Hakamata H, Sakaguchi H, Zhang C, Sakashita N, Suzuki H, Miyazaki A, Takeya M, Takahashi K, Kitamura N, Horiuchi S
Department of Biochemistry, Kumamoto University School of Medicine, Japan.
Biochemistry. 1998 Sep 29;37(39):13720-7. doi: 10.1021/bi980762v.
Apolipoprotein E (apoE)-knockout mice develop severe atherosclerosis associated with high levels of very low-density lipoprotein (VLDL) and intermediate-density lipoprotein (IDL) in plasma. To investigate the atherogenic role of VLDL and IDL, the lipoprotein fraction containing both VLDL and IDL (apoEko-VLDL/IDL) was isolated from plasma of apoE-knockout mice by ultracentrifugation, and its interaction with macrophages was studied. When peritoneal macrophages obtained from apoE-knockout mice were incubated with apoEko-VLDL/IDL, the level of cellular cholesteryl esters (CE) increased with the concentration of apoEko-VLDL/IDL. The level of cellular cholesteryl [3H]oleate formed reached 15.1 nmol/mg of cell protein upon incubation with 50 microg/mL apoEko-VLDL/IDL for 18 h, which was an 8.4-fold increase over the corresponding level induced by low-density lipoprotein (LDL). The cellular CE mass was also significantly increased by apoEko-VLDL/IDL. Morphologically, after exposure to apoEko-VLDL/IDL, macrophages became strongly stained with Sudan black B. The total binding of [125I]apoEko-VLDL/IDL to macrophages was effectively replaced by more than 80% by an excess of the unlabeled ligand. Specific binding, calculated by subtracting the nonspecific binding from the total binding, exhibited a saturation pattern. Similar results were obtained with cell association and degradation experiments. In addition, the endocytic degradation of [125I]apoEko-VLDL/IDL was partially inhibited by LDL, whereas acetyl-LDL did not show any effect. These results indicated that apoEko-VLDL/IDL in its unmodified form produced significant CE accumulation in macrophages through a specific and apoE-independent pathway. This pathway may explain, in part, the mechanisms of foam cell formation in arterial walls and the subsequent development of atherosclerosis in apoE-knockout mice.
载脂蛋白E（apoE）基因敲除小鼠会发展出严重的动脉粥样硬化，其血浆中极低密度脂蛋白（VLDL）和中间密度脂蛋白（IDL）水平较高。为了研究VLDL和IDL的致动脉粥样硬化作用，通过超速离心从apoE基因敲除小鼠的血浆中分离出同时含有VLDL和IDL的脂蛋白组分（apoEko-VLDL/IDL），并研究其与巨噬细胞的相互作用。当将从apoE基因敲除小鼠获得的腹腔巨噬细胞与apoEko-VLDL/IDL一起孵育时，细胞胆固醇酯（CE）水平随apoEko-VLDL/IDL浓度的增加而升高。与50μg/mL apoEko-VLDL/IDL孵育18小时后，形成的细胞胆固醇[3H]油酸酯水平达到15.1 nmol/mg细胞蛋白，这比低密度脂蛋白（LDL）诱导的相应水平增加了8.4倍。apoEko-VLDL/IDL也显著增加了细胞CE总量。形态学上，暴露于apoEko-VLDL/IDL后，巨噬细胞被苏丹黑B强烈染色。[125I]apoEko-VLDL/IDL与巨噬细胞的总结合被过量的未标记配体有效取代了80%以上。通过从总结合中减去非特异性结合计算得出的特异性结合呈现出饱和模式。细胞结合和降解实验也得到了类似的结果。此外，LDL部分抑制了[125I]apoEko-VLDL/IDL的内吞降解，而乙酰化LDL没有任何作用。这些结果表明，未修饰形式的apoEko-VLDL/IDL通过特定的、不依赖apoE的途径在巨噬细胞中产生显著的CE积累。该途径可能部分解释了apoE基因敲除小鼠动脉壁中泡沫细胞形成的机制以及随后动脉粥样硬化的发展。
