Cholesterol Ester Accumulation In Macrophages Research Articles

Fat mass and obesity-associated protein attenuates lipid accumulation in macrophage foam cells and alleviates atherosclerosis in apolipoprotein E-deficient mice.

Genome-wide association studies have linked variants of fat mass and obesity-associated protein (FTO) to obesity. However, the molecular function of FTO remains unclear. In this study, we sought to investigate the potential effects of FTO in modulating cholesterol deposition in macrophage foam cells, as well as whether FTO exerts antiatherosclerotic properties in apolipoprotein E-deficient mice. We transfected RAW264.7 cells with plasmids encoding a wild-type or mutant FTO gene (I367F). The upregulation of FTO markedly attenuated cholesterol ester accumulation in macrophages loaded with oxidized LDL, whereas the downregulation of FTO reversed this effect. Moreover, FTO attenuated the mRNA and protein expression of a scavenger receptor, CD36, which was accompanied by the decline of peroxisome proliferator-activated receptor γ protein. In addition, FTO enhanced the phosphorylation of AMP-activated protein kinase (AMPK)α and acetyl-CoA carboxylase, which was effectively suppressed by FTO small interfering RNA. Pretreatment with compound C or transfection with a dominant-negative AMPKα blocked the FTO-mediated upregulation of ATP-binding cassette transporter A1 and ATP-binding cassette transporter G1. Furthermore, FTO suppressed IL-1β secretion independent of the activation of the nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 inflammasome. In-vivo experiments were performed using apolipoprotein E-deficient mice that were infected with adeno-associated virus serotype 9-derived vectors encoding a wild-type or mutant FTO gene. FTO overexpression prevented the formation of atherosclerotic plaques and markedly reduced the content of plasma total cholesterol and LDL cholesterol. Notably, the antiatherosclerotic properties of FTO were observed only in male mice. We propose that the FTO-dependent control of cholesterol deposition may provide avenues for the treatment of atherosclerosis.

Inhibitors for cholesterol ester accumulation in macrophages from Chinese cabbage.

The cholesterol ester accumulates in macrophages in the early stage of atherosclerotic lesions, leading to the formation of foam cells. We examined the inhibitory effects of the crude extracts of 22 edible plants on foam cell formation and isolated nine chlorophyll derivatives as potent inhibitors from Chinese cabbage. The results of the present study suggest that the chlorophyll derivatives contained in edible plants may be useful for the prevention and treatment of atherosclerosis.

Role of renin-angiotensin system in activation of macrophages by modified lipoproteins

Angiotensin II favors the development of atherosclerosis. Our goal was to determine if foam cell formation increases angiotensin II generation by the endogenous renin-angiotensin system (RAS) and if endogenously produced angiotensin II promotes lipid accumulation in macrophages. Differentiated THP-1 cells were treated with acetylated low-density lipoproteins (ac-LDL), native LDL (n-LDL), or no LDL. Expression of RAS genes was assessed and angiotensin I/II levels were quantified in media and cell lysate. Ac-LDL increased angiotensin I/II levels and the angiotensin II/I ratio in cells and media after foam cell formation. Renin mRNA or activity did not change, but renin blockade completely inhibited the increase in angiotensin II. Angiotensinogen mRNA but not protein level was increased. Angiotensin-converting enzyme (ACE) and cathepsin G mRNA and activities were enhanced by ac-LDL. Inhibition of renin, ACE, or the angiotensin II receptor 1 (AT1-receptor) largely abolished cholesteryl ester formation in cells exposed to ac-LDL and decreased scavenger receptor A (SR-A) and acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT-1) protein levels. Inhibition of renin or the AT1-receptor in cells treated with oxidized LDL also decreased SR-A and ACAT-1 protein and foam cell formation. ac-LDL also increased angiotensin II by human peripheral blood monocyte-derived macrophages, whereas blockade of renin decreased cholesterol ester formation in these macrophages. These findings indicate that, during foam cell formation, angiotensin II generation by the endogenous RAS is stimulated and that endogenously generated angiotensin II is crucial for cholesterol ester accumulation in macrophages exposed to modified LDL.

