Targeting the Foam Cell to Prevent Atherosclerosis

描述(由申请人提供)：富含脂质的巨噬细胞或“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，都起着至关重要的作用。然而，我们目前的治疗手段缺乏主要针对泡沫细胞的药物来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)并在细胞质脂滴(LDs)中积聚其脂质，主要是胆固醇(Chol)酯(CE)，从而成为泡沫细胞。LDS由磷脂、游离胆固醇(Fc)和蛋白质稳定和限制的中性脂核组成。与LD相关的主要结构蛋白是PAT家族成员，在巨噬细胞中含量最丰富的PAT蛋白是脂肪分化相关蛋白(ADFP)。我们已经证明ADFP在泡沫细胞的形成中起着关键作用，它的缺失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在人和小鼠的动脉粥样硬化病变中表达上调，而在载脂蛋白E缺失(apoE-/-)小鼠中消融ADFP具有动脉粥样硬化保护作用。增加巨噬细胞的Chol外流，例如通过增加Chol受体在血浆中的浓度或上调Chol外流转运体的表达，可以保护动脉粥样硬化。这似乎也是缺乏ADFP的动脉粥样硬化保护机制，因为ADFP缺乏的巨噬细胞不会在LDS中以CE的形式积累大量Chol，并将更多的Chol外流到细胞外受体。这项建议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缺乏的巨噬细胞在炎症和凋亡方面的反应。我们还将检查在普通饮食喂养的ADFP-/-小鼠中观察到的动脉粥样硬化保护作用是否也在高脂肪/高胆固醇饮食挑战小鼠中观察到。在第二个目标中，我们将通过辅助依赖腺病毒(HDAd)载体系统增加Chol受体载脂蛋白A1(ApoA1)的血浆浓度，并评估两种不同的增加Chol外流的方法(即增加细胞外受体和限制细胞储存Chol的能力)是否具有额外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，以寻找在储脂和脂外排条件下培养的巨噬细胞中主要的LD相关蛋白和ADFP相互作用因子。我们还将对直接从ADFP表达或不表达ADFP的小鼠皮损中分离的泡沫细胞进行全球基因表达分析，以确定泡沫细胞如何适应缺乏ADFP。从ADFP在泡沫细胞形成和动脉粥样硬化中的作用的广泛分析中，我们期望获得广泛的理解，这可能有助于建立针对泡沫细胞的新策略的基础，以防止动脉粥样硬化的发展。 与公共卫生相关：通过阐明ADFP缺乏的巨噬细胞对高脂应激条件的反应，防止胆固醇在脂滴中堆积同时增加细胞外环境接受胆固醇的能力是否可以协同作用预防动脉粥样硬化，控制巨噬细胞脂滴形成和降解的分子机制，ADFP如何参与这一过程，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了解，如果开发针对脂滴的药物来防止泡沫细胞形成和动脉粥样硬化，这些研究可能非常有用。

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.