Identification and characterization of novel vesicular organelle appearing hyperlipidemic human macrophages

高脂血症状态下的巨噬细胞不断内化修饰的低密度脂蛋白（LDL），并在动脉壁上转化为载脂（泡沫转化）巨噬细胞。这些富含脂质的巨噬细胞通过产生多种趋化因子和细胞因子促进动脉粥样硬化，因此巨噬细胞泡沫转化的机制是研究动脉粥样硬化发生的关键问题。内化的修饰LDL在lamp2阳性的水解室中水解，由修饰LDL产生的游离胆固醇被转移到内质网（ER），内质网驻留酶ACAT1将胆固醇酯化，酯化后的胆固醇被储存为脂滴，从而使巨噬细胞变成脂质负载巨噬细胞。我们发现泡沫转化巨噬细胞产生er来源的acat1阳性囊泡细胞器（中华病理学杂志，156:227- 233,2000）。为了研究这些囊泡的性质，我们用亚细胞分离的方法分析了含脂的人巨噬细胞。在低胆固醇情况下，ACAT1大部分出现在ACAT酶活性可忽略的中密度馏分，而向ACAT酶活性显著的低密度馏分转移。低密度部分含有反式高尔基网络标记物syntaxin 6，来自ACAT1和syntaxin 6的免疫荧光信号在脂质负载的巨噬细胞中部分共混，而在低胆固醇巨噬细胞中不存在。在富含胆固醇的巨噬细胞中，使用含有syntaxin 6的ACAT1特异性抗体，通过免疫吸附法纯化ACAT1阳性组分。由于syntaxin 6分布反式高尔基网络和晚期内体成分，我们进一步探索了ACAT1在富含胆固醇的巨噬细胞中转位到晚期内体的可能性。正如预期的那样，泡沫转化的人巨噬细胞产生acat1阳性的晚期内体成分，这些特殊的细胞器有效地将水解的游离胆固醇重新酯化。这些结果表明，人巨噬细胞通过内质网破碎产生ACAT1阳性囊泡，这些囊泡使ACAT1易位到反式高尔基网络/晚期核内体，从而使富含胆固醇的巨噬细胞发生有效的胆固醇酯化和泡沫转化。少

The macrophages under hyperlipidemic condition continuously internalize modified low density lipoprotein (LDL) and transformed to lipid laden (foamy transformed) macrophages in the arterial wall. These lipid-laden macrophages promote atherosclerosis via producing various chemokines and cytokines, therefore, mechanism of foamy transformation of the macrophages is key issue to investigate atherogenesis. Internalized modified LDL is hydrolyzed in the LAMP2-positive hydrolytic compartment, free cholesterol derived from modified LDL is transferred to the endoplasmic reticulum (ER), ACAT1, an ER resident enzyme, esterifies cholesterol, esterified cholesterol is stored as lipid droplets, thus macrophages turn into lipid laden macrophages. We discovered foamy transformed macrophages produce ER-derived, ACAT1-positive vesicular organelle (Am J Pathol, 156:227-236, 2000). To investigate the nature of these vesicles, we analyzed lipid-laden human macrophages by means of subcellular fractionation … More assay. Most of ACAT1 appeared in middle density fractions that have negligible ACAT enzymatic activity in cholesterol poor situation, while it moved to low density fractions with significant ACAT activity. Low density fractions contained trans-Golgi network marker syntaxin 6, and immunofluorescent signals from ACAT1 and syntaxin 6 were partially colocolized in lipid-laden macrophages, not in cholesterol poor macrophages. In cholesterol-rich macrophages, purified ACAT1-positive fractions by means of immunoadsorption method using ACAT1 specific antibodies contained syntaxin 6. Since syntaxin 6 distributes trans-Golgi network as well as late endosomal components, we furtuher explored the possibility of a translocation of ACAT1 to late endosomes in cholesterol-rich macrophages. As expected, foamy transformed human macrophages produced ACAT1-positive late endosomal components and these special organelles efficiently re-esterify hydrolyzed free cholesterol. These results indicate that human macrophages produce ACAT1-positive vesicles via ER fragmentation, and these vesicles cause ACAT1 translocation to trans-Golgi network/late endosomes, resulting in effective cholesterol esterification and foamy transformation of the cholesterol-rich macrophages. Less