P38 Mitogen-activated Protein Kinase (MAPK) Promotes Cholesterol Ester Accumulation in Macrophages through Inhibition of Macroautophagy

p38 MAPK has been strongly implicated in the development of atherosclerosis, but its role in cholesterol ester accumulation in macrophages and formation of foam cells, an early step in the development of atherosclerosis, has not been investigated. We addressed this issue and made some brand new observations. First, elevated intracellular cholesterol level induced by the exposure to LDL-activated p38 MAPK and activation of p38 MAPK with anisomycin increased the ratio of cholesterol esters over free cholesterol, whereas inhibition of p38 MAPK with SB203580 or siRNA reduced the LDL loading-induced intracellular accumulation of free cholesterol and cholesterol esters in macrophages. Second, exposure to LDL cholesterol inhibited autophagy in macrophages, and inhibition of autophagy with 3-methyladenine increased intracellular accumulation of cholesterol (free cholesterol and cholesterol esters), whereas activation of autophagy with rapamycin decreased intracellular accumulation of free cholesterol and cholesterol esters induced by the exposure to LDL cholesterol. Third, LDL cholesterol loading-induced inhibition of autophagy was prevented by blockade of p38 MAPK with SB203580 or siRNA. Neutral cholesterol ester hydrolase was co-localized with autophagosomes. Finally, LDL cholesterol loading and p38 activation suppressed expression of the key autophagy gene, ulk1, in macrophages. Together, our results provide brand new insight about cholesterol ester accumulation in macrophages and foam cell formation.

P38 Mitogen-activated Protein Kinase (MAPK) Promotes Cholesterol Ester Accumulation in Macrophages through Inhibition of Macroautophagy

p38 Mitogen-activated Protein Kinase (MAPK) Promotes Cholesterol Ester Accumulation in Macrophages through Inhibition of Macroautophagy

Uptake of dexamethasone incorporated into liposomes by macrophages and foam cells and its inhibitory effect on cellular cholesterol ester accumulation

To confirm the efficacy of dexamethasone incorporated into liposomes in the treatment of atherosclerosis, the uptake of dexamethasone-liposomes by macrophages and foam cells and its inhibitory effect on cellular cholesterol ester accumulation in these cells were investigated in-vitro. Dexamethasone-liposomes were prepared with egg yolk phosphatidylcholine, cholesterol and dicetylphosphate in a lipid molar ratio of 7/2/1 by the hydration method. This was adjusted to three different particle sizes to clarify the influence of particle size on the uptake by the macrophages and foam cells, and the inhibitory effect on cellular cholesterol ester accumulation. The distribution of particle sizes of dexamethasone-liposomes were 518.7+/-49.5 nm (L500), 202.2+/-23.1 nm (L200), and 68.6+/-6.5 nm (L70), respectively. For each size, dexamethasone concentration and dexamethasone/lipid molar ratio in dexamethasone-liposome suspension were 1 mg dexamethasone mL-1 and 0.134 mol dexamethasone mol-1 total lipids, respectively. The zeta potential was approximately -70 mV for all sizes. Dexamethasone-liposomes or free dexamethasone were added to the macrophages in the presence of oxidized low density lipoprotein (oxLDL) and foam cells, and then incubated at 37 degrees C. The uptake amount of dexamethasone by the macrophages and foam cells after a 24-h incubation was L500>L200>free dexamethasone>L70. The macrophages in the presence of oxLDL and foam cells were incubated with dexamethasone-liposomes or free dexamethasone for 24 h at 37 degrees C to evaluate the inhibitory effect on the cellular cholesterol ester accumulation. The cellular cholesterol ester level in the macrophages treated with oxLDL was significantly increased compared with that in macrophages without additives. L500, L200 and free dexamethasone significantly inhibited this cholesterol ester accumulation. L500, L200 and free dexamethasone also significantly reduced cellular cholesterol ester accumulation in foam cells. In addition, the relationship between the area under the uptake amount of dexamethasone-time curve (AUC) and the inhibition rate of cholesterol ester accumulation in macrophages and foam cells was evaluated. The inhibition rate of cholesterol ester accumulation (%) was related to the AUC in both types of cell. These results suggested that dexamethasone-liposomes would be a useful approach to the development of a novel drug delivery system for atherosclerotic therapy. Furthermore, the prediction of the inhibitory effect of dexamethasone on cellular cholesterol ester accumulation may become possible by using the results of this study.

