HDL Phospholipids Research Articles

Abstract 226: Serum Amyloid A Isoforms and the Capacity to Mediate Cholesterol Efflux

Serum Amyloid A (SAA) is an acute phase reactant protein that exists in multiple isoforms, can form HDL, and participates in cholesterol efflux. In vitro studies suggest that the SAA 2.1 isoform has an increased capacity to mediate cholesterol efflux compared to the other isoforms. We examined SAA isoforms using a novel mass spectrometric immunoassay (MSIA) and HDL’s cholesterol efflux capacity (via ABCA-1 and SR-BI) in samples from 59 subjects with (n=33) and without type 2 diabetes (n=26). SAA 1.1 levels were detectable in 58, SAA 2.1 in 14 and SAA 2.2 in 36 of the 59 subjects. SAA 2.1 levels significantly correlated with SR-BI cholesterol efflux (r=0.71, p=0.01, n=14), but not ABCA-1 mediated efflux (r=0.1, P=0.1). This correlation was not explained by changes in HDL phospholipids, Apo A-I or HDL cholesterol levels. In contrast, SAA 2.2 or 1.1 levels did not correlate with changes in SR-BI or ABCA-1 mediated efflux. Although the SAA 2.1 isoform is less frequently detected in plasma, our data confirm that it is closely linked with HDL mediated cholesterol efflux, particularly that is SR-BI mediated.

Elderly individuals with high IL‐6 levels have proinflammatory HDL (832.16)

Introduction: High density lipoprotein removes the oxidatively modified lipids and other toxic molecules from peripheral cells for elimination and thus it is anti inflammatory and protective. Under chronic acute phase pressure, unsaturated fatty acids and phospholipids in HDL undergo oxidative modification and that results in HDL losing its anti inflammatory and protective capacity. Objective: we sought to determine if HDL from individuals with inflammatory background can retain its protective ability. Methods: We recruited a group of elderly, visiting the UCLA clinics (n=40), who had normal (Group 1) or higher than normal plasma IL‐6 levels (Group 2). We followed the UCLA Medical Center IRB rules and instructions and analyzed their plasma sample for HDL anti inflammatory capacity. This was done using monocyte chemotactic activity toward human artery wall cells in co culture. Results: While the plasma HDL cholesterol levels of the two groups was similar (59.2+ 7.6 and 61+6.9 mg per dL respectively) with no significant difference, the HDL protective capacity of the group with high serum IL‐6 level was significantly lower than the group 1 who did not have high circulating IL‐6 levels (‐ 35% vs + 67% respectively, p <0.01). Conclusion: The data obtained indicates that in the elderly with high plasma IL‐6 levels the inflammatory pressure results in the loss of the protective capacity of HDL and this in turn moves the organism toward a vicious cycle. HDL protects lipids and other substances form oxidative modification. Los of HDL protective capacity increases the modification of lipids and other macromolecules in LDL, in cell membrane and basically throughout the organism with the pursuing undesirable structural and functional consequences.Grant Funding Source: NIH

Evidence for the presence of lipid-free monomolecular apolipoprotein A-1 in plasma

The first step in reverse cholesterol transport is a process by which lipid-free or lipid-poor apoA-1 removes cholesterol from cells through the action of ATP binding cassette transporter A1 at the plasma membrane. However the structure and composition of lipid-free or -poor apoA-1 in plasma remains obscure. We previously obtained a monoclonal antibody (MAb) that specifically recognizes apoA-1 in preβ1-HDL, the smallest apoA-1-containing particle in plasma, which we used to establish a preβ1-HDL ELISA. Here, we purified preβ1-HDL from fresh normal plasma using said antibody, and analyzed the composition and structure. ApoA-1 was detected, but neither phospholipid nor cholesterol were detected in the purified preβ1-HDL. Only globular, not discoidal, particles were observed by electron microscopy. In nondenaturing PAGE, no difference in the mobility was observed between the purified preβ1-HDL and original plasma preβ1-HDL, or between the preβ1-HDL and lipid-free apoA-1 prepared by delipidating HDL. In sandwich ELISA using two anti-preβ1-HDL MAbs, reactivity with intact plasma preβ1-HDL was observed in ELISA using two MAbs with distinct epitopes but no reactivity was observed in ELISA using a single MAb, and the same phenomenon was observed with monomolecular lipid-free apoA-1. These results suggest that plasma preβ1-HDL is lipid-free monomolecular apoA-1.

