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Background Exercise is associated with a reduced risk of cardiovascular disease. Increased high-density lipoprotein cholesterol (HDL-C) levels are thought to contribute to these benefits, but much of the research in this area has been limited by lack of well-controlled subject selection and exercise interventions. We sought to study the effect of moderate and high-intensity exercise on HDL function, lipid/lipoprotein profile, and other cardiometabolic parameters in a homogeneous population where exercise, daily routine, sleep patterns, and living conditions were carefully controlled. Methods and Results Male Army recruits (n=115, age 22±0.3 years) completed a 12-week moderate-intensity exercise program. A subset of 51 subsequently completed a 15-week high-intensity exercise program. Fitness increased and body fat decreased after moderate- and high-intensity exercise ( P <0.001). Moderate-intensity exercise increased HDL-C and apolipoprotein A-I levels (6.6%, 11.6% respectively), and decreased low-density lipoprotein cholesterol and apolipoprotein B levels (7.2%, 4.9% respectively) (all P <0.01). HDL-C and apolipoprotein A-I levels further increased by 8.2% ( P <0.001) and 6.3% ( P <0.05) after high-intensity exercise. Moderate-intensity exercise increased ABCA-1 (ATP-binding cassette transporter A1) mediated cholesterol efflux by 13.5% ( P <0.001), which was sustained after high-intensity exercise. In a selected subset the ability of HDLs to inhibit ICAM-1 (intercellular adhesion molecule-1) expression decreased after the high ( P <0.001) but not the moderate-intensity exercise program. Conclusions When controlling for exercise patterns, diet, and sleep, moderate-intensity exercise improved HDL function, lipid/lipoprotein profile, fitness, and body composition. A sequential moderate followed by high-intensity exercise program showed sustained or incremental benefits in these parameters. Improved HDL function may be part of the mechanism by which exercise reduces cardiovascular disease risk.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with over 80% of cases already disseminated at diagnosis and facing a dismal five-year survival rate of 35%. EOC cells often spread to the greater omentum where they take-up cholesterol. Excessive amounts of cholesterol can be cytocidal, suggesting that cholesterol efflux through transporters may be important to maintain homeostasis, and this may explain the observation that high expression of the ATP-binding cassette A1 (ABCA1) cholesterol transporter has been associated with poor outcome in EOC patients., Methods: ABCA1 expression was silenced in EOC cells to investigate the effect of inhibiting cholesterol efflux on EOC biology through growth and migration assays, three-dimensional spheroid culture and cholesterol quantification., Results: ABCA1 suppression significantly reduced the growth, motility and colony formation of EOC cell lines as well as the size of EOC spheroids, whilst stimulating expression of ABCA1 reversed these effects. In serous EOC cells, ABCA1 suppression induced accumulation of cholesterol. Lowering cholesterol levels using methyl-B-cyclodextrin rescued the effect of ABCA1 suppression, restoring EOC growth. Furthermore, we identified FDA-approved agents that induced cholesterol accumulation and elicited cytocidal effects in EOC cells., Conclusions: Our data demonstrate the importance of ABCA1 in maintaining cholesterol balance and malignant properties in EOC cells, highlighting its potential as a therapeutic target for this disease.

Objective: Patients with schizophrenia have increased long-term mortality attributable to cardiovascular disease and commonly demonstrate features of mixed dyslipidemia with low HDL-C (high-density lipoprotein cholesterol). The removal of cholesterol from cells by HDL via specific ATP-binding cholesterol transporters is a major functional property of HDL, and its measurement as cholesterol efflux capacity (CEC) can predict cardiovascular risk. Whether HDL function is impaired in patients with schizophrenia is unknown. Approach and Results: We measured basal and ABCA1 (ATP-binding cassette transporter A1)- and ABCG1 (ATP-binding cassette transporter G1)-dependent CEC, comparing patients with schizophrenia with age- and sex-matched healthy controls, and related our findings to nuclear magnetic resonance analysis of lipoprotein subclasses. Total plasma cholesterol and LDL-C (low-density lipoprotein cholesterol) were comparable between healthy controls (n=51) and patients (n=120), but patients with schizophrenia had increased total plasma triglyceride, low HDL-C and apo (apolipoprotein) A-I concentrations. Nuclear magnetic resonance analysis indicated a marked (15-fold) increase in large triglyceride-rich lipoprotein particle concentration, increased small dense LDL particles, and fewer large HDL particles. Despite lower HDL-C concentration, basal CEC was 13.7±1.6% higher, ABCA1-specific efflux was 35.9±1.6% higher, and ABCG1 efflux not different, in patients versus controls. In patients with schizophrenia, ABCA1-specific efflux correlated with the abundance of small 7.8 nm HDL particles but not with serum plasminogen or triglyceride levels., Conclusions: Patients with schizophrenia have increased concentrations of atherogenic apoB-containing lipoproteins, decreased concentrations of large HDL particles, but enhanced ABCA1-mediated CEC. In this population, preventative strategies should focus on reducing atherogenic lipoproteins rather than increasing CEC.

Circulating tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) levels are reduced in patients with cardiovascular disease, and TRAIL gene deletion in mice exacerbates atherosclerosis and inflammation. How TRAIL protects against atherosclerosis and why levels are reduced in disease is unknown. Here, multiple strategies were used to identify the protective source of TRAIL and its mechanism(s) of action. Samples from patients with coronary artery disease and bone-marrow transplantation experiments in mice lacking TRAIL revealed monocytes/macrophages as the main protective source. Accordingly, deletion of TRAIL caused a more inflammatory macrophage with reduced migration, displaying impaired reverse cholesterol efflux and efferocytosis. Furthermore, interleukin (IL)-18, commonly increased in plasma of patients with cardiovascular disease, negatively regulated TRAIL transcription and gene expression, revealing an IL-18-TRAIL axis. These findings demonstrate that TRAIL is protective of atherosclerosis by modulating monocyte/macrophage phenotype and function. Manipulating TRAIL levels in these cells highlights a different therapeutic avenue in the treatment of cardiovascular disease., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

