Mechanism Of Cholesterol Efflux Research Articles

The benzoate plant metabolite ethyl gallate prevents cellular- and vascular-lipid accumulation in experimental models of atherosclerosis

Ethyl gallate (EG) is a well-known constituent of medicinal plants, but its effects on atherosclerosis development are not clear. In the present study, the anti-atherosclerosis effects of EG and the underlying mechanisms were explored using macrophage cultures, zebrafish and apolipoprotein (apo) E deficient mice. Treatment of macrophages with EG (20 μM) enhanced cellular cholesterol efflux to HDL, and reduced net lipid accumulation in response to oxidized LDL. Secretion of monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6) from activated macrophages was also blunted by EG. Fluorescence imaging techniques revealed EG feeding of zebrafish reduced vascular lipid accumulation and inflammatory responses in vivo. Similar results were obtained in apoE-/- mice 6.5 months of age, where plaque lesions and monocyte infiltration into the artery wall were reduced by 70% and 42%, respectively, after just 6 weeks of injections with EG (20 mg/kg). HDL-cholesterol increased 2-fold, serum cholesterol efflux capacity increased by ∼30%, and the levels of MCP-1 and IL-6 were reduced with EG treatment of mice. These results suggest EG impedes early atherosclerosis development by reducing the lipid and macrophage-content of plaque. Underlying mechanisms appeared to involve HDL cholesterol efflux mechanisms and suppression of pro-inflammatory cytokine secretion.

Probucol treatment is associated with an ABCA1-independent mechanism of cholesterol efflux to lipid poor apolipoproteins from foam cell macrophages

ObjectiveProbucol is a cholesterol-lowering agent whose ability to prevent atherosclerosis is currently under study. Herein, we investigate the putative mechanism of probucol by observation of changes in cellular cholesterol efflux and lipid droplet morphology in macrophages. ResultsThe inhibitory activity of probucol was assessed in non-foam or foam cell macrophages expressing ABCA1 generated by treatment with fetal calf serum (FCS) alone or in combination with acetylated LDL, respectively. Probucol inhibited cholesterol efflux to apolipoprotein A-I (apoA-I) by 31.5±0.1% in THP-1 non-foam cells and by 18.5±0.2% in foam cells. In probucol-treated non-foam THP-1 cells, nascent high density lipoprotein (nHDL) particles with a diameter < 7 nm were generated, while in probucol-treated THP-1 foam cells nHDL particles of > 7 nm in diameter containing cholesterol were produced. Foam cells also displayed a significant accumulation of free cholesterol at the plasma membrane, as measured by percent cholestenone formed. Intracellularly, there was a significant decrease in lipid droplet number and an increase in size in probucol-treated THP-1 foam cells when compared to non-treated cells. ConclusionsWe report for the first time that probucol is unable to completely inhibit cholesterol efflux in foam cells to the same extent as in non-foam cells. Indeed, functional nHDL is released from foam cells in the presence of probucol. This difference in inhibitory effect could potentially be explained by changes in the plasma membrane pool as well as intracellular cholesterol storage independently of ABCA1.

Regulation of ABCA1-mediated cholesterol efflux by sphingosine-1-phosphate signaling in macrophages

Sphingolipid and cholesterol metabolism are closely associated at the structural, biochemical, and functional levels. Although HDL-associated sphingosine-1-phosphate (S1P) contributes to several HDL functions, and S1P signaling regulates glucose and lipid metabolism, no study has addressed the involvement of S1P in cholesterol efflux. Here, we show that sphingosine kinase (Sphk) activity was induced by the LXR agonist 22(R)-hydroxycholesterol and required for the stimulation of ABCA1-mediated cholesterol efflux to apolipoprotein A-I. In support, pharmacological Sphk inhibition and Sphk2 but not Sphk1 deficiency abrogated efflux. The involved mechanism included stimulation of both transcriptional and functional ABCA1 regulatory pathways and depended for the latter on the S1P receptor 3 (S1P3). Accordingly, S1P3-deficient macrophages were resistant to 22(R)-hydroxycholesterol-stimulated cholesterol efflux. The inability of excess exogenous S1P to further increase efflux was consistent with tonic S1P3 signaling by a pool of constitutively generated Sphk-derived S1P dynamically regulating cholesterol efflux. In summary, we have established S1P as a previously unrecognized intermediate in LXR-stimulated ABCA1-mediated cholesterol efflux and identified S1P/S1P3 signaling as a positive-feedback regulator of cholesterol efflux. This constitutes a novel regulatory mechanism of cholesterol efflux by sphingolipids.

