CD36 is a receptor for oxidized high density lipoprotein: implications for the development of atherosclerosis

Atherosclerotic plaques result from the excessive deposition of cholesterol esters derived from lipoproteins and lipoprotein fragments. Tissue macrophage within the intimal space of major arterial vessels have been shown to play an important role in this process. We demonstrate in a transfection system using two human cell lines that the macrophage scavenger receptor CD36 selectively elicited lipid uptake from Cu(2+)-oxidized high density lipoprotein (HDL) but not from native HDL or low density lipoprotein (LDL). The uptake of oxHDL displayed morphological and biochemical similarities with the CD36-dependent uptake of oxidized LDL. CD36-mediated uptake of oxidized HDL by macrophage may therefore contribute to atheroma formation.     

Predictive value of different HDL particles for the protection against or risk of coronary heart disease

The inverse relationship between plasma HDL levels and the risk of developing coronary heart disease is well established. The underlying mechanisms of this relationship are poorly understood, largely because HDL consist of several functionally distinct subpopulations of particles that are continuously being interconverted from one to another. This review commences with an outline of what is known about the origins of individual HDL subpopulations, how their distribution is regulated, and describes strategies that are currently available for isolating them. We then summarise what is known about the functionality of specific HDL subpopulations, and how these findings might impact on cardiovascular risk. The final section highlights major gaps in existing knowledge of HDL functionality, and suggests how these deficiencies might be addressed. This article is part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).     

MD simulations suggest important surface differences between reconstituted and circulating spherical HDL

Since spheroidal HDL particles (sHDL) are highly dynamic, molecular dynamics (MD) simulations are useful for obtaining structural models. Here we use MD to simulate sHDL with stoichiometries of reconstituted and circulating particles. The hydrophobic effect during simulations rapidly remodels discoidal HDL containing mixed lipids to sHDL containing a cholesteryl ester/triglyceride (CE/TG) core. We compare the results of simulations of previously characterized reconstituted sHDL particles containing two or three apoA-I created in the absence of phospholipid transfer protein (PLTP) with simulations of circulating human HDL containing two or three apoA-I without apoA-II. We find that circulating sHDL compared with reconstituted sHDL with the same number of apoA-I per particle contain approximately equal volumes of core lipid but significantly less surface lipid monolayers. We conclude that in vitro reconstituted sHDL particles contain kinetically trapped excess phospholipid and are less than ideal models for circulating sHDL particles. In the circulation, phospholipid transfer via PLTP decreases the ratio of phospholipid to apolipoprotein for all sHDL particles. Further, sHDL containing two or three apoA-I adapt to changes in surface area by condensation of common conformational motifs. These results represent an important step toward resolving the complicated issue of the protein and lipid stoichiometry of circulating HDL.     

Effects of cholesteryl ester transfer protein inhibition on apolipoprotein A-II-containing HDL subspecies and apolipoprotein A-II metabolism

This study was designed to establish the mechanism responsible for the increased apolipoprotein (apo) A-II levels caused by the cholesteryl ester transfer protein inhibitor torcetrapib. Nineteen subjects with low HDL cholesterol (<40 mg/dl), nine of whom were also treated with 20 mg of atorvastatin daily, received placebo for 4 weeks, followed by 120 mg of torcetrapib daily for the next 4 weeks. Six subjects in the nonatorvastatin cohort participated in a third phase, in which they received 120 mg of torcetrapib twice daily for 4 weeks. At the end of each phase, subjects underwent a primed-constant infusion of [5,5,5-²H₃]l-leucine to determine the kinetics of HDL apoA-II. Relative to placebo, torcetrapib significantly increased apoA-II concentrations by reducing HDL apoA-II catabolism in the atorvastatin (−9.4%, P < 0.003) and nonatorvastatin once- (−9.9%, P = 0.02) and twice- (−13.2%, P = 0.02) daily cohorts. Torcetrapib significantly increased the amount of apoA-II in the α-2-migrating subpopulation of HDL when given as monotherapy (27%, P < 0.02; 57%, P < 0.003) or on a background of atorvastatin (28%, P < 0.01). In contrast, torcetrapib reduced concentrations of apoA-II in α-3-migrating HDL, with mean reductions of −14% (P = 0.23), −18% (P < 0.02), and −18% (P < 0.01) noted during the atorvastatin and nonatorvastatin 120 mg once- and twice-daily phases, respectively. Our findings indicate that CETP inhibition increases plasma concentrations of apoA-II by delaying HDL apoA-II catabolism and significantly alters the remodeling of apoA-II-containing HDL subpopulations.     