Serum total and high-density lipoprotein phospholipids: Independent predictive value for cardiometabolic risk

Given that serum phospholipids (PL) may serve as inflammation mediators, we studied whether they predicted metabolic syndrome (MetS), type-2 diabetes or coronary heart disease (CHD) risk in people prone to enhanced low-grade inflammation. We analyzed unselected middle-aged Turkish adults with available serum total (n = 852) and HDL-PL (n = 428) measurements and follow-up (mean 6.6 years) by Cox or logistic regression, after exclusion of prevalent cases of outcome disorder. The enzymatic method used measured total content of phosphatidylcholine, sphingomyelin and lyso-phosphatidylcholine. Most lipid and non-lipid variables were significantly different in the upper two compared with the lowest total PL tertile, whereby apolipoprotein (apo)A-I and HDL-cholesterol were higher (not lower). ApoA-I, HDL-cholesterol and uric acid were uniformly positive independent linear covariates of total and HDL PL, apoA-I even in participants without MetS. After adjustment for sex, age, waist circumference, HDL-cholesterol and systolic blood pressure, logistic regression for incident MetS disclosed a 3-fold risk (RR [95% CI 1.28; 6.81]) in the upper HDL-pl tertile. In Cox regression models, while the combined two higher HDL-pl tertiles significantly protected against CHD risk in males (HR 0.29 [95% CI 0.10; 0.89]), they weakly tended to impart risk in females: upper two total PL tertiles tended to increased risk of diabetes and CHD. Excess total PL may mediate inflammatory properties to apoA-I, HDL and uric acid. Excess HDL-pl independently predict risk for MetS in each gender, but are protective against CHD risk in men, possibly because oxidized PL content mediated by total PL is sex-dependent, as reviewed elsewhere.

Furin Is the Primary in Vivo Convertase of Angiopoietin-like 3 and Endothelial Lipase in Hepatocytes

The proprotein convertases (PCs) furin, PC5/6, and PACE4 exhibit unique and/or complementary functions. Their knock-out (KO) in mice resulted in strong and specific phenotypes demonstrating that, in vivo, these PCs are unique and essential during development. However, they also exhibit redundant functions. Liver angiopoietin-like 3 (ANGPTL3) inhibits lipolysis by binding to lipoprotein lipases. It is found in the plasma as full length and truncated forms. The latter is more active and generated by cleavage at a furin-like site. Endothelial lipase (EL) binds heparin sulfate proteoglycans on cell surfaces and catalyzes the hydrolysis of HDL phospholipids. EL activity is regulated by two endogenous inhibitors, ANGPTL3 and ANGPTL4, and by PCs that inactivate EL through cleavage releasing the N-terminal catalytic and C-terminal lipid-binding domains. Herein, because furin and PC5/6 complete KOs are lethal, we used mice lacking furin or PC5/6 specifically in hepatocytes (hKO) or mice completely lacking PACE4. In primary hepatocytes, ANGPTL3 was processed into a shorter form of ANGPTL3 intracellularly by furin only, and extracellularly mainly by PACE4. In vivo, the absence of furin in hepatocytes reduced by ∼50% the circulating levels of cleaved ANGPTL3, while the lack of PACE4 had only a minor effect. Analysis of the EL processing in primary hepatocytes and in vivo revealed that it is mostly cleaved by furin. However, the lack of furin or PC5/6 in hepatocytes and complete PACE4 KO did not appreciably modify plasma HDL levels or EL activity. Thus, inhibition of furin in liver would not be expected to modify the plasma lipid profiles.