Marit Westerterp: Communication Is Key.

As an associate research scientist in Alan Tall’s group at Columbia University, New York, Marit Westerterp’s research has been focused on atherosclerosis and the molecular mechanisms of cholesterol efflux and transport1–5—a subject she will continue to pursue in her upcoming role as associate professor at the University of Groningen in the Netherlands. Circulation Research caught up with Westerterp during her busy transition period and discovered that although this young researcher is a little reserved when talking about her personal life, she is forthcoming and enlightening on the subjects of research and career. In fact, she stresses how important conversations with colleagues are for success in one’s scientific life. Communicating with fellow scientists, Westerterp says, has helped her to cope with tough times, to find new career opportunities, and to expand her research in new directions. And she now fosters such communications and exchanges of ideas in others through her work for the Netherland America Foundation, which promotes cultural and student exchanges between the 2 countries. In her conversation with Circulation Research , Westerterp did share a few personal details. For instance, she spoke of growing up in the Netherlands, of being a keen violinist, and of the fact that both her mother and father are themselves research biologists. Marit Westerterp No. Not really. I mean my parents always stimulated me, but it was not that I already knew at a very young age that I wanted to be a scientist. I had a lot of other creative interests. I was interested in arts and architecture. But at school, I was always interested in chemistry and scientific topics, so I knew I wanted to do something in that direction. I just have an interest in solving complex problems and in …

HDL Cholesterol Efflux Capacity: Cardiovascular Risk Factor and Potential Therapeutic Target.

Low high-density lipoprotein cholesterol (HDL-C) levels are associated with incident cardiovascular events; however, many therapies targeting increases in HDL-C have failed to show consistent clinical benefit. Thus, focus has recently shifted toward measuring high-density lipoprotein (HDL) function. HDL is the key mediator of reverse cholesterol transport, the process of cholesterol extraction from foam cells, and eventual excretion into the biliary system. Cholesterol efflux from peripheral macrophages to HDL particles has been associated with atherosclerosis in both animals and humans. We review the mechanism of cholesterol efflux and the emerging evidence on the association between cholesterol efflux capacity and cardiovascular disease in human studies. We also focus on the completed and ongoing trials of novel therapies targeting different aspects of HDL cholesterol efflux.

Abstract 224: Formation of Cholesterol-Enriched Nascent HDL Requires Helices 5 and 7

Heart disease claims nearly 400,000 individuals in the US per year. Large population studies have repeatedly demonstrated an inverse relationship between HDL concentration and risk. Despite the strong correlation, pharmacological trials that raise plasma HDL have shown little efficacy, suggesting that HDL function should be monitored. Studies were initiated into the mechanism of cholesterol efflux leading to nascent HDL (nHDL) formation. The most productive cholesterol removal occurs when lipid-poor apoA-I, the main structural protein in nHDL, forms particles after interacting with ABCA1. ApoA-I acquires cholesterol and phospholipid at the cell surface forming 10-12 nm diameter nHDL whose composition resembles lipid rafts. Because lipidated apoA-I has a significantly different 3-D conformation compared to its lipid-poor counterpart, the structural modifications leading to nHDL formation were investigated. First, we completed a 3-D solution structure of lipid-poor apoA-I using lysine specific chemical cross-linking in conjunction with specifically engineered double cysteine-containing apoA-I mutants. Strategically placed cysteines form disulfide bonds (locked) when residues are within ~0.5 nm. If no disulfide bond formed (unlocked) then the two cysteines were separated by more than ~0.5 nm. Both methods rely on mass spectrometry to identify amino acid sequence and distance constraints within each 10 repeating helices comprising 80% of full-length apoA-I. By comparing lipid-poor apoA-I conformation to our previously described 3-D structure of apoA-I on 10-12 nm nHDL, we formulated a series of hypothetical helical domain transitions that might drive protein-lipid interaction and particle formation. To test, 10 different double cysteine mutants were employed in either their “locked” (oxidized) or “unlocked” (reduced) state by evaluating their ability to form recombinant HDL using synthetic phospholipid or to form nHDL using HEK cells. After determining HDL particle yield, size and composition from all locked and unlocked double cysteine apoA-I mutants we conclude that both the N- and C-terminal ends of apoA-I are essential for the first steps in lipid acquisition, but that the central helices 5, 6 and 7 are essential for nHDL formation.

