HDL & CELLULAR CHOLESTEROL METABOLISM

该项目将专注于影响细胞和血清中胆固醇转运的细胞和血清因素。 高密度脂蛋白/载脂蛋白。将使用几种细胞类型，但将对巨噬细胞进行研究 强调。在特定的目标1，将研究影响胆固醇外流的细胞因素。假设是为了 被测试的是转运蛋白的表达水平和水扩散在胆固醇中的贡献 供体细胞和受体高密度脂蛋白颗粒的浓度/分布对这些途径是最佳的 测定外排效率。我们将量化水扩散和SR-BI、ABCA1的贡献 和Abcg1转运蛋白从暴露于血清、分离的脂蛋白和重组的高密度脂蛋白的细胞外流。 同时使用遗传操作和药物抑制剂来测量通路贡献的方案 到总流出量，将使用在当前赠款周期内开发的流出量。我们还将研究是否 巨噬细胞泡沫细胞中胆固醇酯(CE)水解产生的胆固醇被清除 通过特定的外流途径，以及脂氧合酶对CE的氧化如何影响清除。血清因子 将在特定目标中考虑对胆固醇流量的影响2.这些实验将检验假设 LCAT、CETP和PLTP引起的高密度脂蛋白重塑改变了不同途径的相对贡献。我们预测 在增加小/血脂差的血清因素的影响之间将存在相互关系 高密度脂蛋白和血清因素增加大量富含磷脂的高密度脂蛋白。《特定目标3》旨在 证明人血清高密度脂蛋白的外流能力取决于高密度脂蛋白“质量”，而不仅仅是总高密度脂蛋白。 胆固醇。我们将使用总高密度脂蛋白胆固醇相似但不同的样本的血清高密度脂蛋白部分 外排能力，以了解巨噬细胞胆固醇外流如何受高密度脂蛋白组成的影响。在……里面 具体目标4我们将使用我们开发的一种方法来同时测量胆固醇的流出和流入 当巨噬细胞与血清或脂蛋白孵育时。这些化验将得出流入、流出的数值 以及当巨噬细胞暴露于各种胆固醇受体时的净通量。建议进行的研究 项目1源于过去与项目2和3建立的密切合作，并将继续 与这些项目中提出的研究很好地结合在一起。 载脂巨噬细胞在动脉壁的积聚是高血压形成的早期事件。 动脉粥样硬化斑块。我们知道这一过程会因为血液中高密度脂蛋白水平的存在而减少，但是 我不明白为什么。这些研究将增加我们对高密度脂蛋白功能的理解。

This project will focus on the cellular and serum factors that affect cholesterol transport between cells and high density lipoprotein (HDL)/ apoproteins. Several cell types will be used but study of macrophages will be emphasized. In Specific Aim 1 cellular factors affecting cholesterol efflux will be studied. The hypothesis to be tested is that the expression level of transporters and the contribution of aqueous diffusion in cholesterol donor cells together with the concentration/distribution of acceptor HDL particles optimal for these pathways determine efflux efficiency. We will quantify the contributions of aqueous diffusion and the SR-BI, ABCA1 and ABCG1 transporters to efflux from cells exposed to serum, isolated lipoproteins and reconstituted HDL. Protocols using both genetic manipulation and pharmacological inhibitors to measure pathway contributions to total efflux that were developed during the current grant cycle will be employed. We will also study if removal of the cholesterol produced by cholesteryl ester (CE) hydrolysis in macrophage foam cells occurs via specific efflux pathways and how oxidation of CE by lipoxygenases affects clearance. Serum factors that affect cholesterol flux will be considered in Specific Aim 2. These experiments will test the hypothesis that HDL remodeling by LCAT, CETP and PLTP alters the relative contribution of different pathways. We predict that there will be a reciprocal relationship between the effects of serum factors that increase small/lipid poor HDL and serum factors that increase large phospholipid-rich HDL. Specific Aim 3 is designed to demonstrate that the efflux capacity of human serum HDL depends on HDL "quality" and not just total HDL cholesterol. We will use the serum HDL fraction from specimens having similar total HDL-C but different efflux capacities to understand how macrophage cholesterol efflux is affected by HDL composition. In Specific Aim 4 we will use an assay we developed to simultaneously measure efflux and influx of cholesterol when macrophages are incubated with serum or lipoproteins. These assays will yield values for influx, efflux and net flux when macrophages are exposed to a variety of cholesterol acceptors. The studies proposed in Project 1 arise from close collaborations established in the past with Projects 2 and 3, and will continue to be well integrated with studies proposed in those projects. The accumulation of cholesterol-loaded macrophages in the arterial wall is an early event in formation of atherosclerotic plaques. We know this process is reduced by the presence of high circulating HDL levels but do not understand why. These studies will add to our understanding of HDL function.