The 'cholesterol efflux capacity (CEC)' assay is a simple in vitro measure of the capacities of individual sera to promote the first step of the reverse cholesterol transport pathway, the delivery of cellular cholesterol to plasma HDL. This review describes the cell biology of this model and critically assesses its application as a marker of cardiovascular risk. We describe the pathways for cell cholesterol export, current cell models used in the CEC assay with their limitations and consider the contribution that measurement of serum CEC provides to our understanding of HDL function in vivo., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) is a recently identified phosphodiesterase, which is a secreted N -linked glycoprotein. SMPDL3A is highly homologous to acid sphingomyelinase (aSMase), but unlike aSMase cannot cleave sphingomyelin. Rather, SMPDL3A hydrolyzes nucleotide tri- and diphosphates and their derivatives. While recent structural studies have shed light on these unexpected substrate preferences, many other aspects of SMPDL3A biology, which may give insight into its function in vivo , remain obscure. Here, we investigate the roles of N-glycosylation in the expression, secretion and activity of human SMPDL3A, using inhibitors of N-glycosylation and site-directed mutagenesis, with either THP-1 macrophages or CHO cells expressing human SMPDL3A. Tunicamycin (TM) treatment resulted in expression of non-glycosylated SMPDL3A that was not secreted, and was largely degraded by the proteasome. Proteasomal inhibition restored levels of SMPDL3A in TM-treated cells, although this non-glycosylated protein lacked phosphodiesterase activity. Enzymatic deglycosylation of purified recombinant SMPDL3A also resulted in significant loss of phosphodiesterase activity. Site-directed mutagenesis of individual N-glycosylation sites in SMPDL3A identified glycosylation of Asn69 and Asn222 as affecting maturation of its N -glycans and secretion. Glycosylation of Asn356 in SMPDL3A, an N- linked site conserved throughout the aSMase-like family, was critical for protection against proteasomal degradation and preservation of enzymatic activity. We provide the first experimental evidence for a predicted 22 residue N-terminal signal peptide in SMPDL3A, which is essential for facilitating glycosylation and is removed from the mature protein secreted from CHO cells. In conclusion, site-specific N-glycosylation is essential for the intracellular stability, secretion and activity of human SMPDL3A., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Reverse cholesterol transport (RCT) is one of the main processes that is thought to protect against cardiovascular disease. RCT constitutes the removal of cholesterol from peripheral sites, its transport through the plasma compartment for delivery to the liver for excretion. Here, we describe an in vivo RCT method that incorporates these steps, measuring movement of cholesterol from macrophages to the plasma, the liver, and finally to the feces in mice.

Apolipoprotein A-I (apoA-I) is the major component of HDL and central to the ability of HDL to stimulate ATP-binding cassette transporter A1 (ABCA1)-dependent, antiatherogenic export of cholesterol from macrophage foam cells, a key player in the pathology of atherosclerosis. Cell-mediated modifications of apoA-I, such as chlorination, nitration, oxidation, and proteolysis, can impair its antiatherogenic function, although it is unknown whether macrophages themselves contribute to such modifications. To investigate this, human monocyte-derived macrophages (HMDMs) were incubated with human apoA-I under conditions used to induce cholesterol export. Two-dimensional gel electrophoresis and Western blot analysis identified that apoA-I is cleaved (∼20-80%) by HMDMs in a time-dependent manner, generating apoA-I of lower MW and isoelectric point. Mass spectrometry analysis identified a novel C-terminal cleavage site of apoA-I between Ser 228 -Phe 229 Recombinant apoA-I truncated at Ser 228 demonstrated profound loss of capacity to solubilize lipid and to promote ABCA1-dependent cholesterol efflux. Protease inhibitors, small interfering RNA knockdown in HMDMs, mass spectrometry analysis, and cathepsin B activity assays identified secreted cathepsin B as responsible for apoA-I cleavage at Ser 228 Importantly, C-terminal cleavage of apoA-I was also detected in human carotid plaque. Cleavage at Ser 228 is a novel, functionally important post-translational modification of apoA-I mediated by HMDMs that limits the antiatherogenic properties of apoA-I.-Dinnes, D. L. M., White, M. Y., Kockx, M., Traini, M., Hsieh, V., Kim, M.-J., Hou, L., Jessup, W., Rye, K.-A., Thaysen-Andersen, M., Cordwell, S. J., Kritharides, L. Human macrophage cathepsin B-mediated C-terminal cleavage of apolipoprotein A-I at Ser 228 severely impairs antiatherogenic capacity., (© FASEB.)

Epidemiologic studies have shown an inverse correlation between high-density lipoprotein (HDL) cholesterol (HDL-C) levels and cardiovascular disease outcomes. However, the hypothesis of a causal relationship between HDL-C and cardiovascular disease has been challenged by genetic and clinical studies. Serum cholesterol efflux capacity (CEC) is an important measure of HDL function in humans. Recent large clinical studies have shown a correlation between in vitro CEC and cardiovascular disease prevalence and incidence, which appears to be independent of HDL-C concentration. The present review summarizes recent large clinical studies and introduces important methodological considerations. Further studies are required to standardize and establish the reproducibility of this measure of HDL function and clarify whether modulating CEC will emerge as a useful therapeutic target., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: Cyclosporin A (CsA) is an immunosuppressant commonly used to prevent organ rejection but is associated with hyperlipidemia and an increased risk of cardiovascular disease. Although studies suggest that CsA-induced hyperlipidemia is mediated by inhibition of low-density lipoprotein receptor (LDLr)-mediated lipoprotein clearance, the data supporting this are inconclusive. We therefore sought to investigate the role of the LDLr in CsA-induced hyperlipidemia by using Ldlr-knockout mice (Ldlr(-/-))., Approach and Results: Ldlr(-/-) and wild-type (wt) C57Bl/6 mice were treated with 20 mg/kg per d CsA for 4 weeks. On a chow diet, CsA caused marked dyslipidemia in Ldlr(-/-) but not in wt mice. Hyperlipidemia was characterized by a prominent increase in plasma very low-density lipoprotein and intermediate-density lipoprotein/LDL with unchanged plasma high-density lipoprotein levels, thus mimicking the dyslipidemic profile observed in humans. Analysis of specific lipid species by liquid chromatography-tandem mass spectrometry suggested a predominant effect of CsA on increased very low-density lipoprotein-IDL/LDL lipoprotein number rather than composition. Mechanistic studies indicated that CsA did not alter hepatic lipoprotein production but did inhibit plasma clearance and hepatic uptake of [(14)C]cholesteryl oleate and glycerol tri[(3)H]oleate-double-labeled very low-density lipoprotein-like particles. Further studies showed that CsA inhibited plasma lipoprotein lipase activity and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9., Conclusions: We demonstrate that CsA does not cause hyperlipidemia via direct effects on the LDLr. Rather, LDLr deficiency plays an important permissive role for CsA-induced hyperlipidemia, which is associated with abnormal lipoprotein clearance, decreased lipoprotein lipase activity, and increased levels of apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9. Enhancing LDLr and lipoprotein lipase activity and decreasing apolipoprotein C-III and proprotein convertase subtilisin/kexin type 9 levels may therefore provide attractive treatment targets for patients with hyperlipidemia receiving CsA., (© 2016 American Heart Association, Inc.)