Persistence of high density lipoprotein particles in obese mice lacking apolipoprotein A-I

Obese mice without leptin (ob/ob) or the leptin receptor (db/db) have increased plasma HDL levels and accumulate a unique lipoprotein referred to as LDL/HDL1. To determine the role of apolipoprotein A-I (apoA-I) in the formation and accumulation of LDL/HDL1, both ob/ob and db/db mice were crossed onto an apoA-I-deficient (apoA-I(-/-)) background. Even though the obese apoA-I(-/-) mice had an expected dramatic decrease in HDL levels, the LDL/HDL1 particle persisted. The cholesterol in this lipoprotein range was associated with both alpha- and beta-migrating particles, confirming the presence of small LDLs and large HDLs. Moreover, in the obese apoA-I(-/-) mice, LDL particles were smaller and HDLs were more negatively charged and enriched in apoE compared with controls. This LDL/HDL1 particle was rapidly remodeled to the size of normal HDL after injection into C57BL/6 mice, but it was not catabolized in obese apoA-I(-/-) mice even though plasma hepatic lipase (HL) activity was increased significantly. The finding of decreased hepatic scavenger receptor class B type I (SR-BI) protein levels may explain the persistence of LDL/HDL1 in obese apoA-I(-/-) mice. Our studies suggest that the maturation and removal of large HDLs depends on the integrity of a functional axis of apoA-I, HL, and SR-BI. Moreover, the presence of large HDLs without apoA-I provides evidence for an apoA-I-independent pathway of cholesterol efflux, possibly sustained by apoE.     

Rapid purification and activity of apolipoprotein CI on the proliferation of bovine vascular endothelial cells in vitro

The growth-promoting activity of human high-density lipoproteins (HDL) and of their apolipoprotein components on bovine vascular endothelial cells in vitro has been compared. When maintained on plastic culture dishes and exposed to medium containing lipoprotein-deficient serum and fibroblast growth factor, these cells do not proliferate. Addition of either HDL or the total HDL apolipoproteins induces significant cell proliferation. Apolipoprotein C_I, purified by chromatography on the ion-exchanger resin Polybuffer exchanger 94, has an effect on the cell growth similar to that of the total apolipoproteins of HDL.     

SR-BI-mediated selective lipid uptake segregates apoA-I and apoA-II catabolism

The HDL receptor scavenger receptor class B type I (SR-BI) binds HDL and mediates the selective uptake of cholesteryl ester. We previously showed that remnants, produced when human HDL(2) is catabolized in mice overexpressing SR-BI, become incrementally smaller, ultimately consisting of small alpha-migrating particles, distinct from pre-beta HDL. When mixed with mouse plasma, some remnant particles rapidly increase in size by associating with HDL without the mediation of cholesteryl ester transfer protein, LCAT, or phospholipid transfer protein. Here, we show that processing of HDL(2) by SR-BI-overexpressing mice resulted in the preferential loss of apolipoprotein A-II (apoA-II). Short-term processing generated two distinct, small alpha-migrating particles. One particle (8.0 nm diameter) contained apoA-I and apoA-II; the other particle (7.7 nm diameter) contained only apoA-I. With extensive SR-BI processing, only the 7.7 nm particle remained. Only the 8.0 nm remnants were able to associate with HDL. Compared with HDL(2), this remnant was more readily taken up by the liver than by the kidney. We conclude that SR-BI-generated HDL remnants consist of particles with or without apoA-II and that only those containing apoA-II associate with HDL in an enzyme-independent manner. Extensive SR-BI processing generates small apoA-II-depleted particles unable to reassociate with HDL and readily taken up by the liver. This represents a pathway by which apoA-I and apoA-II catabolism are segregated.     

