Cholesteryl Ester Hydrolase Research Articles

Cytoskeleton disruption in J774 macrophages: Consequences for lipid droplet formation and cholesterol flux

Macrophages store excess unesterified cholesterol (free, FC) in the form of cholesteryl ester (CE) in cytoplasmic lipid droplets. The hydrolysis of droplet-CE in peripheral foam cells is critical to HDL-promoted reverse cholesterol transport because it represents the first step in cellular cholesterol clearance, as only FC is effluxed from cells to HDL. Cytoplasmic lipid droplets move within the cell utilizing the cytoskeletal network, but, little is known about the influence of the cytoskeleton on lipid droplet formation. To understand this role we employed cytochalasin D (cyt.D) to promote actin depolymerization in J774 macrophages. Incubating J774 with acetylated LDL creates foam cells having a 4-fold increase in cellular cholesterol content (30–40% cholesterol present as cholesteryl ester (CE)) in cytoplasmic droplets. Lipid droplets formed in the presence of cyt.D are smaller in diameter. CE-deposition and -hydrolysis are decreased when cells are cholesterol-enriched in the presence of cyt.D or latrunculin A, another cytoskeleton disrupting agent. However, when lipid droplets formed in the presence of cyt.D are isolated and incubated with an exogenous CE hydrolase, the CE is more rapidly metabolized compared to droplets from control cells. This is apparently due to the smaller size and altered lipid composition of the droplets formed in the presence of cyt.D. Cytoskeletal proteins found on CE droplets influence droplet lipid composition and maturation in model foam cells. In J774 macrophages, cytoskeletal proteins are apparently involved in facilitating the interaction of lipid droplets and a cytosolic neutral CE hydrolase and may play a role in foam cell formation. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

Estrogen-dependent activation of neutral cholesterol ester hydrolase underlying gender difference of atherogenesis in apoE−/− mice

ObjectiveMechanisms underlying gender difference of atherogenesis were investigated focusing on direct effects of estrogen on the artery. MethodsFirst, male and female apoE−/− mice were fed an atherogenic diet for 16 weeks from 10 weeks of age. Second, female apoE−/− mice were ovariectomized (ovx) or sham operated at 8 weeks of age, and 2-weeks afterwards, one-third of each ovx-group received conjugated equine estrogens (CEE) (0, 2.5 or 5.0μg/day) for 16 weeks. Atherosclerotic lesions were examined after experimental periods. To clarify anti-atherogenic effect of 17β-estradiol (E2) on artery, neutral cholesteryl ester hydrolase (N-CEase) activity in aorta and peritoneal macrophages, and E2-treated J774A.1 cells were measured. ResultsFirst, atherosclerotic lesion in female mice was significantly less than male mice without any changes in serum lipids and lipoprotein profile. N-CEase activity in aorta and peritoneal macrophages in female mice was significantly higher than male mice. Second, atherosclerotic lesion in ovx-group was significantly greater than sham-group. CEE-replacement to ovx-group decreased atherosclerotic lesion in a dose-dependent manner. N-CEase activity in aorta and peritoneal macrophages was decreased in ovx-group compared to sham-group, and restored by CEE-replacement in macrophages. To study detailed mechanisms, J774A.1 cells were treated with E2. E2 significantly increased N-CEase activity, and cAMP-dependent protein kinase (A-kinase) type II activity and the protein in cytosol fraction without any changes of total protein of A-kinase type II. ConclusionThese results suggest that gender difference of atherogenesis is partly accounted for activation of N-CEase through estrogen-dependent translocation of A-kinase type II in macrophages.

