Miranda Van Eck

Leukocyte ABCA1 controls susceptibility to atherosclerosis and macrophage recruitment into tissues

The ATP-binding cassette transporter 1 (ABCA1) has recently been identified as a key regulator of high-density lipoprotein (HDL) metabolism, which is defective in familial HDL-deficiency syndromes such as Tangier disease. ABCA1 functions as a facilitator of cellular cholesterol and phospholipid efflux, and its expression is induced during cholesterol uptake in macrophages. To assess the role of macrophage ABCA1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr−/−) mice that are selectively deficient in leukocyte ABCA1 (ABCA1−/−) by using bone marrow transfer (ABCA1−/− → LDLr−/−). Here we demonstrate that ABCA1−/− → LDLr−/− chimeras develop significantly larger and more advanced atherosclerotic lesions compared with chimeric LDLr−/− mice with functional ABCA1 in hematopoietic cells. Targeted disruption of leukocyte ABCA1 function did not affect plasma HDL cholesterol levels. The amount of macrophages in liver and spleen and peripheral blood leukocyte counts is increased in the ABCA1−/− → LDLr−/− chimeras. Our results provide evidence that leukocyte ABCA1 plays a critical role in the protection against atherosclerosis, and we identify ABCA1 as a leukocyte factor that controls the recruitment of inflammatory cells.

Genetic variant of the scavenger receptor BI in humans

BACKGROUND In mice, the scavenger receptor class B type I (SR-BI) is essential for the delivery of high-density lipoprotein (HDL) cholesterol to the liver and steroidogenic organs. Paradoxically, elevated HDL cholesterol levels are associated with increased atherosclerosis in SR-BI-knockout mice. It is unclear what role SR-BI plays in human metabolism. METHODS We sequenced the gene encoding SR-BI in persons with elevated HDL cholesterol levels and identified a family with a new missense mutation (P297S). The functional effects of the P297S mutation on HDL binding, cellular cholesterol uptake and efflux, atherosclerosis, platelet function, and adrenal function were studied. RESULTS Cholesterol uptake from HDL by primary murine hepatocytes that expressed mutant SR-BI was reduced to half of that of hepatocytes expressing wild-type SR-BI. Carriers of the P297S mutation had increased HDL cholesterol levels (70.4 mg per deciliter [1.8 mmol per liter], vs. 53.4 mg per deciliter [1.4 mmol per liter] in noncarriers; P<0.001) and a reduced capacity for efflux of cholesterol from macrophages, but the carotid artery intima-media thickness was similar in carriers and in family noncarriers. Platelets from carriers had increased unesterified cholesterol content and impaired function. In carriers, adrenal steroidogenesis was attenuated, as evidenced by decreased urinary excretion of sterol metabolites, a decreased response to corticotropin stimulation, and symptoms of diminished adrenal function. CONCLUSIONS We identified a family with a functional mutation in SR-BI. The mutation carriers had increased HDL cholesterol levels and a reduction in cholesterol efflux from macrophages but no significant increase in atherosclerosis. Reduced SR-BI function was associated with altered platelet function and decreased adrenal steroidogenesis. (Funded by the European Community and others.).

Differential effects of scavenger receptor BI deficiency on lipid metabolism in cells of the arterial wall and in the liver

Scavenger receptor class B, type I (SRBI) is a key regulator of high density lipoprotein (HDL) metabolism. It facilitates the efflux of cholesterol from cells in peripheral tissues to HDL and mediates the selective uptake of cholesteryl esters from HDL in the liver. We investigated the effects of SRBI deficiency in the arterial wall and in the liver using SRBI-deficient mice and wild-type littermates fed a Western-type diet. The SRBI-deficient mice showed massive accumulation of cholesterol-rich HDL in the circulation, reflecting impaired delivery to the liver. Strikingly, SRBI deficiency did not alter hepatic cholesterol (ester) content nor did it affect the expression of key regulators of hepatic cholesterol homeostasis, including HMG-CoA reductase, the low density lipoprotein receptor, and cholesterol 7α-hydroxylase. However, a ∼40% reduction in biliary cholesterol content was observed, and the expression of ABCG8 and ABCG5, ATP half-transporters implicated in the transport of sterols from the liver to the bile, was attenuated by 70 and 35%, respectively. In contrast to the situation in the liver, SRBI deficiency did result in lipid deposition in the aorta and atherosclerosis. Vascular mRNA analysis showed increased expression of inflammatory markers as well as of genes involved in cellular cholesterol homeostasis. Our data show that, although hepatic cholesterol homeostasis is maintained upon feeding a Western-type diet, SRBI deficiency is associated with de-regulation of cholesterol homeostasis in the arterial wall that results in an increased susceptibility to atherosclerosis.

