Mediated Cholesterol Efflux Research Articles

Cholesterol Accumulation in Dendritic Cells Links the Inflammasome to Acquired Immunity

Autoimmune diseases such as systemic lupus erythematosus (SLE) are associated with increased cardiovascular disease and reduced plasma high-density lipoprotein (HDL) levels. HDL mediates cholesterol efflux from immune cells via the ATP binding cassette transporters A1 and G1 (ABCA1/G1). The significance of impaired cholesterol efflux pathways in autoimmunity is unknown. We observed that Abca1/g1-deficient mice develop enlarged lymph nodes (LNs) and glomerulonephritis suggestive of SLE. This lupus-like phenotype was recapitulated in mice with knockouts of Abca1/g1 in dendritic cells (DCs), but not in macrophages or Tcells. DC-Abca1/g1 deficiency increased LN and splenic CD11b+ DCs, which displayed cholesterol accumulation and inflammasome activation, increased cell surface levels of thegranulocyte macrophage-colony stimulating factor receptor, and enhanced inflammatory cytokine secretion. Consequently, DC-Abca1/g1 deficiency enhanced Tcell activation and Th1 and Th17 cell polarization. Nlrp3 inflammasome deficiency diminished the enlarged LNs and enhanced Th1 cell polarization. These findings identify an essential role of DC cholesterol efflux pathways in maintaining immune tolerance.

Short-term cooling increases serum triglycerides and small high-density lipoprotein levels in humans

Cold exposure and β3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influence lipoprotein metabolism by enhancing lipoprotein lipase-mediated catabolism of triglyceride-rich lipoproteins and increasing plasma high-density lipoprotein (HDL) levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive. The objective was to assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men. Body mass index-matched young, lean men were exposed to a personalized cooling protocol for 2hours. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1H-nuclear magnetic resonance. Adenosine triphosphate-binding cassette A1 (ABCA1)-mediated cholesterol efflux capacity of HDL was measured using [3H]cholesterol-loaded ABCA1-transfected Chinese hamster ovary cells. Short-term cooling increased serum levels of free fatty acids, triglycerides, and cholesterol. Cooling increased the concentration of large very low-density lipoprotein (VLDL) particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux invitro. Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.

Abstract 43: Cholesterol Efflux Pathways Suppress Inflammasome Activation in Mice and Humans

Plasma high-density-lipoprotein (HDL) has several anti-atherogenic properties, including its key role in functioning as acceptor for ATP-binding cassette A1 and G1 (ABCA1 and ABCG1) mediated cholesterol efflux. We have shown previously that macrophage Abca1/g1 deficiency accelerates atherosclerosis, by enhancing foam cell formation and inflammatory cytokine expression in atherosclerotic plaques. Macrophage cholesterol accumulation activates the inflammasome, leading to caspase-1 cleavage, required for IL-1β and IL-18 secretion. Several studies have suggested that inflammasome activation accelerates atherogenesis. We hypothesized that macrophage Abca1/g1 deficiency activates the inflammasome. In Ldlr -/- mice fed a Western type diet (WTD), macrophage Abca1/g1 deficiency increased IL-1β and IL-18 plasma levels (2-fold; P <0.001), and induced caspase-1 cleavage. Deficiency of the inflammasome components Nlrp3 or caspase-1 in macrophage Abca1/g1 knockouts reversed the increase in plasma IL-18 levels ( P <0.001), indicating these changes were inflammasome dependent. We found that macrophage Abca1/g1 deficiency induced caspase-1 cleavage in splenic CD115 + monocytes and CD11b + macrophages. While mitochondrial ROS production or lysosomal function were not affected, macrophage Abca1/g1 deficiency led to an increased splenic population of monocytes (2.5-fold; P <0.01). Monocytes secrete ATP, and as a result, ATP secretion from total splenic cells was increased (2.5-fold; P <0.01), likely contributing to inflammasome activation. Caspase-1 deficiency decreased atherosclerosis in macrophage Abca1/g1 deficient Ldlr -/- mice fed WTD for 8 weeks (225822 vs 138606 μm 2 ; P <0.05). Of therapeutic interest, one injection of reconstituted HDL (100 mg/kg) in macrophage Abca1/g1 knockouts decreased plasma IL-18 levels ( P <0.05). Tangier disease patients, with a homozygous loss-of-function for ABCA1, showed increased IL-1β and IL-18 plasma levels (3-fold; P <0.001), suggesting that cholesterol efflux pathways also suppress inflammasome activation in humans. These findings suggest that macrophage cholesterol efflux pathways suppress inflammasome activation, possibly contributing to the anti-atherogenic effects of HDL treatment.

