Differential Effect of apoE isoform on cholesterol-loaded macrophage

载脂蛋白E （apoE）在脂蛋白代谢和动脉粥样硬化疾病中起关键作用。人类apoE存在三种主要的共同亚型，即E2、E3和E4。已知这些亚型对脂蛋白代谢和动脉粥样硬化有不同的影响。在本研究中，为了研究这些异构体在巨噬细胞的逆向胆固醇转运和vldl -甘油三酯脂解中的作用，我们利用腺病毒载体表达了这些异构体。不内源性表达apoE的RAW264.7小鼠巨噬细胞是胆固醇负荷的。在装载胆固醇后，用腺病毒载体感染细胞以表达apoE亚型。与感染LacZ腺病毒的对照细胞相比，apoE2的表达显著降低了细胞的酯化胆固醇水平。ApoE3和E4也能降低细胞胆固醇含量，但其效果不如apoE2有效。在接下来的实验中，为了阐明外源性apoE在逆转胆固醇转运中的作用，我们将装载胆固醇的RAW264.7细胞与先前感染apoE腺病毒的HeLa细胞中收获的培养基一起培养。本实验表明apoE3在胆固醇逆向转运中是有效的，而apoE2和E4作用不大。最后，为了阐明apoE对VLDL-甘油三酯脂解的作用，我们将腺病毒载体注射到apoE/ ldl受体双缺陷小鼠体内，获得含apoE的VLDL。改变载脂蛋白e /TG的比例，用牛脂蛋白脂肪酶体外脂解VLDL颗粒。本实验发现，无论其异构体如何，apoE存在于VLDL颗粒上均可抑制脂解，且apoE2/TG比值的增加比E3和E4对脂解的抑制作用更大。综上所述，apoE亚型对巨噬细胞的逆向胆固醇转运有不同的影响，无论是内源性表达还是外源性添加。无论apoE的异构体是什么，它都能抑制vldl -脂解，并且与其他两种异构体相比，apoE2具有更实质性的抑制作用。少

Apolipoprotein E (apoE) plays a key role in the lipoprotein metabolism and atherosclerotic diseases. There exist three major common isoforms in human apoE, i.e., E2, E3 and E4. These isoforms have been known to have differential effect on lipoprotein metabolism and atherosclerosis. In the present study, in order to investigate the roles of these isoforms on 1) the reverse cholesterol transport from the macrophages and 2) VLDL-triglycerides lipolysis, we utilized adenoviral vector for the expression of these isoforms.The RAW264.7 mouse macrophage cell line, which does not express apoE endogenously, was cholesterol-loaded. After loading cholesterol, the cells were infected with adenoviral vectors to express apoE isoforms. The expression of apoE2 reduced cellular esterified-cholesterol levels significantly compared with control cells infected with LacZ adenovirus. ApoE3 and E4 also reduced the cellular cholesterol content, however, their effect was not so much effective as apoE2. In the n … More ext experiment, cholesterol-loaded RAW264.7 cells were incubated with the medium harvested from the HeLa cells previously infected with apoE adenovirus, in order to elucidate the role of exogenous apoE on reverse cholesterol transport. This experiment revealed that apoE3 is effective in the reverse cholesterol transport, however, apoE2 and E4 have little effect. Finally, to elucidate the role of apoE on VLDL-triglycerides lipolysis, adenoviral vectors were injected to apoE/LDL-receptor double deficient mice and apoE-containing VLDL was obtained. These VLDL particles were subjected to in vitro lipolysis assay with bovine lipoprotein lipase, with changing the ratio of apoE/TG. This experiment revealed that the existence of apoE on VLDL particles inhibits lipolysis regardless of its isoform, and increasing ratio of apoE2/TG had more inhibitory effect on the lipolysis compared with E3 and E4.In summary, apoE isoforms have differential effect on reverse cholesterol transport from macrophages not only when they were expressed endogenously but also when they were added exogenously. The VLDL-lipolysis is inhibited by apoE regardless of its isoform, and apoE2 has more substantial inhibitory effect compared with the other two isoforms. Less