Phospholipase A2 Mediates Apolipoprotein-Independent Uptake of Chylomicron Remnant-Like Particles by Human Macrophages

Apolipoprotein E-receptor-mediated pathways are the main routes by which macrophages take up chylomicron remnants, but uptake may also be mediated by receptor-independent routes. To investigate these mechanisms, triacylglycerol (TG) accumulation induced by apolipoprotein-free chylomicron remnant-like particles (CRLPw/o) in human monocyte-derived macrophages was evaluated. Macrophage TG content increased about 5-fold after incubation with CRLPw/o, and this effect was not reduced by the inhibition of phagocytosis, macropinocytosis, apolipoprotein E function, or proteoglycan bridging. The role of lipases, including lipoprotein lipase, cholesteryl ester hydrolase, and secretory (sPLA2) and cytosolic phospholipase A2, was studied using [3H]TG-labelled CRLPw/o. Total cell radioactivity after incubation with [3H]TG CRLPw/o was reduced by 15–30% by inhibitors of lipoprotein lipase and cholesteryl ester hydrolase and by about 45% by inhibitors of sPLA2 and cytosolic PLA2 . These results suggest that macrophage lipolytic enzymes mediate the internalization of postprandial TG-rich lipoproteins and that sPLA2 and cytosolic PLA2, play a more important role than extracellular lipoprotein lipase-mediated TG hydrolysis.

Atherosclerotic lesion progression is attenuated by reconstitution with bone marrow from macrophage-specific cholesteryl ester hydrolase transgenic mice

Accumulation of cholesteryl ester (CE)-enriched macrophage foam cells is central to the development of atherosclerotic lesions. Intracellular CE hydrolysis is the rate-limiting step in the removal of free cholesterol from macrophage foam cells. Enhancing this process by transgenic overexpression of CE hydrolase (CEH) resulted in a significant decrease in diet-induced atherosclerosis in LDL receptor-deficient (LDLR-/-) mice. However, for development of this step as an antiatherosclerotic target it is imperative to demonstrate that increase in CE hydrolysis after initiation of lesion formation will also attenuate further lesion progression. The objective of the present study was to directly address this issue using an animal model. LDLR-/- mice were fed a high-fat high-cholesterol diet (Western Diet) for 8 wk to initiate lesion formation and were then divided into three groups. Group 1 mice were killed to determine baseline lesion development. Mice in groups 2 and 3 were irradiated and transplanted with either LDLR-/- or LDLR-/-CEH transgenic bone marrow and maintained on Western Diet. Atherosclerotic lesion progression was assessed after 12 wk. While a more than fourfold increase in total lesions (compared to group 1) was seen in group 2 receiving LDLR-/- marrow, a significantly lower increase (<2-fold) was noted in mice reconstituted with CEH transgenic marrow (group 3). Lesions in group 3 mice were also more cellular with smaller necrotic cores. Lesion progression is associated with a switch in macrophage phenotype from anti-inflammatory M2 to proinflammatory M1 phenotype and is consistent with reduced lesion progression. Aortas from group 3 mice contained a significantly higher percentage of macrophages in M2 phenotype (Ly6C(lo)). These data demonstrate for the first time that enhancing macrophage CE hydrolysis even after lesion initiation can still attenuate further lesion progression and also switches the phenotype of lesion-associated macrophages to anti-inflammatory M2 phenotype establishing intracellular CE hydrolysis as an anti-atherosclerotic as well as anti-inflammatory target.

Important Considerations for Evaluating the Data Presented by Igarashi et al

To The Editor: The major conclusion drawn in the article by Igarashi et al,1 that cholesteryl ester hydrolase (gene symbol CES1) does not contribute to neutral cholesterol ester (CE) hydrolase activity, is based on the inability of the authors to detect CE hydrolase activity associated with the cDNA clone that they obtained by PCR amplification and by the inability of the authors to demonstrate change in intracellular CE hydrolytic activity by short hairpin (sh)RNA-mediated knockdown of CES1. The authors have not provided the sequence of the clone obtained and do not unequivocally show its identity to the reported CES1 with established role not only in intracellular CE hydrolysis,2 mobilization,3 and cholesterol efflux4 but also in attenuation of diet-induced atherosclerosis and lesion necrosis.5 It is noteworthy that the sense primer used in this study also shows a 95% homology to human CES4 (17/18 bp match; accession no. NR_003276). Single amino …