Hepatic lipase- and endothelial lipase-deficiency in mice promotes macrophage-to-feces RCT and HDL antioxidant properties

Hepatic lipase (HL) and endothelial lipase (EL) are negative regulators of plasma HDL cholesterol (HDLc) levels and presumably could affect two main HDL atheroprotective functions, macrophage-to-feces reverse cholesterol transport (RCT) and HDL antioxidant properties. In this study, we assessed the effects of both HL and EL deficiency on macrophage-specific RCT process and HDL ability to protect against LDL oxidation. HL- and EL-deficient and wild-type mice were injected intraperitoneally with [3H]cholesterol-labeled mouse macrophages, after which the appearance of [3H]cholesterol in plasma, liver, and feces was determined. The degree of HDL oxidation and the protection of oxidative modification of LDL co-incubated with HDL were evaluated by measuring conjugated diene kinetics. Plasma levels of HDLc, HDL phospholipids, apoA-I, and platelet-activated factor acetyl-hydrolase were increased in both HL- and EL-deficient mice. These genetically modified mice displayed increased levels of radiolabeled, HDL-bound [3H]cholesterol 48h after the label injection. The magnitude of macrophage-derived [3H]cholesterol in feces was also increased in both the HL- and EL-deficient mice. HDL from the HL- and EL-deficient mice was less prone to oxidation and had a higher ability to protect LDL from oxidation, compared with the HDL derived from the wild-type mice. These changes were correlated with plasma apoA-I and apoA-I/HDL total protein levels. In conclusion, targeted inactivation of both HL and EL in mice promoted macrophage-to-feces RCT and enhanced HDL antioxidant properties.

Measurement of the phospholipase activity of endothelial lipase in mouse plasma

Endothelial lipase (EL) is a major negative regulator of plasma HDL levels in mice, rabbits, and most probably, humans. Although this regulatory function is critically dependent on EL's hydrolysis of HDL phospholipids, as yet there is no phospholipase assay specific for EL in plasma. We developed such an assay for the mouse enzyme using a commercially available phospholipid-like fluorescent substrate in combination with an EL neutralizing antibody. The specificity of the assay was established using EL knockout mice and its utility demonstrated by detection of an increase in plasma EL phospholipase activity following exposure of wild-type mice to lipopolysaccharide. The assay revealed that murine pre-heparin plasma does not contain measurable EL activity, indicating that the hydrolysis of HDL phospholipids by EL in vivo likely occurs on the cell surface.

Effect of quercetin against lindane induced alterations in the serum and hepatic tissue lipids in wistar rats

ObjectiveTo assess the effect of quercetin (flavonoid) against lindane induced alterations in lipid profile of wistar rats. MethodsRats were administered orally with lindane (100 mg/kg body weight) and quercetin (10 mg/kg body weight) for 30 days. After the end of treatment period lipid profile was estimated in serum and tissue. ResultsElevated levels of serum cholesterol, triglycerides, low density lipoprotein (LDL), very Low Density Lipoprotein (VLDL) and tissue triglycerides, cholesterol with concomitant decrease in serum HDL and tissue phospholipids were decreased in lindane treated rats were found to be significantly decreased in the quercetin and lindane co-treated rats. ConclusionsOur study suggests that quercetin has hypolipidemic effect and offers protection against lindane induced toxicity in liver by restoring the altered levels of lipids. The quercetin cotreatment along with lindane for 30 days reversed these biochemical alterations in lipids induced by lindane.

