The Role of Collagen Uptake in Regulating the Severity of Pulmonary Fibrosis

描述（由申请人提供）：肺纤维化是一种常见疾病，无有效的药物治疗。纤维化是一种病理性组织反应，其特征在于富含胶原的基质的细胞外沉积，其破坏正常的肺泡结构，阻止有效的气体交换。胶原蛋白是纤维化组织的主要成分，其代谢是一个动态过程，胶原蛋白的产生和降解之间的平衡决定了组织结构。虽然已经有相当多的研究致力于确定负责肺纤维化中胶原蛋白产生的途径，但对负责去除纤维化区域中积聚的胶原蛋白的途径了解甚少。目前尚不清楚旨在增加胶原蛋白摄取的疗法是否会使已确诊的纤维化疾病患者受益。我们最近发现糖蛋白乳脂肪球表皮生长因子样8（Mfge 8）结合并靶向胶原蛋白，供巨噬细胞在细胞内摄取和降解。由于体内胶原降解缺陷，Mfge 8缺陷的小鼠对博来霉素损伤产生过度的纤维化反应。本提案的总体目标是研究巨噬细胞介导的胶原吞噬作用在限制肺纤维化严重程度中的作用。我们将通过三个具体目标来实现这一目标。在所提出的实验中，我们将确定Mfge 8的哪些结构域对于胶原蛋白结合和胶原蛋白摄取是关键的。我们将绘制Mfge 8结合的胶原蛋白被细胞吸收的分子途径。我们将通过一系列的巨噬细胞耗竭实验来研究巨噬细胞在体内Mfge 8依赖性和非依赖性肺纤维化重塑中的作用。以这种方式，我们将研究巨噬细胞降解胶原蛋白的途径。更好地了解这些途径将为旨在治疗已建立的纤维化疾病的疗法提供新的靶点。我们的具体目标是：第一个目标是确定Mfge 8的哪些结构域对胶原结合和摄取至关重要。我们将创建一系列的Mfge 8结构与截短和/或突变的序列，并评估其功能，在体外和体内测定胶原蛋白的结合和摄取。我们将使用从Mfge 8-/-和野生型小鼠获得的细胞。第二个目的是确定EGFR受体家族和巨胞饮在Mfge 8依赖性胶原摄取中的作用。我们将通过一系列体外实验来实现这一目标，这些实验描绘了Mfge 8结合的胶原蛋白激活EGFR的机制以及EGFR激活如何通过巨胞饮诱导胶原蛋白摄取。最终目的是确定肺巨噬细胞在体内胶原再吸收中的作用。我们将使用药理学方法（氯膦酸盐和抗F4/80抗体）和巨噬细胞耗竭的转基因方法（Mafia小鼠和CD11b-DTR小鼠）来确定巨噬细胞介导的胶原降解在肺纤维化中的体内作用。我们将在肺中建立博莱霉素治疗诱导的纤维化后消耗巨噬细胞，然后评估巨噬细胞消耗对富含胶原的瘢痕的正常重塑的影响。我们还将通过一系列骨髓消融和免疫重建实验确定对肺重建重要的Mfge 8的细胞来源。这些研究有可能确定新的途径和介质的胶原蛋白摄取，可以有针对性地治疗已建立的肺纤维化。 公共卫生相关性：肺纤维化是一种常见疾病，没有有效的药物治疗选择。旨在发现从肺部去除纤维化瘢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瘢痕重塑的正常途径，目的是发现新的治疗肺纤维化的治疗靶点。

DESCRIPTION (provided by applicant): Pulmonary fibrosis is a common disorder without effective pharmacological treatment. Fibrosis is a pathological tissue response characterized by extracellular deposition of collagen-rich matrix that disrupts normal alveolar architecture preventing effective gas exchange. 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. While there has been considerable research directed toward identifying the pathways responsible for collagen production in pulmonary fibrosis, the pathways responsible for removal of collagen 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 found the glycoprotein Milk Fat Globule Epidermal Growth Factor like 8 (Mfge8) binds and targets collagen for intracellular uptake and degradation by macrophages. Mice deficient in Mfge8 develop an exaggerated fibrotic response to bleomycin injury due to an in vivo defect in collagen degradation. The overall objective of this proposal is to investigate the role of macrophage-mediated collagen phagocytosis in limiting the severity of pulmonary fibrosis. We will pursue this objective through 3 specific aims. In the proposed experiments, we will determine which domains of Mfge8 are critical for collagen binding and collagen uptake. We will map the molecular pathway by which Mfge8-bound collagen is taken up by cells. We will examine the role of macrophages in Mfge8-dependent and independent remodeling of pulmonary fibrosis in vivo through a series of macrophage depletion experiments. In this fashion, we will investigate the pathways by which macrophages degrade collagen. A better understanding of these pathways will provide novel targets for therapies aimed at treating established fibrotic disease. Our specific aims are: The first aim is to determine which domains of Mfge8 are critical for collagen binding and uptake. We will create a series of Mfge8 constructs with truncated and/or mutated sequences and evaluating their function in in vitro and in vivo assays of collagen binding and uptake. We will use cells obtained from Mfge8-/- and wild type mice. The second aim is to determine the role EGFR receptor family and macropinocytosis in Mfge8-dependent collagen uptake. We will accomplish this aim through a series of in vitro experiments delineating the mechanism by which Mfge8-bound collagen activates EGFR and how EGFR activation induces collagen uptake through macropinocytosis. The final aim is to determine the role of pulmonary macrophages in collagen resorption in vivo. We will use both pharmacological approaches (Clodronate and anti-F4/80 antibody) and transgenic approaches of macrophage depletion (Mafia mice and CD11b-DTR mice) to determine the in vivo role of macrophage-mediated collagen degradation in pulmonary fibrosis. We will deplete macrophages after fibrosis induced by bleomycin treatment is established in the lung and then evaluate the effect of macrophage depletion on the normal remodeling of collagen-rich scar. We will also determine the cellular source of Mfge8 important for lung remodeling through a series of bone marrow ablation and immune reconstitution experiments. These studies have the potential to identify novel pathways and mediators of collagen uptake that can be targeted to treat established pulmonary fibrosis. 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.