Circulating monocytes and atherogenesis: From animal experiments to human studies

Despite significant improvements in the prevention and management of atherosclerosis, its consequences still remain a major cause of disability and mortality. Better understanding of pathophysiological processes implicated in the initial stages of atherogenesis is critical for the discovery of effective approaches to prevent ischaemic stage of the disease and to reduce huge economic and social burden currently associated with atherosclerosis (1). According to the current paradigm the atherosclerotic process is primarily initiated in the vascular wall itself where monocyte-derived macrophages transform to ‘foam’ cells by accumulating lipids (2). Consequent apoptosis or death of macrophages is largely responsible for necrotic core generation, progressive accumulation of free cholesterol and expansion of the necrotic core within plaques. Often, unbalanced generation of inflammatory cytokines by plaque monocyte-derived macrophages (e.g. via up-regulation of Toll-like receptors) as well as angiogenic promoting pathological plaque neovascularisation, and enzymes degrading extracellular matrix results in destabilisation of atherosclerotic plaques (3–5). Whilst a critical role of vascular wall monocyte-derived macrophages in atherosclerotic plaque formation is widely recognised, the impact of circulating monocytes on atherogenesis is less established. For example, accumulation of modified (mainly oxidised) lipids via scavenger receptors was, until recently, exclusively attributed to vascular wall resident macrophages rather than circulating monocytes (6). Oxidised low-density lipoproteins (LDL) taken up via scavenger receptors (e.g. CD36 and CD204) are delivered to lysosomes, where they undergo esterification by cholesterol esterase and are largely converted into free cholesterol and fatty acids (6). It has been reported that CD36 contributes 60–70% of cholesterol ester accumulation in macrophages exposed to oxidised LDL (7). Deletion of the genes encoding CD36 and CD204 retards the development of atherosclerotic lesions in animal models of atherosclerosis (6). However, circulating monocytes are equipped with these same scavenger receptors and may thus start accumulating lipids even prior to their migration to tissues and differentiation to macrophages (8). Exposure to modified LDL induces rapid ‘foam’ cell formation from freshly isolated peripheral blood monocytes, accompanied by upregulation of CD204 and CD36 (8). The clinical relevance of these findings is supported by the observation that an absence of CD36 on circulating monocytes in individuals from a Japanese population results in 40–50% reduction in oxidised LDL binding to monocyte-derived-macrophages compared to normal subjects (9). Certainly, monocyte counts are increased in patients with stenotic coronary artery disease and the Intermountain Heart Collaborative Study Group recently reported a population of 3,227 patients with or without coronary artery disease whereby high monocyte counts were independent predictors of future myocardial infarction and death (10). However, the impact of monocytes in atherogenesis may go far beyond direct lipid accumulation as they are also actively involved in inflammatory responses attributable to their principal function within the innate immune system. Indeed, Correspondence to: Dr. Eduard Shantsila University of Birmingham Centre for Cardiovascular Sciences, City Hospital Birmingham, B18 7QH, UK Tel: +44 121 554 3801, Fax: +44 121 554 4083 E-mail: e.shantsila@bham.ac.uk

Modification of LDL with oxidized 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphorylcholine (oxPAPC) results in a novel form of minimally modified LDL that modulates gene expression in macrophages