Seeking Novel Targets for Improving In Vivo Macrophage-Specific Reverse Cholesterol Transport: Translating Basic Science into New Therapies for the Prevention and Treatment of Atherosclerosis

Epidemiologic studies have demonstrated that increased high-density lipoprotein cholesterol (HDL-C) is a protective factor against cardiovascular disease. However, the beneficial therapeutic effects of raising HDL-C are proving difficult to confirm in humans. Macrophage-specific reverse cholesterol transport (RCT) is thought to be one of the most important HDL-mediated cardioprotective mechanisms. A new approach was developed to measure in vivo RCT from labeled cholesterol macrophages to liver and feces in mice. Since its original publication, this method has been extensively used to assess the effects of genetic manipulation of pivotal genes involved in HDL metabolism on this major HDL antiatherogenic function in mice. These studies indicate that in vivo macrophage-specific RTC is a strong predictor of atherosclerosis susceptibility compared with steady-state plasma HDL-C levels or other global RCT measurements. This review aims to identify the best molecular targets for improving this HDL antiatherogenic function. Strong evidence supports a positive effect of interventions on macrophage adenosine triphosphate-binding cassette transporter (ABC) A1 and neutral cholesteryl ester hydrolase, apolipoprotein (apo) A-I, apoE, liver scavenger receptor class B type I and ABCG5/G8 on in vivo macrophage-specific RCT and atherosclerosis susceptibility. However, other genetic modifications have yielded conflicting results. Several preclinical studies tested the effects on macrophage-specific RCT in vivo of promising new HDL-based therapeutic agents, which include cholesteryl ester transfer protein inhibitors, apoA-I-directed therapies, liver X receptor and peroxisome proliferator-activated receptor agonists, intestinal cholesterol absorption inhibitors, fish oil and phenolic acid intake, inflammatory modulation and non-nucleoside reverse transcriptase inhibitors. This review also discusses recent findings on the potential effects of these therapeutic approaches on macrophage RCT in mice and cardiovascular risk in humans.

Volume-Sensitive Chloride Current in Macrophages is Regulated by High-Cholesterol Diet, Cholesteryl Ester Hydrolase and ROS

Cholesteryl ester (CE) accumulation in macrophages activates proinflammatory mediators and underlies atherosclerotic plaque formation. We tested whether dietary cholesterol regulates volume-sensitive Cl- current (ICl,swell) via reactive oxygen species (ROS) and determined whether depleting CE by macrophage-specific transgenic over-expression of human cholesteryl ester hydrolase (CEHTg) suppresses ICl,swell. ICl,swell was recorded in peritoneal macrophages from LDL receptor-null (Ldlr-/-) and Ldlr-/-CEHTg mice feed chow or a Western-type high-fat, high-cholesterol diet (16 wk). In macrophages from non-fasting Ldlr-/- on chow, ICl,swell at +60 mV was 6.7 ± 1.4 pA/pF under isosmotic control conditions, increased to 112.6 ± 11.4 pA/pF on exposure to 100-μM H2O2 and to 102.9 ± 13.1 pA/pF upon osmotic swelling (0.85T), and was fully blocked by the ICl,swell-inhibitor DCPIB (10 μM). CEHTg expression did not alter ICl,swell under control conditions, but significantly reduced ICl,swell elicited by H2O2 to 33.7 ± 5.0 pA/pF and by 0.85T to 24.7 ± 6.3 pA/pF; ICl,swell remained DCPIB-sensitive. By contrast, ICl,swell already was strongly activated, 113.9 ± 24.1 pA/pF, under isosmotic control conditions in macrophages from Ldlr-/- fed a high-cholesterol diet; H2O2 did not elicit additional current, 126.8 ± 37.7 pA/pF, and DCPIB fully blocked ICl,swell. High-cholesterol-induced ICl,swell was ROS-dependent. Both ebselen (15 μM), a ROS scavenger, and rotenone (10 μM), a mitochondrial e- transport blocker, fully suppressed ICl,swell. Furthermore, CEHTg expression reversed basal ICl,swell activation by high-cholesterol diet. ICl,swell in Ldlr-/-CEHTg macrophages was 2.7 ± 0.8 pA/pF under control conditions, was increased to only 27.4 ± 3.7 and 23.1 ± 3.6 pA/pF by H2O2 and 0.85T, and was fully blocked by DCPIB. These data suggest that macrophage CE regulate both ICl,swell under control conditions and its response to stimulation. Furthermore, mitochondrial ROS are required for cholesterol-dependent ICl,swell activation.