Effect of peroxisome proliferator-activated receptor γ on the cholesterol efflux of peritoneal macrophages in inflammation

Atherosclerosis, a chronic inflammatory disorder characterized by lipid and cholesterol accumulation, is the principal contributing factor to the pathology of cardiovascular disease. Macrophages contribute to plaque development by internalizing native and modified lipoproteins that convert them into cholesterol-rich foam cells. With multiple factors, including hypercholesterolemia and inflammation, promoting atherosclerosis, it is of great significance to elucidate how the mechanism of cholesterol efflux from the macrophages changes and the role of peroxisome proliferator-activated receptor γ (PPARγ) in these situations. Following isolation and culture of peritoneal macrophages from C57BL/6 mice in the present study, the cells were divided into three groups: The control group, the ciglitazone group and the PPARγ antisense oligonucleotide group. The expression of PPARγ and nuclear factor of κ light polypeptide gene enhancer in B‑cells inhibitorα (IκBα) in each group was observed through the levels of protein and mRNA, and then the cholesterol efflux of each group was investigated. In addition, the same experiments were repeated following stimulation of each group with lipopolysaccharide (LPS). No significant difference in the expression levels of PPARγ between the control group and ciglitazone group was observed. The expression levels of PPARγ in the PPARγ antisense oligonucleotide group were evidently lower than those in the control group. Subsequent to stimulation with LPS, the expression levels of PPARγ in the three groups were higher than those of each group prior to stimulation. The cholesterol efflux of the PPARγ antisense oligonucleotide group was clearly suppressed following stimulation with LPS in comparison with that of the other groups. PPARγ contributes to anti-inflammation by protecting IκBα from being phosphorylated and degraded and promoting cholesterol efflux from peritoneal macrophages in inflammation.

In vitro steatosis hepatic cell model to compare the lipid-lowering effects of pomegranate peel polyphenols with several other plant polyphenols as well as its related cholesterol efflux mechanisms

This study was aimed to compare the relative activities of the purified pomegranate peels polyphenols (PPPs) with some other plant polyphenols including punicalagin, ellagic acid, gallic acid, phlorizin, and epigallocatechin gallate (EGCG) on the lipid metabolism regulation, and the cholesterol efflux mechanisms of PPPs and punicalagin were also investigated. In this paper, a convenient and accurate in vitro HL7702 steatosis hepatic cell model was applied to evaluate the lipid-lowering effects of the tested polyphenols. The results showed that PPPs possessed the strongest lipid-lowering effects. Prevention group (treated with polyphenols when establishing of steatosis model) was more effective than treatment group (treated with polyphenols after establishment of steatosis model). Punicalagin displayed the strongest lipid-lowering effects among all the tested components of pomegranate peel polyphenols. Moreover, PPPs and punicalagin (10, 20, 40 μg/mL) significantly increased the mRNA expression of LXRα (Liver X receptor alpha) and its target genes-ABCA1 (ATP-binding cassette transporter A1) in a dose-dependent manner in HL7702 steatosis hepatic cells. The high mRNA expression of LXRα and ABCA1, next to lovastatin, was observed in cells treated with 40 μg/mL of PPPs. These in vitro findings suggested that PPPs might have great potential in the clinic treatment of hyperlipemia.

