Jinghua Bie

Macrophage cholesteryl ester mobilization and atherosclerosis

Accumulation of cholesteryl esters (CE) stored as cytoplasmic lipid droplets is the main characteristic of macrophage foam cells that are central to the development of atherosclerotic plaques. Since only unesterified or free cholesterol (FC) can be effluxed from the cells to extracellular cholesterol acceptors, hydrolysis of CE is the obligatory first step in CE mobilization from macrophages. This reaction, catalyzed by neutral cholesteryl ester hydrolase (CEH), is increasingly being recognized as the rate-limiting step in FC efflux. CEH, therefore, regulates the process of reverse cholesterol transport and ultimate elimination of cholesterol from the body. In this review, we summarize the earlier controversies surrounding the identity of CEH in macrophages, discuss the characteristics of the various candidates recognized to date and examine their role in mobilizing cellular CE and thus regulating atherogenesis. In addition, physiological requirements to hydrolyze lipid droplet-associated substrate and complexities of interfacial catalysis are also discussed to emphasize the importance of evaluating the biochemical characteristics of candidate enzymes that may be targeted in the future to attenuate atherosclerosis.

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-1β and interleukin-6) and chemokine (MCP-1; monocyte chemoattractant protein). Attenuation of NF-κB- 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.

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.

Liver-specific Cholesteryl Ester Hydrolase deficiency attenuates sterol elimination in the feces and increases Atherosclerosis in Ldlr-/- Mice

Objective—Liver is the major organ responsible for the final elimination of cholesterol from the body either as biliary cholesterol or as bile acids. Intracellular hydrolysis of lipoprotein-derived cholesteryl esters (CEs) is essential to generate the free cholesterol required for this process. Earlier, we demonstrated that overexpression of human CE hydrolase (Gene symbol CES1) increased bile acid synthesis in human hepatocytes and enhanced reverse cholesterol transport in mice. The objective of the present study was to demonstrate that liver-specific deletion of its murine ortholog, Ces3, would decrease cholesterol elimination from the body and increase atherosclerosis. Approach and Results—Liver-specific Ces3 knockout mice (Ces3-LKO) were generated, and Ces3 deficiency did not affect the expression of genes involved in cholesterol homeostasis and free cholesterol or bile acid transport. The effects of Ces3 deficiency on the development of Western diet–induced atherosclerosis were examined in low density lipoprotein receptor knock out−/− mice. Despite similar plasma lipoprotein profiles, there was increased lesion development in low density lipoprotein receptor knock out−/−Ces3-LKO mice along with a significant decrease in the bile acid content of bile. Ces3 deficiency significantly reduced the flux of cholesterol from [3H]-CE–labeled high-density lipoproteins to feces (as free cholesterol and bile acids) and decreased total fecal sterol elimination. Conclusions—Our results demonstrate that hepatic Ces3 modulates the hydrolysis of lipoprotein-delivered CEs and thereby regulates free cholesterol and bile acid secretion into the feces. Therefore, its deficiency results in reduced cholesterol elimination from the body, leading to significant increase in atherosclerosis. Collectively, these data establish the antiatherogenic role of hepatic CE hydrolysis.

Identification of a novel intracellular cholesteryl ester hydrolase (carboxylesterase 3) in human macrophages: compensatory increase in its expression after carboxylesterase 1 silencing

Cholesteryl ester (CE) hydrolysis is the rate-limiting step in the removal of free cholesterol (FC) from macrophage foam cells, and several enzymes have been identified as intracellular CE hydrolases in human macrophages. We have previously reported the antiatherogenic role of a carboxylesterase [carboxylesterase 1 (CES1)], and the objective of the present study was to determine the contribution of CES1 to total CE hydrolytic activity in human macrophages. Two approaches, namely, immune depletion and short hairpin (sh)RNA-mediated knockdown, were used. Immuneprecipitation by a CES1-specific antibody resulted in a 70-80% decrease in enzyme activity, indicating that CES1 is responsible for >70% of the total CE hydrolytic activity. THP1-shRNA cells were generated by stably transfecting human THP1 cells with four different CES1-specific shRNA vectors. Despite a significant (>90%) reduction in CES1 expression both at the mRNA and protein levels, CES1 knockdown neither decreased intracellular CE hydrolysis nor decreased FC efflux. Examination of the underlying mechanisms for the observed lack of effects of CES1 knockdown revealed a compensatory increase in the expression of a novel CES, CES3, which is only expressed at <30% of the level of CES1 in human macrophages. Transient overexpression of CES3 led to an increase in CE hydrolytic activity, mobilization of intracellular lipid droplets, and a reduction in cellular CE content, establishing CES3 as a bona fide CE hydrolase. This study provides the first evidence of functional compensation whereby increased expression of CES3 restores intracellular CE hydrolytic activity and FC efflux in CES1-deficient cells. Furthermore, these data support the concept that intracellular CE hydrolysis is a multienzyme process.

