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.).

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 Ϫ/Ϫ ) mice that are selectively deficient in macrophage ABCG1 by using bone marrow transfer (ABCG1 Ϫ/Ϫ 3 LDLr Ϫ/Ϫ ). Peritoneal macrophages isolated from donor ABCG1 Ϫ/Ϫ mice exhibited a 22% (Pϭ0.0007) 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 Ϫ/Ϫ 3 LDLr Ϫ/Ϫ mice and controls. In lungs of ABCG1 Ϫ/Ϫ 3 LDLr Ϫ/Ϫ 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 49Ϯ12ϫ10 3 m 2 for ABCG1 ϩ/ϩ 3 LDLr Ϫ/Ϫ mice versus 65Ϯ15ϫ10 3 m 2 for ABCG1 Ϫ/Ϫ 3 LDLr Ϫ/Ϫ mice and after 12 weeks of high-cholesterol diet feeding 124Ϯ17ϫ10 3 m 2 for ABCG1 ϩ/ϩ 3 LDLr Ϫ/Ϫ mice versus 168Ϯ17ϫ10 3 m 2 for ABCG1 Ϫ/Ϫ 3 LDLr Ϫ/Ϫ mice. Atherosclerotic lesion size depended on both time and the macrophage ABCG1 genotype (Pϭ0.038 by 2-way ANOVA, nՆ8), 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.

We have previously identified the E3 ubiquitin ligase-inducible degrader of the low density lipoprotein receptor (LDLR) (Idol) as a post-translational modulator of LDLR levels. Idol is a direct target for regulation by liver X receptors (LXRs), and its expression is responsive to cellular sterol status independent of the sterol-response element-binding proteins. Here we demonstrate that Idol also targets two closely related LDLR family members, VLDLR and ApoE receptor 2 (ApoER2), proteins implicated in both neuronal development and lipid metabolism. Idol triggers ubiquitination of the VLDLR and ApoER2 on their cytoplasmic tails, leading to their degradation. We further show that the level of endogenous VLDLR is sensitive to cellular sterol content, Idol expression, and activation of the LXR pathway. Pharmacological activation of the LXR pathway in mice leads to increased Idol expression and to decreased Vldlr levels in vivo. Finally, we establish an unexpected functional link between LXR and Reelin signaling. We demonstrate that LXR activation results in decreased Reelin binding to VLDLR and reduced Dab1 phosphorylation. The identification of VLDLR and ApoER2 as Idol targets suggests potential roles for this LXR-inducible E3 ligase in the central nervous system in addition to lipid metabolism.

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 156Ϯ19ϫ10 3 m 2 after only 6 weeks of WTD feeding. Lesions, however, were smaller than single ABCA1 KO transplanted animals (226Ϯ30ϫ10 3 m 2 ; PϽ0.05) and not significantly different from single ABCG1 KO (117Ϯ22ϫ10 3 m 2 ) and WT transplanted mice (112Ϯ15ϫ10 3 m 2 ). 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. Key Words: ABCA1 Ⅲ ABCG1 Ⅲ atherosclerosis Ⅲ macrophage Ⅲ transplantation Ⅲ cholesterol T he transport of excess cholesterol by HDL from macrophages in the periphery back to the liver for catabolism and excretion in bile and feces, called reverse cholesterol transport (RCT), plays an important protective role in the development of atherosclerosis. 1 Several ATP-binding cassette (ABC) transporters have been implicated in macrophage lipid metabolism, RCT, and atherosclerosis. 2,3 The fulltransporter ABCA1 is highly induced in lipid-laden macrophages where it facilitates cellular cholesterol and phospholipid efflux to lipid-poor apoproteins like apoAI or ApoE. 4 As cholesterol efflux from macrophages results in decreased cellular lipid accumulation, macrophage ABCA1 expression has been suggested to protect against atherosclerosis. Information on the critical role of macrophage ABCA1 in lesion formation and progression was provided by bone marrow transplantation studies, using mostly LDL receptor (LDLr) KO mice as recipients with ABCA1 KO mice or ABCA1 overexpressor mice as donors leading to the anticipated induction and inhibition of lesion formation, respectively. [5][6][7] More recently, in macrophages a second ABC-transporter, ABCG1, was identified as a transporter for cholesterol from cells to HDL. 8 -10 Because, similarly to ABCA1, ABCG1 is highly induced in lipid-laden macrophages and able to facilitate efflux of cholesterol from macrophages, 8,9 it was anticipated that ABCG1 would add to the protective function of ABCA1 in lesion formation. This hypothesis was strengthened by data from Kennedy et al who showed th...

