Macrophage-Lipoprotein Interactions

与细胞外基质紧密交联的脂蛋白聚集体是主要类型 动脉粥样硬化病变中的脂蛋白。这些聚集物中的大部分胆固醇是未酯化的，但它 目前尚不清楚保留和聚集的细胞外 LDL 核心中的胆固醇酯是如何变化的 水解——特别是因为据报道涉及溶酶体水解酶。我们最近的研究 证明了保留和聚集核心中胆固醇酯水解的新机制 低密度脂蛋白 (LDL)，其中巨噬细胞 (M) 形成紧密密封的隔室，围绕聚集的部分 低密度脂蛋白。然后，它们酸化这些隔室并向其中分泌溶酶体酶，形成溶酶体 突触。研究表明，溶酶体酸性脂肪酶可在胞外水解胆固醇酯， 称为消化外噬的过程，导致细胞外产生未酯化的胆固醇，并且 将这种胆固醇运输到细胞中会导致泡沫细胞的形成。体内胆固醇浓度很高 初步研究中观察到聚集的低密度脂蛋白会导致细胞外胆固醇的形成 晶体，可引起 M 的炎症反应。该提案的总体假设是 这种降解脂蛋白的机制与传统的吞噬细胞相比有显着差异 或内吞机制，这些差异在病理生理学中具有重要影响 动脉粥样硬化。此外，了解这一过程可能会改善治疗干预措施。 第一个目标的工作将描述消化外食的分子机制。这将 包括对溶酶体胞吐作用所需的 Rab 和 SNARE 蛋白的研究。由 Tlr4 发出信号， Myd88、PI3 激酶、Akt、Syk、Vav、Cdc42 和其他分子已被证明对消化很重要 将探索外噬以及其他信号分子的作用。 高浓度的胆固醇在聚集的 LDL 中产生，并在 Aim 2 的形成中产生 将检查胆固醇晶体和由此产生的 Mf 炎症激活。 第三个目标的工作将使用光学成像和三维电子显微镜（FIB-SEM）来检查 小鼠动脉粥样硬化模型中溶酶体突触的 3D 结构。溶酶体突触的形成， 胆固醇晶体与保留和聚集的低密度脂蛋白的关联以及炎症激活将是 通过光学显微镜对各种动脉粥样硬化小鼠模型进行了研究。 更好地了解动脉粥样硬化病变中发生的细胞和分子事件可以导致 更好的风险评估和潜在的新疗法。

Aggregates of lipoproteins that are tightly crosslinked to the extracellular matrix are the major type of lipoprotein in atherosclerotic lesions. The majority of the cholesterol in these aggregates is unesterified, but it has been unclear how the cholesteryl esters in the core of retained and aggregated extracellular LDL are hydrolyzed – especially because a lysosomal hydrolase has been reported to be involved. Our recent studies demonstrate a novel mechanism for the hydrolysis of cholesteryl esters in the core of retained and aggregated LDL in which macrophages (M) create tightly sealed compartments that surround portions of the aggregated LDL. They then acidify these compartments and secrete lysosomal enzymes into them, creating a lysosomal synapse. It has been shown that the extracellular hydrolysis of cholesteryl esters by lysosomal acid lipase, in a process called digestive exophagy, leads to production of unesterified cholesterol outside the cell, and transport of this cholesterol into the cell leads to foam cell formation. The high concentrations of cholesterol in the aggregated LDL were observed in a preliminary study to lead to the formation of extracellular cholesterol crystals, which can cause inflammatory responses in M . The overarching hypothesis of this proposal is that this mechanism for degrading lipoproteins has significant differences as compared to conventional phagocytic or endocytic mechanisms and that these differences have important consequences in the pathophysiology of atherosclerosis. Furthermore, understanding this process may lead to improved therapeutic interventions. Work in the first aim will characterize the molecular mechanisms of digestive exophagy. This will include a study of the Rab and SNARE proteins that are required for lysosomal exocytosis. Signaling by Tlr4, Myd88, PI3-kinase, Akt, Syk, Vav, Cdc42, and other molecules has been shown to be important for digestive exophagy, and the roles of additional signaling molecules will be explored. High concentrations of cholesterol are generated in aggregated LDL, and in Aim 2 formation of cholesterol crystals and resulting inflammatory activation of M will be examined. Work in the third aim will use optical imaging and 3-D electron microscopy (FIB-SEM) to examine the 3D structures of lysosomal synapses in a mouse atherosclerosis model. Formation of lysosomal synapses, association of cholesterol crystals with retained and aggregated LDL, and inflammatory activation will be studied in various mouse models of atherosclerosis by optical microscopy. Better understanding of the cellular and molecular events occurring in atherosclerotic lesions can lead to better risk assessments and potentially new therapies.