Unstable lipid-rich plaques in atherosclerosis are characterized by the accumulation of macrophage foam cells loaded with cholesterol ester (CE). Although hormone-sensitive lipase and cholesteryl ester hydrolase (CEH) have been proposed to mediate the hydrolysis of CE in macrophages, circumstantial evidence suggests the presence of other enzymes with neutral cholesterol ester hydrolase (nCEH) activity. Here we show that the murine orthologue of KIAA1363, designated as neutral cholesterol ester hydrolase (NCEH), is a microsomal nCEH with high expression in murine and human macrophages. The effect of various concentrations of NaCl on its nCEH activity resembles that on endogenous nCEH activity of macrophages. RNA silencing of NCEH decreases nCEH activity at least by 50%; conversely, its overexpression inhibits the CE formation in macrophages. Immunohistochemistry reveals that NCEH is expressed in macrophage foam cells in atherosclerotic lesions. These data indicate that NCEH is responsible for a major part of nCEH activity in macrophages and may be a potential therapeutic target for the prevention of atherosclerosis.Atherosclerotic cardiovascular diseases are the leading causes of mortality in industrialized countries, despite advances in the management of coronary risk factors. Heart attacks arise from thrombotic occlusion of coronary arteries following the rupture of plaques. Lipid-rich plaques, which are characterized by a plethora of CE 3 -laden macrophage foam cells, are prone to rupture (1). Thus, it is important to clarify the mechanism that eliminates CE from macrophage-derived foam cells. Foam cells are generated by the unlimited uptake of modified lipoproteins through scavenger receptors (2). Cholesterol in the lipoproteins is stored in lipid droplets as CE after re-esterification by acyl-CoA:cholesterol acyltransferase 1 (ACAT1) (3). Hydrolysis of CE is the initial step toward elimination of cholesterol from foam cells (4). Free cholesterol thus generated is re-esterified or is released from the cells primarily through ATP-binding cassette transporters (5). Thus, the balance between synthesis and hydrolysis of CE conceivably governs the level of CE in macrophages.Hydrolysis of CE in macrophages has been known for over 40 years (6). However, its molecular mechanism has yet to be fully understood. Circumstantial evidence suggests that the hydrolysis of CE in foam cell macrophages is mediated by hormonesensitive lipase (HSL), a multifunctional enzyme that catalyzes the hydrolysis of triacylglyerol (TG), diacylglycerol, CE, and retinyl ester in various organs such as adipose tissue, muscle, and testis (7,8). This belief is supported by the following facts. First, it has been demonstrated that various lines of macrophages express HSL (9 -12). Second, HSL expression is regulated coordinately with nCEH activity in murine macrophages (13). Third, we (14) and others (15) demonstrated that overexpression of HSL is associated with increased hydrolysis of CE stores in THP-1 and RAW264.7 macrophages.However, recent ...

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.

Rationale: Hydrolysis of intracellular cholesterol ester (CE) is the key step in the reverse cholesterol transport in macrophage foam cells. We have recently shown that neutral cholesterol ester hydrolase (Nceh)1 and hormone-sensitive lipase (Lipe) are key regulators of this process in mouse macrophages. However, it remains unknown which enzyme is critical in human macrophages and atherosclerosis.Objective: We aimed to identify the enzyme responsible for the CE hydrolysis in human macrophages and to determine its expression in human atherosclerosis. Methods and Results:We compared the expression of NCEH1, LIPE, and cholesterol ester hydrolase (CES1) in human monocyte-derived macrophages (HMMs) and examined the effects of inhibition or overexpression of each enzyme in the cholesterol trafficking. The pattern of expression of NCEH1 was similar to that of neutral CE hydrolase activity during the differentiation of HMMs. Overexpression of human NCEH1 increased the hydrolysis of CE, thereby stimulating cholesterol mobilization from THP-1 macrophages. Knockdown of NCEH1 specifically reduced the neutral CE hydrolase activity. Pharmacological inhibition of NCEH1 also increased the cellular CE in HMMs. In contrast, LIPE was barely detectable in HMMs, and its inhibition did not decrease neutral CE hydrolase activity. Neither overexpression nor knockdown of CES1 affected the neutral CE hydrolase activity. NCEH1 was expressed in CD68-positive macrophage foam cells of human atherosclerotic lesions. A therosclerotic cardiovascular diseases are the leading cause of mortality in industrialized countries, despite advances in the management of coronary risk factors. Heart attacks arise from the thrombotic occlusion of coronary arteries following the rupture of plaques. Lipid-rich plaques, which are characterized by a plethora of cholesterol ester (CE)-laden macrophage foam cells, are prone to rupture. 1 Esterification of cholesterol in macrophages is mediated by acyl-coenzyme A cholesterol acyltransferase 1 or sterol Oacyltransferase 1 (SOAT1). 2 Conflicting results have been reported as to the effects of genetic ablation of SOAT1 on atherosclerosis in mice. 3,4 Furthermore, it has not been successful to demonstrate the efficacy of nonselective inhibitors of SOAT to clinically prevent the atherosclerosis in humans. 5,6 On the other hand, the hydrolysis of intracellular CE is the initial step of reverse cholesterol transport. 7 As the hydrolysis of CE preceding reverse cholesterol transport takes place at neutral pH, the enzymes catalyzing it have been collectively called neutral CE hydrolases. Because this step is rate-limiting, particularly in macrophage foam cells, 8,9 it is important to clarify the mechanisms that mediate the hydrolysis of CE in foam cells. Conclusions: NCEH1 is expressed in human atheromatousTo date, 3 enzymes have been proposed to serve as neutral CE hydrolases in macrophages: hormone-sensitive lipase (LIPE) 10 ; cholesteryl ester hydrolase (CEH), 11 which is identical to human liver carboxylesterase 1 ...