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363−/− but unchanged in HSL−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.

Activity-based Protein Profiling Identifies a Host Enzyme, Carboxylesterase 1, Which Is Differentially Active during Hepatitis C Virus Replication

Hepatitis C virus (HCV) relies on many interactions with host cell proteins for propagation. Successful HCV infection also requires enzymatic activity of host cell enzymes for key post-translational modifications. To identify such enzymes, we have applied activity-based protein profiling to examine the activity of serine hydrolases during HCV replication. Profiling of hydrolases in Huh7 cells replicating HCV identified CES1 (carboxylesterase 1) as a differentially active enzyme. CES1 is an endogenous liver protein involved in processing of triglycerides and cholesterol. We observe that CES1 expression and activity were altered in the presence of HCV. The knockdown of CES1 with siRNA resulted in lower levels of HCV replication, and up-regulation of CES1 was observed to favor HCV propagation, implying an important role for this host cell protein. Experiments in HCV JFH1-infected cells suggest that CES1 facilitates HCV release because less intracellular HCV core protein was observed, whereas HCV titers remained high. CES1 activity was observed to increase the size and density of lipid droplets, which are necessary for the maturation of very low density lipoproteins, one of the likely vehicles for HCV release. In transgenic mice containing human-mouse chimeric livers, HCV infection also correlates with higher levels of endogenous CES1, providing further evidence that CES1 has an important role in HCV propagation.

Cellular cholesterol delivery, intracellular processing and utilization for biosynthesis of steroid hormones

Steroid hormones regulate diverse physiological functions such as reproduction, blood salt balance, maintenance of secondary sexual characteristics, response to stress, neuronal function and various metabolic processes. They are synthesized from cholesterol mainly in the adrenal gland and gonads in response to tissue-specific tropic hormones. These steroidogenic tissues are unique in that they require cholesterol not only for membrane biogenesis, maintenance of membrane fluidity and cell signaling, but also as the starting material for the biosynthesis of steroid hormones. It is not surprising, then, that cells of steroidogenic tissues have evolved with multiple pathways to assure the constant supply of cholesterol needed to maintain optimum steroid synthesis. The cholesterol utilized for steroidogenesis is derived from a combination of sources: 1) de novo synthesis in the endoplasmic reticulum (ER); 2) the mobilization of cholesteryl esters (CEs) stored in lipid droplets through cholesteryl ester hydrolase; 3) plasma lipoprotein-derived CEs obtained by either LDL receptor-mediated endocytic and/or SR-BI-mediated selective uptake; and 4) in some cultured cell systems from plasma membrane-associated free cholesterol. Here, we focus on recent insights into the molecules and cellular processes that mediate the uptake of plasma lipoprotein-derived cholesterol, events connected with the intracellular cholesterol processing and the role of crucial proteins that mediate cholesterol transport to mitochondria for its utilization for steroid hormone production. In particular, we discuss the structure and function of SR-BI, the importance of the selective cholesterol transport pathway in providing cholesterol substrate for steroid biosynthesis and the role of two key proteins, StAR and PBR/TSO in facilitating cholesterol delivery to inner mitochondrial membrane sites, where P450scc (CYP11A) is localized and where the conversion of cholesterol to pregnenolone (the common steroid precursor) takes place.