Research Advances of Intracellular Transport and Function of ABCA1

ATP-binding cassette transporter 1(ABCA1) mediated lipid efflux and evolved to maintain cholesterol homeostasis.Newborn ABCA1 should through intracellular transport and chemical modification,ultimately being a mature functional transporter with its function of lipid transport.The intracellular transport and correct localization of ABCA1 plays an important role in its function.ABCA1 researches focused on the lipid transport,and proposed the model of the various cholesterol efflux mechanisms,such as channel transport model,mushroom-like protuberances model and retroendocytosis model.Recent studies have shown,ABCA1 can regulate the plasma membrane lipid rafts,and involve in immune and inflammatory regulation.This review focuses on the current views on intracellular transport and various functions of ABCA1,in order to provide the new therapeutic targets for atherosclerosis-related diseases.

151 ECTOPIC EXPRESSION OF THE TERE1 (UBIAD1) PROTEIN INHIBITS GROWTH OF RENAL CLEAR CELL CARCINOMA CELLS AND REDUCES CHOLESTEROL

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2012151 ECTOPIC EXPRESSION OF THE TERE1 (UBIAD1) PROTEIN INHIBITS GROWTH OF RENAL CLEAR CELL CARCINOMA CELLS AND REDUCES CHOLESTEROL William Fredericks, Priti Lal, Nathaniel Fredericks, Stephen Malkowicz, John Tomaszewski, and S. Bruce Malkowicz William FredericksWilliam Fredericks Philadelphia, PA More articles by this author , Priti LalPriti Lal Philadelphia, PA More articles by this author , Nathaniel FredericksNathaniel Fredericks Philadelphia, PA More articles by this author , Stephen MalkowiczStephen Malkowicz Philadelphia, PA More articles by this author , John TomaszewskiJohn Tomaszewski Philadelphia, PA More articles by this author , and S. Bruce MalkowiczS. Bruce Malkowicz Philadelphia, PA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.201AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Elevated intracellular cholesterol is an emerging mechanism of tumor progression in several cancers, including renal clear cell carcinoma (RCC) and is based on the pleiotropic effects of cholesterol on metabolism, growth signaling, and apoptosis. Recently, we found that expression of the TERE1 protein can significantly reduce cholesterol levels in bladder and prostate cancer cell lines. This is consistent with the role of TERE1 in synthesis of vitamin K2, which is known to activate SXR-driven cholesterol efflux mechanisms. We also identified TBL2 as a TERE1-interacting protein that co-localizes to mitochondria. Our objective was to examine the relationship between TERE1, cell growth and the elevated cholesterol phenotype of RCC cells to support a potential tumor suppressor role for TERE1 as has been demonstrated with bladder and prostate cancer cells. METHODS A TMA panel of 100 RCC lesions was evaluated for TERE1 expression by immunohistochemistry. TERE1 and TBL2 dosage was manipulated in the RCC cell lines: Caki-1 and Caki-2 and in HEK 293 cells by adenovirus and nucleofection-mediated ectopic expression. Cell proliferation was examined via ATP luminometry and cellular cholesterol was determined by Amplex Red assay. Vitamins K2 and K3 were used as positive controls. RESULTS In 57% of RCC lesions, TERE1 expression was reduced (36%) or absent (21%). Ectopic TERE1 or TBL2 expression in Caki-1, Caki-2, and HEK 293 cells reduced cholesterol by up to 40%. Vitamins K2 and K3 (30μM) reduced cholesterol up to 50%. Over-expression of TERE1 resulted in an 80% decrease in growth of Caki-1 and Caki-2 cell lines over 10-days. CONCLUSIONS Current theories recognize that reduced cholesterol efflux may contribute to oxidative stress associated with malignant progression. Loss of TERE1 in RCC may contribute to the elevated cholesterol phenotype associated with progression. TERE1-mediated synthesis of the redox-cycling and alkylating quinones, vitamin K-2 and K-3, suggests that loss of TERE1 expression may be a mechanism tumors use to uncouple oxidative stress signaling in mitochondria from apoptosis or negative growth signaling by elevation of cholesterol. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e62-e63 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information William Fredericks Philadelphia, PA More articles by this author Priti Lal Philadelphia, PA More articles by this author Nathaniel Fredericks Philadelphia, PA More articles by this author Stephen Malkowicz Philadelphia, PA More articles by this author John Tomaszewski Philadelphia, PA More articles by this author S. Bruce Malkowicz Philadelphia, PA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Astaxanthin Enhances ATP-Binding Cassette Transporter A1/G1 Expressions and Cholesterol Efflux from Macrophages