Aims: Macrophage apoptosis is a prominent feature of atherosclerosis, yet whether cell death-protected macrophages would favour the resolution of already established atherosclerotic lesions, and thus hold therapeutic potential, remains unknown., Methods and Results: We irradiated then transplanted into Apoe(-/-) or LDLr(-/-) recipient mice harbouring established atherosclerotic lesions, bone marrow cells from mice displaying enhanced macrophage survival through overexpression of the antiapoptotic gene hBcl-2 (Mø-hBcl2 Apoe(-/-) or Mø-hBcl2 Apoe(+/+) LDLr(-/-)). Both recipient mice exhibited decreased lesional apoptotic cell content and reduced necrotic areas when repopulated with Mø-hBcl2 mouse-derived bone marrow cells. In contrast, only LDLr(-/-) recipients showed a reduction in plasma cholesterol levels and in atherosclerotic lesions. The absence of significant reduction of plasma cholesterol levels in the context of apoE deficiency highlighted macrophage-derived apoE as key in both the regulation of plasma and tissue cholesterol levels and the progression of pre-existing lesion. Accordingly, hBcl2 expression in macrophages was associated with larger pools of Kupffer cells and Ly-6C(low) monocytes, both high producers of apoE. Additionally, increased Kupffer cells population was associated with improved clearance of apoptotic cells and modified lipoproteins., Conclusion: Collectively, these data show that promoting macrophage survival provides a supplemental source of apoE, which hinders pre-existing plaque progression., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Rationale: High-density lipoprotein (HDL) is a heterogeneous population of particles. Differences in the capacities of HDL subfractions to remove cellular cholesterol may explain variable correlations between HDL-cholesterol and cardiovascular risk and inform future targets for HDL-related therapies. The ATP binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux to lipid-free apolipoprotein A-I, but the majority of apolipoprotein A-I in the circulation is transported in a lipidated state and ABCA1-dependent efflux to individual HDL subfractions has not been systematically studied., Objective: Our aims were to determine which HDL particle subfractions are most efficient in mediating cellular cholesterol efflux from foam cell macrophages and to identify the cellular cholesterol transporters involved in this process., Methods and Results: We used reconstituted HDL particles of defined size and composition, isolated subfractions of human plasma HDL, cell lines stably expressing ABCA1 or ABCG1, and both mouse and human macrophages in which ABCA1 or ABCG1 expression was deleted. We show that ABCA1 is the major mediator of macrophage cholesterol efflux to HDL, demonstrating most marked efficiency with small, dense HDL subfractions (HDL3b and HDL3c). ABCG1 has a lesser role in cholesterol efflux and a negligible role in efflux to HDL3b and HDL3c subfractions., Conclusions: Small, dense HDL subfractions are the most efficient mediators of cholesterol efflux, and ABCA1 mediates cholesterol efflux to small dense HDL and to lipid-free apolipoprotein A-I. HDL-directed therapies should target increasing the concentrations or the cholesterol efflux capacity of small, dense HDL species in vivo., (© 2015 American Heart Association, Inc.)

Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcgl-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ- tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macro- phages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux.

Cholesterol-loaded foam cell macrophages are prominent in atherosclerotic lesions and play complex roles in both inflammatory signaling and lipid metabolism, which are underpinned by large scale reprogramming of gene expression. We performed a microarray study of primary human macrophages that showed that transcription of the sphingomyelin phosphodiesterase acid-like 3A (SMPDL3A) gene is up-regulated after cholesterol loading. SMPDL3A protein expression in and secretion from primary macrophages are stimulated by cholesterol loading, liver X receptor ligands, and cyclic AMP, and N-glycosylated SMPDL3A protein is detectable in circulating blood. We demonstrate for the first time that SMPDL3A is a functional phosphodiesterase with an acidic pH optimum. We provide evidence that SMPDL3A is not an acid sphingomyelinase but unexpectedly is active against nucleotide diphosphate and triphosphate substrates at acidic and neutral pH. SMPDL3A is a major source of nucleotide phosphodiesterase activity secreted by liver X receptor-stimulated human macrophages. Extracellular nucleotides such as ATP may activate pro-inflammatory responses in immune cells. Increased expression and secretion of SMPDL3A by cholesterol-loaded macrophage foam cells in lesions may decrease local concentrations of pro-inflammatory nucleotides and potentially represent a novel anti-inflammatory axis linking lipid metabolism with purinergic signaling in atherosclerosis., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Dynamins are fission proteins that mediate endocytic and exocytic membrane events and are pharmacological therapeutic targets. These studies investigate whether dynamin II regulates constitutive protein secretion and show for the first time that pharmacological inhibition of dynamin decreases secretion of apolipoprotein E (apoE) and several other proteins constitutively secreted from primary human macrophages. Inhibitors that target recruitment of dynamin to membranes (MiTMABs) or directly target the GTPase domain (Dyngo or Dynole series), dose- and time- dependently reduced the secretion of apoE. SiRNA oligo's targeting all isoforms of dynamin II confirmed the involvement of dynamin II in apoE secretion. Inhibition of secretion was not mediated via effects on mRNA or protein synthesis. 2D-gel electrophoresis showed that inhibition occurred after apoE was processed and glycosylated in the Golgi and live cell imaging showed that inhibited secretion was associated with reduced post-Golgi movement of apoE-GFP-containing vesicles. The effect was not restricted to macrophages, and was not mediated by the effects of the inhibitors on microtubules. Inhibition of dynamin also altered the constitutive secretion of other proteins, decreasing the secretion of fibronectin, matrix metalloproteinase 9, Chitinase-3-like protein 1 and lysozyme but unexpectedly increasing the secretion of the inflammatory mediator cyclophilin A. We conclude that pharmacological inhibitors of dynamin II modulate the constitutive secretion of macrophage apoE as a class effect, and that their capacity to modulate protein secretion may affect a range of biological processes.

The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes.

