Plasma membrane cholesterol and monocyte /macrophage function

在动脉粥样硬化发病的早期，聚集的氧化脂蛋白颗粒附着于受影响动脉的内皮下基质中的蛋白质。穿过上覆内皮的单核细胞（Mos）与这些基质结合的脂蛋白颗粒接触，吞噬它们，并最终摄取大量脂质，发育成脂质充盈的巨噬细胞泡沫细胞，其促进动脉粥样硬化斑块的进一步生长。我们推测，与这些基质结合脂蛋白的相互作用引起的Mos和巨噬细胞质膜胆固醇水平的改变可能是部分的。 负责病变的生长。基于我们的初步研究，我们假设，保留和聚集的脂蛋白改变肌动蛋白的动力学在莫斯和巨噬细胞在内皮下的空间，因此抑制这些细胞的迁移和吞噬功能。这可能会促进组织中细胞的保留，从而促进动脉粥样硬化斑块的生长。这项赠款的具体目的是为了检验这一假设。许多研究已经调查了降低膜胆固醇水平对细胞功能的影响， 但是很少有人关注增加膜胆固醇水平这一潜在的更具有生理学意义的事件的影响。我们的初步数据表明，过载的Mo的质膜与胆固醇降低其迁移率在三维（3D）胶原凝胶。同样，我们对中性粒细胞的研究表明，胆固醇消耗抑制Rac GTP酶介导的肌动蛋白重组和迁移。我们建议首先量化刺激肌动蛋白重组在莫斯和巨噬细胞的程度是敏感的膜胆固醇水平的升高或降低，然后我们将研究这些变化如何影响肌动蛋白依赖的迁移和吞噬功能。接下来，我们将直接测试 对迁移和吞噬功能的影响可归因于Rho GTP酶（即Rho、Rac和Cdc 42）的激活和/或靶向的破坏。最初，我们将使用药理学手段来改变膜胆固醇，后来我们将逐步使用更多的生理胆固醇调节方案，从分离的脂蛋白到基质包埋的脂蛋白，模拟致动脉粥样硬化的环境。

Early in the pathogenesis of atherosclerosis, aggregated, oxidized lipoprotein particles become attached to proteins in the subendothelial matrix of the affected arteries. Monocytes (Mos) that traverse the overlying endothelium come into contact with these matrix-bound lipoprotein particles, engulf them, and eventually take up large amounts of lipid, developing into lipid-engorged macrophage foam cells, which promote further growth of the atherosclerotic plaque. We hypothesize that alterations in the plasma membrane cholesterol levels in Mos and macrophages caused by interactions with these matrix-bound lipoproteins may be partially responsible for lesion growth. Based on our preliminary studies, we postulate that retained and aggregated lipoproteins alter actin dynamics in Mos and macrophages in the subendothelial space, and therefore inhibit the migration and phagocytic function of these cells. This may promote the retention of cells in the tissue and thus the growth of atherosclerotic plaques. The specific aims of this grant are designed to test this hypothesis. Numerous studies have investigated the effects of reducing membrane cholesterol levels on cellular functions, but far fewer have looked at the effects of the potentially more physiologically significant event of increasing membrane cholesterol levels. Our preliminary data indicate that overloading a Mo's plasma membrane with cholesterol decreases its migration rate in a three dimensional (3D) collagen gel. Similarly, our studies with neutrophils showed that cholesterol depletion inhibited Rac GTPase-mediated actin reorganization and migration. We propose to first quantify the extent to which stimulated actin reorganization in Mos and macrophages is sensitive to raising or lowering membrane cholesterol levels, and then we will study how these changes affect the actin-dependent functions of migration and phagocytosis. Next, we will directly test the idea that effects on migration and phagocytic function can be attributed to disruption of activation and/or targeting of Rho GTPases (i.e. Rho, Rac, and Cdc42). Initially we will use pharmacological means to alter membrane cholesterol, and later we will use progressively more physiological cholesterol modulating scenarios from isolated lipoproteins to matrix-embedded lipoproteins that mimic an atherogenic environment.