Oxidized phospholipids (oxPL) have been found in atherosclerotic plaques and have been associated with the development of atherosclerosis. To investigate the LDL modifying effects of oxPL and subsequent consequences for macrophages, murine bone marrow macrophages were treated with LDL modified with the oxidation products of 1-palmitoyl-2-arachidonoyl- sn-glycero-3-phosphorylcholine, oxPAPC. Effects of oxPAPC-modified LDL (oxPAPC-LDL) on macrophages were compared to native LDL, copper oxidized LDL (Cu-oxLDL) and acetylated LDL (acLDL). LDL treatment with oxPAPC or CuSO 4 induced active oxidation of the LDL particles, resulting in significant increase in F2-isoprostanes, typical for LDL oxidation, compared with native LDL, acLDL or PAPC-LDL. Uptake of oxPAPC-LDL in macrophages was mediated by CD36 pathways as shown by SR-A and CD36 inhibitors. Surprisingly, both Cu-oxLDL and acLDL induced cholesterol ester accumulation in macrophages while oxPAPC-LDL did not. Microarray analysis showed a pronounced similarity between Cu-oxLDL and oxPAPC-LDL gene expression induction, whereas acLDL and oxPAPC-LDL did not overlap. The main feature shared by oxPAPC-LDL and Cu-oxLDL was the induction of the glutathione dependent antioxidant response, indicating an important role in protecting against both types of modified LDL induced stress. Our experiments show that oxPL modifies LDL, resulting in a minimally oxidized particle that shares many features of classically copper oxidized LDL and that may have relevant in vivo properties.

Group V Secretory Phospholipase A2-modified Low Density Lipoprotein Promotes Foam Cell Formation by a SR-A- and CD36-independent Process That Involves Cellular Proteoglycans

Accumulating evidence indicates that secretory phospholipase A2 (sPLA2) enzymes promote atherogenic processes. We have previously showed the presence of Group V sPLA2 (GV sPLA2) in human and mouse atherosclerotic lesions, its hydrolysis of low density lipoprotein (LDL) particles, and the ability of GV sPLA2-modified LDL (GV-LDL) to induce macrophage foam cell formation in vitro. The goal of this study was to investigate the mechanisms involved in macrophage uptake of GV-LDL. Peritoneal macrophages from C57BL/6 mice (wild type (WT)), C57BL/6 mice deficient in LDL receptor (LDLR-/-), or SR-A and CD36 (DKO) were treated with control LDL, GV-LDL, oxidized LDL (ox-LDL) or LDL aggregated by vortexing (vx-LDL). As expected, ox-LDL induced significantly more cholesterol ester accumulation in WT and LDLR-/- compared with DKO macrophages. In contrast, there was no difference in the accumulation of GV-LDL or vx-LDL in the three cell types. 125I-ox-LDL exhibited high affinity, saturable binding to WT cells that was significantly reduced in DKO cells. Vx-LDL and GV-LDL showed low affinity, non-saturable binding that was similar for both cell types, and significantly higher compared with control LDL. GV-LDL degradation in WT and DKO cells was similar. Analyses by confocal microscopy indicated a distinct intracellular distribution of Alexa-568-labeled GV-LDL and Alexa-488-labeled ox-LDL. Uptake of GV-LDL (but not ox-LDL or vx-LDL) was significantly reduced in cells preincubated with heparin or NaClO3, suggesting a role for proteoglycans in GV-LDL uptake. Our data point to a physiological modification of LDL that has the potential to promote macrophage foam cell formation independent of scavenger receptors.

Loss of receptor-mediated lipid uptake via scavenger receptor A or CD36 pathways does not ameliorate atherosclerosis in hyperlipidemic mice

Macrophage internalization of modified lipoproteins is thought to play a critical role in the initiation of atherogenesis. Two scavenger receptors, scavenger receptor A (SR-A) and CD36, have been centrally implicated in this lipid uptake process. Previous studies showed that these receptors mediated the majority of cholesterol ester accumulation in macrophages exposed to oxidized LDL and that mice with deletions of either receptor exhibited marked reductions in atherosclerosis. This work has contributed to an atherosclerosis paradigm: scavenger receptor-mediated oxidized lipoprotein uptake is required for foam cell formation and atherogenesis. In this study, Apoe-/- mice lacking SR-A or CD36, backcrossed into the C57BL/6 strain for 7 generations, were fed an atherogenic diet for 8 weeks. Hyperlipidemic Cd36-/-Apoe-/- and Msr1-/-Apoe-/- mice showed significant reductions in peritoneal macrophage lipid accumulation in vivo; however, in contrast with previous reports, this was associated with increased aortic sinus lesion areas. Characterization of aortic sinus lesions by electron microscopy and immunohistochemistry showed abundant macrophage foam cells, indicating that lipid uptake by intimal macrophages occurs in the absence of CD36 or SR-A. These data show that alternative lipid uptake mechanisms may contribute to macrophage cholesterol ester accumulation in vivo and suggest that the roles of SR-A and CD36 as proatherosclerotic mediators of modified LDL uptake in vivo need to be reassessed.

