|Budget Amount *help
¥3,300,000 (Direct Cost : ¥3,300,000)
Fiscal Year 1999 : ¥1,200,000 (Direct Cost : ¥1,200,000)
Fiscal Year 1998 : ¥2,100,000 (Direct Cost : ¥2,100,000)
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
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