Macrophage expression of peroxisome proliferator-activated receptor-alpha reduces atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPARalpha ligands have been shown to reduce cardiovascular events in high-risk subjects. PPARalpha expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes. METHODS AND RESULTS: To examine the contribution of PPARalpha expression by bone marrow-derived cells in atherosclerosis, male and female low-density lipoprotein receptor-deficient (LDLR(-/-)) mice were reconstituted with bone marrow from PPARalpha(-/-) or PPARalpha(+/+) mice and challenged with a high-fat diet. Although serum lipids and lipoprotein profiles did not differ between the groups, the size of atherosclerotic lesions in the distal aorta of male and female PPARalpha(-/-) --> LDLR(-/-) mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPARalpha(-/-) --> LDLR(-/-) mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPARalpha(+/+) --> LDLR(-/-) mice. Peritoneal macrophages from PPARalpha(-/-) mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPARalpha(-/-) macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-kappaB-regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-kappaB gene expression levels in vivo in atherosclerotic lesions of PPARalpha(-/-) --> LDLR(-/-) mice compared with the lesions of control PPARalpha(+/+) --> LDLR(-/-) mice. CONCLUSIONS: These data demonstrate that PPARalpha expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.

Reduced ABCA1-mediated cholesterol efflux and accelerated atherosclerosis in apolipoprotein E-deficient mice lacking macrophage-derived ACAT1.

BACKGROUND: Macrophage acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) and apolipoprotein E (apoE) have been implicated in regulating cellular cholesterol homeostasis and therefore play critical roles in foam cell formation. Deletion of either ACAT1 or apoE results in increased atherosclerosis in hyperlipidemic mice, possibly as a consequence of altered cholesterol processing. We have studied the effect of macrophage ACAT1 deletion on atherogenesis in apoE-deficient (apoE-/-) mice with or without the restoration of macrophage apoE. METHODS AND RESULTS: We used bone marrow transplantation to generate apoE-/- mice with macrophages of 4 genotypes: apoE+/+/ACAT1+/+ (wild type), apoE+/+/ACAT1-/- (ACAT-/-), apoE-/-/ACAT1+/+ (apoE-/-), and apoE-/-/ACAT1-/- (2KO). When macrophage apoE was present, plasma cholesterol levels normalized, and ACAT1 deficiency did not have significant effects on atherogenesis. However, when macrophage apoE was absent, ACAT1 deficiency increased atherosclerosis and apoptosis in the proximal aorta. Cholesterol efflux to apoA-I was significantly reduced (30% to 40%; P<0.001) in ACAT1-/- peritoneal macrophages compared with ACAT1+/+ controls regardless of apoE expression. 2KO macrophages had a 3- to 4-fold increase in ABCA1 message levels but decreased ABCA1 protein levels relative to ACAT1+/+ macrophages. Microarray analyses of ACAT1-/- macrophages showed increases in proinflammatory and procollagen genes and decreases in genes regulating membrane integrity, protein biosynthesis, and apoptosis. CONCLUSIONS: Deficiency of macrophage ACAT1 accelerates atherosclerosis in hypercholesterolemic apoE-/- mice but has no effect when the hypercholesterolemia is corrected by macrophage apoE expression. However, ACAT1 deletion impairs ABCA1-mediated cholesterol efflux in macrophages regardless of apoE expression. Changes in membrane stability, susceptibility to apoptosis, and inflammatory response may also be important in this process.

ACAT1 deficiency increases cholesterol synthesis in mouse peritoneal macrophages.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) esterifies free cholesterol and stores cholesteryl esters in lipid droplets. Macrophage ACAT1 deficiency results in increased atherosclerotic lesion area in hyperlipidemic mice via disrupted cholesterol efflux, increased lipoprotein uptake, accumulation of intracellular vesicles, and accelerated apoptosis. The objective of this study was to determine whether lipid synthesis is affected by ACAT1. The synthesis, esterification, and efflux of new cholesterol were measured in peritoneal macrophages from ACAT1(-/-) mice. Cholesterol synthesis was increased by 134% (p=0.001) in ACAT1(-/-) macrophages compared to wildtype macrophages. Increased synthesis resulted in a proportional increase in the efflux of newly synthesized cholesterol. Although the esterification of new cholesterol was reduced by 93% (p<0.001) in ACAT1(-/-) macrophages, trace amounts of newly synthesized cholesteryl esters were detectable. Furthermore, the expression of SREBP1a mRNA was increased 6-fold in ACAT1(-/-) macrophages compared to wildtype macrophages, suggesting an up-regulation of cholesterol and fatty acid synthesis in ACAT1(-/-) macrophages. Increased cholesterol synthesis and up-regulation of SREBP in ACAT1(-/-) macrophages suggests that ACAT1 affects the regulation of lipid metabolism in macrophages. This change in cholesterol homeostasis may contribute to the atherogenic potential of ACAT1(-/-) macrophages.