Improved Insulin Sensitivity in High Fat- and High Cholesterol-fed Ldlr−/− Mice with Macrophage-specific Transgenic Expression of Cholesteryl Ester Hydrolase: ROLE OF MACROPHAGE INFLAMMATION AND INFILTRATION INTO ADIPOSE TISSUE

Cellular cholesterol balance induces changes in the inflammatory status of macrophages, and low grade chronic inflammation is increasingly being recognized as one of the key steps in the development of atherosclerosis as well as insulin resistance. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of intracellular stored cholesteryl esters (CEs) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have earlier reported reduced atherosclerosis and lesion necrosis in macrophage-specific CEH transgenic mice on a Ldlr(-/-) background. In the present study, we tested the hypothesis that reduced intracellular accumulation of CE in macrophages from CEH transgenic mice will attenuate expression of proinflammatory mediators, thereby reducing infiltration into adipose tissue, alleviating inflammation, and resulting in improved insulin sensitivity. Western diet fed Ldlr(-/-)CEH transgenic mice showed improved insulin sensitivity as assessed by glucose and insulin tolerance tests. Macrophages from CEH transgenic mice expressed significantly lower levels of proinflammatory cytokines (interleukin-1beta and interleukin-6) and chemokine (MCP-1; monocyte chemoattractant protein). Attenuation of NF-kappaB- and AP-1-driven gene expression was determined to be the underlying mechanism. Infiltration of macrophages into the adipose tissue that increases inflammation and impairs insulin signaling was also significantly reduced in Ldlr(-/-)CEH transgenic mice. In the OP-9 adipocyte peritoneal macrophage co-culture system, macrophages from CEH transgenic mice had a significantly reduced effect on insulin signaling as measured by Akt phosphorylation compared with nontransgenic macrophages. Taken together, these studies demonstrate that macrophage-specific overexpression of CEH decreases expression of proinflammatory mediators and attenuates macrophage infiltration into the adipose tissue, resulting in decreased circulating cytokines and improved insulin sensitivity.

Insulin Suppresses HDL-Mediated Cholesterol Efflux from Macrophages Through Inhibition of Neutral Cholesteryl Ester Hydrolase and ATP-Binding Cassette Transporter G1 Expressions

We studied the effect of insulin on HDL-mediated cholesterol efflux from macrophages. The potential involvement of cholesteryl ester hydrolysis and membrane cholesterol transport was also addressed. Human monocyte-derived THP-1 cells were developed into macrophages. Cholesterol efflux was measured by incubating macrophages, labeled with [³H]-cholesterol, with HDL for 24 h. The cells were treated with insulin (0-500 nM) for 30 min prior to the addition of HDL. To investigate the molecular mechanisms of the effect of insulin, the expressions of neutral cholesteryl ester hydrolase (nCEH) and ATP-binding cassette transporter (ABC) G1 were analyzed. Insulin inhibited, in a concentration-dependent manner, HDL-mediated cholesterol efflux from macrophages. Insulin also inhibited the enzyme activity of nCEH and its mRNA and protein expression in cells. Insulin also suppressed the expressions of mRNA and protein for ABCG1. Insulin inhibits HDL-mediated cholesterol efflux from macrophages, which may result from the suppression of nCEH and ABCG1 expressions. Our findings show part of the potential molecular mechanism of atherogenesis in type 2 diabetes with hyperinsulinemia.

Role of cholesteryl ester hydrolase in atherosclerosis

Hydrolysis of cellular cholesteryl esters by neutral cholesteryl esters hydrolase (CEH) is the obligatory first step in removal of cholesterol from artery wall-associated macrophage foam cells and is increasingly being recognized as the rate-limiting step in the process of reverse cholesterol transport, by which excess cholesterol is ultimately removed from the body. In this review, we recapitulate the earlier controversies surrounding the identity of neutral CEH in macrophages, characterization of several potential candidates and their role in atherogenesis. Since final elimination of cholesterol occurs either as direct secretion into bile or as bile acids that are removed in the feces and the liver represents the major organ responsible for this final elimination, the role of hepatic CEH in the process of reverse cholesterol transport is also summarized. Neutral CEH is identified as a potential antiatherosclerotic target and future directions to manipulate CEH as a means to attenuate atherosclerosis are discussed.

