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

项目总结/摘要 肺纤维化是一种常见的疾病，目前尚无有效的药物治疗。纤维化是一 病理性组织反应，特征为富含胶原的基质的细胞外沉积， 正常的肺泡结构阻止有效的气体交换。胶原蛋白的代谢， 纤维化组织中的胶原蛋白的合成是一个动态的过程， 降解决定组织结构。虽然有相当多的研究针对 确定负责肺纤维化中胶原蛋白产生的途径， 用于去除纤维化区域中积累的胶原蛋白的方法知之甚少。治疗是否针对 增加胶原蛋白摄取是否有益于患有已确定的纤维化疾病的患者是未知的。我们最近 发现糖蛋白乳脂肪球表皮生长因子8（Mfge 8）结合并靶向胶原蛋白， 巨噬细胞的细胞内摄取和降解。Mfge 8缺陷的小鼠发生夸大的纤维化 由于胶原降解的体内缺陷，对博来霉素损伤的反应。本报告的总体目标 建议是研究巨噬细胞介导的胶原吞噬作用在限制严重性 肺纤维化我们将通过三个具体目标来实现这一目标。在实验中，我们将 确定Mfge 8的哪些结构域对胶原结合和胶原摄取至关重要。我们将绘制 Mfge 8结合的胶原蛋白被细胞吸收的分子途径。我们将研究 通过一系列研究，巨噬细胞在体内Mfge 8依赖性和独立性肺纤维化重塑中的作用 巨噬细胞消耗实验的结果以这种方式，我们将研究巨噬细胞 降解胶原蛋白。更好地了解这些途径将为针对以下疾病的治疗提供新的靶点： 治疗已确定的纤维化疾病。 我们的具体目标是： 第一个目的是确定Mfge 8的哪些结构域对胶原结合和摄取至关重要。我们 将创建一系列具有截短和/或突变序列的Mfge 8构建体，并评估它们的功能 在体外和体内测定胶原结合和摄取。我们将使用从Mfge 8-/-和野生型获得的细胞， 型小鼠。第二个目的是确定EGFR受体家族和巨胞饮作用在Mfge 8中的作用。 依赖胶原蛋白摄取。我们将通过一系列体外实验来实现这一目标， Mfge 8结合胶原激活EGFR的机制以及EGFR激活如何诱导胶原 通过巨胞饮作用摄取。最终目的是确定肺巨噬细胞在胶原蛋白中的作用。 体内再吸收。我们将使用两种药理学方法（氯膦酸盐和抗F4/80抗体）， 巨噬细胞耗竭的转基因方法（Mafia小鼠和CD 11b-DTR小鼠），以确定体内 巨噬细胞介导的胶原降解在肺纤维化中的作用我们会在 在肺中建立由博来霉素处理诱导的纤维化，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。我们还将确定Mfge 8的细胞来源 通过一系列骨髓消融和免疫重建对肺重塑很重要 实验这些研究有可能确定胶原蛋白摄取的新途径和介质， 可以靶向治疗已建立的肺纤维化。

Project Summary/Abstract 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.