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

BackgroundMild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low‐density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals.Methods and ResultsCholesterol efflux from THP‐1 macrophages was assessed using plasma obtained from normo‐ and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3–17.1 μmol/L) exogenously added to plasma‐ or apolipoprotein A1–supplemented media also decreased macrophage cholesterol efflux in a concentration‐ and time‐dependent manner. We also showed reduced protein expression of the ATP‐binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1–mediated cholesterol efflux, in THP‐1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP‐1 macrophages.ConclusionsCholesterol efflux from THP‐1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease.

Alteration of high-density lipoprotein functionality in Alzheimer's disease patients.

The aims of the present study were to determine whether high-density lipoprotein (HDL) functionality-mediated cholesterol efflux is altered in Alzheimer's disease and to investigate the role and effect of amyloid-beta (Aβ) in the regulation of the anti-atherogenic activity of HDL. Eighty-seven elderly subjects were recruited, of whom 27 were healthy, 27 had mild cognitive impairment (MCI), and 33 had mild Alzheimer's disease (mAD). Our results showed that total cholesterol levels are negatively correlated with the Mini-Mental State Examination (MMSE) score (r = -0.2602, p = 0.0182). HDL from the mAD patients was less efficient at mediating cholesterol efflux from J774 macrophages (p < 0.05) than HDL from the healthy subjects and MCI patients. While HDL from the MCI patients was also less efficient at mediating cholesterol efflux than HDL from the healthy subjects, the difference was not significant. Interestingly, the difference between the healthy subjects and the MCI and mAD patients with respect to the capacity of HDL to mediate cholesterol efflux disappeared when ATP-binding cassette transporter A1 (ABCA1)-enriched J774 macrophages were used. HDL fluidity was significantly inversely correlated with the MMSE scores (r = -0.4137, p < 0.009). In vitro measurements of cholesterol efflux using J774 macrophages showed that neither Aβ1-40 nor Aβ1-42 stimulate cholesterol efflux from unenriched J774 macrophages in basal or ABCA1-enriched J774 macrophages.

Effects of Saturated Fatty Acids from Butter and Cheese on High‐density Lipoprotein (HDL)‐Mediated Cholesterol Efflux Capacity

OBJECTIVEThe association between dietary saturated fat (SFA) intake and cardiovascular disease risk remains controversial. Emerging data suggest that SFA from different sources may show different association with cardiovascular and cardiometabolic risk. The objective of this study was to examine how SFA from cheese (CHEESE) influences HDL‐mediated cholesterol efflux capacity (CEC), a key anti‐atherogenic property of HDL, compared with a low fat/high carbohydrate diet (CHO), a diet rich in SFA from butter (BUTTER) and diets either high in monounsaturated (MUFA) or polyunsaturated fatty acids (PUFA).METHODSA total of 46 men and women with abdominal obesity and with relatively low HDL cholesterol (HDL‐C) completed this randomized crossover controlled trial consisting of 5 predetermined isoenergetic diets of 4 weeks each (Table 1): 1) CHEESE, 2) BUTTER, 3) MUFA, 4) PUFA, 5) CHO. All foods were provided to participants during each experimental diet. CEC was assessed with radiolabelled J774 macrophages using apolipoprotein B depleted serum samples taken from participants at the end of each dietary phase. HDL lipid composition (gas chromatography) and HDL particle size distribution (polyacrylamide gradient gel electrophoresis) were also assessed.RESULTSAll higher fat diets increased HDL‐C concentrations compared to CHO (all p&lt;0.001). BUTTER and CHEESE increased low‐density lipoprotein cholesterol (LDL‐C) concentrations more than CHO, MUFA and PUFA diets, particularly among individuals with high baseline LDL‐C (all p&lt;0.001). LDL‐C concentrations tended to be greater after BUTTER than after CHEESE (p=0.07). BUTTER, PUFA and CHEESE significantly increased large HDL2b levels compared to CHO (all p≤0.01). BUTTER also increased HDL2b levels more than MUFA (p=0.04). There was no difference in HDL‐mediated CEC between CHEESE and CHO (p=NS). BUTTER significantly increased HDL‐mediated CEC compared to CHEESE and CHO (both p&lt;0.05). MUFA also increased CEC compared to CHEESE and CHO (both p&lt;0.05). PUFA had no significant effect on HDL CEC compared to the others diets (p=NS). The increase in LDL‐C after BUTTER (vs. CHO) was significantly correlated with concurrent increase in CEC in men (r=0.44, p=0.04), but not in women (r=0.27, p=0.20).CONCLUSIONSThese results provide evidence of a matrix effect modulating the effects of dairy SFA on HDL characteristics and function, with greater changes seen with SFA from butter than with SFA from cheese in men and women with abdominal obesity and with relatively low HDL‐C levels. The increase in HDL‐mediated CEC seen with SFA from butter parallels the increase in LDL‐C among men, but not among women. Additional studies assessing other HDL functions will provide further insights on how the food source may modify the effects of SFA on cardiometabolic risk.Support or Funding InformationDairy Research Cluster Initiative (Dairy Farmers of Canada, Agriculture and Agri‐Food Canada, the Canadian Dairy Network and the Canadian Dairy Commission) and the National Dairy Council. Macronutrients (% energy), calcium, sodium and fibers content of experimental diets CHEESE BUTTER MUFA PUFA CHO Lipids (%) 32.0 32.0 32.0 32.0 25.0 SFA (%) 12.6 12.4 5.8 5.8 5.8 MUFA (%) 12.5 12.3 19.6 12.6 12.6 PUFA (%) 4.8 4.8 4.8 11.5 4.8 Carbohydrates (%) 51.9 52.0 51.9 51.9 58.9 Proteins (%) 16.0 16.0 16.0 16.0 16.0 Calcium (mg/2500 kcal) 1261 811 812 812 842 Sodium (mg/2500 kcal) 2482 2480 2479 2479 2485 Fibers (g/2500 kcal) 31 31 31 31 31