In both mice (1, 2) and humans (3) there is a strong inverse relation between the blood levels of HDL and the development of atherosclerosis. The atheroprotective effect of HDL is ascribed to its role in reverse cholesterol transport (RCT), as first proposed by Glomset (4), in which HDL accepts cholesterol from peripheral cells, including those in the arterial wall, and delivers it to the liver for biliary secretion (reviewed in Refs. 4-10). In addition, HDL can deliver its cholesteryl ester (CE) to the adrenals and testis or ovary for steroid hormone synthesis (11,12). At both the liver and steroidogenic tissues, cholesterol delivery occurs via selective cellular uptake of HDL-CE without stoichiometric degradation of HDL protein (13,14). Acton and coworkers (15) provided the first evidence that scavenger receptor class B type I (SR-BI) can mediate the selective uptake of HDL-CE in Chinese hamster ovary cells stably transfected with mouse SR-BI. Furthermore, treatment of the adrenocortical cell line Y1-BS1 with antibodies directed against mouse SR-BI resulted in a dramatic decrease in the selective uptake of HDL-CE (16).In vivo, the expression levels of rat and mouse SR-BI mRNA and protein are highest in liver and steroidogenic tissues (adrenal gland, testis, and ovary) (17, 18), all tissues that display selective uptake of HDL-CE. We showed earlier (19) that changes in SR-BI expression in rat liver, induced by estradiol treatment or a high-cholesterol diet, correlated with changes in the selective uptake of HDL-CE in vivo, supporting a function of SR-BI in mediating the selective uptake of HDL-CE. Adenovirus-mediated hepatic overexpression of SR-BI in mice on both sinusoidal and canalicular surfaces of hepatocytes resulted in the virtual disappearance of plasma HDL and a substantial increase in biliary cholesterol (20). A similar decrease in plasma HDL cholesterol levels was found in transgenic mice overexpressing SR-BI under the control of the apolipoprotein A-I promoter (21). These studies indicated that SR-BI expression in the liver can regulate blood HDL me-

Foam cell formation due to excessive accumulation of cholesterol by macrophages is a pathological hallmark of atherosclerosis. Macrophages cannot limit the uptake of cholesterol and therefore depend on cholesterol efflux pathways for preventing their transformation into foam cells. Several ABC-transporters, including ABCA1 and ABCG1, facilitate the efflux of cholesterol from macrophages. These transporters, however, also affect membrane lipid asymmetry which may have important implications for cellular endocytotic pathways. We propose that in addition to the generally accepted role of these ABC-transporters in the prevention of foam cell formation by induction of cholesterol efflux from macrophages, they also influence the macrophage endocytotic uptake.