Abstract 9179: Free Oxidized Fatty Acids Contribute to Pro-Inflammatory HDL

Background: Based on previous studies we thought it likely that the content of oxidized phospholipids would be elevated in pro-inflammatory HDL. While total plasma levels of oxidized phospholipids were increased in mouse models of atherosclerosis and in humans with coronary heart disease (CHD), the content of these oxidized phospholipids in HDL was NOT increased. This led us to look for other lipid components of HDL that might contribute to pro-inflammatory HDL. Methods &Results: HDL was isolated from sucrose-cryopreserved plasma by ultracentrifugation in the presence of anti-oxidants. The content of free 5-HETE, 9-HETE, 12-HETE, 15-HETE, 9-HODE, and 13-HODE were determined by LC-MS/MS and found to be significantly (p<0.0001; p = 0.0008; p = 0.0289; p = 0.0028; p = 0.0052; and p = 0.0029, respectively) elevated in HDL of 10 patients with CHD (all of whom had pro-inflammatory HDL as determined by cell-based assay) compared to 10 healthy controls (all of whom had anti-inflammatory HDL as determined by cell-based assay). To determine if free oxidized fatty acids contributed to the formation of pro-inflammatory HDL these oxidized fatty acids were added to anti-inflammatory HDL in amounts similar to that found in the CHD patients. The addition of 5-HETE, 9-HETE, and 15-HETE significantly (p = 0.0199; p = 0.0249; p = 0.0382, respectively) reduced the ability of the anti-inflammatory HDL to inhibit cytokine production in cultures of human aortic endothelial cells exposed to oxidized phospholipids. In contrast, addition of saturated non-oxidized free fatty acids such as decanoic or stearic acid did not alter the anti-inflammatory properties of HDL. Conclusions: These results suggest that i) HDL from CHD patients contains elevated levels of free oxidized fatty acids; ii) Free oxidized fatty acids can convert anti-inflammatory HDL to pro-inflammatory HDL.

High-density lipoprotein phospholipids interfere with dendritic cell Th1 functional maturation

Lipoproteins are both lipid carriers in the blood and regulators of essential biological processes. Several studies demonstrated that lipoproteins modified during pathological conditions could alter dendritic cell (DC) maturation. Here the immune function of non-pathological lipoproteins is addressed by analysing their impact on human DC maturation triggered by TLR ligands. Upon TLR4 stimulation, low- and high-density lipoproteins (LDL and HDL) strongly inhibited the ability of DC to induce a Th1 response of T cells, characterized by high levels of IFNγ secretion, whereas the effect of very low-density lipoprotein was subject to variations. HDL also inhibited the Th1 function of DC stimulated by TLR1/2 and TLR2/6 ligands. The phospholipid fraction from HDL retained the inhibitory activity of the lipoprotein. We identified the 1-palmitoyl-2-linoleyl-phosphatidylcholine (PLPC) as one active phospholipid that inhibited the Th1 function of mature DCs whereas the dipalmitoyl-phosphatidylcholine had no significant effect. The treatment of DC by PLPC, 24h before TLR4 stimulation, resulted in reduced activation of NF-κB. This study shows that some HDL phospholipids have a direct immunoregulatory function, by modulating DC ability to activate a Th1 response of T cells.

Relationship of adiponectin with metabolic syndrome components in pubertal children

ObjectiveAdiponectin is an adipose tissue-derived adipocytokine which appears in decreased concentrations in obese patients and in several processes related to cardiovascular disease, such as type 2 diabetes or metabolic syndrome. The aim of this study was to analyze the relationship between adiponectin and components of metabolic syndrome (lipid profile, blood pressure, insulin and insulin resistance) in pubertal Spanish children. MethodsThe population-based sample included 810 healthy children (382 boys and 428 girls) 12–16 years of age. Anthropometric parameters and blood pressure were measured. Lipid levels were determined by standard methods, and insulin and adiponectin concentrations were measured by ELISA. Insulin resistance index was assessed by HOMA-IR. ResultsAdiponectin levels were negatively correlated with insulin and HOMA in both boys and girls, and remained significant after adjustment for BMI z-score in girls. After this adjustment, adiponectin maintained a positive correlation with HDL–cholesterol and HDL–phospholipids in both genders, and correlated with triglycerides in girls. Multiple linear regression analysis showed that, after adjustment for BMI z-score, adiponectin accounted for 15.8% of the variation of HDL–cholesterol in girls and for 5% of its variation in boys; meanwhile, it accounted for 15.8% and 12.7% of the variation of HDL–phospholipids in girls and boys, respectively. ConclusionsAdiponectin levels in 12- to 16-year-old children appear to be more strongly related to better lipid profile and insulin sensitivity in girls than in boys. Our study shows, for the first time to our knowledge, a significant positive correlation between adiponectin and HDL–phospholipids in pubertal children.