Human paraoxonase 1 overexpression in mice stimulates HDL cholesterol efflux and reverse cholesterol transport.

This study was aimed to investigate the effect of human PON1 overexpression in mice on cholesterol efflux and reverse cholesterol transport. PON1 overexpression in PON1-Tg mice induced a significant 3-fold (p<0.0001) increase in plasma paraoxonase activity and a significant ~30% (p<0.0001) increase in the capacity of HDL to mediate cholesterol efflux from J774 macrophages compared to wild-type mice. It also caused a significant 4-fold increase (p<0.0001) in the capacity of macrophages to transfer cholesterol to apoA-1, a significant 2-fold (p<0.0003) increase in ABCA1 mRNA and protein expression, and a significant increase in the expression of PPARγ (p<0.0003 and p<0.04, respectively) and LXRα (p<0.0001 and p<0.01, respectively) mRNA and protein compared to macrophages from wild-type mice. Moreover, transfection of J774 macrophages with human PON1 also increased ABCA1, PPARγ and LXRα protein expression and stimulates macrophages cholesterol efflux to apo A1. In vivo measurements showed that the overexpression of PON1 significantly increases the fecal elimination of macrophage-derived cholesterol in PON1-Tg mice. Overall, our results suggested that the overexpression of PON1 in mice may contribute to the regulation of the cholesterol homeostasis by improving the capacity of HDL to mediate cholesterol efflux and by stimulating reverse cholesterol transport.

ABCA1 Deficiency Affects Basal Cognitive Deficits and Dendritic Density in Mice.

ATP-binding cassette transporter A1 (ABCA1) mediates cholesterol efflux to lipid-free apolipoproteins and regulates the generation of high density lipoproteins. Previously, we have shown that lack of Abca1 significantly increases amyloid deposition and cognitive deficits in Alzheimer’s disease model mice expressing human amyloid-β protein precursor (APP). The goal of this study was to determine if ABCA1 plays a role in memory deficits caused by amyloid-β (Aβ) oligomers and examine neurite architecture of pyramidal hippocampal neurons. Our results confirm previous findings that Abca1 deficiency significantly impairs spatial memory acquisition and retention in the Morris water maze and long-term memory in novel object recognition of APP transgenic mice at a stage of early amyloid pathology. Neither test demonstrated a significant difference between Abca1ko and wild-type (WT) mice. We also examined the effect of intra-hippocampal infused Aβ oligomers on cognitive performance of Abca1ko mice, compared to control infusion of scrambled Aβ peptide. Age-matched WT mice undergoing the same infusions were also used as controls. In this model system, we found a statistically significant difference between WT and Abca1ko mice infused with scrambled Aβ, suggesting that Abca1ko mice are vulnerable to the effect of mild stresses. Moreover, examination of neurite architecture in the hippocampi revealed a significant decrease in neurite length, number of neurite segments, and branches in Abca1ko mice when compared to WT mice. We conclude that mice lacking ABCA1 have basal cognitive deficits that prevent them from coping with additional stressors, which is in part due to impairment of neurite morphology in the hippocampus.