Objective: This EAS Consensus Panel critically appraised evidence relevant to the benefit to risk relationship of functional foods with added plant sterols and/or plant stanols, as components of a healthy lifestyle, to reduce plasma low-density lipoprotein-cholesterol (LDL-C) levels, and thereby lower cardiovascular risk., Methods and Results: Plant sterols/stanols (when taken at 2 g/day) cause significant inhibition of cholesterol absorption and lower LDL-C levels by between 8 and 10%. The relative proportions of cholesterol versus sterol/stanol levels are similar in both plasma and tissue, with levels of sterols/stanols being 500-/10,000-fold lower than those of cholesterol, suggesting they are handled similarly to cholesterol in most cells. Despite possible atherogenicity of marked elevations in circulating levels of plant sterols/stanols, protective effects have been observed in some animal models of atherosclerosis. Higher plasma levels of plant sterols/stanols associated with intakes of 2 g/day in man have not been linked to adverse effects on health in long-term human studies. Importantly, at this dose, plant sterol/stanol-mediated LDL-C lowering is additive to that of statins in dyslipidaemic subjects, equivalent to doubling the dose of statin. The reported 6-9% lowering of plasma triglyceride by 2 g/day in hypertriglyceridaemic patients warrants further evaluation., Conclusion: Based on LDL-C lowering and the absence of adverse signals, this EAS Consensus Panel concludes that functional foods with plant sterols/stanols may be considered 1) in individuals with high cholesterol levels at intermediate or low global cardiovascular risk who do not qualify for pharmacotherapy, 2) as an adjunct to pharmacologic therapy in high and very high risk patients who fail to achieve LDL-C targets on statins or are statin- intolerant, 3) and in adults and children (>6 years) with familial hypercholesterolaemia, in line with current guidance. However, it must be acknowledged that there are no randomised, controlled clinical trial data with hard end-points to establish clinical benefit from the use of plant sterols or plant stanols., (Copyright © 2013 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Macrophage-specific apolipoprotein E (apoE) secretion plays an important protective role in atherosclerosis. However, the precise signaling mechanisms regulating apoE secretion from primary human monocyte-derived macrophages (HMDMs) remain unclear. Here we investigate the role of protein kinase C (PKC) in regulating basal and stimulated apoE secretion from HMDMs. Treatment of HMDMs with structurally distinct pan-PKC inhibitors (calphostin C, Ro-31-8220, Go6976) and a PKC inhibitory peptide all significantly decreased apoE secretion without significantly affecting apoE mRNA or apoE protein levels. The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated apoE secretion, and both PMA-induced and apoAI-induced apoE secretion were inhibited by PKC inhibitors. PKC regulation of apoE secretion was found to be independent of the ATP binding cassette transporter ABCA1. Live cell imaging demonstrated that PKC inhibitors inhibited vesicular transport of apoE to the plasma membrane. Pharmacological or peptide inhibitor and knockdown studies indicate that classical isoforms PKCα/β and not PKCδ, -ε, -θ, or -ι/ζ isoforms regulate apoE secretion from HMDMs. The activity of myristoylated alanine-rich protein kinase C substrate (MARCKS) correlated with modulation of PKC activity in these cells, and direct peptide inhibition of MARCKS inhibited apoE secretion, implicating MARCKS as a downstream effector of PKC in apoE secretion. Comparison with other secreted proteins indicated that PKC similarly regulated secretion of matrix metalloproteinase 9 and chitinase-3-like-1 protein but differentially affected the secretion of other proteins. In conclusion, PKC regulates the secretion of apoE from primary human macrophages.

It is recognized that the development of atherosclerosis involves many elements of an inflammatory process, involving components of both the innate and adaptive immune systems. The presence and roles of macrophages and T-cells in atherogenesis are well-established. More recently dendritic cells have been identified in the vasculature and in atherosclerotic lesions. This review summarises our current understanding of the roles of dendritic cells in the development and regression of atherosclerosis.

ABCG1 is an ABC half-transporter that exports cholesterol from cells to HDL. This study set out to investigate differences in posttranslational processing of two human ABCG1 protein isoforms, termed ABCG1(+12) and ABCG1(-12), that differ by the presence or absence of a 12 amino acid peptide. ABCG1(+12) is expressed in human cells and tissues, but not in mice. We identified two protein kinase A (PKA) consensus sites in ABCG1(+12), absent from ABCG1(-12). Inhibition of PKA with either of two structurally unrelated inhibitors resulted in a dose-dependent increase in cholesterol export from cells expressing ABCG1(+12), whereas ABCG1(-12)-expressing cells were unaffected. This was associated with stabilization of the ABCG1(+12) protein, and ABCG1(+12)-S389 was necessary to mediate these effects. Mutation of this serine to aspartic acid, simulating a constitutively phosphorylated state, resulted in accelerated degradation of ABCG1(+12) and reduced cholesterol export. Engineering an equivalent PKA site into ABCG1(-12) rendered this isoform responsive to PKA inhibition, confirming the relevance of this sequence. Together, these results demonstrate an additional level of complexity to the posttranslational control of this human ABCG1 isoform that is absent from ABCG1(-12) and the murine ABCG1 homolog.

Cholesterol excess is typical of various diseases including atherosclerosis. We have investigated whether cholesterol accumulation in the ER (endoplasmic reticulum) can inhibit exit of vesicular cargo and secretion of proteins by studying apoE (apolipoprotein E), a significant glycoprotein in human health and disease. CHO (Chinese hamster ovary) cells expressing human apoE under a cholesterol-independent promoter incubated with cholesterol-cyclodextrin complexes showed increased levels of cellular free and esterified cholesterol, inhibition of SREBP-2 (sterol-regulatory-element-binding protein 2) processing, and a mild induction of ER stress, indicating significant accumulation of cholesterol in the ER. Secretion of apoE was markedly inhibited by cholesterol accumulation, and similar effects were observed in cells enriched with lipoprotein-derived cholesterol and in primary human macrophages. Removal of excess cholesterol by a cyclodextrin vehicle restored apoE secretion, indicating that the transport defect was reversible. That cholesterol impaired protein trafficking was supported by the cellular accumulation of less sialylated apoE glycoforms, and by direct visualization of altered ER to Golgi transport of thermo-reversible VSVG (vesicular stomatitis virus glycoprotein) linked to GFP (green fluorescent protein). We conclude that intracellular accumulation of cholesterol in the ER reversibly inhibits protein transport and secretion. Strategies to correct ER cholesterol may restore homoeostatic processes and intracellular protein transport in conditions characterized by cholesterol excess.

How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties.