Specific Binding of ApoA-I, Enhanced Cholesterol Efflux, and Altered Plasma Membrane Morphology in Cells Expressing ABC1

Mutations of the ABC1 transporter have been identified as the defect in Tangier disease, characterized by low HDL and cholesterol ester accumulation in macrophages. A full-length mouse ABC1 cDNA was used to investigate the mechanisms of lipid efflux to apoA-I or HDL in transfected 293 cells. ABC1 expression markedly increased cellular cholesterol and phospholipid efflux to apoA-I but had only minor effects on lipid efflux to HDL. The increased lipid efflux appears to involve a direct interaction between apoA-I and ABC1, because ABC1 expression substantially increased apoA-I binding at the cell surface, and chemical cross-linking and immunoprecipitation analysis showed that apoA-I binds directly to ABC1. In contrast to scavenger receptor BI (SR-BI), another cell surface molecule capable of facilitating cholesterol efflux, ABC1 preferentially bound lipid-free apoA-I but not HDL. Immunofluorescence confocal microscopy showed that ABC1 is primarily localized on the cell surface. In the absence of apoA-I, cells overexpressing ABC1 displayed a distinctive morphology, characterized by plasma membrane protrusions and resembling echinocytes that form when there are excess lipids in the outer membrane hemileaflet. The studies provide evidence for a direct interaction between ABC1 and apoA-I, but not HDL, indicating that free apoA-I is the metabolic substrate for ABC1. Plasma membrane ABC1 may act as a phospholipid/cholesterol flippase, providing lipid to bound apoA-I, or to the outer membrane hemileaflet.

Inhibitory effects of HepG2 cell-derived apolipoprotein A-I-containing lipoproteins on cholesteryl ester accumulation in macrophages.

We investigated the mechanisms of inhibitory effects on foam cell formation of apolipoprotein A-I-containing lipoproteins secreted by HepG2 cells (HepG2-HDL) using mouse peritoneal macrophages. When macrophages were incubated with acetylated low-density lipoprotein (acetyl-LDL) in the presence of HepG2-HDL, cholesterol ester (CE) accumulation in cells was reduced by 63%. This inhibitory capacity was almost similar to that of plasma high-density lipoprotein (HDL). When macrophages were converted to foam cells with acetyl-LDL and then reacted with HepG2-HDL or plasma HDL, the HDL-induced CE reduction was 2.2-fold greater than HepG2-HDL. Similar results were obtained using apo E-free HepG2-HDL. Since the inhibitory effect of HDL on acetyl-LDL-induced CE accumulation in macrophages is due largely to its cholesterol efflux capacity, these results suggest the presence of an additional mechanism for the inhibition of CE accumulation by HepG2-HDL. To investigate the mechanism, acetyl-LDL was reisolated from HepG2-HDL by Sephacryl S-300 gel filtration after incubation in a cell-free system. Reisolated acetyl-LDL showed a significant reduction in electrophoretic mobility. The extent of CE accumulation by reisolated acetyl-LDL was reduced by 20% compared with control acetyl-LDL. Moreover, its endocytic degradation by macrophages was reduced by 28%. HepG2-HDL also inhibited macrophage degradation of acetyl-LDL as well as oxidized LDL, a likely atherogenic lipoprotein. This inhibitory effect was ascribed to the HepG2-HDL subfraction containing pre-beta HDL. Our results indicated that apo A-I-containing lipoproteins as a physiological model of nascent HDL may inhibit foam cell formation by reducing ligand activity of atherogenic lipoproteins. These data possibly suggest inhibitory function of nascent HDL for the formation of foam cells in vivo.

