Macrophage cholesteryl ester mobilization and atherosclerosis

Abstract

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.

Figure 1. Fundamental Difference between ACAT inhibition and CEH over-expression as strategies for reducing macrophage CE content

Intracellular CE are in a dynamic equilibrium with FC and this equilibrium is maintained by the activities of ACAT1 and CEH. FC released by the action of CEH has two fates - indicated as 1 (re-esterification by ACAT1 and 2 (efflux to extracellular cholesterol acceptors). Under conditions of ACAT1 inhibition (bottom right), there is reduction in cellular CE content. However, since FC can no longer be re-esterified, FC has only one fate (2) – efflux to extracellular acceptors. This leads to an increase in intracellular FC. Accumulation of FC in plasma membrane or in endoplasmic reticulum leads to cellular toxicity. On the other hand, if CEH is over-expressed, the released FC still has the two fates (1 and 2). The cgellular FC remaining after efflux can be re-esterified by functional ACAT1 preventing any increase in cellular FC although reducing the net intracellular CE content.

Figure 2. Over-expression of CEH in cells with stable over-expression of ACAT1 (agmACAT1 cells) reduces cellular CE content without increasing the cellular FC levels

agmACAT1 cells were transiently transfected either with an empty vector pCMV or with CEH expression vector in the presence or absence of ACAT1 inhibitor. Twenty four hours post-transfection, medium was replaced with either serum free medium supplemented with 0.5% BSA (limiting acceptor) or with complete growth medium containing 10% FBS (sufficient acceptor). Total lipids were extracted after additional 48 hours and cellular free (FC) and esterified cholesterol (EC) content determined by GLC. It should be noted that content of FC and EC is higher when 10% FBS is included in the medium due to lipoprotein uptake. An increase in FC is only seen when ACAT1 was inhibited. CEH over-expression led to a slight decrease and not an increase in cellular FC. Cellular EC content was reduced with CEH over-expression and the magnitude of this decrease was greater in ACAT1 inhibited cells. *p<0.05

Figure 3

Intracellular localization of candidate CE hydrolases and their potential association with lipid droplets – the physiological substrate.

Figure 4

Regulation of interfacial catalysis by a lipolytic enzyme.

Figure 5. Activation of CEH promoter activity by LXRα agonist (TO-901317)

COS-7 cells were co-transfected with either Luciferase reporter construct driven tandem LXR response elements (LXRE-Luc) or by 1686 bp long CEH proximal promoter (CEH-1686-Luc) along with an empty vector or LXRα expression vector. Twenty four post-transfection, cells were exposed to LXRα ligand TO-901317. Luciferase activity was measured in cell lysates after an additional 24 hours and normalized to β-galactosidase. Fold increase in promoter activity by ligand dependent activation of LXRα are indicated.