Liver-specific transgenic expression of cholesteryl ester hydrolase reduces atherosclerosis in Ldlr−/− mice

The liver plays a central role in the final elimination of cholesterol from the body either as bile acids or as free cholesterol (FC), and lipoprotein-derived cholesterol is the major source of total biliary cholesterol. HDL is the major lipoprotein responsible for removal and transport of cholesterol, mainly as cholesteryl esters (CEs), from the peripheral tissues to the liver. While HDL-FC is rapidly secreted into bile, the fate of HDL-CE remains unclear. We have earlier demonstrated the role of human CE hydrolase (CEH, CES1) in hepatic hydrolysis of HDL-CE and increasing bile acid synthesis, a process dependent on scavenger receptor BI expression. In the present study, we examined the hypothesis that by enhancing the elimination of HDL-CE into bile/feces, liver-specific transgenic expression of CEH will be anti-atherogenic. Increased CEH expression in the liver significantly increased the flux of HDL-CE to bile acids. In the LDLR−/− background, this enhanced elimination of cholesterol led to attenuation of diet-induced atherosclerosis with a consistent increase in fecal sterol secretion primarily as bile acids. Taken together with the observed reduction in atherosclerosis by increasing macrophage CEH-mediated cholesterol efflux, these studies establish CEH as an important regulator in enhancing cholesterol elimination and also as an anti-atherogenic target.

Macrophage-specific transgenic expression of cholesteryl ester hydrolase attenuates hepatic lipid accumulation and also improves glucose tolerance in ob/ob mice

Cellular cholesterol homeostasis is increasingly being recognized as an important determinant of the inflammatory status of macrophages, and a decrease in cellular cholesterol levels polarizes macrophages toward an anti-inflammatory or M2 phenotype. Cholesteryl ester hydrolase (CEH) catalyzes the hydrolysis of stored intracellular cholesteryl esters (CE) and thereby enhances free cholesterol efflux and reduces cellular CE content. We have reported earlier reduced atherosclerosis as well as lesion necrosis and improved insulin sensitivity (due to decreased adipose tissue inflammation) in macrophage-specific CEH transgenic (CEHTg) mice in the LDLR(-/-) background. In the present study, we examined the effects of reduced intracellular accumulation of CE in CEHTg macrophages in an established diabetic mouse model, namely the leptin-deficient ob/ob mouse. Macrophage-specific transgenic expression of CEH improved glucose tolerance in ob/ob-CEHTg mice significantly compared with ob/ob nontransgenic littermates, but with no apparent change in macrophage infiltration into the adipose tissue. However, there was a significant decrease in hepatic lipid accumulation in ob/ob-CEHTg mice. Consistently, decreased [(14)C]acetate incorporation into total lipids and triglycerides was noted in precision-cut liver slices from ob/ob-CEHTg mice. In the primary hepatocyte-macrophage coculture system, macrophages from CEHTg mice significantly reduced the incorporation of [(14)C]acetate into triglycerides in hepatocytes, indicating a direct effect of macrophages on hepatocyte triglyceride biosynthesis. Kupffer cells isolated from ob/ob-CEHTg mice were polarized toward an anti-inflammatory M2 (Ly6C(lo)) phenotype. Taken together, these studies demonstrate that transgenic overexpression of CEH in macrophages polarizes hepatic macrophages (Kupffer cells) to an anti-inflammatory M2 phenotype that attenuates hepatic lipid synthesis and accumulation.

Cholesterol removal from plaques and elimination from the body: change in paradigm to reduce risk for heart disease

Abstract Accumulation of lipid-laden macrophage foam cells in arterial wall is the hallmark of atherosclerosis, the underlying cause of cardiovascular disease (CVD). Increased uptake of cholesteryl ester-rich modified low-density lipoprotein (LDL) is thought to be responsible for lipid accumulation and strong inverse correlation exists between plasma LDL and CVD. However, despite reaching the target LDL levels significant residual risk for CVD remains. Furthermore, current therapeutic strategies do not lead to regression of existing plaques. This review discusses a change in paradigm emphasizing the importance of removal of cholesterol from macrophage foam cells and final elimination of cholesterol from the body. Intracellular processes involved in this process are described to provide insight into the development of new therapies for CVD.

Role of cholesteryl ester hydrolase in atherosclerosis

Abstract 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.

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.