Scavenger receptor BI and ATP-binding cassette transporter A1 in reverse cholesterol transport and atherosclerosis.

Purpose of review The appearance of scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) in macrophages and liver implicates these transporters in different stages of reverse cholesterol transport. This review focuses on the role of SR-BI and ABCA1 in reverse cholesterol transport in the context of atherosclerotic lesion development. Recent findings Recent studies indicate that hepatic expression of ABCA1 and SR-BI is important for the generation of nascent HDL and the delivery of HDL cholesteryl esters to the liver, respectively. Although macrophage SR-BI and ABCA1 do not contribute significantly to circulating HDL levels, the perpetual cycle of HDL lipidation and delipidation by the liver ensures the availability of acceptors for cholesterol efflux that maintain cholesterol homeostasis in arterial macrophages, thereby reducing atherogenesis. In addition to its established role in the selective uptake of HDL cholesteryl esters, there is now evidence that hepatic SR-BI facilitates postprandial lipid metabolism, and that hepatic secretion of VLDL is dependent on ABCA1-mediated nascent HDL formation. Thus, remnant and HDL metabolism are more intimately intertwined in hepatic lipid metabolism than has previously been appreciated. Summary Recent advances in the understanding of the role of ABCA1 and SR-BI in HDL metabolism and their atheroprotective properties indicate the significant potential of modulating ABCA1 and SR-BI expression in both arterial wall macrophages and the liver for the treatment of atherosclerotic coronary artery disease.

Macrophage ABCG1 Deletion Disrupts Lipid Homeostasis in Alveolar Macrophages and Moderately Influences Atherosclerotic Lesion Development in LDL Receptor-Deficient Mice

Objective—ABCG1 has recently been identified as a facilitator of cellular cholesterol and phospholipid efflux to high-density lipoprotein (HDL). Its expression in macrophages is induced during cholesterol uptake in macrophages and by liver X receptor (LXR). The role of macrophage ABCG1 in atherosclerotic lesion development is, however, still unknown. Methods and Results—To assess the role of macrophage ABCG1 in atherosclerosis, we generated low-density lipoprotein (LDL) receptor knockout (LDLr−/−) mice that are selectively deficient in macrophage ABCG1 by using bone marrow transfer (ABCG1−/− → LDLr−/−). Peritoneal macrophages isolated from donor ABCG1−/− mice exhibited a 22% (P=0.0007) decrease in cholesterol efflux to HDL. To induce atherosclerosis, transplanted mice were fed a high-cholesterol diet containing 0.25% cholesterol and 15% fat for 6 and 12 weeks. Serum lipid levels and lipoprotein profiles did not differ significantly between ABCG1−/− → LDLr−/− mice and controls. In lungs of ABCG1−/− → LDLr−/− mice a striking accumulation of lipids was observed in macrophages localized to the subpleural region. After 6 weeks of high-cholesterol diet feeding the atherosclerotic lesion size was 49±12×103 &mgr;m2 for ABCG1+/+ → LDLr−/− mice versus 65±15×103 &mgr;m2 for ABCG1−/− → LDLr−/− mice and after 12 weeks of high-cholesterol diet feeding 124±17×103 &mgr;m2 for ABCG1+/+ → LDLr−/− mice versus 168±17×103 &mgr;m2 for ABCG1−/− → LDLr−/− mice. Atherosclerotic lesion size depended on both time and the macrophage ABCG1 genotype (P=0.038 by 2-way ANOVA, n≥8), indicating a moderately 33% to 36% increase in lesion formation in the absence of macrophage ABCG1. Conclusions—Macrophage ABCG1 deficiency does lead to heavy lipid accumulation in macrophages of the lung, and also a moderately significant effect on atherosclerotic lesion development was observed.

