Macrophage Expression of APOAI and Atherosclerosis

描述(申请人提供)：载脂蛋白(Apo)Al是血浆高密度脂蛋白的主要结构成分。血浆高密度脂蛋白和载脂蛋白A1水平与冠心病率呈负相关，其调控是对动脉粥样硬化的治疗干预。载脂蛋白A1是胆固醇反向转运(RCT)的驱动力，RCT是将过量的外周胆固醇输送到肝细胞的途径。RCT促进血管健康的机制尚不清楚，存在载脂蛋白A1和高密度脂蛋白在动脉壁发挥直接保护作用的可能性。从这个角度来看，较高的高密度脂蛋白水平可能只是与动脉壁中较高浓度的高密度脂蛋白有关，这可能是斑块稳定的真正效应。在这笔赠款的前一个周期中，我们已经表明，动脉巨噬细胞独家产生apoAl会导致斑块中apoAl的高浓度，而血浆中的apoAl含量最低，而血浆高密度脂蛋白没有变化。这种干预措施完全预防了血浆高密度脂蛋白水平不同的小鼠模型的动脉粥样硬化，范围从几乎为零到正常的两倍。我们还发现，动脉载脂蛋白A1的表达显著降低了载脂蛋白E缺乏诱导的动脉粥样硬化，提示巨噬细胞载脂蛋白A1可以对抗沉重的脂质负担。我们通过转基因、巨噬细胞特异性载脂蛋白A1的表达和使用载脂蛋白AI表达逆转录病毒载体的骨髓细胞转导相结合的方法实现了这一点。我们现在提出这些研究路线的自然发展，旨在实现机械性发现、技术创新和临床可译性。我们将开发由生理性ME2启动子驱动的表达载脂蛋白A1结构的慢病毒载体，该启动子通过生理性LXR环对胆固醇负荷敏感。在这个系统中，在巨噬细胞转化为泡沫细胞的过程中，巨噬细胞特异性apoA1的表达将上调。我们还将生产由相同的胆固醇敏感启动子驱动的表达apoA1的转基因小鼠。另一项技术进步将是使用纯化的造血干细胞(HSC)和亚致命剂量的辐射。使用这些工具，我们将评估人载脂蛋白A1和单一氨基酸变体在体内和体外对血管脂肪堆积和炎症的影响。已知天然载脂蛋白A1变异体可以降低高密度脂蛋白水平，对动脉粥样硬化的形成有不同的影响。要检验的主要假设是：1)载脂蛋白A1的血管效应可以与血浆高密度脂蛋白水平分离；2)载脂蛋白A1对巨噬细胞胆固醇稳态、炎症和氧化有直接影响。这些研究有可能揭示高密度脂蛋白对动脉斑块的治疗效果的基础。

DESCRIPTION (provided by applicant): Apolipoprotein (apo) Al is the main structural component of plasma HDL. Plasma levels of HDL and apoAl are inversely related to CHD rates, and their manipulation is a therapeutic intervention for atherosclerosis. ApoAl is the driver of reverse cholesterol transport (RCT), the pathway through which excess peripheral cholesterol is delivered to the hepatocyte. The mechanism through which RCT promotes vascular health is not understood, and the possibility exists that apoAl and HDL exert direct protective effects in the artery wall. In this light, higher HDL levels might simply be related to higher concentrations of HDL in the artery wall, which may be the true effector of plaque stabilization. In the previous cycle of this grant we have shown that the exclusive production of apoAl from arterial macrophages leads to high concentrations of apoAl in the plaque, with minimal amounts of apoAl in plasma and no changes in plasma HDL. This intervention resulted in the complete prevention of atherosclerosis in mouse models with different levels of plasma HDL, ranging from almost none to twice normal. We have also shown that arterial expression of apoAl significantly reduces the atherogenesis induced by apoE deficiency, suggesting that macrophage apoAl can counter heavy lipid burden. We have accomplished this through a combined approach of transgenic, macrophage-specific apoAl expression, and bone marrow cell transduction using apoAI-expressing retroviral vectors. We are now proposing the natural progression of these investigative lines, aiming for mechanistic discoveries, technical innovation, and clinical translatability. We will develop lentiviral vectors expressing apoAl constructs driven by the physiologic ME2 promoter, which is sensitive to cholesterol loading through the physiologic LXR loop. In this system, macrophage-specific expression of apoAl will be up-regulated during the transformation of the macrophage into a foam cell. We will also produce transgenic mice that express apoAl driven by the same cholesterol-sensitive promoter. Another technological advance will be the use of purified hematopoietic stem cells (HSC) and sub-lethal doses of radiation. Using these tools, we will evaluate the effects of human apoAl and single amino-acid variants on vascular lipid accumulation and inflammation in vivo and in vitro. The natural apoAl variants are known to lower HDL levels with diverging effects on atherogenesis. The main hypotheses to be tested are: 1) The vascular effects of apoAl can be dissociated from plasma HDL levels; 2) ApoAl has direct effects on macrophage cholesterol homeostasis, inflammation, and oxidation. These studies have the potential to uncover the basis for the therapeutic effect of HDL on the arterial plaque.