Objective: Bcl-x is the most abundantly expressed member of the Bcl-2 gene family in macrophages, but its role in macrophage apoptosis during atherogenesis is unknown., Methods and Results: We previously reported dual pro- and antiatherogenic effects of macrophage survival in early versus advanced atherosclerotic lesions, respectively, potentially reflecting growing impairment of efferocytosis during plaque progression. Here, we specifically inactivated Bcl-x in macrophages and evaluated its impact on atherosclerotic lesion formation in Apoe(-/-) mice at various stages of the disease. Bcl-x deficiency in macrophages increased their susceptibility to apoptosis, resulting in the depletion of tissue macrophages in vivo, including its major pool, Küppfer cells in the liver. We also observed increased cholesterol levels that were, however, not associated with any acceleration of early atherosclerotic plaque progression. This observation suggests that the atheroprotective effect of macrophage apoptosis at that stage of disease was counterbalanced by enhanced cholesterol levels. Bcl-x KO(mac)/Apoe(-/-) mice exhibited significantly larger advanced lesions than control mice. These lesions showed vulnerable traits. Such enhanced lesion size may occur as a result not only of apoptotic cell accumulation but also of elevated cholesterol levels., Conclusions: Modulation of macrophage resistance to apoptosis through targeted deletion of Bcl-x has a major impact on the entire macrophage cell population in the body, including Küpffer cells. Macrophage survival may, therefore, not only influence atherosclerotic plaque development and vulnerability but also cholesterol metabolism.

The growing prevalence of disorders of lipid and sterol homeostasis such as obesity and atherosclerosis in Western societies has increased demand for the development of therapies for their prevention and treatment. Crucial for this development is an understanding of the underlying cellular mechanisms which are involved in both healthy and diseased states. However, gaps remain in our knowledge of fundamental processes, such as intracellular sterol transport, lipid storage and mobilisation. Functional genomic strategies, such as genome-wide gene knockdown screens, are increasingly being exploited as powerful strategies to fill these gaps. This review discusses experimental approaches and findings in recent functional genomics studies of sterol and lipid biology, both in model organisms and human cells.

The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis., (© 2011 John Wiley & Sons A/S.)

High-density lipoprotein (HDL) mediated reverse cholesterol transport (RCT) is regarded to be crucial for prevention of foam cell formation and atherosclerosis. ABC-transporter A1 (ABCA1) and scavenger receptor BI (SR-BI) are involved in the biogenesis of HDL and the selective delivery of HDL cholesterol to the liver, respectively. In the present study, we phenotypically characterized mice lacking these two proteins essential for HDL metabolism. ABCA1×SR-BI double knockout (dKO) mice showed severe hypocholesterolemia mainly due to HDL loss, despite a 90% reduction of HDL cholesterol uptake by liver. VLDL production was increased in dKO mice. However, non-HDL cholesterol levels were reduced, probably due to enhanced clearance via LRP1. Hepatobiliary cholesterol transport and fecal sterol excretion were not impaired in dKO mice. In contrast, the macrophage RCT in dKO mice was markedly impaired as compared to WT mice, associated with the accumulation of macrophage foam cells in the lung and Peyer's patches. Strikingly, no atherosclerotic lesion formation was observed in dKO mice. In conclusion, both ABCA1 and SR-BI are essential for maintaining a properly functioning HDL-mediated macrophage RCT, while the potential anti-atherosclerotic functions of ABCA1 and SR-BI are not evident in dKO mice due to the absence of pro-atherogenic lipoproteins., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Type 2 diabetes and dyslipidemia are risk factors for cardiovascular disease. However, mechanisms by which hypertriglyceridemia influences atherogenesis remain unclear. We examined effects of dyslipidemic diabetic serum on macrophage lipid accumulation as a model of foam cell formation. Normal human macrophages were cultured in media supplemented with 10% serum from non-diabetic normolipidemic or non-diabetic hypercholesterolemic adults versus adults with Type 2 diabetes; diabetes and hypertriglyceridemia; or diabetes and hypercholesterolemia. Exposure to diabetic sera resulted in increased macrophage fatty acids (2-3 fold higher, both saturated and unsaturated). Macrophage expression of CD36, scavenger receptor A (SR-A) and stearoyl-CoA desaturase (SCD) was increased, most prominently in macrophages exposed to hypertriglyceridemic diabetic serum (twofold increase in CD36 and fourfold increase in SCD, p < 0.05). In these conditions, RNA inhibition of CD36 reduced macrophage free cholesterol (163.9 ± 10.5 vs. 221.9 ± 26.2 mmol free cholesterol/g protein, p = 0.04). RNA inhibition of SCD decreased macrophage fatty acid content, increased ABCA1 level and enhanced cholesterol efflux (18.0 ± 3.9 vs. 8.0 ± 0.8% at 48 h, p = 0.03). Diabetic dyslipidemia may contribute to accelerated atherosclerosis via alterations in macrophage lipid metabolism favoring foam cell formation. Increased expression of CD36 and SR-A would facilitate macrophage lipid uptake, while increased expression of SCD could block compensatory upregulation of ABCA1 and cholesterol efflux. Further studies are needed to clarify whether modulation of macrophage lipid metabolism might reduce progression of diabetic atherosclerosis.

Macrophages in arterial walls accumulate lipids leading to the development of atherosclerotic plaques. However, mechanisms underlying macrophage lipid accumulation and foam cell formation are often studied without accounting for risk factors such as dyslipidemia. We investigated the effect of varying concentrations of triglyceride (TG) within physiological range on macrophage fatty acid (FA) accumulation and expression of cholesterol efflux proteins. Human monocytes were cultured in media supplemented with 10% sera containing low (0.7 mmol/L) to high (1.4 mmol/L) TG. The resulting macrophages were harvested after 10 days for analysis of FA content and composition and expression of genes involved in lipid metabolism. Exposure to higher TG and lower HDL concentrations in media increased macrophage lipid content. Macrophages exposed to higher TG had increased total FA content compared with controls (876 μg/mg protein vs. 652 μg/mg protein) and greater proportions of C16:0, C18:1 and C18:2. Macrophage expression of both ABCA1 and ABCG1 cholesterol efflux proteins were reduced when higher TG concentrations were present in the media. Expression of scavenger receptor CD36, involved in lipoprotein uptake, was also downregulated in macrophages exposed to higher TG. Culturing macrophages in conditions of higher versus lower TG influenced macrophage FA content and composition, and levels of regulatory proteins. Replicating in vitro levels of dyslipidemia encountered in vivo may provide an informative model for investigation of atherogenesis.

