Targeting the Foam Cell to Prevent Atherosclerosis

描述（由申请人提供）：概述载脂巨噬细胞或“泡沫细胞”在动脉粥样硬化形成的所有阶段（从病变开始到斑块破裂）中起关键作用。然而，我们目前的治疗武库缺乏主要针对泡沫细胞的药物，以防止动脉粥样硬化的发展。巨噬细胞通过摄取修饰的低密度脂蛋白（mLDL）并将其脂质（主要是胆固醇（CHOL）酯（CE））积聚在细胞质脂滴（LD）中而成为泡沫细胞。LD由磷脂、游离CHOL（FC）和蛋白质稳定和限制的中性脂质核心组成。主要结构LD相关蛋白是PAT家族的成员，并且在巨噬细胞中最丰富的PAT蛋白是脂肪分化相关蛋白（ADFP）。我们已经表明，ADFP在泡沫细胞形成中起着关键作用，它的缺乏严重限制了巨噬细胞在体外和体内成为泡沫细胞的能力。ADFP在人类和小鼠动脉粥样硬化病变中上调，在载脂蛋白E-/-小鼠中ADFP消融具有动脉粥样硬化保护作用。增强来自巨噬细胞的CHOL流出，例如通过增加血浆中CHOL受体的浓度或通过上调CHOL流出转运蛋白的表达，防止动脉粥样硬化。这似乎也是与缺乏ADFP相关的动脉粥样硬化保护机制，因为ADFP缺陷型巨噬细胞在LD中不以CE形式积累同样多的CHOL，并且将更多的CHOL流出到细胞外受体。该提案的广泛目标是深入了解ADFP在LD生物学，泡沫细胞形成和动脉粥样硬化发展中的作用。在第一个目标中，我们将在体外测试在高脂应激条件下培养的ADFP缺陷型巨噬细胞在炎症和凋亡方面的反应。我们还将检查在喂食常规食物的ADFP-/-小鼠中观察到的动脉粥样硬化保护作用是否也在用高脂肪/高CHOL饮食攻击小鼠时观察到。在第二个目标中，我们将用辅助依赖性腺病毒（HDAd）载体系统增加CHOL受体载脂蛋白A1（apoA 1）的血浆浓度，并评估增加CHOL流出的两种不同方法（即增加细胞外受体和限制细胞储存CHOL的能力）是否具有相加的动脉粥样硬化保护作用。在第三个目标中，我们将进行蛋白质组学分析，以找到在脂质储存和脂质流出条件下培养的巨噬细胞中的主要LD相关蛋白和ADFP相互作用物。我们还将在直接从表达或不表达ADFP的小鼠病变中分离的泡沫细胞中进行全局基因表达分析，以确定泡沫细胞如何适应ADFP的缺乏。通过对ADFP在泡沫细胞形成和动脉粥样硬化中作用的广泛分析，我们期望获得广泛的理解，这可能有助于建立靶向泡沫细胞以预防动脉粥样硬化发展的新策略的基础。 公共卫生相关性：通过阐明ADFP缺陷型巨噬细胞对高脂应激条件的反应，是否在增加细胞外环境接受胆固醇的能力的同时防止胆固醇在脂滴中积累可以协同作用以防止动脉粥样硬化，控制巨噬细胞中脂滴发生和降解的分子机制，ADFP如何参与该过程，以及泡沫细胞如何适应ADFP的缺乏，这些研究将提供对泡沫细胞生物学的广泛理解，如果要开发靶向脂滴以防止泡沫细胞形成和动脉粥样硬化的药物，这可能非常有用。

DESCRIPTION (provided by applicant): Summary Lipid-laden macrophages or "foam cells" play crucial roles in all the stages of atherogenesis, from lesion initiation to plaque rupture. However, our current therapeutic arsenal lacks of drugs primarily targeting the foam cell to prevent atherosclerosis development. Macrophages become foam cells by the uptake of modified low- density lipoproteins (mLDL) and accumulation of their lipids, mostly cholesterol (CHOL) esters (CE), in cytoplasmic lipid droplets (LDs). LDs consist of a core of neutral lipid stabilized and circumscribed by phospholipids, free CHOL (FC) and proteins. The main structural LD-associated proteins are the members of the PAT-family, and in macrophages the most abundant PAT-protein is adipose differentiation-related protein (ADFP). We have shown that ADFP plays a key role in foam cell formation, and its absence severely restricts the ability of macrophages to become foam cells in vitro and in vivo. ADFP is upregulated in human and mouse atherosclerotic lesions, and ADFP ablation in apolipoprotein E-null (apoE-/-) mice is atheroprotective. Enhancing CHOL efflux from macrophages, e.g. by increasing the concentration in plasma of CHOL acceptors or by upregulating the expression of CHOL efflux transporters, protects against atherosclerosis. This also seems to be the atheroprotective mechanism associated to the lack of ADFP, since ADFP-deficient macrophages do not accumulate as much CHOL in the form of CE in LDs, and efflux more CHOL to extracellular acceptors. The broad goal of this proposal is to gain insight on the role of ADFP in LD biology, foam cell formation and atherosclerosis development. In the first aim, we will test in vitro the response, in terms of inflammation and apoptosis, of ADFP-deficient macrophages cultured under high lipid stress conditions. We will also check if the atheroprotection observed in ADFP-/- mice fed regular chow is also observed when mice are challenged with a high fat/high CHOL diet. In the second aim, we will increase the plasma concentration of the CHOL acceptor apolipoprotein A1 (apoA1) with a helper-dependent adenovirus (HDAd) vector system, and assess whether two different approaches to increase CHOL efflux (i.e. increasing extracellular acceptors and limiting the cell's ability to store CHOL) will have additive atheroprotective effects. In the third aim, we will perform proteomic analyses to find the main LD-associated proteins and ADFP interactors in macrophages cultured under lipid-storing and lipid-efflux conditions. We will also perform a global gene expression analysis in foam cells directly isolated from lesions of mice that do or do not express ADFP to determine how the foam cell adjusts to the lack of ADFP. From this broad-based analysis of the role of ADFP in foam cell formation and atherosclerosis we expect to obtain a breadth of understanding that may help to set- up the bases for novel strategies to target the foam cell to prevent atherosclerosis development. PUBLIC HEALTH RELEVANCE: By elucidating the response of ADFP-deficient macrophages to high lipid stress conditions, whether preventing cholesterol accumulation in lipid droplets while increasing the ability of the extracellular milieu to accept cholesterol can work synergistically to prevent atherosclerosis, the molecular mechanisms that govern lipid droplet genesis and degradation in macrophages, how ADFP is involved in the processes, and how the foam cell adjust to the lack of ADFP, these studies will provide a breadth of understanding on foam cell biology that may be very useful if drugs to target the lipid droplet to prevent foam cell formation and atheroclerosis are to be developed.