Objective-The interaction of platelets with low density lipoprotein (LDL) contributes to the development of cardiovascular disease. Platelets are activated by native LDL (nLDL) through apoE Receptor 2Ј (apoER2Ј)-mediated signaling to p38 MAPK and by oxidized LDL (oxLDL) through lysophosphatidic acid (LPA) signaling to Rho A and Ca 2ϩ . Here we report a new mechanism for platelet activation by oxLDL. Methods and Results-Oxidation of nLDL increases p38MAPK activation through a mechanism that is (1) independent of LPA, and (2) unlike nLDL-signaling not desensitized by prolonged platelet-LDL contact or inhibited by receptorassociated protein or chondroitinase ABC. Antibodies against scavenger receptors CD36 and SR-A alone fail to block p38 MAPK activation by oxLDL but combined blockade inhibits p38 MAPK by Ͼ40% and platelet adhesion to fibrinogen under flow by Ͼ60%. Mouse platelets deficient in either CD36 or SR-A show normal p38 MAPK activation by oxLDL but combined deficiency of CD36 and SR-A disrupts oxLDL-induced activation of p38 MAPK by Ͼ70%. Key Words: platelets Ⅲ LDL Ⅲ oxidized LDL Ⅲ CD36 Ⅲ scavenger receptor-A A n elevated level of native low density lipoprotein (nLDL) is a risk factor for arterial thrombosis and atherosclerosis as demonstrated in familial hypercholesterolemia, where defective apoB/E receptors fail to remove nLDL from the circulation. Atherogenesis starts when nLDL accumulates in the vessel wall at sites of injury and is oxidized by products from macrophages, smooth muscle cells, and endothelial cells. 1 Oxidized LDL (oxLDL) accumulates in monocytes that have infiltrated the subendothelium and differentiated into macrophages. The resulting foam cells are characteristic for the early atherosclerotic lesion. 2 OxLDL further contributes to atherosclerosis because it contains lysophosphatidic acid (LPA), which starts platelet shape change and aggregation. 3 In healthy individuals, the concentration of oxLDL is low. The normal intima contains little oxLDL (1.86Ϯ0.59 ng/g apolipoprotein B100 [apoB100]) but levels increase 6-fold in atherosclerotic lesions (11.9Ϯ1.7 ng/g apoB100). 4 Blood from atherosclerotic patients contains autoantibodies that react with oxidation-specific epitopes in both the lipid and protein moiety of oxLDL, 5,6 indicating that oxLDL is also present in the circulation. Hence, in the circulation, platelets can come into contact with oxLDL and become activated, thereby contributing to thrombotic occlusion. Conclusion-TheseThe oxidation of nLDL in vivo can be mimicked in vitro by treatment of nLDL with FeSO 4 . These oxLDL preparations resemble in vivo oxidized LDL with respect to electrophoretic mobility, density, LPA content, fragmentation of apoB100, chemotactic activity for monocytes, and susceptibility to degradation by macrophages. 3,7-9 LPA makes oxLDL a potent platelet activating agent 3 through activation of its LPA 1 and LPA 3 receptors, 10 which are members of the endothelial differentiation gene receptor family. At low concentrations, LPA stimulates Rho, Rho-ki...

Receptor-mediated cholesterol uptake has been suggested to play a role in maintaining the adrenal intracellular free cholesterol pool and the ability to produce hormones. Therefore, in the current study, we evaluated the importance of scavenger receptor class B type I (SR-BI)-mediated cholesteryl ester uptake from HDL for adrenal glucocorticoid hormone synthesis in vivo. No difference was observed in the plasma level of corticosterone between SR-BI-deficient and wild-type mice under ad libitum feeding conditions. Overnight fasting (?16 h) stimulated the plasma level of corticosterone by 2-fold in wild-type mice. In contrast, no effect of fasting on plasma corticosterone levels was observed in SR-BI-deficient mice, leading to a 44% lower plasma corticosterone level compared with their wild-type littermate controls. In parallel, an almost complete depletion of lipid stores in the adrenal cortex of fasted SR-BIdeficient mice was observed. Plasma adrenocorticotropic hormone levels were increased by 5-fold in fasted SR-BIdeficient mice. SR-BI deficiency induced marked changes in the hepatic expression of the glucocorticoid-responsive genes cholesterol 7a-hydroxylase, HMG-CoA synthase, apolipoprotein A-IV, corticosteroid binding globulin, interleukin-6, and tumor necrosis factor-a, which coincided with a 42% decreased plasma glucose level under fasting conditions. In conclusion, we show that the absence of adrenal HDL cholesteryl ester uptake in SR-BI-deficient mice impairs the adrenal glucocorticoid-mediated stress response to fasting as a result of adrenal glucocorticoid insufficiency and attenuated liver glucocorticoid receptor signaling, leading to hypoglycemia under fasting conditions.-Hoekstra, M., I. Meurs, M. Koenders, R. Out, R. B. Hildebrand, J. K. Kruijt, M. Van Eck, and T. J. C. Van Berkel. Absence of HDL cholesteryl ester uptake in mice via SR-BI impairs an adequate adrenal glucocorticoid-mediated stress response to fasting. J. Lipid Res. 2008. 49: 738-745.