Caveolin-1 (Cav1), a structural protein required for the formation of invaginated membrane domains known as caveolae, has been implicated in cholesterol trafficking and homeostasis. Here we investigated the contribution of Cav1 to apolipoprotein A-I (apoA-I) cell surface binding and intracellular processing using mouse embryonic fibroblasts (MEFs) derived from wild type (WT) or Cav1-deficient (Cav1(-/-)) animals. We found that cells expressing Cav1 have 2.6-fold more apoA-I binding sites than Cav1(-/-) cells although these additional binding sites are not associated with detergent-free lipid rafts. Further, Cav1-mediated binding targets apoA-I for internalization and degradation and these processes are not correlated to cholesterol efflux. Despite lower apoA-I binding, cholesterol efflux from Cav1(-/-) MEFs is 1.7-fold higher than from WT MEFs. Stimulation of ABCA1 expression with an LXR agonist enhances cholesterol efflux from both WT and Cav1(-/-) cells without increasing apoA-I surface binding or affecting apoA-I processing. Our results indicate that there are at least two independent lipid binding sites for apoA-I; Cav1-mediated apoA-I surface binding and uptake is not linked to cholesterol efflux, indicating that membrane domains other than caveolae regulate ABCA1-mediated cholesterol efflux.

Rationale: macrophages cannot limit the uptake of lipids and rely on cholesterol efflux mechanisms for maintaining cellular cholesterol homeostasis. Important mediators of macrophage cholesterol efflux are ATP-binding cassette transporter 1 (ABCA1), which mediates the efflux of cholesterol to lipid-poor apolipoprotein AI, and scavenger receptor class B type I (SR-BI), which promotes efflux to mature high-density lipoprotein., Objective: the aim of the present study was to increase the insight into the putative synergistic roles of ABCA1 and SR-BI in foam cell formation and atherosclerosis., Methods and Results: low-density lipoprotein receptor knockout (LDLr KO) mice were transplanted with bone marrow from ABCA1/SR-BI double knockout mice, the respective single knockouts, or wild-type littermates. Serum cholesterol levels were lower in ABCA1/SR-BI double knockout transplanted animals, as compared to the single knockout and wild-type transplanted animals on Western-type diet. Despite the lower serum cholesterol levels, massive foam cell formation was found in macrophages from spleen and the peritoneal cavity. Interestingly, ABCA1/SR-BI double knockout transplanted animals also showed a major increase in proinflammatory KC (murine interleukin-8) and interleukin-12p40 levels in the circulation. Furthermore, after 10 weeks of Western-type diet feeding, atherosclerotic lesion development in the aortic root was more extensive in the LDLr KO mice reconstituted with ABCA1/SR-BI double knockout bone marrow., Conclusions: deletion of ABCA1 and SR-BI in bone marrow-derived cells enhances in vivo macrophage foam cell formation and atherosclerotic lesion development in LDLr KO mice on Western diet, indicating that under high dietary lipid conditions, both macrophage ABCA1 and SR-BI contribute significantly to cholesterol homeostasis in the macrophage in vivo and are essential for reducing the risk for atherosclerosis.

Cyclosporin A (CsA) is an immunosuppressant drug widely used in organ transplant recipients and people with autoimmune disorders. Long term treatment with CsA is associated with many side effects including hyperlipidemia and an increased risk of atherosclerosis. While its immunosuppressive effects are closely linked to its effects on T cell activation via the inhibition of the nuclear factor of activated T cells (NFAT) pathway, the precise mechanisms underlying its cardiovascular effects appear to involve multiple pathways additional to those relevant for immunosuppression. These include inhibition of calcineurin activity and intracellular cyclophilin peptidylprolyl isomerase and chaperone activities, inhibition of pro-inflammatory extracellular cyclophilin A, and NFAT-independent transcriptional effects. CsA demonstrates complex effects on lipoprotein metabolism and bile acid production, and affects endothelial cells, smooth muscle cells and macrophages, all of which are critical to the atherosclerotic process. Interpretation of the available data is hampered as many experimental models are used to study the effects of CsA in vivo and in vitro, leading to diverse and often contradictory findings. In this review we will describe the cellular mechanisms related to CsA-induced hyperlipidemia and atherosclerosis, with a focus on identifying pro-atherogenic pathways that are distinct from those relevant to its immunosuppressant effects. The potential of CsA analogues to avoid such sequelae will be discussed., (2010 Elsevier Inc. All rights reserved.)

Objective: Infusions of apoA-I in the lipid-free form or as a constituent of discoidal reconstituted high-density lipoproteins, (A-I)rHDL, markedly inhibit acute vascular inflammation in normocholesterolemic New Zealand White (NZW) rabbits. This effect is apparent even when apoA-I is administered 24h prior to the inflammatory insult. The present study asks if this benefit is related to an improved anti-inflammatory capacity of the high-density lipoprotein (HDL) fraction, or to increased arterial expression of genes that inhibit inflammation., Methods and Results: The ability of apoA-I to increase the anti-inflammatory capacity of HDL was assessed by infusing normocholesterolemic NZW rabbits with saline, lipid-free apoA-I or (A-I)rHDL. The infused apoA-I incorporated rapidly into the rabbit HDL fraction. The animals were sacrificed at 5 or 360 min post-infusion and plasma was collected. HDL were isolated by ultracentrifugation and incubated with cytokine-activated cultured human coronary artery endothelial cells. HDL from animals sacrificed at 5 min post-apoA-I infusion had a slightly enhanced anti-inflammatory capacity relative to HDL from the saline-infused animals. The anti-inflammatory capacity of HDL from the animals sacrificed at 360 min post-apoA-I infusion was comparable to that of HDL from the saline-infused animals. The effect of (A-I)rHDL infusions on arterial 3β-hydroxysteroid-Δ24 reductase (DHCR24) and endothelial adhesion molecule expression was investigated in cholesterol-fed NZW rabbits. Relative to animals infused with saline, (A-I)rHDL infusions decreased aortic VCAM-1 and ICAM-1 protein expression by 73 and 54%, respectively (p<0.05), and increased DHCR24 mRNA levels by 56% (p<0.0001)., Conclusion: ApoA-I inhibits vascular inflammation in NZW rabbits, at least in part, by increasing DHCR24 expression., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Apolipoprotein E (apoE) is a 34-kDa glycoprotein secreted from various cells including hepatocytes and macrophages and plays an important role in remnant lipoprotein clearance, immune responses, Alzheimer disease, and atherosclerosis. Cellular apoE and plasma apoE exist as multiple glycosylated and sialylated glycoforms with plasma apoE being less glycosylated/sialylated than cell-derived apoE. Some of the glycan structures on plasma apoE are characterized; however, the more complicated structures on plasma and cellular/secreted apoE remain unidentified. We investigated glycosylation and sialylation of cellular and secreted apoE from primary human macrophages by one- and two-dimensional gel electrophoresis and mass spectrometry. Our results identify eight different glycoforms with (HexNAc)(2)-Hex(2)-(NeuAc)(2) being the most complex glycan detected on Thr(194) in both cellular and secreted apoE. Four additional glycans were identified on apoE(283-299), and using beta-elimination/alkylation by methylamine in vitro, we identified Ser(290) as a novel site of glycan attachment. Comparison of plasma and cellular/secreted apoE from the same donor confirmed that cell-derived apoE is more extensively sialylated than plasma apoE. Given the importance of the C terminus of apoE in regulating apoE solubility, stability, and lipid binding, these results may have important implications for our understanding of apoE biochemistry.

