Macrophage-lipoprotein interactions

描述(申请人提供)：动脉粥样硬化的部分原因是脂蛋白在动脉壁中积聚，单核/巨噬细胞聚集到该区域，并形成富含脂质的巨噬细胞，称为泡沫细胞。泡沫细胞的形成依赖于与脂蛋白的相互作用以及随后细胞内特定隔室之间的胆固醇运输。本项目将研究脂蛋白和巨噬细胞的特性如何导致泡沫细胞的形成和动脉粥样硬化的发病机制。我们将研究这一过程的几个关键方面。首先，血管壁上的许多脂蛋白聚集在一起，与细胞外基质成分紧密结合，这阻止了它们通过简单的内吞过程内化。当巨噬细胞遇到这些聚集的脂蛋白时，在脂蛋白内化之前有一段较长的接触时间。初步数据表明，在接触期间，巨噬细胞形成了一个不寻常的细胞外酸性消化间隔室。这个胞外隔室的酸化将用定量荧光显微镜进一步表征，并将研究酸化的机制。细胞外隔室的结构将使用中压电子显微镜和断层成像进行检查。我们还将研究酸性水解酶进入细胞室的机制，包括钙信号和特定的SNAR分子在介导溶酶体与质膜融合中的作用。溶酶体酸性脂肪酶的存在会导致胆固醇酯的水解和游离甾醇的释放，后者可以被局部插入质膜。具有良好特性的荧光甾醇及其酯类化合物将被掺入脂蛋白中，以直接监测这一过程。将研究该隔间中的其他分解代谢过程(例如，蛋白质降解)。胆固醇转运到细胞膜导致信号转导机制的激活，包括小的GTP酶，RAC，导致肌动蛋白聚合增加。我们将研究从聚集的脂蛋白向巨噬细胞输送类固醇对RAC、Rho和PI3-激酶活性以及肌动蛋白组装的影响。肌动蛋白组装增加扩大细胞外、酸性、消化室的可能性将使用这些信号蛋白的抑制剂进行研究。不同类别的巨噬细胞对聚集的脂蛋白的反应可能不同。来自经典和非经典单核细胞的巨噬细胞与聚集的脂蛋白的相互作用将在组织培养中进行比较。在动脉粥样硬化的小鼠模型中，来自经典和非经典Mo亚群的巨噬细胞和泡沫细胞的丰度也将被检测。公共卫生相关性：该项目研究了免疫系统细胞、巨噬细胞与血管壁脂蛋白相互作用导致动脉粥样硬化病变的早期步骤。这些动脉粥样硬化病变可能导致心脏病和中风，这是美国人残疾和死亡的主要原因。基于这些研究的研究结果可能会导致新的治疗方法来预防或逆转动脉粥样硬化病变的形成。

DESCRIPTION (provided by applicant): Atherosclerosis results in part from the accumulation of lipoproteins in the arterial wall, recruitment of monocytes/macrophages to the region, and formation of lipid-laden macrophages, known as foam cells. Foam cell formation is dependent on interactions with lipoproteins and subsequent cellular cholesterol transport among specific intracellular compartments. This project will examine how characteristics of both the lipoprotein and the macrophage lead to foam cell formation and the pathogenesis of atherosclerosis. Several key aspects of this process will be examined. First, many of the lipoproteins in the wall of blood vessels become aggregated and tightly bound to extracellular matrix components, and this prevents their internalization by simple endocytic processes. When macrophages encounter these aggregated lipoproteins, there is a prolonged period of contact before the lipoprotein is internalized. Preliminary data indicate that macrophages form an unusual extracellular, acidic, digestive compartment during this contact period. The acidification of this extracellular compartment will be characterized further using quantitative fluorescence microscopy, and the mechanism for acidification will be examined. The structure of the extracellular compartment will be examined using intermediate voltage electron microscopy and tomographic imaging. The mechanism for delivering acid hydrolases into the compartment will also be examined, including the role of calcium signaling and specific SNARE molecules in mediating lysosomal fusion with the plasma membrane. The presence of lysosomal acid lipase in this compartment would lead to hydrolysis of cholesterol esters and release of free sterol, which can be inserted locally in to the plasma membrane. Well- characterized fluorescent sterols and their ester derivatives will be incorporated into lipoproteins to monitor this process directly. Other catabolic processes (e.g., protein degradation) in this compartment will be investigated. Delivery of cholesterol to the plasma membrane leads to activation of signal transduction mechanisms, including the small GTPase, Rac, leading to increased actin polymerization. The consequences of sterol delivery from aggregated lipoproteins to macrophages on Rac, Rho and PI3-kinase activity and actin assembly will be examined. The possibility that increased actin assembly enlarges the extracellular, acidic, digestive compartment will be examined using inhibitors of these signaling proteins. Different classes of macrophages may respond differently to aggregated lipoproteins. The interactions of macrophages derived from classical and nonclassical monocytes with aggregated lipoproteins will be compared in tissue culture. The abundance of macrophages and foam cells derived from classical and nonclassical Mo subsets in atherosclerotic lesions in mouse models of atherosclerosis will also be examined. PUBLIC HEALTH RELEVANCE: This project examines the early steps leading to an atherosclerotic lesion as a consequence of the interaction between an immune system cell, the macrophage, with lipoproteins in the wall of blood vessels. These atherosclerotic lesions can lead to heart disease and stroke, which are major causes of disability and death of Americans. Research findings based on these studies may lead to new treatments to prevent or reverse the formation of atherosclerotic lesions.