Macrophage cholesterol homeostasis and metabolic diseases: critical role of cholesteryl ester mobilization

Abstract

Atherogenic dyslipidemia, including low HDL levels, is the major contributor of residual risk of cardiovascular disease that remains even after aggressive statin therapy to reduce LDL-cholesterol. Currently, distinction is not made between HDL-cholesterol and HDL, which is a lipoprotein consisting of several proteins and a core containing cholesteryl esters (CEs). The importance of assessing HDL functionality, specifically its role in facilitating cholesterol efflux from foam cells, is relevant to atherogenesis. Since HDLs can only remove unesterified cholesterol from macrophages while cholesterol is stored as CEs within foam cells, intracellular CE hydrolysis by CE hydrolase is vital. Reduction in macrophage lipid burden not only attenuates atherosclerosis but also reduces inflammation and linked pathologies such as Type 2 diabetes and chronic kidney disease. Targeting reduction in macrophage CE levels and focusing on enhancing cholesterol flux from peripheral tissues to liver for final elimination is proposed.

Figure 1. Current concepts in high-density lipoprotein metabolism.

ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1; ACAT1: Acyl coenzyme A:cholesterol acyltransferase-1; ApoA1: Apolipoprotein A1; CE: Cholesteryl ester; CEH: Cholesteryl ester hydrolase; FC: Free cholesterol; LDLR: LDL receptor; SR-A: Scavenger receptor A; SR-BI: Scavenger receptor BI; TG: Triglyceride.

Figure 2. Essential components of the cell system for the determination of cholesterol efflux potential/capacity of serum or high-density lipoprotein from human subjects.

(A) Since CEH-mediated hydrolysis is the obligatory first and also the rate-limiting step in efflux of unesterified or free cholesterol, the cell system used should overexpress CEH to ensure that efflux of cholesterol is truly dependent on the capacity of the extracellular acceptor (serum or HDL). (B) The cells should express the cholesterol transporters (ABCA1 and ABCG1) relevant to macrophage foam cells. (C) Cholesterol that is effluxed and monitored should arise from a pool of CE, a storage form that is present in macrophage foam cells under physiological conditions. ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding cassette transporter G1; ACAT1: Acyl coenzyme A:cholesterol acyltransferase-1; ApoA1: Apolipoprotein A1; CE: Cholesteryl ester; CEH: Cholesteryl ester hydrolase; FC: Free cholesterol; SR-BI: Scavenger receptor BI.

Figure 3. Decreased ability of serum from patients with established cardiovascular disease to mediate cholesterol efflux from THP1-cholesteryl ester hydrolysis cells.

THP1-CEH cells were differentiated into macrophages by the addition of PMA. Differentiated macrophages were labeled with [³H]-cholesterol (2 μCi/ml) and loaded by incubating with acetylated LDL (50 μg/ml) for 48 h. Following a 24-h equilibration in serum-free medium, cholesterol efflux was initiated by the addition of either 1 or 4% of serum obtained from normal subjects or patients with established cardiovascular disease to the culture medium. Efflux of [³H]-cholesterol in the culture medium was determined at 3 and 6 h. Cells were lysed at the end of 6 h and total incorporation of radioactivity was determined. Percent efflux was calculated (dpm in culture medium/total incorporation × 100) and data (mean ± SD) for 6 h is shown; number of subjects in each group is indicated within the bars. The HDL-cholesterol levels were 62.9 ± 10.3 mg/dl for subjects without disease and 52.3 ± 7.67 mg/dl for subjects with disease. *p < 0.05.

Figure 4. Macrophage cholesterol homeostasis.

The influx pathways include uptake of native LDL either via LDLR or by macropinocytosis; uptake of aggregated LDL by phagocytosis (not shown); and receptor-mediated uptake of mLDL via scavenger receptors CD36 and SR-A. Following degradation in the lysosomes, the released FC is re-esterified by ACAT-1 and stored in the cytoplasm as CEs. For efflux, stored CEs are hydrolyzed by a neutral CEH and the release FC is effluxed to extracellular acceptors ApoA1 (via ABCA1) or to nascent HDL (via ABCG1). SR-BI facilitates bidirectional movement of cholesterol. FC within the macrophage, therefore, has two fates – fate 1 is the re-esterification by ACAT1 and fate 2 is the efflux to extracellular acceptors. ABCG1: ATP-binding cassette transporter G1; ACAT1: Acyl coenzyme A:cholesterol acyltransferase-1; ApoA1: Apolipoprotein A1; CE: Cholesteryl ester; CEH: Cholesteryl ester hydrolase; FC: Free cholesterol; LDLR: LDL receptor; mLDL: modified LDL; SR-A: Scavenger receptor A; SR-BI: Scavenger receptor BI.

Figure 5. Unchanged levels of cholesterol associated with plasma lipoproteins in macrophage-specific cholesteryl ester hydrolase transgenic mice despite a significant (>50%) reduction in atherosclerotic plaque formation.

Plasma was obtained from non-fasted LDLR^−/− (light blue bars) and LDLR^−/−CEHTg (dark blue bars) mice after 16 weeks of Western high-fat high-cholesterol diet feeding. An aliquot of the plasma from each animal was applied to Superose-6 FPLC column and cholesterol content of individual lipoproteins determined by an on-line assay. Data are expressed as mean ± SD, for n = 6. IDL: Intermediate-density lipoprotein; LDLR: LDL receptor.

Figure 6. Decreased expression of cholesteryl ester hydrolase in lipid-laden foam cells associated with human atherosclerotic plaque.

Human carotid artery lesions were stained for CEH (red) and macrophages (CD68, green). The region outlined in yellow shows staining for CEH as well as macrophages, and was not associated with lipid accumulation. The area outlined in white showed minimal staining for CEH, but was stained for macrophages and showed high lipid content. CEH: Cholesteryl ester hydrolase.