ATP-binding cassette transporters (ABC) A1 and G1 are key molecules in cholesterol efflux from macrophages, which is an initial step of reverse cholesterol transport (RCT), a major anti-atherogenic property of high-density lipoprotein (HDL). Astaxanthin is one of the naturally occurring carotenoids responsible for the pink-red pigmentation in a variety of living organisms. Although astaxanthin is known to be a strong antioxidant, it remains unclear through what mechanism of action it affects cholesterol homeostasis in macrophages. We therefore investigated the effects of astaxanthin on cholesterol efflux and ABCA1/G1 expressions in macrophages. Astaxanthin enhanced both apolipoprotein (apo) A-I- and HDL-mediated cholesterol efflux from RAW264.7 cells. In supporting these enhanced cholesterol efflux mechanisms, astaxanthin promoted ABCA1/G1 expression in various macrophages. In contrast, peroxisome proliferator-activated receptor γ, liver X receptor (LXR) α and LXRβ levels remained unchanged by astaxanthin. An experiment using actinomycin D demonstrated that astaxanthin transcriptionally induced ABCA1/G1 expression, and oxysterol depletion caused by overexpression of cholesterol sulfotransferase further revealed that these inductions in ABCA1/G1 were independent of LXR-mediated pathways. Finally, we performed luciferase assays using human ABCA1/G1 promoter-reporter constructs to reveal that astaxanthin activated both promoters irrespective of the presence or absence of LXR-responsive elements, indicating LXR-independence of these activations. In conclusion, astaxanthin increased ABCA1/G1 expression, thereby enhancing apoA-I/HDL-mediated cholesterol efflux from the macrophages in an LXR-independent manner. In addition to the anti-oxidative properties, the potential cardioprotective properties of astaxanthin might therefore be associated with an enhanced anti-atherogenic function of HDL.

Mechanism of cholesterol efflux in humans after infusion of reconstituted high-density lipoprotein

Infusion of reconstituted HDL (rHDL) leads to changes in HDL metabolism as well as to an increased capacity of plasma to support cholesterol efflux providing an opportunity to investigate mechanisms linking cholesterol efflux to changes in plasma HDL. Patient plasmas after infusion of rHDL were tested ex vivo for their capacity to stimulate cholesterol efflux. Reconstituted HDL enhanced mobilization of cholesterol from tissues in vivo as shown by rising HDL cholesterol concentrations over the infusion period. Infusion of rHDL in vivo led to increased cholesterol efflux ex vivo; surprisingly, removing apoB-containing lipoproteins while preserving all HDL subfractions eliminated this increase. Infusion of rHDL led to the remodelling of plasma HDL; however, the capacity of plasma to support cholesterol efflux did not correlate with changes in the concentrations of any of HDL subfractions. Unmodified rHDL accounted for only a proportion of the increment in cholesterol efflux capacity. Furthermore, studies using HeLa and BHK cells overexpressing ABCA1, ABCG1, and SR-B1 showed that the contribution of these cellular mediators of cholesterol efflux to the enhanced capacity of plasma for the efflux was minimal. Enhanced cholesterol efflux from tissues requires the presence of apoB-containing lipoproteins and may involve enhanced flow of cholesterol through multiple components of the reverse cholesterol transport pathway rather than being determined by a specific HDL subfraction.

Enhanced Foam Cell Formation, Atherosclerotic Lesion Development, and Inflammation by Combined Deletion of ABCA1 and SR-BI in Bone Marrow–Derived Cells in LDL Receptor Knockout Mice on Western-Type Diet

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. 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. 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. 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.