Formation of high density lipoproteins containing both apolipoprotein A-I and A-II in the rabbit

Human plasma HDLs are classified on the basis of apolipoprotein composition into those that contain apolipoprotein A-I (apoA-I) without apoA-II [(A-I)HDL] and those containing apoA-I and apoA-II [(A-I/A-II)HDL]. ApoA-I enters the plasma as a component of discoidal particles, which are remodeled into spherical (A-I)HDL by LCAT. ApoA-II is secreted into the plasma either in the lipid-free form or as a component of discoidal high density lipoproteins containing apoA-II without apoA-I [(A-II)HDL]. As discoidal (A-II)HDL are poor substrates for LCAT, they are not converted into spherical (A-II)HDL. This study investigates the fate of apoA-II when it enters the plasma. Lipid-free apoA-II and apoA-II-containing discoidal reconstituted HDL [(A-II)rHDL] were injected intravenously into New Zealand White rabbits, a species that is deficient in apoA-II. In both cases, the apoA-II was rapidly and quantitatively incorporated into spherical (A-I)HDL to form spherical (A-I/A-II)HDL. These particles were comparable in size and composition to the (A-I/A-II)HDL in human plasma. Injection of lipid-free apoA-II and discoidal (A-II)rHDL was also accompanied by triglyceride enrichment of the endogenous (A-I)HDL and VLDL as well as the newly formed (A-I/A-II)HDL. We conclude that, irrespective of the form in which apoA-II enters the plasma, it is rapidly incorporated into spherical HDLs that also contain apoA-I to form (A-I/A-II)HDL.     

Hepatic uptake and metabolism of phosphatidylcholine associated with high density lipoproteins

Background

Phosphatidylcholine (PC) is the predominant phospholipid associated with high density lipoproteins (HDL). Although the hepatic uptake of cholesteryl esters from HDL is well characterized, much less is known about the fate of PC associated with HDL. Thus, we investigated the uptake and subsequent metabolism of HDL-PC in primary mouse hepatocytes.

Methods and results

The absence of scavenger receptor-BI resulted in a 30% decrease in cellular incorporation of [³H]PC whereas [³H]cholesteryl ether uptake was almost completely abolished. Although endocytosis is not involved in the uptake of cholesteryl esters from HDL, we demonstrate that HDL internalization accounts for 40% of HDL-PC uptake. Extracellular remodeling of HDL by secretory phospholipase A₂ significantly enhances HDL lipid uptake. HDL-PC taken up by hepatocytes is partially converted to triacylglycerols via PC-phospholipase C-mediated hydrolysis of PC and incorporation of diacylglycerol into triacylglcyerol. The formation of triacylglcerol is independent of scavenger receptor-BI and occurs in extralysosomal compartments.

Conclusions and general significance

These findings indicate that HDL-associated PC is incorporated into primary hepatocytes via a pathway that differs significantly from that of HDL-cholesteryl ester, and shows that HDL-PC is more than a framework molecule, as evidenced by its partial conversion to hepatic triacylglycerol.     

Inhibition of MMP-2 activation and release as a novel mechanism for HDL-induced cardioprotection

High density lipoproteins (HDL) protect the heart against ischemia/reperfusion (I/R) injury, and matrix metalloproteinase-2 (MMP-2) directly contributes to cardiac contractile dysfunction after I/R. To investigate the possible involvement of MMP-2 inhibition in HDL-mediated cardioprotection, isolated rat hearts underwent 20 min of low-flow ischemia and 30 min of reperfusion. Plasma-derived and synthetic HDL attenuated the I/R-induced cardiac MMP-2 activation and release in a dose-dependent way. The attenuation of I/R-induced MMP-2 activation by HDL correlated with the reduction of post-ischemic contractile dysfunction and cardiomyocyte necrosis. These results indicate prevention of MMP-2 activation as a novel mechanism for HDL-mediated cardioprotection.     