Subcellular localization of carboxylesterase‐4 in rat liver cells and its role as neutral cholesteryl ester hydrolase

The rat carboxylesterases, ES‐2 (serum esterase), ES‐3 (pI 5.6 esterase), ES‐4 (pI 6.2/6.4 esterase), ES‐10 (pI 6.0/6.1 esterase), and ES‐15 (pI 5.0/5.2 esterase) comprise a multi‐gene family, and function in hydrolysis of a variety of ester‐ and amide‐ containing chemicals to their respective free acids. Using realtime PCR, in the rat hepatic cell line McArdle‐RH7777 (McA) we observed ES‐4 mRNA had the highest level of expression compared to ES‐2 and ES‐3, while ES‐10 was not detectable. The subcellular location of ES‐4 in McA cells was investigated using confocal microscopy. ES4 co‐localized with ER Tracker dye, a maker for the endoplasmic reticulum (ER). Immunohistochemical studies with rat liver demonstrated the presence of ES‐4 within hepatocytes. ES‐4 also functions as a rat hepatic cholesteryl ester hydrolase. McA cells overexpressing ES‐4 showed decreased cholesteryl ester (CE) levels compared to control cells indicating that ES‐4 can hydrolyze CE in intact cells. In cells where ES‐4 levels were suppressed by 75% using siRNA there were increased CE levels. Confocal microscopy, using Oil Red O and ES‐4 antibodies, revealed areas of association of ES‐4 with the CE droplets. The results show ES‐4 is expressed in hepatocytes and specifically in endoplasmic reticulum. ES‐4 is directly associated with CE droplets and functions as cholesteryl ester hydrolase. Supported by NIH‐DK044498.

Identification of Neutral Cholesterol Ester Hydrolase, a Key Enzyme Removing Cholesterol from Macrophages

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.

Abstract 5476: Hepatic Over-Expression of Cholesteryl Ester Hydrolase Enhances Cholesterol Elimination and in vivo Reverse Cholesterol Transport

Neutral Cholesteryl ester hydrolase (CEH) mediated hydrolysis of cellular cholesteryl esters (CE) is required not only to generate free cholesterol for efflux from macrophages but also to release FC from lipoprotein delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH will regulate the hydrolysis of lipoprotein derived CE and enhance reverse cholesterol transport (RCT). Adenoviral mediated CEH over-expression led to a significant increase (&gt;3 fold) in bile acid output (see Figure ). To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol loaded and [ 3 H]-cholesterol labeled J774 macrophages were injected intraperitoneally into mice and appearance of [ 3 H]-cholesterol in gall bladder bile and feces over 48 hours was quantified. Mice over-expressing CEH had significantly higher [ 3 H]-cholesterol in bile and feces and it was associated with bile acids. This CEH-mediated increased movement of [ 3 H]-cholesterol from macrophages to bile and feces was significantly attenuated in SR-BI−/−mice. These studies demonstrate that similar to macrophage CEH that rate limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output. This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia &amp; Washington, DC).

Hepatic overexpression of cholesteryl ester hydrolase enhances cholesterol elimination and in vivo reverse cholesterol transport

