Macrophage-lipoprotein Interactions

描述（由申请人提供）：在动脉粥样硬化的早期阶段，血管壁中的巨噬细胞（MF）遇到已被广泛修饰的脂蛋白沉积。这些脂蛋白受到脂肪酶的作用，使它们聚集起来，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无法捕捉到MF与这些保留和聚集的脂蛋白（agLDL）相互作用的基本方面，这些相互作用导致泡沫细胞形成。先进的显微镜方法将用于描述一种新型的相互作用，其中MF形成细胞外溶酶体（溶酶体突触），其消化agLDL。以下目标将追求表征溶酶体突触形成的MF：目的1。继续描述聚集脂蛋白细胞外水解的新机制。A.确定溶酶体分泌的分子机制B.分析甾醇和其他代谢物向细胞转移的机制（例如，NPC2的作用）C.在模拟内膜下空间的细胞外基质模型中检查MF与agLDL的相互作用D.对溶酶体突触进行3D电镜分析E.确定树突状细胞是否也与agLDL Aim 2形成溶酶体突触。分析MF与agLDL相互作用中涉及的受体和信号转导途径A.利用敲除小鼠细胞、RNAi和药理学试剂检测PI3激酶、Akt和其他信号分子在溶酶体突触形成和功能中的作用B.检测TLR4、Syndecan 4和其他潜在表面受体的作用C.检测胆固醇和脂质代谢产物在激活信号传导Aim 3中的作用检查小鼠动脉粥样硬化病变中溶酶体突触的形成A.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B.使用快速活检和高压冷冻，然后使用3D电子显微镜获得动脉粥样硬化病变中MF与agLDL相互作用的高分辨率图像。虽然免疫系统细胞，特别是MFs，在斑块发育中的重要性多年来已经被认识到，但这些免疫系统细胞的作用的许多基本方面仍然知之甚少。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。这可能会提高对动脉粥样硬化和炎症之间关系的理解。这些见解可能有助于开发治疗方法，并为心血管危险因素（如炎症生物标志物升高）的潜在基础提供更深入的了解。

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.