Macrophage-lipoprotein Interactions

巨噬细胞-脂蛋白相互作用

• 批准号: 8299476
• 负责人: Frederick R. Maxfield
• 金额: $ 42.25万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8299476
• 关键词: 1-Phosphatidylinositol 3-Kinase; Acid Lipase; Actins; American; Arterial Fatty Streak; Atherosclerosis; Binding; Biological Markers; Biopsy; Blood; Blood Vessels; Cell Maintenance; Cell Surface Receptors; Cells; Cessation of life; Cholesterol; Cholesterol Esters; Dendritic Cells; Deposition; Development; Electron Microscopy; Endosomes; Enzymes; Event; Extracellular Matrix; Foam Cells; Freezing; Goals; Heart Diseases; Hydrolysis; Image; Immune system; Inflammation; Injection of therapeutic agent; Knockout Mice; Label; Lead; Lipase; Lipids; Lipoproteins; Low-Density Lipoproteins; Membrane; Methods; Microscopy; Modeling; Molecular; Mus; Phospholipids; Process; RNA Interference; Receptor Signaling; Research; Resolution; Role; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Signaling Molecule; Staging; Sterols; Stream; Stroke; Surface; Synapses; TLR4 gene; Tissues; base; cardiovascular risk factor; disability; extracellular; improved; insight; macrophage; monocyte; novel; polymerization; pressure; prevent; receptor; rho GTP-Binding Proteins; synaptogenesis; syndecan-4; therapy development; vacuolar H+-ATPase

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.

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物。这些脂蛋白受到脂肪酶的作用，导致它们聚集，并与细胞外基质紧密结合。它们还经过化学修饰（例如氧化）。使用可溶性脂蛋白的研究无法捕获 MF 与这些保留和聚集的脂蛋白 (agLDL) 相互作用的重要方面，从而导致泡沫细胞形成。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），消化 agLDL。通过 MF 来表征溶酶体突触形成，将追求以下目标： 目标 1. 继续表征聚集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. 分析甾醇和其他代谢物转移至细胞的机制（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 与 agLDL A 相互作用的受体和信号转导途径。使用来自敲除小鼠的细胞、RNAi 和药物制剂检查 PI3 激酶、Akt 和其他信号分子在溶酶体突触的形成和功能中的作用 B. 检查TLR4、Syndecan 4 和其他潜在表面受体的作用 C. 检查胆固醇和脂质代谢物在激活信号传导中的作用 目的 3. 检查小鼠动脉粥样硬化病变中的溶酶体突触 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 电子显微镜以获得高分辨率MF 与 agLDL 在动脉粥样硬化病变中相互作用的图像。 虽然多年来人们已经认识到免疫系统细胞（尤其是 MF）在斑块形成中的重要性，但人们对这些免疫系统细胞作用的许多基本方面知之甚少。主要目标是表征 MF 与 agLDL 相互作用触发的信号事件。这可能会导致人们更好地了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，并有助于更深入地了解心血管危险因素（例如炎症生物标志物升高）的根本基础。