High density lipoprotein (HDL) particles from end-stage renal disease patients are defective in promoting reverse cholesterol transport

Atherosclerotic cardiovascular disease (CVD) represents the largest cause of mortality in end-stage renal disease (ESRD). CVD in ESRD is not explained by classical CVD risk factors such as HDL cholesterol mass levels making functional alterations of lipoproteins conceivable. HDL functions in atheroprotection by promoting reverse cholesterol transport (RCT), comprising cholesterol efflux from macrophage foam cells, uptake into hepatocytes and final excretion into the feces. ESRD-HDL (n = 15) were compared to healthy control HDL (n = 15) for their capacity to promote in vitro (i) cholesterol efflux from THP-1 macrophage foam cells and (ii) SR-BI-mediated selective uptake into ldla[SR-BI] cells as well as (iii) in vivo RCT. Compared with HDL from controls, ESRD-HDL displayed a significant reduction in mediating cholesterol efflux (p < 0.001) and SR-BI-mediated selective uptake (p < 0.01), two key steps in RCT. Consistently, also the in vivo capacity of ESRD-HDL to promote RCT when infused into wild-type mice was significantly impaired (p < 0.01). In vitro oxidation of HDL from healthy controls with hypochloric acid was able to fully mimic the impaired biological activities of ESRD-HDL. In conclusion, we demonstrate that HDL from ESRD patients is dysfunctional in key steps as well as overall RCT, likely due to oxidative modification.

ATP binding cassette A1 (ABCA1) mediates microparticle formation during high-density lipoprotein (HDL) biogenesis

Background and aimsMicro-particles (MP) are secreted by various cells. Their biological roles in health and in disease remain unknown. Here we describe formation of MP in the process of ABCA1-dependent cholesterol efflux in different cell types. MethodsThe ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) is the rate-limiting step in the biogenesis of high-density lipoproteins (HDL). We have found that ABCA1 and apoA-I contribute to the formation of MP. Using cell-based systems with overexpression and selective inactivation of ABCA1, pharmacological blockade and modulation of membrane cholesterol content, we characterized MP release from various cell lines. We studied MP release in BHK cells stably expressing ABCA1 under mifepristone control, human THP-1 macrophages and HepG2 cells without, or with incubation with human apoA-I. ResultsABCA1 mediates the production of MPs containing cholesterol. This was also confirmed in primary human monocyte-derived macrophages (MDMs). Adding apoA-I markedly increases MP release from cells. Inhibition of ABCA1 with probucol or decreasing plasma membrane cholesterol with methyl-β cyclodextrin (CDX) markedly reduced MP release and nascent HDL formation. MPs do not contain apoA-I, but contain flotilin-2, a marker of plasma membrane, and CD63, an exosome marker. MPs exhibit considerable size heterogeneity (50–250 nm). ConclusionsWe show that MPs are lipoprotein-sized structures created by the ABCA1 transporter, and contribute approximately 30% of ABCA1-and apoA-I mediated cholesterol efflux. In addition, we found that MPs release from cells consists, in part, of exosomes and depends on the same pathway used for HDL biogenesis.

Multifunctional Dextran Sulfate-Coated Reconstituted High Density Lipoproteins Target Macrophages and Promote Beneficial Antiatherosclerotic Mechanisms.

An atorvastatin calcium (AT)-loaded dextran sulfate (DXS)-coated core-shell reconstituted high density lipoprotein (rHDL), termed AT-DXS-LP-rHDL, was developed for targeted drug delivery to macrophages and suppression of inflammation via the high affinity of DXS with scavenge receptor class AI (SR-AI) as well as depletion of intracellular cholesterol by apolipoprotein A-I (apoA-I)-mediated cholesterol efflux. These core-shell nanoparticles comprising an AT-loaded negatively charged poly(lactide-co-glycolide) (PLGA) core and a cationic lipid bilayer shell were prepared by nanoprecipitation method followed by thin film hydration and extrusion. The nanoparticles were further functionalized with apoA-I and DXS via sodium cholate mediation and electrostatic interaction, respectively. The core-shell structure and the surface coating of apoA-I and DXS were verified by the increased particle size, inverted zeta potential, and reduced in vitro drug release rate. The TEM image further confirmed the entrapment of the PLGA nanoparticles in the aqueous interior of the liposomes. In vitro cell viability assay showed the biocompatibility of the AT-loaded nanocarriers. The cellular uptake study illustrated that the targeting efficacy to macrophages increased in the following order: PLGA nanoparticles (P-NP), core-shell nanoparticles (LP-NP), core-shell rHDL (LP-rHDL), and DXS-LP-rHDL. Moreover, cellular drug efficacy of AT-loaded nanoparticles in preventing macrophage-derived foam cell formation and inflammation such as intracellular lipid deposition, cholesterol esters content, DiI-oxLDL uptake, cholesterol efflux, and secretion of TNF-α, IL-6, and IL-10 was much better than that of the drug-free nanoparticles, consistent with the results of cellular uptake study. Collectively, AT-DXS-LP-rHDL, as multifunctional carriers, could not only deliver more drug to macrophages, but also present antiatherogenic actions of the biofunctional nanocarriers through damping oxidized low density lipoproteins (oxLDL) uptake and promoting cholesterol efflux.

