Macrophage Expression of APOAI and Atherosclerosis

描述（由申请人提供）：载脂蛋白（apo） Al是血浆HDL的主要结构成分。血浆HDL和apoAl水平与冠心病发病率呈负相关，对其进行调节是动脉粥样硬化的治疗干预措施。ApoAl是逆向胆固醇转运（RCT）的驱动因素，通过这一途径，过量的外周胆固醇被传递到肝细胞。RCT促进血管健康的机制尚不清楚，apoAl和HDL可能在动脉壁中发挥直接保护作用。由此看来，较高的HDL水平可能仅仅与动脉壁中较高浓度的HDL有关，这可能是斑块稳定的真正效应。在本研究的前一个周期中，我们已经证明，动脉巨噬细胞产生的apoAl会导致斑块中高浓度的apoAl，而血浆中只有少量的apoAl，血浆HDL没有变化。在血浆HDL水平从几乎为零到两倍正常的小鼠模型中，这种干预完全预防了动脉粥样硬化。我们还发现apoAl的动脉表达可显著降低apoE缺乏引起的动脉粥样硬化，这表明巨噬细胞apoAl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apoAl表达和使用表达apoai的逆转录病毒载体进行骨髓细胞转导的联合方法实现了这一目标。我们现在建议这些研究线的自然进展，旨在机制发现，技术创新和临床可翻译性。我们将开发由生理性ME2启动子驱动的表达apoAl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。在该系统中，巨噬细胞向泡沫细胞转化过程中，巨噬细胞特异性表达apoAl会上调。我们还将生产转基因小鼠，表达由相同的胆固醇敏感启动子驱动的apoAl。另一个技术进步将是使用纯化的造血干细胞（HSC）和亚致死剂量的辐射。利用这些工具，我们将在体内和体外评估人类apoAl和单氨基酸变异对血管脂质积累和炎症的影响。已知天然apoAl变异可以降低HDL水平，对动脉粥样硬化有不同的影响。拟验证的主要假设有：1)apoAl的血管效应可以与血浆HDL水平分离；2) ApoAl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HDL对动脉斑块治疗作用的基础。

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.