Native high-density lipoproteins inhibit platelet activation via scavenger receptor BI: Role of negatively charged phospholipids

ObjectivesHIGH-density lipoproteins (HDL) are a negative predictor of platelet-dependent thrombus formation and reduced platelet activation has been observed in vitro in the presence of HDL3, a major HDL fraction. However, mechanisms underlying the anti-thrombotic effects of HDL3 are poorly understood. Scavenger receptors class B represent possible HDL3 binding partners on platelets. We here investigated the role of scavenger receptor class B type I (SR-BI) and CD36 in mediating inhibitory effects of native HDL3 on thrombin-induced platelet activation. Methods and resultsRhodamine isothiocyanate-labeled HDL3 bound specifically to platelets and HDL3 binding was inhibited by scavenger receptor class B ligands such as phosphatidylserine (PS)- or phosphatidylinositol (PI)-containing liposomes or maleylated albumin (mBSA). By contrast, scavenger receptor class A ligands failed to displace HDL3 from platelets. HDL3, PS- and PI-liposomes, and mBSA inhibited thrombin-induced platelet aggregation, fibrinogen binding, P-selectin expression and mobilization of intracellular Ca2+. In addition, PS- and PI-liposomes emulated HDL3-induced intracellular signaling cascades including diacylglycerol production and protein kinase C activation. The reduction of platelet activation by liposomes was related to their PS or PI content. Moreover, inhibitory effects of native HDL3 were enhanced after enriching lipoproteins with PS, while PS- and PI-poor HDL2 failed to inhibit platelet aggregation and Ca2+ mobilization. Both, HDL3 and PS-containing liposomes failed to inhibit thrombin-induced activation of platelets obtained from SR-BI-deficient mice but not CD36-deficient mice. ConclusionWe suggest that SR-BI is a functional receptor for native HDL3 on platelets that generates an inhibitory signal for platelet activation. The content of negatively charged phospholipids (PS, PI) in HDL may be an important determinant of their anti-thrombotic potential.

Differential effects of gemfibrozil and fenofibrate on reverse cholesterol transport from macrophages to feces in vivo

Gemfibrozil and fenofibrate, two of the fibrates most used in clinical practice, raise HDL cholesterol (HDLc) and are thought to reduce the risk of atherosclerotic cardiovascular disease. These drugs act as PPARα agonists and upregulate the expression of genes crucial in reverse cholesterol transport (RCT). In the present study, we determined the effects of these two fibrates on RCT from macrophages to feces in vivo in human apoA-I transgenic (hApoA-ITg) mice. [3H]cholesterol-labeled mouse macrophages were injected intraperitoneally into hApoA-ITg mice treated with intragastric doses of fenofibrate, gemfibrozil or a vehicle solution for 17days, and radioactivity was determined in plasma, liver and feces. Fenofibrate, but not gemfibrozil, enhanced [3H]cholesterol flux to plasma and feces of female hApoA-ITg mice. Fenofibrate significantly increased plasma HDLc, HDL phospholipids, hApoA-I levels and phospholipid transfer protein activity, whereas these parameters were not altered by gemfibrozil treatment. Unlike gemfibrozil, fenofibrate also induced the generation of larger HDL particles, which were more enriched in cholesteryl esters, together with higher potential to generate preβ-HDL formation and caused a significant increase in [3H]cholesterol efflux to plasma. Our findings demonstrate that fenofibrate promotes RCT from macrophages to feces in vivo and, thus, highlight a differential action of this fibrate on HDL.

The apolipoprotein A5 (APOA5) gene predisposes Caucasian children to elevated triglycerides and vitamin E (Four Provinces Study)