Down-regulation of hormone-sensitive lipase in sterol ester-laden J774.2 macrophages

The development of atherosclerotic plaques in arteries is a key step in atherogenesis, with cholesterol ester accumulation in macrophage-derived foam cells being recognized as a major pathogenic event in this process. In this study, the mouse macrophage cell line J774.2 was induced to accumulate intracellular sterol esters by incubation with 25-hydroxycholesterol in the presence of oleic acid. The accumulation of sterol esters in these cells was found to be accompanied by a marked decrease in the activity of the enzyme responsible for their hydrolysis, namely hormone-sensitive lipase (HSL); Western blotting studies revealed a corresponding decrease in the levels of the HSL polypeptide. Similar findings were obtained after incubation with oxidized low-density lipoprotein or very-low-density lipoprotein. These findings suggest that down-regulation of the expression of HSL is important in cholesterol ester accumulation in macrophages.

Beta-VLDL in hepatic lipase deficiency induces apoE-mediated cholesterol ester accumulation in macrophages.

Hepatic lipase-deficient subjects in the Ontario kindred are compound heterozygotes for hepatic lipase mutations (Ser267-->Phe and Thr383-->Met). Cholesteryl ester-rich beta-very-low-density lipoprotein (beta-VLDL) accumulates in plasma and such subjects have premature atherosclerosis. To determine a possible mechanism, we hypothesized that hepatic lipase-deficient beta-VLDL, homozygous for apolipoprotein (apo) E3, would cause cholesteryl ester accumulation and foam cell formation in macrophages. beta-VLDL and pre-beta-VLDL were isolated by Pevikon electrophoresis and incubated with J774 macrophages, cells that do not secrete apoE. beta-VLDL increased cellular cholesteryl ester content 13-fold, whereas pre-beta-VLDL increased cholesteryl ester sevenfold. beta-VLDL increased acyl CoA:cholesterol acyltransferase activity fourfold (measured as [14C]oleate incorporation into cholesteryl ester). Preincubation of hepatic lipase-deficient beta-VLDL with the anti-apoE monoclonal antibody 1D7, which inhibits binding of apoE to low-density lipoprotein receptors, inhibited cellular cholesteryl ester accumulation by 75%, whereas the anti-apoB blocking monoclonal antibody 5E11 failed to inhibit cellular cholesteryl ester accumulation. In contrast to hepatic lipase deficiency, beta-VLDL from type III subjects (E2/E2) failed to increase cellular cholesteryl ester or acyl CoA:cholesterol acyltransferase more than 1.5-fold. Thus, hepatic lipase-deficient beta-VLDL readily induces cholesteryl ester accumulation in J774 macrophages, a process mediated by functional apoE3. This may explain the premature atherosclerosis observed in this kindred.

Analysis of cholesterol ester accumulation in macrophages by the use of digital imaging fluorescence microscopy

Low density lipoprotein (LDL) induced accumulation of cholesterol esters was analyzed by the digital imaging fluorescence microscopy (DIFM) in murine tumor macrophages. To analyze cholesterol ester accumulation, P388D 1 macrophages were incubated with increasing quantities of unmodified or acetylated human LDL, washed, and live stained with a lipophylic fluorescent dye Nile Red. The increase in fluorescence intensity was quantitatively determined by the interactive laser cytometer (ACAS 470) and compared with the accumulation of cellular cholesterol esters determined by the gas liquid chromatography. Correlation between the two methods was highly significant ( r > 0.9, P < 0.001). A good agreement between the two methods was also found in terms of sensitivity and reproducibility. With the use of 589 nm narrowband interference filter in the light path of emitted light the intensity of fluorescence correlated well with cellular cholesterol ester content even in the presence of relatively high concentrations of triglycerides. Therefore, digital imaging fluorescence microscopy appears to be a reliable method for quantification of cholesterol ester accumulation at the single cell level offering new possibilities of studying interactions between cells and cholesterol ester rich lipoproteins.