| Budget Amount *help |
¥14,800,000 (Direct Cost: ¥14,800,000)
Fiscal Year 2006: ¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥11,300,000 (Direct Cost: ¥11,300,000)
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| Research Abstract |
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
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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
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