Relative roles of various efflux pathways in net cholesterol efflux from macrophage foam cells in atherosclerotic lesions

Cholesterol efflux mechanisms are essential for macrophage cholesterol homeostasis. HDL, an important cholesterol efflux acceptor, comprises a class of heterogeneous particles that induce cholesterol efflux via distinct pathways. This review focuses on the understanding of the different cholesterol efflux pathways and physiological acceptors involved, and their regulation in atherosclerotic lesions. The synergistic interactions of ATP-binding cassette transporters A1 and G1 as well as ATP-binding cassette transporter A1 and scavenger receptor class B type I are essential for cellular cholesterol efflux and the prevention of macrophage foam cell formation. However, the importance of aqueous diffusion should also not be underestimated. Significant progress has been made in understanding the mechanisms underlying ATP-binding cassette A1-mediated cholesterol efflux and regulation of its expression and trafficking. Conditions locally in the atherosclerotic lesion, for example, lipids, cytokines, oxidative stress, and hypoxia, as well as systemic factors, including inflammation and diabetes, critically influence the expression of cholesterol transporters on macrophage foam cells. Furthermore, HDL modification and remodeling in atherosclerosis, inflammation, and diabetes impairs its function as an acceptor for cellular cholesterol. Recent advances in the understanding of the regulation of cholesterol transporters and their acceptors in atherosclerotic lesions indicate that HDL-based therapies should aim to enhance the activity of cholesterol transporters and improve both the quantity and quality of HDL.

Interleukin-10 Facilitates Both Cholesterol Uptake and Efflux in Macrophages

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by scavenger receptor in macrophages initiates the chronic proinflammatory cascade and necrosis core formation that characterize atherosclerosis. We report here that a cytokine considered to be anti-atherogenic, interleukin-10 (IL10), promotes cholesterol uptake from modified lipoproteins in macrophages and its transformation into foam cells by increasing the expression of scavenger receptor CD36 and scavenger receptor A. Although uptake of modified lipoproteins is considered proatherogenic, we found that IL10 also increases cholesterol efflux from macrophages to protect against toxicity of free cholesterol accumulation in the cell. This process was PPARgamma-dependent and was mediated through up-regulation of ABCA1 (ATP-binding cassette transporter A1) protein expression. Importantly, expression of inflammatory molecules, such as tumor necrosis factor-alpha, intercellular adhesion molecule-1, and MMP9 as well as apoptosis were dramatically suppressed in lipid-laden foam cells treated with IL10. The notion that IL10 can mediate both the uptake of cholesterol from modified lipoproteins and the efflux of stored cholesterol suggests that the process of foam cell formation is not necessarily detrimental as long as mechanisms of cholesterol efflux and transfer to an exogenous acceptor are functioning robustly. Our results present a comprehensive antiatherogenic role of IL10 in macrophages, including enhanced disposal of harmful lipoproteins, inhibition of inflammatory molecules, and reduced apoptosis.

Effects of acceptor composition and mechanism of ABCG1-mediated cellular free cholesterol efflux

Among the known mechanisms of reverse cholesterol transport (RCT), ATP binding cassette transporter G1 (ABCG1)-mediated free cholesterol (FC) transport is the most recent and least studied. Here, we have characterized the efficiencies of different acceptors using baby hamster kidney (BHK) cells transfected with human ABCG1 cDNA, which is inducible upon treatment with mifepristone. When normalized on particle number and particle surface area, the acceptor efficiency for FC efflux was as follows: small unilamellar vesicles (SUV)>LDL>reconstituted HDL>HDL2 = HDL3. Based on phospholipid content, the order was reversed. ABCG1 also mediated phospholipid efflux to human serum and HDL3. ABCG1-mediated FC efflux correlated significantly with a number of HDL subfractions and components in serum collected from 25 normolipidemic individuals: apolipoprotein A-II (apoA-II) (r2 = 0.7), apolipoprotein A-I (apoA-I) (r2 = 0.5), HDL-C (r2 = 0.4), HDL-PL (r2 = 0.4), α-2 HDL (r2 = 0.4), and preβ HDL (r2 = 0.2). ABCG1 did not enhance influx of FC or cholesteryl oleyl ether (COE) when cells were incubated with radiolabeled HDL3. ABCG1 expression did not increase the association of HDL3 with cells. Compared with control cells, ABCG1 expression significantly increased the FC pool available for efflux and the rate constant for efflux. In conclusion, composition and particle size determine the acceptor efficiency for ABCG1-mediated efflux. ABCG1 increases cell membrane FC pools and changes its rate of desorption into the aqueous phase without enhancing the association with the acceptor.

