纤维细胞标志物，并表达促纤维化介质，如透明质酸合成酶2（Has2）。我还表明，这些细胞支持小鼠AEC 2在三维小鼠培养系统中的克隆生长和分化，这表明它们是肺泡上皮细胞生态位的关键组成部分。我假设Pdgfra+成纤维细胞是博来霉素肺损伤后肌成纤维细胞的来源，这些细胞在触发时产生促纤维化介质，如透明质酸（HA）和Has2，并且这些细胞是稳态期间人AEC 2生态位的关键组分。目的1：确定博来霉素肺损伤后Pdgfra+成纤维细胞是否产生肌成纤维细胞。目标2：检验Pdgfra+成纤维细胞是博来霉素诱导的肺纤维化发展中促纤维化介质HA和Has2的主要来源的假设。目标3：检验原代人PDGFRa+成纤维细胞支持人AEC 2的存活、增殖和分化，从而充当AEC 2生态位的组分的假设。

DESCRIPTION (provided by applicant): The goal of this project is to provide support and continued mentorship as I develop into an independent physician-scientist studying pulmonary fibrosis and the role of epithelial- mesenchymal interaction in the pathogenesis of the disease. The curriculum is specifically designed to strengthen my theoretical and practical knowledge base in murine models of lung fibrosis, genetic and phenotypic analysis, and human lung cell biology. This will be achieved through hands on laboratory experience, didactic training, presentation of data and results, interactions with experts in related fields, and mentorship from a dedicated advisory committee. As outlined in this proposal, I will have at least 75% of time protected for research. During this time, I will be able to gain practical expertise in a number of new techniques, including flow cytometry, fluorescence activated cell sorting (FACS), assessment of murine respiratory mechanics, and human lung cell isolation and culture. I will also build my knowledge base in relevant areas of genomics and gene expression analysis. I am well positioned to successfully complete the work outlined in the proposal given my experienced mentors and the rich and dynamic research environment in which I will perform the work. Research Project: Idiopathic pulmonary fibrosis (IPF) is a disease characterized by severe and progressive alveolar fibrosis that occurs most often in older individuals and that has no effective treatment short of lung transplant. Based upon human genetic studies revealing a link between mutations in genes specific to type 2 alveolar epithelial cells (AEC2s) and familial forms of pulmonary fibrosis, it is accepted that abnormalities in the alveolar epithelium are associated with disease development. We lack knowledge, however, of how these epithelial abnormalities trigger the exuberant response from the underlying mesenchyme that leads to significant and progressive scar formation. We also are unaware of which cells give rise to the pathologic myofibroblasts that are characteristic of the disease. I have identified a unique population of fibroblasts in the lung mesenchyme that are likely to be a source of myofibroblasts. These cells, which express PDGF receptor alpha (Pdgfra), lie in close proximity to AEC2s and, after bleomycin lung injury, proliferate, up-regulate myofibroblast markers, and express pro-fibrotic mediators such as hyaluronic acid synthase 2 (Has2). I have also shown that these cells support the clonal growth and differentiation of murine AEC2s in a 3-dimensional murine culture system, suggesting that they are critical components of the alveolar epithelial cell niche. I hypothesize that Pdgfra+ fibroblasts are a source of myofibroblasts following bleomycin lung injury, that these cells produce pro-fibrotic mediators such as hyaluronan (HA) and Has2 when triggered, and that these cells are critical components of the human AEC2 niche during homeostasis. I will test these hypotheses in the following specific aims: AIM 1: To determine if Pdgfra+ fibroblasts give rise to myofibroblasts after bleomycin lung injury. AIM 2: To test the hypothesis that Pdgfra+ fibroblasts are a major source of the pro-fibrotic mediators HA and Has2 in the development of bleomycin-induced pulmonary fibrosis. AIM 3: To test the hypothesis that primary human PDGFRa+ fibroblasts support the survival, proliferation, and differentiation of human AEC2s, thereby serving as components of the AEC2 niche.