Macrophage ATP-binding cassette transporter A1 overexpression inhibits atherosclerotic lesion progression in low-density lipoprotein receptor knockout mice.

Background—ATP-binding cassette transporter A1 (ABCA1) is a key regulator of cellular cholesterol and phospholipid transport. Previously, we have shown that inactivation of macrophage ABCA1 induces atherosclerosis in low-density lipoprotein receptor knockout (LDLr−/−) mice. However, the possibly beneficial effects of specific upregulation of macrophage ABCA1 on atherogenesis are still unknown. Methods and Results—Chimeras that specifically overexpress ABCA1 in macrophages were generated by transplantation of bone marrow from human ABCA1 bacterial artificial chromosome (BAC) transgenic mice into LDLr−/− mice. Peritoneal macrophages isolated from the ABCA1 BAC → LDLr−/− chimeras exhibited a 60% (P=0.0006) increase in cholesterol efflux to apolipoprotein AI. To induce atherosclerosis, the mice were fed a Western-type diet containing 0.25% cholesterol and 15% fat for 9, 12, and 15 weeks, allowing analysis of effects on initial lesion development as well as advanced lesions. No significant effect of macrophage ABCA1 overexpression was observed on atherosclerotic lesion size after 9 weeks on the Western-type diet (245±36×103 &mgr;m2 in ABCA1 BAC → LDLr−/− mice versus 210±20×103 &mgr;m2 in controls). However, after 12 weeks, the mean atherosclerotic lesion area in ABCA1 BAC → LDLr−/− mice remained only 164±15×103 &mgr;m2 (P=0.0008) compared with 513±56×103 &mgr;m2 in controls (3.1-fold lower). Also, after 15 weeks on the diet, lesions in mice transplanted with ABCA1 overexpressing bone marrow were still 1.6-fold smaller (393±27×103 &mgr;m2 compared with 640±59×103 &mgr;m2 in control transplanted mice; P=0.0015). Conclusion—ABCA1 upregulation in macrophages inhibits the progression of atherosclerotic lesions.

Apolipoprotein E Protects Against Bacterial Lipopolysaccharide-induced Lethality A NEW THERAPEUTIC APPROACH TO TREAT GRAM-NEGATIVE SEPSIS

Septic shock is the most common cause of death in intensive care units and no effective treatment is available at present. Lipopolysaccharide (LPS) is the primary mediator of Gram-negative sepsis by inducing the production of macrophage-derived cytokines. Previously, we showed that apolipoprotein E (apoE), an established modulator of lipid metabolism, can bind LPS, thereby redirecting LPS from macrophages to hepatocytes in vivo. We now report that intravenously administered LPS strongly increases the serum levels of apoE. In addition, apoE can prevent the LPS-induced production of cytokines and subsequent death in rodents. Finally, apoE-deficient mice show a significantly higher sensitivity toward LPS than control wild-type mice. These findings indicate that apoE may have a physiological role in the protection against sepsis, and recombinant apoE may be used therapeutically to protect against LPS-induced endotoxemia.

Dual Role for Scavenger Receptor Class B, Type I on Bone Marrow-Derived Cells in Atherosclerotic Lesion Development