Relationship of Cholesterol Efflux Capacity with Coronary Plaque Lipid Composition on Near Infrared Spectroscopy

Background: The capacity of high-density lipoprotein (HDL) to promote cell cholesterol efflux associates with cardiovascular events. Near Infrared Spectroscopy (NIRS) quantitates lipid content within plaque. However, little is known about the impact of HDL functionality on coronary plaques. Methods: 74 patients who underwent NIRS imaging were examined. Coronary plaque lipid burden [lipid core burden index (LCBI) and LCBI at the 4 mm maximal segment (LCBI4mm max)] were assessed. We measured ATP-binding cassette A1 and G1 (ABCA1 and ABCG1)-mediated cholesterol efflux capacity (CEC) of serum in cellular systems. Results: The mean age was 60 years and 27% were female. Patients with LCBI4mm max >median demonstrated greater levels of triglyceride (142 vs. 124 mg/dL, p = 0.04). In the whole cohort, LCBI4mm max did not correlate with CEC via ABCA1 (r = 0.06, p = 0.60) or ABCG1 (r = 0.05, p = 0.66). Similarly, mean LCBI did not correlate with CEC via ABCA1 (r = 0.02, p = 0.84) or ABCG1 (r = 0.09, p = 0.42). However, ABCA1 mediated CEC inversely correlated with LCBI4mm max in patients with baseline LCBI4mm max ≤median (r = -0.34, p = 0.04), but not in those with greater LCBI4mm max levels (r = 0.05, p = 0.78). In contrast, HDL-C did not correlate with LCBI4mm max at any level. Conclusions: ABCA1 mediated cholesterol efflux capacity appears to correlate with plaque lipid content in smaller, less lipidic lesions, but not in larger plaques containing greater amounts of lipid. This may have implications for understanding the impact of HDL functionality on plaque at different stages. The impact of these findings on therapeutic interventions targeting HDL functionality remains to be determined.

Effects of garlic on brachial endothelial function and capacity of plasma to mediate cholesterol efflux in patients with coronary artery disease.

Objective:This study investigated the effects of garlic on brachial endothelial function and THP-1 macrophage cholesterol efflux (CE) and examined whether garlic modulates ATP-binding cassette (ABC) A1 and ABCG1 mRNA expressions in peripheral blood mononuclear cells (PBMCs) isolated from patients with coronary artery disease (CAD).Methods:In this randomized, placebo-controlled trial, patients with CAD were randomly divided into two groups: those receiving garlic powder or placebo tablets twice daily for 3 months. Brachial flow-mediated dilation (FMD) was assessed using ultrasound. Fasting blood samples were collected before and after period and PBMC and plasma were isolated. Human THP-1 monocytes were differentiated into macrophages, labeled with 3H-cholesterol, and incubated with plasma samples, and CE was assessed. ABCA1 and ABCG1 mRNA expressions were determined in PBMCs.Results:After 3 months, brachial FMD values significantly improved (50.7%) in the garlic group compared with those in the placebo group (p=0.016). High-sensitive C-reactive protein (hs-CRP) levels significantly decreased in the garlic group, but the difference between the two groups was not statistically significant. No significant difference was observed with regard to CE and ABCA1 and ABCG1 mRNA expressions in PBMCs. CE was negatively correlated with hs-CRP levels.Conclusion:Short-term treatment with garlic may improve the endothelial function and may affect hs-CRP levels; however, it could neither significantly improve THP-1 macrophage CE nor affect ABCA1 or ABCG1 expressions in PBMCs.

Association of Cholesterol Efflux Capacity With Clinical Features of Metabolic Syndrome: Relevance to Atherosclerosis.

