Macrophage-lipoprotein Interactions

描述(申请人提供)：在动脉粥样硬化的早期阶段，血管壁上的巨噬细胞(MF)会遇到脂蛋白沉积，这种沉积已经被广泛修改。这些脂蛋白被脂肪酶作用，导致它们聚集在一起，并与细胞外基质紧密结合。它们也经过化学修饰(例如，氧化)。使用可溶性脂蛋白的研究不能捕捉到MF与这些滞留和聚集的脂蛋白(AgLDL)相互作用的基本方面，这些相互作用导致泡沫细胞的形成。先进的显微镜方法将被用来描述一种新型的相互作用，在这种相互作用中，MF形成一个细胞外溶酶体(溶酶体突触)，消化agLDL。以下目的将被用来描述溶酶体突触的形成：目的1.继续描述聚集的脂蛋白的细胞外水解的新机制。A.确定溶酶体分泌的分子机制B.分析类固醇和其他代谢物向细胞转移的机制(如NPC2的作用)C.在模拟内膜下间隙的细胞外基质模型中检测MF和agLDL的相互作用D.对溶酶体突触进行3D电子显微镜分析E.确定树突状细胞是否也与agLDL形成溶酶体突触目标2.分析参与MF和agLDL相互作用的受体和信号转导途径2.利用来自基因敲除小鼠的细胞RNAi，检测PI3Kinase、Akt和其他信号分子在溶酶体突触形成和功能中的作用以及药理学试剂B。检查TLR4、Syndecan 4和其他潜在的表面受体C的作用。检查胆固醇和脂质代谢产物在激活信号目的中的作用3.检查小鼠动脉粥样硬化病变中溶酶体突触的形成。使用多光子显微镜、注射荧光脂蛋白和标记单核细胞来确定在切除的动脉粥样硬化组织中是否可以看到溶酶体突触。使用快速活检和高压冷冻，然后使用3D电子显微镜获得高分辨率图像，以了解MF和agLDL在动脉粥样硬化病变中的相互作用。虽然免疫系统细胞，特别是MFS在斑块发展中的重要性已经被认识到多年，但这些免疫系统细胞的许多基本方面的作用却知之甚少。一个主要的目标是表征由MF与agLDL相互作用触发的信号事件。这可能有助于更好地理解动脉粥样硬化和炎症之间的关系。这些见解可能有助于开发治疗方法，并提供对心血管风险因素潜在基础的更深层次理解，例如炎症生物标志物的升高。 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): In early stages of atherosclerosis, macrophages (MF) in the wall of blood vessels encounter lipoprotein deposits that have been extensively modified. These lipoproteins have been acted upon by lipases, causing them to become aggregated, and they are tightly bound to the extracellular matrix. They are also chemically modified (e.g., oxidized). Studies that have used soluble lipoproteins cannot capture essential aspects of the interactions of MF with these retained and aggregated lipoproteins (agLDL) that lead to foam cell formation. Advanced microscopy methods will be used to describe a novel type of interaction in which MF form an extracellular lysosome (a lysosomal synapse), which digests the agLDL. The following aims will be pursued to characterize lysosomal synapse formation by MF: Aim 1. Continue to characterize the novel mechanisms for extracellular hydrolysis of aggregated lipoproteins. A. Determine the molecular mechanisms for lysosomal secretion B. Analyze the mechanisms for transfer of sterol and other metabolites to cells (e.g., role of NPC2) C. Examine the interaction of MF with agLDL in extracellular matrix models that mimic the subintimal space D. Carry out 3D electron microscopy analysis of lysosomal synapses E. Determine whether dendritic cells also form lysosomal synapses with agLDL Aim 2. Analyze the receptors and signal transduction pathways involved in interaction of MF with agLDL A. Examine the role of PI3 Kinase, Akt, and other signaling molecules in the formation and function of the lysosomal synapse using cells from knockout mice, RNAi, and pharmacological agents B. Examine the role of TLR4, Syndecan 4 and other potential surface receptors C. Examine the role of cholesterol and lipid metabolites in activating signaling Aim 3. Examine the formation of lysosomal synapses in mouse atherosclerotic lesions A. Use multiphoton microscopy, injection of fluorescent lipoproteins, and labeled monocytes to determine whether lysosomal synapses can be seen in excised atherosclerotic tissue B. Use rapid biopsies and high pressure freezing followed by 3D electron microscopy to obtain high resolution images of the interaction of MF with agLDL in atherosclerotic lesions. While the importance of immune system cells, especially MFs, in plaque development has been recognized for years, there are many fundamental aspects of the role of these immune system cells that are poorly understood. A major goal is to characterize the signaling events that are triggered by the interaction of MF with agLDL. This may lead to improved understanding of the relationship between atherosclerosis and inflammation. Such insights may help in developing therapies as well as providing deeper understanding of the underlying basis for cardiovascular risk factors such as elevated biomarkers for inflammation. 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.