Macrophage-lipoprotein Interactions

我们描述了保留和聚集的低密度脂蛋白中胆固醇酯的一种新的水解机制。 (AgLDL)，这是动脉粥样硬化病变中脂蛋白的主要形式。巨噬细胞(MΦ)创造 围绕着agLDL的紧密密封的隔间。它们酸化这些隔间并分泌 溶酶体酶进入其中，形成溶酶体突触。这会导致形成未酯化的 细胞外的胆固醇，可被输送到质膜，导致信号变化 转导和泡沫细胞的形成。我们假设这种降解agLDL的机制 与吞噬或内吞机制相比有显著差异，这些差异有 对动脉粥样硬化的病理生理学和治疗有重要影响。我们将研究细胞 调节这一过程的机制(我们称之为外噬)；更好地描述体内外噬的特征；以及 探索高密度脂蛋白和环糊精在清除外噬产生的胆固醇方面的新作用。(1) 描述ag低密度脂蛋白的胞外水解机制。我们已经使用了来自基因敲除小鼠的MΦ， RNAi，以及确定TLR4、MyD88、SYK、Akt、PI3激酶和其他酶的作用的药物 外噬中的信号分子。我们主要使用三种定量方法：溶酶体分泌、F-形成。 肌动蛋白在MΦ接触agLDL时，形成脂滴。我们将鉴定Rab和SNARE蛋白 溶酶体分泌所需的基因，我们将确定在外噬过程中被激活的基因。(二)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来 分析动脉粥样硬化小鼠模型中溶酶体突触的活性。我们将使用骨髓 移植到低密度脂蛋白受体基因敲除小鼠中，以确定信号传递过程的重要性。(3) 描述高密度脂蛋白与与M-Φ接触的agLDL的相互作用。与M-Φ接触的AgLDL值非常高 未酯化的胆固醇。高密度脂蛋白或胆固醇平衡的环糊精可以去除多余的胆固醇，而不是 细胞中胆固醇的流失。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.