The function of scavenger receptor class B, type I (SR-BI) in the liver as a high-density lipoprotein receptor that promotes the selective uptake of cholesteryl esters is well defined. Its role in macrophages, however, is primarily unknown, because it functions in the uptake of (modified) lipoproteins as well as the secretion of cholesterol to high-density lipoproteins. In this study, the biological role of SR-BI on bone marrow-derived cells, including macrophages, in lipid metabolism and atherosclerosis was assessed by selective disruption of SR-BI in bone marrow in two established models of atherosclerosis: low-density lipoprotein (LDL) receptor-deficient mice that develop extensive atherosclerosis on a Western-type diet and wild-type mice that develop fatty streak lesions when fed a high-cholesterol diet containing 0.5% cholate. The presence of SR-BI in bone marrow-derived cells in LDLr-/- mice decreased lesion development after 9 and 12 weeks of Western-type diet feeding, indicating that macrophage SR-BI protects against lesion development. At 6 weeks, no significant effect of SR-BI in bone marrow-derived cells on lesion development was observed. Interestingly, after only 4 weeks of Western-type diet feeding of transplanted LDLr-/- mice and in wild-type mice on a high-cholesterol/cholate diet, the presence of SR-BI in bone marrow-derived cells increased the development of small fatty streak lesions. It thus appears that, depending on the stage of atherosclerotic lesion development, SR-BI in bone marrow-derived cells is either pro-atherogenic or anti-atherogenic, indicating a unique dual role in the pathogenesis of atherosclerosis.

Stimulation of Cholesterol Efflux by LXR Agonists in Cholesterol-Loaded Human Macrophages Is ABCA1-Dependent but ABCG1-Independent

Objective—Maintenance of cholesterol homeostasis in human macrophages is essential to prevent foam cell formation. We evaluated the relative contribution of the ABCA1 and ABCG1 transporters to cholesterol efflux from human macrophages, and of the capacity of LXR agonists to reduce foam cell formation by stimulating export of cellular cholesterol. Methods and Results—ABCG1 mRNA levels were strongly increased in acLDL-loaded THP-1 macrophages and in HMDM on stimulation with LXR agonists. However, silencing of ABCG1 expression using ABCG1-specific siRNA indicated that ABCG1 was not essential for cholesterol efflux to HDL in cholesterol-loaded human macrophages stimulated with LXR agonists. Indeed, ABCA1 was solely responsible for the stimulation of cholesterol efflux to HDL on LXR activation, as this effect was abolished in HMDM from Tangier patients. Furthermore, depletion of cellular ATP indicated that the LXR-induced export of cholesterol was an ATP-dependent transport mechanism in human macrophages. Finally, use of an anti–Cla-1 blocking antibody identified the Cla-1 receptor as a key component in cholesterol efflux to HDL from cholesterol-loaded human macrophages. Conclusion—Our data indicate that stimulation of cholesterol efflux to HDL by LXR agonists in human foam cells involves an ATP-dependent transport mechanism mediated by ABCA1 that it appears to be independent of ABCG1 expression.

Combined Deletion of Macrophage ABCA1 and ABCG1 Leads to Massive Lipid Accumulation in Tissue Macrophages and Distinct Atherosclerosis at Relatively Low Plasma Cholesterol Levels

Objective—The purpose of this study was to evaluate the effect of the combined deletion of ABCA1 and ABCG1 expression in macrophages on foam cell formation and atherosclerosis. Methods and Results—LDL receptor knockout (KO) mice were transplanted with bone marrow from ABCA1/ABCG1 double KO (dKO) mice. Plasma cholesterol levels after 6 weeks of Western-type diet (WTD) feeding were significantly lower in dKO transplanted mice than ABCA1 KO, ABCG1 KO, and control transplanted animals. Extreme foam cell formation was present in macrophages of various tissues and the peritoneal cavity of dKO transplanted animals. Furthermore, severe hypoplasia of the thymus and a significant decrease in CD4-positive T cells in blood was observed. Despite relatively low plasma cholesterol levels dKO transplanted animals developed lesion sizes of 156±19×103 &mgr;m2 after only 6 weeks of WTD feeding. Lesions, however, were smaller than single ABCA1 KO transplanted animals (226±30×103 &mgr;m2; P<0.05) and not significantly different from single ABCG1 KO (117±22×103 &mgr;m2) and WT transplanted mice (112±15×103 &mgr;m2). Conclusions—Macrophage ABCA1 and ABCG1 play a crucial role in the prevention of macrophage foam cell formation, whereas combined deletion only modestly influences atherosclerosis which is associated with an attenuated increase in WTD-induced plasma cholesterol and decreased proinflammatory CD4-positive T cell counts.