Neutral cholesteryl ester hydrolase (CEH)-mediated hydrolysis of cellular cholesteryl esters (CEs) is required not only to generate free cholesterol (FC) for efflux from macrophages but also to release FC from lipoprotein-delivered CE in the liver for bile acid synthesis or direct secretion into the bile. We hypothesized that hepatic expression of CEH would regulate the hydrolysis of lipoprotein-derived CE and enhance reverse cholesterol transport (RCT). Adenoviral-mediated CEH overexpression led to a significant increase in bile acid output. To assess the role of hepatic CEH in promoting flux of cholesterol from macrophages to feces, cholesterol-loaded and [3H]cholesterol-labeled J774 macrophages were injected intraperitoneally into mice and the appearance of [3H]cholesterol in gallbladder bile and feces over 48 h was quantified. Mice overexpressing CEH had significantly higher [3H]cholesterol radiolabel in bile and feces, and it was associated with bile acids. This CEH-mediated increased movement of [3H]cholesterol from macrophages to bile acids and feces was significantly attenuated in SR-BI(-/-) mice. These studies demonstrate that similar to macrophage CEH that rate-limits the first step, hepatic CEH regulates the last step of RCT by promoting the flux of cholesterol entering the liver via SR-BI and increasing hepatic bile acid output.

Inhibition of carboxylesterase 1 is associated with cholesteryl ester retention in human THP-1 monocyte/macrophages

Cholesteryl esters are hydrolyzed by cholesteryl ester hydrolase (CEH) yielding free cholesterol for export from macrophages. Hence, CEH has an important regulatory role in macrophage reverse cholesterol transport (RCT). CEH and human carboxylesterase 1 (CES1) appear to be the same enzyme. CES1 is inhibited by oxons, the bioactive metabolites of organophosphate (OP) pesticides. Here, we show that CES1 protein is robustly expressed in human THP-1 monocytes/macrophages and its biochemical activity inhibited following treatment of cell lysates and intact cells with chlorpyrifos oxon, paraoxon, or methyl paraoxon (with nanomolar IC50 values) or after immunodepletion of CES1 protein. CES1 protein expression in cells is unaffected by a 24-h paraoxon treatment, suggesting that the reduced hydrolytic activity is due to covalent inhibition of CES1 by oxons and not down-regulation of expression. Most significantly, treatment of cholesterol-loaded macrophages with either paraoxon (a non-specific CES inhibitor) or benzil (a specific CES inhibitor) caused enhanced retention of intracellular cholesteryl esters and a “foamy” phenotype, consistent with reduced cholesteryl ester mobilization. Thus, exposure to OP pesticides, which results in the inhibition of CES1, may also inhibit macrophage RCT, an important process in the regression of atherosclerosis.

Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis

Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lep(ob/ob)/HSL(-/-)) showed massive accumulation of CE in the liver compared with Lep(ob/ob)/HSL(+/+) mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL(-/-) mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL(-/-) mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL(-/-) mice showed reduced activity of CE hydrolase and contained more CE than those from HSL(+/+) mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes.

Epoxycholesterol Impairs Cholesteryl Ester Hydrolysis in Macrophage Foam Cells, Resulting in Decreased Cholesterol Efflux

Strategies to inhibit or reverse cholesterol accumulation in macrophages have been shown to be atheroprotective. Notably, the administration of LXR agonists upregulates key players in the reverse cholesterol transport pathway, including the ABCA1 and ABCG1 transporters. However, the effects of natural LXR activators, oxysterols, on lipid-laden macrophages remains elusive. We assessed the ability of 2 oxysterols, 22(R)-hydroxycholesterol (22-OH) and 24(S),25-epoxycholesterol (epoxycholesterol), to promote cholesterol efflux to apoA-I from LDL- and modified LDL-labeled and loaded macrophages and thus rescue the phenotype associated with the accumulation of cellular cholesterol in these cells. In macrophages labeled with LDL-derived cholesterol, epoxycholesterol treatment enhances ABCA1-mediated cholesterol efflux. In contrast, in AcLDL-loaded macrophages, epoxycholesterol treatment decreases cholesterol efflux to apoA-I, despite a dramatic increase in the expression of ABCA1 in response to epoxycholesterol treatment. We show that the decreased efflux is attributable to impaired cholesterol mobilization from lipid droplets, resulting from decreased cholesteryl ester hydrolase activity. Epoxycholesterol impairs cholesteryl ester hydrolysis activity in macrophage foam cells, thus reducing the availability of cholesterol for efflux to cholesterol acceptors.