Objective Apolipoprotein A-V plays an important role in lipid metabolism regulation, particularly modulating triglyceride levels, as has been shown by many association studies in adults. The aim of this study was to analyse the effect of APOA5 on lipid profiles and fat-soluble vitamins (due to its strong relationship with triglyceride metabolism) in children. Methods We determined polymorphisms −1131T>C and S19W in the APOA5 gene in 964 6–8-year-old participants of the 4P study and analysed the influence of the APOA5 gene on plasma lipid levels (total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides), apolipolipoproteins (apo A-I and apo B) and fat-soluble antioxidant vitamin (α-tocopherol, γ-tocopherol, lycopene, α-carotene, β-carotene and retinol) levels. Results The allele frequencies of both polymorphisms were comparable to those described in adult Caucasian populations (0.08 and 0.07 for −1131T>C and S19W, respectively). Boys carrying the −1131C allele have a 12% increase in circulating triglyceride levels ( p = 0.016) and a 7% decrease in HDL phospholipid levels ( p = 0.016). Linked to its effect on triglycerides, boys with the −1131C allele also have a 5% increase in plasma α-tocopherol levels ( p = 0.032). This effect was not observed in female participants. Boys carrying the rare allele for the S19W polymorphism have a 4% increase in circulating cholesterol levels ( p = 0.045), whereas girls have a 9% increase in circulating triglyceride levels ( p = 0.029). Linked to its effect on triglycerides, female carriers of the rare allele for S19W also have a 6% increase in α-tocopherol levels ( p = 0.009). Conclusion In children, the effect of APOA5 gene variants on triglyceride levels is related to gender, and because of the strong relationship between lipid metabolism and fat-soluble antioxidant vitamins, it also involves a significant elevation in α-tocopherol concentrations.

HDL superphospholipidation enhances key steps in reverse cholesterol transport

HDL-phospholipids (HDL-PL) play an important role in reverse cholesterol transport (RCT). Phosphatidylcholine (PC) is the most important phospholipid in RCT because it is the essential cholesterol-binding component of lipoproteins and is the acyl donor in the esterification of FC by lecithin:cholesterol acyltransferase (LCAT). FC efflux to sera is a positive anti-atherogenic function of HDL-PL. Although PC has long been recognized as an anti-atherogenic agent, development of new HDL therapies based on PC has been fraught with issues of efficacy, cost, and safety. Moreover, some methods to increase HDL-PC perturb HDL and release lipid-free apolipoproteins (apo) A-I. We developed a new method, HDL SPLn (SPLn) using a modified detergent removal method that obviates these concerns. SPLn can incorporate PC into HDL and increase HDL-PC > 10-fold. This is achieved with no loss of apo A-I. According to size exclusion chromatography and native gradient gel electrophoresis, SPLn raises the HDL particle weight in a dose-dependent way, from ∼120 to ∼350 kDa. Kinetic analysis of FC efflux to the resulting SPLn particles shows that K m and V max for SPLn HDL are lower and higher respectively than for native HDL. As a consequence, the catalytic efficiency, V max/ K m, increases by more than 400%. Clinically, small increases in serum HDL-PL are associated with significant and profound increases in FC efflux to serum. Treatment of relatively small amounts of plasma by SPLn is a potential method of improving at least one step in RCT.

High density lipoprotein (HDL) particle size and composition predicts embryo fragmentation

OBJECTIVE: HDL particles are the predominant lipoprotein in human follicular fluid (FF) and only small HDLs cross the blood follicle barrier. We previously showed that FF-HDL cholesterol and its component protein, ApoAI, negatively predict embryo fragmentation (EF). We hypothesized that: (1) FF-HDL particle size and composition are different from plasma HDL particles; and (2) FF-HDL particle composition is an important determinant of embryo quality. DESIGN: Cross-sectional study MATERIALS AND METHODS: Characterization of homologous human plasma and FF lipoprotein particle sizes using NMR spectrometry and HDL2/HDL3 subclass fractionated biochemical analysis was performed in 9 IVF patients. Prospective clinical correlations were made between FF HDL lipid compositions (by HPLC) and EF in 38 study patients in whom individual mature follicles were aspirated and embryos tracked. RESULTS: We confirmed that EF is inversely correlated with levels of FF HDL cholesterol (p<0.003). Compared to serum, FF contains a significantly higher proportion of small relative to large HDL particles with a conspicuous lack of medium-sized HDL particles. The HDL phospholipid to cholesterol molar ratio (PL/C ratio) is positively correlated with a higher proportion of small HDL particles as measured by NMR (n=9, r=0.77, p<0.01) and HDL2/HDL3 subclass composition. The PL/C ratio is further positively correlated with embryo fragmentation during IVF (n=38, r=0.67, p<0.01). CONCLUSIONS: This is the first study to demonstrate the existence of different HDL particle size distributions in FF compared to blood by NMR. We further show a correlation between parameters of HDL particle lipid composition and size with embryo fragmentation. Future work will focus on molecular characterization of FF HDL components. This work suggests the possibity that remodeling of FF HDL is important to oocyte and embryo development which requires further investigation.

