Macrophage-lipoprotein Interactions

我们描述了一种新的机制，水解胆固醇酯在保留和聚集的LDL （agLDL），其是动脉粥样硬化病变中脂蛋白的主要形式。宏程序（MΦ）创建 紧密密封的隔室包围着agLDL。它们酸化这些隔间并分泌 溶酶体酶进入其中，产生溶酶体突触。这导致形成未酯化的 胆固醇在细胞外，可以被传递到质膜，导致信号的变化 转导和泡沫细胞形成。我们假设这种降解agLDL的机制 与吞噬或内吞机制相比存在显著差异，这些差异 对动脉粥样硬化的病理生理学和治疗具有重要意义。我们将研究细胞 调节这一过程的机制（我们称之为外食）;更好地表征体内的外食;以及 探索HDL和环糊精在清除由外食产生的胆固醇中的新作用。（一） 描述agLDL胞外水解的机制。我们已经使用了来自敲除小鼠的MΦ， RNAi和药理学试剂以鉴定TLR 4、Myd 88、SYK、Akt、PI 3激酶和其他激酶的作用。 信号分子。我们使用三种主要的定量分析：溶酶体分泌，F- 肌动蛋白，其中MΦ接触agLDL，并形成脂滴。我们将鉴定Rab和SNARE蛋白 我们还将鉴定在外食过程中被激活的基因。(2)确定 溶酶体突触在动脉粥样硬化病变中的作用。我们将使用光学和电子显微镜， 分析动脉粥样硬化小鼠模型中溶酶体突触的活性。我们会用骨髓 移植到LDL受体敲除小鼠，以确定信号传导过程的重要性。（三） 表征HDL与agLDL与MΦ接触时的相互作用。与MΦ接触的AgLDL水平很高 未酯化的胆固醇高密度脂蛋白或胆固醇平衡环糊精可以去除多余的胆固醇， 从细胞中损失胆固醇。我们将描述这一过程及其对泡沫细胞形成的影响。

We described a novel mechanism for the hydrolysis of cholesteryl esters in retained and aggregated LDL (agLDL), which is the predominant form of lipoprotein in atherosclerotic lesions. Macrophages (MΦ) create tightly sealed compartments that surround the agLDL. They acidify these compartments and secrete lysosomal enzymes into them, creating a lysosomal synapse. This leads to formation of unesterified cholesterol outside the cell, which can be delivered to the plasma membrane leading to changes in signal transduction and foam cell formation. We hypothesize that this mechanism for degrading agLDL has significant differences compared to phagocytic or endocytic mechanisms and that these differences have important consequences for the pathophysiology and treatment of atherosclerosis. We will study the cellular mechanisms that regulate this process (which we call exophagy); better characterize exophagy in vivo; and explore a novel role for HDL and cyclodextrins in clearing the cholesterol produced by exophagy. (1) Characterize the mechanisms for extracellular hydrolysis of agLDL. We have used MΦ from knockout mice, RNAi, and pharmacological agents to identify a role for TLR4, Myd88, SYK, Akt, PI3 kinases, and other signaling molecules in exophagy. We use three main quantitative assays: lysosome secretion, formation of F- actin where MΦ contact agLDL, and formation of lipid droplets. We will identify the Rab and SNARE proteins required for lysosome secretion, and we will identify genes that are activated during exophagy. (2) Determine the role of lysosomal synapses in atherosclerotic lesions. We will use optical and electron microscopy to analyze the activity of lysosomal synapses in mouse models of atherosclerosis. We will use bone marrow transplants into LDL receptor knockout mice to determine the importance of signaling processes. (3) Characterize HDL interactions with agLDL in contact with MΦ. AgLDL in contact with MΦ has very high levels of unesterified cholesterol. HDL or cholesterol-balanced cyclodextrins can remove excess cholesterol with no loss of cholesterol from cells. We will characterize this process and its impact on foam cell formation.