BackgroundThe contribution of high‐density lipoprotein to cardiovascular benefit is closely linked to its role in the cellular cholesterol efflux process; however, various clinical and biochemical variables are known to modulate the overall cholesterol efflux process. The aim of this study was to evaluate the extent to which clinical and biological anomalies associated with the establishment of the metabolic syndrome modulate cholesterol efflux capacity and contribute to development of atherosclerosis.Methods and ResultsThis study involved patients (n=1202) displaying atherogenic dyslipidemia in primary prevention who were referred to our prevention center. Among these patients, 25% presented at least 3 criteria of the metabolic syndrome, as defined by the National Cholesterol Education Program Adult Treatment Panel III. We measured the capacity of 40‐fold diluted serum to mediate cholesterol efflux from cholesterol‐loaded human THP‐1 macrophages. Cholesterol efflux capacity was reduced progressively by 4% to 11% (P<0.0001) as a function of the increasing number of coexisting criteria for the metabolic syndrome from 1 to 5. This observation was primarily related to reductions in scavenger receptor class B member 1 and ATP binding cassette subfamily G member 1–dependent efflux. Multivariate analyses indicate that serum efflux capacity was significantly associated with established metabolic syndrome (odds ratio 0.45; 95% CI 0.28–0.72; P=0.009) independent of age, low‐density lipoprotein cholesterol, status with regard to lipid‐lowering therapy, smoking status, and alcohol consumption.ConclusionsOur study revealed that individual criteria of metabolic syndrome are closely related synergistically to cholesterol efflux capacity. In addition, established metabolic syndrome and cholesterol efflux capacity were independently associated with clinical features of atherosclerosis.

Inhibitory effect of PCSK9 on Abca1 protein expression and cholesterol efflux in macrophages

Background and aimsProprotein convertase subtilisin/kexin type 9 (PCSK9) may have extra-hepatic effects on cholesterol homeostasis of vascular macrophages. In this study, we aimed to investigate PCSK9 role on the anti-atherogenic process of ATP binding cassette transporter A1 (Abca1)-mediated cholesterol efflux. MethodsAbca1-mediated cholesterol efflux was evaluated by a radioisotopic technique in mouse peritoneal macrophages (MPM) from wild-type (WT) or LDL receptor knock-out (Ldlr−/−) mice exposed to human recombinant PCSK9, in the presence of liver X receptor/retinoid X receptor (LXR/RXR) ligands or acetylated LDL (AcLDL) to stimulate Abca1 expression. Protein and gene expression was evaluated by Western blot and quantitative real time PCR, respectively. ResultsPCSK9 inhibited Abca1-mediated cholesterol efflux induced by LXR/RXR agonists in WT MPM (−55%, p < 0.05) but not in Ldlr−/− MPM. This effect was fully abrogated by the co-incubation with an anti-PCSK9 antibody. The inhibition of Abca1-dependent efflux induced by PCSK9 was associated with a reduction of Abca1 protein expression only in WT cells. Abca1 gene expression was significantly downregulated by PCSK9 in WT macrophages (−64%, p < 0.001) and, to a lesser extent, in MPM lacking Ldlr (−35%, p < 0.001). The inhibitory effect on Abca1-mediated efflux was also confirmed in AcLDL-treated macrophages. PCSK9 had a marginal or no effect on the expression of the lipid transporters Sr-b1 and Abcg1. ConclusionsPCSK9 plays a direct role on Abca1-mediated cholesterol efflux through a downregulation of Abca1 gene and Abca1 protein expression. This extrahepatic effect may influence relevant steps in the pathogenesis of atherosclerosis, such as foam cell formation.

Functionality of High-Density Lipoprotein as Antiatherosclerotic Therapeutic Target.