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.

Reverse modulation of the HDL Anionic Peptide Factor and phospholipid transfer protein activity in coronary artery disease and type 2 diabetes mellitus

Objectives The high density lipoprotein Anionic Peptide Factor (HDL 3-APF) was previously described as an apolipoprotein that promotes the reverse cholesterol transport. Since phospholipid transfer protein (PLTP) is involved in such mechanism we attempted to focus on the two APF and PLTP proteins. Design and methods We recruited 56 type 2 diabetic patients with ( n = 36) or without ( n = 20) coronary artery disease (CAD) and 19 CAD patients. The three groups were compared to 39 healthy control subjects. In all groups, lipid profile was determined and plasma APF concentrations and PLTP activity were measured. Results In all patients, the PLTP activity was significantly increased in comparison with controls ( p < 0.01), in concomitance with a plasma APF level decrease in groups with CAD (with and without type 2 diabetes) ( p < 0.01). Multiple linear regression analysis demonstrated that, when apoA-I, HDL-C, HDL-phospholipids and PLTP activity were taken into account as independent variables (after univariate regression analysis), HDL-PL was positively and independently related to APF ( p < 0.0001 in whole population; p = 0.0090 in controls) and PLTP activity was negatively and independently related to APF in whole population and all patients' groups (all p < 0.05), but positively and independently associated to APF in controls ( p = 0.0005). Conclusions APF could be considered as a specific marker against CAD and type 2 diabetes mellitus and our results confirm the atherogenic behavior of PLTP in CAD. Thus, these two proteins are likely to be regulated in a reverse manner.

HDL Composition Regulates Displacement of Cell Surface‐Bound Hepatic Lipase

HDL is able to displace cell surface-bound hepatic lipase (HL) and stimulate vascular triglyceride (TG) hydrolysis, much like heparin. Displacement appears to be a result of a high-affinity association of HL and apoA-I. HDL varies in its ability to displace HL, and therefore experiments were undertaken to evaluate the impact of HDL composition and structure on HL displacement from cell surface proteoglycans. HDL apolipoprotein and lipid composition directly affect HL displacement. ApoA-II and apoC-I significantly increase HL displacement from the cell surface. While changes in HDL cholesteryl ester and fatty acid content have no effect on HL displacement, increases in HDL phospholipid and TG content significantly inhibit HL displacement. HDL fractions from hyperlipidemic patients are unable to displace HL from the cell surface. These results indicate that the structure and composition of HDL particles in plasma are central to regulation of HL displacement and the hydrolytic activity of HL.

The roles of different pathways in the release of cholesterol from macrophages

Cholesterol efflux occurs by different pathways, including transport mediated by specific proteins. We determined the effect of enriching cells with free cholesterol (FC) on the release of FC to human serum. Loading Fu5AH cells with FC had no effect on fractional efflux, whereas enriching mouse peritoneal macrophages (MPMs) resulted in a doubling of fractional efflux. Efflux from cholesterol-normal MPM and Fu5AH cells to 15 human sera correlated well with HDL parameters. However, these relationships were reduced or lost with cholesterol-loaded MPMs. Using macrophages from scavenger receptor class B type I (SR-BI)-, ABCA1-, and ABCG1-knockout mice, together with inhibitors of SR-BI- and ABCA1-mediated efflux, we were able to quantitate efflux upon loading macrophages with excess cholesterol and to establish the contributions of the various efflux pathways in cholesterol-normal and -enriched cells. The removal of ABCA1 had essentially no effect on the total efflux when cell cholesterol levels were normal. However, in cholesterol-enriched cells, the removal of ABCA1 reduced efflux by 50%. Approximately 20% of the efflux stimulated by FC-loading MPM is attributable to ABCG1. The SR-BI contribution to efflux was small. Another pathway that is present in all cells is aqueous diffusion. Our studies demonstrate that this mechanism is one of the major contributors to efflux, particularly in cholesterol-normal cells.