Background— The peroxisome proliferator–activated receptor-α (PPARα) plays important roles in lipid metabolism, inflammation, and atherosclerosis. PPARα ligands have been shown to reduce cardiovascular events in high-risk subjects. PPARα expression by arterial cells, including macrophages, may exert local antiatherogenic effects independent of plasma lipid changes. Methods and Results— To examine the contribution of PPARα expression by bone marrow–derived cells in atherosclerosis, male and female low-density lipoprotein receptor–deficient (LDLR −/− ) mice were reconstituted with bone marrow from PPARα −/− or PPARα +/+ 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 PPARα −/− →LDLR −/− mice was significantly increased (44% and 46%, respectively) compared with controls. Male PPARα −/− →LDLR −/− mice also had larger (44%) atherosclerotic lesions in the proximal aorta than male PPARα +/+ →LDLR −/− mice. Peritoneal macrophages from PPARα −/− mice had increased uptake of oxidized LDL and decreased cholesterol efflux. PPARα −/− macrophages had lower levels of scavenger receptor B type I and ABCA1 protein expression and an accelerated response of nuclear factor-κB–regulated inflammatory genes. A laser capture microdissection analysis verified suppressed scavenger receptor B type I and increased nuclear factor-κB gene expression levels in vivo in atherosclerotic lesions of PPARα −/− →LDLR −/− mice compared with the lesions of control PPARα +/+ →LDLR −/− mice. Conclusions— These data demonstrate that PPARα expression by macrophages has antiatherogenic effects via modulation of cell cholesterol trafficking and inflammatory activity.

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.

Abstract-The concentration of apolipoprotein (apo) AI in the artery wall is thought to enhance cellular cholesterol efflux and protect against atherosclerosis. It has been shown that although macrophages do not make apoAI, they respond to it by increased cholesterol efflux. We hypothesized that macrophage production of apoAI would increase cholesterol efflux and reduce atherogenesis. In this study, we produced mice expressing human apoAI under the control of the macrophage-specific scavenger receptor-A promoter (m-AI). Human apoAI was detectable in the serum HDL fraction of m-AI transgenic mice at concentrations too low to affect serum cholesterol or HDL levels. Immunoblotting showed the presence of human apoAI in transgenic macrophage culture supernatants, mostly as lipoprotein-free protein, with a small component associated with HDL-like particles. Atherosclerosis studies using apoAI (Ϫ/Ϫ) mice transplanted with m-AI bone marrow showed that in the absence of macrophage-derived apoE, local expression of apoAI reduced diet-induced lesions in the proximal aorta. Additionally, m-AI macrophages showed a 40% increase in cholesterol efflux compared with control macrophages. These data support the hypothesis that apoAI production by macrophages in the artery wall is protective against atherosclerosis. This protection is likely mediated by increased cholesterol efflux and decreased foam cell formation in vivo.

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 (Ϫ/Ϫ) 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 lipoproteinderived cholesterol and propose a pathway for the pro-atherogenic transformation of ACAT1 (Ϫ/Ϫ) macrophages. isoform is found at high levels in macrophages and steroidogenic tissues. The ACAT2 isoform is expressed only in the liver and the small intestine and is involved in the assembly of lipoproteins. Inhibition of cholesterol esterification in macrophages is expected to slow down foam cell formation and decrease lesion size by blocking the storage of cholesterol and facilitating cholesterol efflux. However, recent studies in our laboratory have shown that the deletion of the ACAT1 gene in macrophages results in an increase in atherosclerotic lesion area in the aortas of hyperlipidemic mice. 2 Apolipoproteins (apo) E and AI, and the ATP-binding cassette (ABC) A1 are the main modulators of cholesterol efflux in macrophages. 3-5 ABCA1 is responsible for the movement of phospholipids and cholesterol to apoAI, which results in the formation of nascent high-density lipoprotein. 6 Systemically, ABCA1 is a critical part of the reverse cholesterol transport system. Tangier disease, caused by mutations in the ABCA1 gene, results in severely decreased highdensity lipoprotein and apoAI levels, and accelerates atherosclerosis. 5,7 Both CE storage and FC efflux are important physiological markers of cholesterol balance in macrophages. Pharmacological inhibition of ACAT has been shown to increase cholesterol efflux in many studies. 8 -10 However, there are conflicting reports of increased and decreased atherosclerosis in animal models after the administration of ACAT inhibitors. 1 In our previous study, the worsening effect of ACAT1 gene deletion in hyperlipidemic mice may have been related to increased macrophage apoptosis induced by FC accumulation. 2,9,11 In the current studies, we labele...

mediated cholesterol efflux from macrophages: separation of autocrine and paracrine effects. Am J Physiol Cell Physiol 288: C586 -C592, 2005. First published October 27, 2004 doi:10.1152/ajpcell. 00210.2004.-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. autocrine/paracrine system; cholesterol acceptor; extracellular space; distribution volume AN IMPORTANT CHALLENGE in atherosclerosis research is the characterization of the effects of locally synthesized apolipoprotein E (apoE) within the vessel wall. Endogenous synthesis and secretion of apoE by macrophages in the vessel wall have been shown to protect against atherosclerosis (22). Arterial macrophages participate in inflammation, tissue remodeling, and lipid metabolism. ApoE, which is synthesized by hepatocytes, adipocytes, and macrophages, mediates lipoprotein metabolism and affects cellular cholesterol homeostasis. apoE from macrophages accepts cholesterol from cells in the vessel wall and transports it back to the liver, where the cholesterol can be excreted as bile (3,9). This pathway is called the reverse cholesterol transport (RCT) system. The effect of apoE can be due to its cellular or extracellular positioning, and therefore an apoE molecule can mediate cholesterol efflux from the macrophage that originally secreted it (autocrine effect) or from surrounding macrophages (paracrine effect). The terms "autocrine" and "paracrine," traditionally applied to signaling peptides and hormones, have also been used to describe the actions of mediators with a broad range of functions besides signal transduction (1,7,25). It can be assumed that for the sake of cholesterol efflux an individual macrophag...