A great majority of the morbidity and mortality worldwide can still be attributed to cardiovascular diseases, such as ischemic (coronary) heart disease, angina pectoris, and myocardial and cerebral infarction. Atherosclerosis, narrowing of the arteries because of arterial cholesterol deposition in macrophage foam cells, is the driving force behind the cardiovascular disease pathology. Water-soluble protein/lipid complexes called lipoproteins mediate the transport of cholesterol and other lipoid substances through the blood compartment. Relatively high levels of cholesterol associated with apolipoprotein B–containing low-density lipoprotein (LDL) particles predispose human subjects to the development of atherosclerosis and, thereby, increase the risk for cardiovascular disease.1,2 Apolipoprotein B–containing lipoproteins are, therefore, generally regarded as being proatherogenic factors. Cholesterol ester–rich high-density lipoprotein (HDL) particles use apolipoprotein A1 (apoA1) as their primary protein component. In sharp contrast to LDL, HDL is considered a potent anti-atherogenic agent. This notion is based on the fact that, in the general population, a strong inverse correlation exists between plasma levels of HDL cholesterol and the risk of cardiovascular disease.1 Of note, this inverse association seems to be independent of the level of cholesterol associated with proatherogenic LDL particles. As such, increasing plasma levels of HDL cholesterol has long been regarded a promising alternative therapy to supplement classical statin–based LDL cholesterol–lowering strategies that are able to reduce cardiovascular disease by only ≈30%.3 However, over the last decade, the enthusiasm for HDL as an interesting therapeutic target has been challenged by the HDL hypothesis critics because genetic association studies have excluded HDL cholesterol levels as determinants for cardiovascular disease risk.2,4 Furthermore, several therapeutic HDL-targeting approaches have proven insufficient to secure benefit for cardiovascular disease patients. Niacin is the most effective drug available in the clinic to raise plasma HDL cholesterol levels. Despite the fact that niacin is …

Persistent changes in lipoprotein lipids after a single infusion of ascending doses of MDCO-216 (apoA-IMilano/POPC) in healthy volunteers and stable coronary artery disease patients

Background and aimsEffects of single ascending doses of MDCO-216 on plasma lipid and lipoprotein levels were assessed in human healthy volunteers and in patients with stable coronary artery disease (CAD). MethodsMDCO-216 was infused at a single dose of 5, 10, 20, 30 or 40 mg/kg over 2 h and blood was collected at 2, 4, 8, 24, 48, 168 and 720 h after start of infusion (ASOI). Lipoprotein lipids were assessed by FLPC and by 1H NMR. ResultsPlasma concentrations of free cholesterol (FC) displayed a rapid and dose-dependent rise, peaking at 8 h, but remaining above baseline until 48 h ASOI, whereas levels of esterified cholesterol (CE) increased at lower doses but not at higher doses, and even decreased below baseline at the highest dose. Plasma cholesterol esterification rate (CER) decreased with a first nadir between 4 and 8 h and a second nadir at 48 h ASOI. Taken over all subjects receiving MDCO-216, the increase in FC at 8 h correlated inversely with the drop in CER at 4 h but positively with the increase in basal and scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux capacities at 2 h ASOI.Upon FPLC analysis, FC was found to increase first in high density lipoproteins (HDL) and very low density lipoproteins (VLDL) and later (at 48 or 168 h ASOI) in low density lipoproteins (LDL). CE initially decreased in LDL and HDL but after 24 h started to increase in VLDL and LDL whereas HDL-CE was still below baseline at 48 h. Phospholipids (PL) showed the same pattern as FC. Triglycerides (TG) also rose rapidly, most prominently in VLDL, but also in LDL and HDL. Apolipoprotein E (Apo-E) in VLDL increased at 4–8 h but returned to baseline at 24 h ASOI.1H NMR analysis showed a rapid and dose-dependent increase in HDL particle size, peaking at 2 h and returning to baseline at 24 h, and a small increase in HDL particle concentration. After infusion of the 40 mg/kg dose, LDL and VLDL-particles also increased in number and size. ConclusionsA single administration of MDCO-216 caused rapid changes in lipid levels and lipoprotein composition, some of which persisted for at least 7 days.

HDL function is impaired in acute myocardial infarction independent of plasma HDL cholesterol levels

High-density lipoproteins (HDLs) protect against the development of atherosclerotic cardiovascular disease. HDL function represents an emerging concept in cardiovascular research. This study investigated the association between HDL functionality and acute myocardial infarction (MI) independent of HDL-cholesterol plasma levels. Participants (non-ST-segment elevation MI, non-STEMI, n=41; STEMI, n=37; non-MI patients, n=33) from a prospective follow-up study enrolling patients with acute chest pain were matched for age and plasma HDL cholesterol. The invitro capacity of HDL to (1) mediate cholesterol efflux from macrophage foam cells, (2) prevent low-density lipoprotein oxidation, and (3) inhibit TNF-α-induced vascular adhesion molecule-1 expression in endothelial cells was determined. STEMI-HDL displayed reduced cholesterol efflux (P<.001) and anti-inflammatory functionality (P=.001), whereas the antioxidative properties were unaltered. Cholesterol efflux correlated with the anti-inflammatory HDL activity (P<.001). Not C-reactive protein levels, a marker of systemic inflammation, but specifically plasma myeloperoxidase levels were independently associated with impaired HDL function (efflux: P=.022; anti-inflammation: P<.001). Subjects in the higher risk quartile of efflux (odds ratio [OR], 5.66; 95% confidence interval [CI], 1.26-25.00; P=.024) as well as anti-inflammatory functionality of HDL (OR, 5.53; 95% CI, 1.83-16.73; P=.002) had a higher OR for MI vs those in the three lower risk quartiles combined. Independent of plasma HDL cholesterol levels, 2 of 3 antiatherogenic HDL functionalities tested were significantly impaired in STEMI patients, namely cholesterol efflux and anti-inflammatory properties. Increased myeloperoxidase levels might represent a major contributing mechanism for decreased HDL functionality in MI patients.