Objective: To evaluate the expression of two ABCG1 isoforms that differ in the presence or absence of a 12 amino acid (AA) peptide between the ABC cassette and the transmembrane region, termed ABCG1(+12) and ABCG1(-12), respectively, in human vascular cells and tissues., Methods and Results: mRNA for both isoforms was expressed in human macrophages, vascular endothelial and smooth muscle cells as well as whole human spleen, lung, liver and brain tissue. However, ABCG1(+12) was not expressed in mouse tissues. 2D gel electrophoresis of ABCG1 protein indicated that both protein isoforms were expressed in human macrophages. Furthermore the half-lives of the two ABCG1 protein isoforms, stably expressed in CHOK1 cells, measured under basal conditions were different, suggesting the presence of a degradation or stabilising signal in or near the 12AA region of ABCG1(+12)., Conclusion: ABCG1(+12) is an isoform of ABCG1 exclusively expressed in human cells at the RNA and protein level. As ABCG1(+12) is not expressed in mice, although mouse models are widely used to elucidate the function of ABCG1, further investigations into the importance of this human ABCG1 isoform are warranted., (Copyright (c) 2009 Elsevier Ireland Ltd. All rights reserved.)

The aim of this study was to correlate the lipid content and size of discoidal reconstituted HDL particles with their ability to promote cellular cholesterol efflux. Homogeneous discoidal rHDL particles containing apoA-I and POPC, with diameters of 7.8, 9.6, 10.8, 12.5, and 17.0 nm, were prepared by the cholate dialysis technique. Cholesterol efflux to rHDL was evaluated in pathway-specific cell models for ABCA1-, ABCG1-, and SR-BI-mediated efflux. ABCA1-mediated efflux was efficiently promoted by the 7.8 nm rHDL containing 82 POPC molecules per particle. This rHDL also promoted ABCG1, but not SR-BI, cholesterol efflux. All large and lipid-rich rHDLs, with a diameter of >or=9.6 nm and a phospholipid content of >/=202 molecules per particle, promoted both SR-BI- and ABCG1-mediated efflux. Our results indicated that the ABCA1-mediated cell cholesterol efflux can be efficiently driven not only by monomolecular lipid free/poor apoA-I but also by a small discoidal phospholipid-containing particle resembling plasma pre-beta1 HDL. This same particle also promotes ABCG1- but not SR-BI-mediated efflux. These results help to clarify the role of plasma pre-beta1 HDL in reverse cholesterol transport.

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., Conclusions: 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.

Cyclosporin A (CsA) is an immunosuppressant that inhibits protein phosphatase 2B (PP2B/calcineurin) and is associated with hyperlipidemia, decreased cholesterol efflux via ATP-binding cassette transporter A1 (ABCA1), and increased risk of atherosclerosis. Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, the secretion of which from human macrophages is regulated by the serine/threonine protein kinase A (PKA) and intracellular calcium (Ca(2+)) (Kockx, M., Guo, D. L., Huby, T., Lesnik, P., Kay, J., Sabaretnam, T., Jary, E., Hill, M., Gaus, K., Chapman, J., Stow, J. L., Jessup, W., and Kritharides, L. (2007) Circ. Res. 101, 607-616). As PP2B is Ca(2+)-dependent and has been linked to PKA-dependent processes, we investigated whether CsA modulated apoE secretion. CsA dose- and time-dependently inhibited secretion of apoE from primary human macrophages and from Chinese hamster ovary cells stably transfected with human apoE and increased cellular apoE levels without affecting apoE mRNA. [(35)S]Met kinetic modeling studies showed that CsA inhibited both secretion and degradation of apoE, increasing the half-life of cellular apoE 2-fold. CsA also inhibited secretion from primary human Tangier disease macrophages and from mouse macrophages deficient in ABCA1, indicating that the effect is independent of the known inhibition of ABCA1 by CsA. The role of PP2B in mediating apoE secretion was confirmed using additional peptide and chemical inhibitors of PP2B. Importantly, kinetic modeling, live-cell imaging, and confocal microscopy all indicated that CsA inhibited apoE secretion by mechanisms quite distinct from those of PKA inhibition, most likely inducing accumulation of apoE in the endoplasmic reticulum compartment. Taken together, these results establish a novel mechanism for the pro-atherosclerotic effects of CsA, and establish for the first time a role for PP2B in regulating the intracellular transport and secretion of apoE.

Objective: Oxidized low-density lipoprotein (oxLDL) induces survival of colony stimulating factor-1 (CSF-1)-dependent macrophages in vitro. Because atherosclerotic lesion-associated macrophages take up large amounts of glucose, we investigated whether, and how, oxLDL promotes glucose uptake and how glucose metabolism regulates oxLDL-induced macrophage survival., Methods and Results: OxLDL-induced macrophage survival required glucose metabolism. OxLDL stimulated 2 phases of glucose uptake, namely acute and chronic, which required PI3K but not MEK1/2 activity. PI3K appeared to regulate glucose transport via glucose transporter affinity and/or mobilization. OxLDL also maintained levels of the prosurvival proteins, Bcl-2 and Bcl-x(L), after CSF-1 had been removed through a combination of mechanisms including transcription, translation, and protein stabilization. Significantly, inhibition of glucose metabolism reduced Bcl-2 and Bcl-x(L) protein levels. MEK1/2 and PI3K activities were also required for oxLDL-induced Bcl-2 and Bcl-x(L) mRNA upregulation., Conclusions: These results suggest that oxLDL enhances macrophage survival in the absence of CSF-1 by inducing PI3K-dependent glucose uptake, which is metabolized to maintain Bcl-2 and Bcl-x(L) protein levels.