ACAT1 deficiency disrupts cholesterol efflux and alters cellular morphology in macrophages.

OBJECTIVE: Acyl-coenzyme A: cholesterol acyltransferase (ACAT) converts intracellular free cholesterol (FC) into cholesteryl esters (CE) for storage in lipid droplets. Recent studies in our laboratory have shown that the deletion of the macrophage ACAT1 gene results in apoptosis and increased atherosclerotic lesion area in the aortas of hyperlipidemic mice. The objective of the current study was to elucidate the mechanism of the increased atherosclerosis. METHODS AND RESULTS: CE storage and FC efflux were studied in ACAT1(-/-) peritoneal macrophages that were treated with acetylated low-density lipoprotein (acLDL). Our results show that efflux of cellular cholesterol was reduced by 25% in ACAT1-deficient cells compared with wild-type controls. This decrease occurred despite the upregulated expression of ABCA1, an important mediator of cholesterol efflux. In contrast, ACAT1 deficiency increased efflux of the cholesterol derived from acLDL by 32%. ACAT1-deficient macrophages also showed a 26% increase in the accumulation of FC derived from acLDL, which was associated with a 75% increase in the number of intracellular vesicles. CONCLUSIONS: Together, these data show that macrophage ACAT1 influences the efflux of both cellular and lipoprotein-derived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1(-/-) macrophages.

ApoE-mediated cholesterol efflux from macrophages: separation of autocrine and paracrine effects.

Macrophages in the vessel wall secrete high levels of apolipoprotein E (apoE). Cholesterol efflux from macrophages to apoE has been shown to decrease foam cell formation and prevent atherosclerosis. An apoE molecule can mediate cholesterol efflux from the macrophage that originally secreted it (autocrine effect) or from surrounding macrophages (paracrine effect). Traditional methodologies have not been able to separate these serial effects. The novel methodology presented here was developed to separate autocrine and paracrine effects by using a simple mathematical model to interpret the effects of dilution on apoE-mediated cholesterol efflux. Our results show that, at very dilute concentrations, the paracrine effect of apoE is not evident and the autocrine effect becomes the dominant mediator of efflux. However, at saturating concentrations, paracrine apoE causes 80-90% of the apoE-mediated cholesterol efflux, whereas autocrine apoE is responsible for the remaining 10-20%. These results suggest that the relative importance of autocrine and paracrine apoE depends on the size of the local distribution volume, a factor not considered in previous in vitro studies of apoE function. Furthermore, autocrine effects of apoE could be critical in the prevention of foam cell formation in vivo. This novel methodology may be applicable to other types of mixed autocrine/paracrine systems, such as signal transduction systems.

Macrophage apolipoprotein A-I expression protects against atherosclerosis in ApoE-deficient mice and up-regulates ABC transporters.

The antiatherogenic effect of high-density lipoprotein (HDL) and its major protein component apolipoprotein A-I (apoA-I) has been largely attributed to their key roles in reverse cholesterol transport (RCT) and cellular cholesterol efflux. Substantial evidence shows that overexpression of human apoA-I reduces atherosclerosis in animal models. However, it is uncertain whether this protection is due to an increase in plasma HDL level or to a local effect in the artery wall. To test the hypothesis that expression of human apoA-I in macrophages can promote RCT in the artery wall, we used a retroviral construct expressing human apoA-I cDNA (MFG-HAI) to transduce ApoE(-/-) bone marrow cells and then transplanted these cells into ApoE(-/-) mice with preexisting atherosclerosis. ApoE(-/-) mice reconstituted with MFG-HAI marrow had a significant reduction (30%) in atherosclerotic lesions in the proximal aorta compared to control mice that received marrow expressing MFG parental virus. Peritoneal macrophages isolated from MFG-HAI mice showed a four- to fivefold increase in mRNA expression levels of both ATP-binding cassette (ABC) A1 and ABCG1 compared to controls. Our data demonstrate that gene transfer-mediated expression of human apoA-I in macrophages can compensate in part for apoE deficiency and delay the progression of atherosclerotic lesions by stimulating ABC-dependent cholesterol efflux and RCT.