Abstract 307: Apoa-1 Binding Protein Regulates Retinal Angiogenesis via Control of Notch Signaling

ApoA-1 binding protein (AIBP), a mediator of cholesterol efflux from endothelia cells to HDL, plays a vital role in zebrafish angiogenesis. Although it is evolutionary conserved from Drosophila to human, its role of angiogenesis in higher vertebrate has not been studied. Thus, we propose that the role of AIBP in angiogenesis is also conserved from zebrafish to mice to humans. Mouse models display tremendous power in the study of human cardiovascular disease as the murine and human vasculatures are highly similar. We therefore generated Aibp -/- mice, which are viable and fertile. We then compared retinal vasculature development in Aibp -/- mice and wild-type control mice by whole mount immunostaining. Our data show that AIBP deficiency in mice results in significantly enhanced angiogenesis, including an increase in the radial extension of vascular plexus, the development of a denser upper capillary layer, and more tip cells as well as filopodia in the retinas. Notch signaling pathway plays an essential role for proper angiogenesis. However, the role of cholesterol metabolism in the Notch pathway is poorly understood. We found the expressions of the Notch downstream targets Hes1, Hey1 and Hey2 were upregulated in Aibp -/- retinas compared to controls. Furthermore, the protein level of Notch intracellular domain (NICD) was decreased in Aibp -/- retinas. These results suggest that the activity of Notch signaling is decreased in retinas from Aibp -/- mice. Mechanistically, we found AIBP positively regulates Notch signaling by affect on γ-secretase activity. Moreover, we performed Matrigel plug assay. Comparing that of wild-type, Matrigel plugs from Aibp -/- mice has more VE-cadherin staining and higher VE-cadherin mRNA expression level; and shows lower Hey1 expression as well as decreased NICD protein levels. It indicates that Matrigel plugs from Aibp -/- mice expresses more blood vessels and show lower Notch signaling activity. Our study demonstrated that AIBP inhibits angiogenesis by activating the Notch pathway and are the first to connect AIBP, a cholesterol metabolism regulator, to Notch signaling.

ABCG1 and ABCG4 Suppress γ-Secretase Activity and Amyloid β Production.

ATP-binding cassette G1 (ABCG1) and ABCG4, expressed in neurons and glia in the central nervous system, mediate cholesterol efflux to lipid acceptors. The relationship between cholesterol level in the central nervous system and Alzheimer’s disease has been reported. In this study, we examined the effects of ABCG1 and ABCG4 on amyloid precursor protein (APP) processing, the product of which, amyloid β (Aβ), is involved in the pathogenesis of Alzheimer’s disease. Expression of ABCG1 or ABCG4 in human embryonic kidney 293 cells that stably expressed Swedish-type mutant APP increased cellular and cell surface APP levels. Products of cleavage from APP by α-secretase and by β-secretase also increased. The levels of secreted Aβ, however, decreased in the presence of ABCG1 and ABCG4, but not ABCG4-KM, a nonfunctional Walker-A lysine mutant. In contrast, secreted Aβ levels increased in differentiated SH-SY5Y neuron-like cells in which ABCG1 and ABCG4 were suppressed. Furthermore, Aβ42 peptide in the cerebrospinal fluid from Abcg1 null mice significantly increased compared to the wild type mice. To examine the underlying mechanism, we analyzed the activity and distribution of γ-secretase. ABCG1 and ABCG4 suppressed γ-secretase activity and disturbed γ-secretase localization in the raft domains where γ-secretase functions. These results suggest that ABCG1 and ABCG4 alter the distribution of γ-secretase on the plasma membrane, leading to the decreased γ-secretase activity and suppressed Aβ secretion. ABCG1 and ABCG4 may inhibit the development of Alzheimer’s disease and can be targets for the treatment of Alzheimer’s disease.