Objective: ATP-binding cassette transporter G1 (Abcg1) and apolipoprotein E (Apoe) play a role in macrophage cholesterol efflux and consequently the development of atherosclerosis. A possible interaction between Abcg1 and Apoe in cholesterol efflux was postulated, but the potential combined action of these proteins on atherosclerotic lesion formation is unclear., Methods: LDL receptor knockout (KO) mice were transplanted with bone marrow from Abcg1/Apoe double KO (dKO) mice, their respective single knockouts, and wild-type (WT) controls and challenged with a high-fat/high-cholesterol diet for 6 weeks to induce atherosclerosis., Results: No differences were found in serum lipid levels. The mean atherosclerotic lesion area in dKO transplanted animals (187+/-18x10(3)microm(2)) was 1.4-fold (p<0.01) increased compared to single knockouts (Abcg1 KO: 138+/-5x10(3)microm(2); Apoe KO: 131+/-7x10(3)microm(2)) and 1.9-fold (p<0.001) as compared to WT controls (97+/-15x10(3)microm(2)). In vitro cholesterol efflux experiments established that combined deletion of Abcg1 and Apoe leads to a larger attenuation of macrophage cholesterol efflux to HDL as compared to single knockouts., Conclusions: Single deletion of macrophage Abcg1 or Apoe does lead to a moderate non-significant increase in atherosclerotic lesion development as tested by ANOVA, while combined deletion of Abcg1 and Apoe induces a more dramatic and significant increase in atherosclerosis. Our results indicate an additive, independent effect for both macrophage Abcg1 and Apoe in the prevention of atherosclerosis.

Lipid rafts defined as cholesterol- and sphingomyelin-rich domains have been isolated from different cell types that vary greatly in their lipid profiles. Here, we investigated the contribution of the structural protein caveolin-1 (Cav1) to the overall lipid composition and domain abundance in mouse embryonic fibroblasts (MEFs) from wild-type (WT) or Cav1-deficient (Cav1(-/-)) animals. Our findings show that Cav1 expression had no effect on free (membrane-associated) cholesterol levels. However, Cav1(-/-)-deficient cells did have a higher proportion of sphingomyelin, decreased abundance of unsaturated phospholipids, and a trend toward shorter fatty acid chains in phosphatidylcholine. We isolated detergent-resistant membranes (DRMs), nondetergent raft domains (NDR), and cholesterol oxidase (CO)-sensitive domains and assessed the abundance of ordered domains in intact cells using the fluorescent dye Laurdan. Despite differences in phospholipid composition, we found that cholesterol levels in DRMs, NDR, and CO-sensitive domains were similar in both cell types. The data suggest that Cav1 is not required to target cholesterol to lipid rafts and that CO does not specifically oxidize caveolar cholesterol. In contrast, the abundance of ordered domains in adherent cells is reduced in Cav1(-/-) compared with WT MEFs, suggesting that cell architecture is critical in maintaining Cav1-induced lipid rafts.

Macrophages, cytokines, and matrix metalloproteinases (MMP) play important roles in atherogenesis. The Ca(2+)-binding protein S100A12 regulates monocyte migration and may contribute to atherosclerosis by inducing proinflammatory cytokines in macrophages. We found significantly higher S100A12 levels in sera from patients with coronary artery disease than controls and levels correlated positively with C-reactive protein. S100A12 was released into the coronary circulation from ruptured plaque in acute coronary syndrome, and after mechanical disruption by percutaneous coronary intervention in stable coronary artery disease. In contrast to earlier studies, S100A12 did not stimulate proinflammatory cytokine production by human monocytes or macrophages. Similarly, no induction of MMP genes was found in macrophages stimulated with S100A12. Because S100A12 binds Zn(2+), we studied some functional aspects that could modulate atherogenesis. S100A12 formed a hexamer in the presence of Zn(2+); a novel Ab was generated that specifically recognized this complex. By chelating Zn(2+), S100A12 significantly inhibited MMP-2, MMP-9, and MMP-3, and the Zn(2+)-induced S100A12 complex colocalized with these in foam cells in human atheroma. S100A12 may represent a new marker of this disease and may protect advanced atherosclerotic lesions from rupture by inhibiting excessive MMP-2 and MMP-9 activities by sequestering Zn(2+).

Oxysterols are structurally identical to cholesterol, but with one or more additional oxygen containing functional groups (such as alcohol, carbonyl or epoxide groups). The wide array of oxysterols encountered in human health and disease vary in their origin (either enzymic or non-enzymic), and their putative effects and/or function(s). Some are thought to be damaging, whereas others may play important physiological roles, including in the regulation of cholesterol homeostasis. In this review, we will concentrate on the major cellular oxysterols. We summarise their location, generation, metabolism and elimination, as well as providing insights into the latest research into their regulatory roles in cholesterol homeostasis.

Objectives: To evaluate the capacity of serum from carriers of LCAT gene mutations to promote cell cholesterol efflux through the ABCA1, ABCG1, and SR-BI pathways., Methods: Serum was obtained from 41 carriers of mutant LCAT alleles (14 carriers of two mutant LCAT alleles and 27 heterozygotes) and 10 non-carrier relatives (controls). The capacity of serum to promote cholesterol efflux was tested in pathway-specific cell models., Results: LCAT deficient sera were significantly more efficient than control sera in promoting cell cholesterol efflux via ABCA1 (3.1+/-0.3% for carriers of two mutant LCAT alleles and 2.6+/-0.2% for heterozygotes vs. 1.5+/-0.4% for controls), and less efficient in promoting ABCG1- and SR-BI-mediated cholesterol efflux. The enhanced capacity of LCAT deficient serum for ABCA1 efflux is explained by the increased content of prebeta-HDL, as indicated by the significant positive correlation between ABCA1 efflux and serum prebeta-HDL content (R=0.468, P<0.001). Moreover, chymase treatment of LCAT deficient serum selectively degraded prebeta-HDL and completely abolished ABCA1 efflux. Despite the remarkable reductions in serum HDL levels, LCAT deficient sera were as effective as control sera in removing mass cholesterol from cholesterol-loaded macrophages., Conclusions: Serum from carriers of LCAT gene mutations has the same capacity of control serum to decrease the cholesterol content of cholesterol-loaded macrophages due to a greater cholesterol efflux capacity via ABCA1.