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

项目摘要/摘要 肺纤维化是一种常见病，缺乏有效的药物治疗。纤维化是一种 病理组织反应，其特征是细胞外富含胶原的基质沉积，破坏 正常的肺泡结构阻碍了有效的气体交换。胶原蛋白的代谢，主要是 纤维组织的成分，是胶原合成和胶原合成之间平衡的动态过程。 降解决定组织结构。虽然有相当多的研究针对 确定在肺纤维化中负责胶原产生的途径，负责的途径 对于清除纤维化区堆积的胶原蛋白，人们知之甚少。无论治疗的目的是 增加胶原摄取是否会使已确诊的纤维性疾病患者受益尚不清楚。我们最近做了 发现糖蛋白乳脂球样表皮生长因子8(Mfge8)结合并靶向胶原蛋白 巨噬细胞的细胞内摄取和降解。缺乏Mfge8的小鼠出现夸大的纤维化 由于体内胶原降解缺陷引起的博莱霉素损伤的反应。这样做的总体目标是 建议研究巨噬细胞介导的胶原吞噬作用在限制糖尿病严重程度中的作用 肺纤维化。我们将通过三个具体目标来实现这一目标。在拟议的实验中，我们将 确定Mfge8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 Mfge8结合的胶原蛋白被细胞吸收的分子途径。我们将研究这一角色 巨噬细胞通过一系列途径参与Mfge8依赖和独立的体内肺纤维化重塑 巨噬细胞耗尽实验。通过这种方式，我们将研究巨噬细胞 降解胶原蛋白。对这些途径的更好理解将为治疗提供新的靶点 治疗已确定的纤维性疾病。 我们的具体目标是： 第一个目标是确定Mfge8的哪些结构域对胶原结合和摄取至关重要。我们 我将创建一系列带有截断和/或突变序列的Mfge8构建体并评估它们的功能 体外和体内胶原结合和摄取的测定。我们将使用从Mfge8-/-和Wild获得的细胞 输入老鼠。第二个目的是确定EGFR受体家族和巨噬细胞吞噬在Mfge8- 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，描绘出 Mfge8结合的胶原激活EGFR的机制及EGFR激活如何诱导胶原 通过巨噬细胞吞噬而摄取。最终目的是确定肺巨噬细胞在胶原蛋白中的作用。 体内吸收。我们将使用两种药理方法(氯屈膦酸盐和抗F4/80抗体)和 转基因去巨噬细胞方法(黑色素瘤小鼠和CD11b-DTR小鼠)体内检测 巨噬细胞介导的胶原降解在肺纤维化中的作用。我们将耗尽巨噬细胞 博莱霉素性肺纤维化模型的建立及巨噬细胞的作用 耗竭对正常重塑的富胶原瘢痕。我们还将确定Mfge8的细胞来源 通过一系列骨髓切除和免疫重建对肺重塑的重要作用 实验。这些研究有可能确定胶原摄取的新途径和媒介 可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.