Human Endothelial Cells of the Placental Barrier Efficiently Deliver Cholesterol to the Fetal Circulation via ABCA1 and ABCG1

Although maternal-fetal cholesterol transfer may serve to compensate for insufficient fetal cholesterol biosynthesis under pathological conditions, it may have detrimental consequences under conditions of maternal hypercholesterolemia leading to preatherosclerotic lesion development in fetal aortas. Maternal cholesterol may enter fetal circulation by traversing syncytiotrophoblast and endothelial layers of the placenta. We hypothesized that endothelial cells (ECs) of the fetoplacental vasculature display a high and tightly regulated capacity for cholesterol release. Using ECs isolated from human term placenta (HPECs), we investigated cholesterol release capacity and examined transporters involved in cholesterol efflux pathways controlled by liver-X-receptors (LXRs). HPECs demonstrated 2.5-fold higher cholesterol release to lipid-free apolipoprotein (apo)A-I than human umbilical vein ECs (HUVECs), whereas both cell types showed similar cholesterol efflux to high-density lipoproteins (HDLs). Interestingly, treatment of HPECs with LXR activators increased cholesterol efflux to both types of acceptors, whereas no such response could be observed for HUVECs. In line with enhanced cholesterol efflux, LXR activation in HPECs increased expression of ATP-binding cassette transporters ABCA1 and ABCG1, while not altering expression of ABCG4 and scavenger receptor class B type I (SR-BI). Inhibition of ABCA1 or silencing of ABCG1 decreased cholesterol efflux to apoA-I (-70%) and HDL(3) (-57%), respectively. Immunohistochemistry localized both transporters predominantly to the apical membranes of placental ECs in situ. Thus, ECs of human term placenta exhibit unique, efficient and LXR-regulated cholesterol efflux mechanisms. We propose a sequential pathway mediated by ABCA1 and ABCG1, respectively, by which HPECs participate in forming mature HDL in the fetal blood.

Brefeldin A inhibits cholesterol efflux without affecting the rate of cellular uptake and re-secretion of apolipoprotein A-I in adipocytes

A possible role of cellular uptake and re-secretion of apoA-I in the mechanism of cholesterol efflux induced by apoA-I was investigated using a novel experimental approach. Incubation of adipocytes with a recombinant human apoA-I containing a consensus PKA phosphorylation site, pka-ApoA-I, leads to the appearance of phosphorylated protein in the cell culture medium unambiguously proving cellular uptake and re-secretion of pka-ApoA-I. Phosphorylation of apoA-I is abolished by PKA inhibitors and enhanced by PKA activators demonstrating the specific involvement of PKA. Studies on the concentration dependence of pka-apoA-I phosphorylation and competition experiments with human apoA-I suggest that apolipoprotein uptake is a receptor mediated process. A possible role of apoA-I recycling in the mechanism of cholesterol efflux was investigated by determining the rates of apoA-I induced cholesterol efflux and apoA-I recycling in the presence and in the absence of Brefeldin A (BFA). The studies showed that BFA strongly inhibits cholesterol efflux without affecting the rate of apoA-I recycling. Since BFA affects vesicular trafficking of ABCA1, this study suggests that the interaction of apoA-I with ABCA1 does not mediate apolipoprotein uptake and re-secretion. This result suggests that lipidation of apoA-I and apolipoprotein uptake/re-secretion are independent processes.

34 Investigation on mechanism of cholesterol efflux in human monocytes/macrophages THP-1

34 Investigation on mechanism of cholesterol efflux in human monocytes/macrophages THP-1

Molecular mechanism of cholesterol efflux; involvement of a member of the Rho GTPASE family CDC42

Molecular mechanism of cholesterol efflux; involvement of a member of the Rho GTPASE family CDC42