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性疾病中起关键作用。人类载脂蛋白E有三种主要的同工酶，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1)巨噬细胞胆固醇逆向转运和2)极低密度脂蛋白-甘油三酯脂解中的作用，我们使用腺病毒载体表达这些异构体。加载胆固醇后，用腺病毒载体感染细胞表达载脂蛋白E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也能降低细胞内胆固醇含量，但效果不如APOE2。在The n…中进一步用载脂蛋白E腺病毒感染的HeLa细胞培养载脂蛋白的RAW264.7细胞，研究外源载脂蛋白E在胆固醇逆向转运中的作用。本实验表明，apoE3在胆固醇逆向转运方面是有效的，而apoE2和E4的作用不大。最后，为了阐明载脂蛋白E在极低密度脂蛋白-甘油三酯脂解中的作用，将腺病毒载体注射到载脂蛋白E/低密度脂蛋白受体双缺陷小鼠，获得了含载脂蛋白E的极低密度脂蛋白。改变载脂蛋白E/甘油三酯的比例，用牛脂蛋白脂酶进行体外脂解实验。本实验表明，载脂蛋白E在VLDL颗粒上的存在，无论其异构体如何，都能抑制脂解作用，并且与E3和E4相比，增加载脂蛋白2/TG对脂解的抑制作用更强。综上所述，载脂蛋白E异构体不仅在内源性表达时对巨噬细胞胆固醇的反向转运有不同的作用，在外源性表达时也有差异。不同亚型的载脂蛋白E均能抑制极低密度脂蛋白的脂解，其中载脂蛋白2的抑制作用比其他两种异构体更为显著。较少

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