Cholesterol ester (CE)-laden macrophage foam cells are the hallmark of atherosclerosis, and the hydrolysis of intracellular CE is one of the key steps in foam cell formation. Although hormone-sensitive lipase (LIPE) and cholesterol ester hydrolase (CEH), which is identical to carboxylsterase 1 (CES1, hCE1), were proposed to mediate the neutral CE hydrolase (nCEH) activity in macrophages, recent evidences have suggested the involvement of other enzymes. We have recently reported the identification of a candidate, neutral cholesterol ester hydrolase 1(Nceh1). Here we demonstrate that genetic ablation of Nceh1 promotes foam cell formation and the development of atherosclerosis in mice. We further demonstrate that Nceh1 and Lipe mediate a comparable degree of nCEH activity in macrophages and together account for most of the activity. Mice lacking both Nceh1 and Lipe aggravated atherosclerosis in an additive manner. Thus, Nceh1 is a promising target for the treatment of atherosclerosis.

The selectivity of microbial inhibitors of acylCoA : cholesterol acyltransferase (ACAT) toward the two isozymes, ACAT1 and ACAT2, was assessed in cell-based assays. Purpactin A (IC 50 values of ACAT1 vs. IC 50 values of ACAT2; 2.5 mM vs. 1.5 mM), terpendole C (10 mM vs. 2.0 mM), C (32 mM vs. 0.36 mM) and D (38 mM vs. 1.5 mM) showed selective inhibition against ACAT2. In particular, pyripyropene A was found to be the most selective ACAT2 inhibitor with a selective index of more than 1,000.Keywords acyl-CoA : cholesterol acyltransferase, isozyme, microbial inhibitors, lipid droplet accumulation, pyripyropene, beauveriolide, atherosclerosis Introduction Acyl-CoA : cholesterol acyltransferase (ACAT), an ER membrane protein, is responsible for many functions in the body. ACAT has been recognized as a target for inhibition by a new type of antiatherosclerotic agents [1]. Many pharmaceutical laboratories have developed synthetic ACAT inhibitors. However, almost none of them could be successfully developed because of side effects or low in vivo efficacy [2]. Recent molecular biological studies revealed the existence in mammals of two different ACAT isozymes, ACAT1 and ACAT2 [3ϳ6]. ACAT1 is ubiquitously expressed in tissues and cells, while ACAT2 is expressed predominantly in the liver (hepatocytes) and intestine [7]. Therefore, it is important to determine the selectivity of inhibitors toward the two ACAT isozymes for their development as new antiatherosclerotic agents [8]. However, such data have rarely been reported so far [9,10].Our research group discovered a number of microbial ACAT inhibitors with an enzyme assay using rat liver microsomes; these inhibitors included pyripyropenes [11ϳ13], purpactins [14,15], glisoprenins [16ϳ18] and terpendoles [19ϳ21] (Fig. 1). The activities of these inhibitors in cell-based assays and their selectivity toward the two ACAT isozymes have not been fully studied. We also developed a cell-based assay of lipid droplet accumulation in mouse macrophages, and discovered beauveriolides [22ϳ25], phenochalasins [26,27], spylidone [28], sespendole [29] and K97-0239s [30] with this assay (Fig. 2). We studied the molecular targets of beauveriolides I and III in macrophages and found that they inhibited cholesteryl ester (CE) synthesis by blocking ACAT activity in macrophages, leading to the inhibition of lipid droplet accumulation. More importantly, they proved orally active in atherogenic mouse models and are expected to provide [31,32]. However, the molecular targets of the other inhibitors are not yet well defined, and their effect on ACAT activity as a potential target should be studied.Thus, we studied microbial inhibitors discovered by screening against ACAT ( Fig. 1) and lipid droplet accumulation ( Fig. 2) by examining their inhibitory activity against ACAT1 and ACAT2 isozymes, and their selectivity toward the isozymes. For the examination, two cell lines, CHO cells expressing African Green monkey ACAT1 (ACAT1-CHO) and ACAT2 (ACAT2-CHO) were used [9]. was purchased from Per...

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Objective-Pyripyropene A (PPPA) of fungal origin is the first compound that has been found to strongly and selectively inhibit acyl-coenzyme A:cholesterol acyltransferase 2 (ACAT2) isozyme activity in vitro. The purpose of the present study was to investigate in vivo efficacy of the ACAT2-selective inhibitor in atherosclerosis. Methods and Results-PPPA treatment (10 to 100 mg/kg) caused 30.5Ϯ4.7% to 55.8Ϯ3.3% inhibition of the cholesterol absorption from the mouse intestine. When PPPA (10 to 50 mg/kg per day) was orally administered to apolipoprotein E-knockout mice for 12 weeks, the levels of plasma cholesterol, very-low-density lipoprotein (VLDL), and low-density lipoprotein (LDL) and hepatic cholesterol content were lowered. Furthermore, the ratio of cholesteryl oleate (exclusively synthesized in hepatic ACAT2) to cholesteryl linoleate in VLDL-and LDL-derived cholesteryl ester decreased, indicating that hepatic ACAT2 activity was inhibited by PPPA. PPPA-treated mice had reduced atherogenic lesion areas that were lowered by 26.2Ϯ3.7% to 46Ϯ3.8% in the aortae and by 18.9Ϯ3.6% to 37.6Ϯ6.0% in the hearts. Conclusion-Our findings indicate that ACAT2-selective inhibition in the intestine and the liver can be effective against atherosclerosis and that PPPA appears to be a potential antiatherogenic lead compound. This study is the first demonstration of the in vivo efficacy of PPPA, an ACAT2-selective inhibitor, in atherosclerosis. PPPA-treated atherogenic mice showed a decrease in intestinal cholesterol absorption and cholesterol and cholesteryl oleate levels in both LDL and VLDL, resulting in protection of atherosclerosis development. Key Words: atherosclerosis Ⅲ hypercholesterolemia Ⅲ inhibitors C ardiovascular diseases are a group of disorders of the heart and blood vessels. They represent the leading cause of death globally, with more individuals dying each year from cardiovascular diseases than from any other cause. Statins, which are inhibitors of 3-hydroxy-3-methylglutarylcoenzyme A reductase, reduce the risk of complications and death from cardiovascular events by approximately 30%. 1 Consequently, the quest for novel pharmacological agents that target specific steps of atherogenesis has intensified significantly in recent years. The enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT), which catalyzes the synthesis of cholesteryl ester (CE) from free cholesterol and long-chain fatty acyl-coenzyme A, has been considered a promising therapeutic target. 2 Although a number of synthetic ACAT inhibitors have been developed, they have failed to reduce cardiovascular events in clinical trials.Two ACAT isozymes have been identified in mammals: ACAT1 and ACAT2. [3][4][5][6] Each isozyme has a distinct pattern of expression among tissues. Although ACAT1 is ubiquitously expressed at a high level in sebaceous glands, steroidogenic tissues, and macrophages, ACAT2 is expressed predominantly in the liver and intestine. 7 ACAT1-selective inhibition may cause detrimental effects, 8 -10 whereas ACAT2-selective inhibitio...