Dehydroepiandrosterone fatty acyl esters in high density lipoprotein: interaction with human vascular endothelial cells and vascular responses ex vivo

Dehydroepiandrosterone (DHEA) fatty acyl esters once incorporated in high density lipoprotein (HDL) induce a stronger vasodilatory response in rat mesenteric arteries ex vivo compared to native HDL. We studied the role of HDL receptor, scavenger receptor class B, type 1 (SR-B1), as well as estrogen and androgen receptors in the vasodilatory response of HDL-associated DHEA fatty acyl esters. Using cultured human vascular endothelial cells (HUVEC), we investigated the possible internalization and cellular response of HDL-associated DHEA esters. We prepared DHEA ester-enriched HDL by incubating human plasma in the presence of DHEA. After isolation and purification, HDL was added in cumulative doses to arterial rings precontracted with noradrenaline. Inhibition of the function of SR-B1 almost completely abolished maximal vasorelaxation by DHEA-enriched HDL while estrogen or androgen receptor blockage had no significant effect. When HUVECs were incubated in the presence of [³H]DHEA ester-enriched HDL, the amount of intracellular [³H]-radioactivity increased steadily during 24 h. Blocking of SR-B1 reduced this uptake by a mean of 30%. The proportion of unesterified [³H]DHEA, as analyzed by thin-layer chromatography, increased intracellularly and in the cell culture media after several hours of incubation of the cells in the presence of [³H]DHEA ester-enriched HDL. This indicated slow hydrolysis of DHEA fatty acyl esters and subsequent excretion of unesterified DHEA by the cells. In conclusion, DHEA-enriched HDL induced vasorelaxation via the SR-B1-facilitated pathway. However, this vasodilation is not likely to be attributed to rapid hydrolysis of HDL-associated DHEA esters by the vascular endothelium.     

Mechanisms involved in vitamin E transport by primary enterocytes and in vivo absorption

It is generally believed that vitamin E is absorbed along with chylomicrons. However, we previously reported that human colon carcinoma Caco-2 cells use dual pathways, apolipoprotein B (apoB)-lipoproteins and HDLs, to transport vitamin E. Here, we used primary enterocytes and rodents to identify in vivo vitamin E absorption pathways. Uptake of [(3)H]alpha-tocopherol by primary rat and mouse enterocytes increased with time and reached a maximum at 1 h. In the absence of exogenous lipid supply, these cells secreted vitamin E with HDL. Lipids induced the secretion of vitamin E with intermediate density lipoproteins, and enterocytes supplemented with lipids and oleic acid secreted vitamin E with chylomicrons. The secretion of vitamin E with HDL was not affected by lipid supply but was enhanced when incubated with HDL. Microsomal triglyceride transfer protein inhibition reduced vitamin E secretion with chylomicrons without affecting its secretion with HDL. Enterocytes from Mttp-deficient mice also secreted less vitamin E with chylomicrons. In vivo absorption of [(3)H]alpha-tocopherol by mice after poloxamer 407 injection to inhibit lipoprotein lipase revealed that vitamin E was associated with triglyceride-rich lipoproteins and small HDLs containing apoB-48 and apoA-I. These studies indicate that enterocytes use two pathways for vitamin E absorption. Absorption with chylomicrons is the major pathway of vitamin E absorption. The HDL pathway may be important when chylomicron assembly is defective and can be exploited to deliver vitamin E without increasing fat consumption.     

Influence of HDL-cholesterol-elevating drugs on the in vitro activity of the HDL receptor SR-BI

Treatment of atherosclerotic disease often focuses on reducing plasma LDL-cholesterol or increasing plasma HDL-cholesterol. We examined in vitro the effects on HDL receptor [scavenger receptor class B type I (SR-BI)] activity of three classes of clinical and experimental plasma HDL-cholesterol-elevating compounds: niacin, fibrates, and HDL376. Fenofibrate (FF) and HDL376 were potent (IC(50) approximately 1 microM), direct inhibitors of SR-BI-mediated lipid transport in cells and in liposomes reconstituted with purified SR-BI. FF, a prodrug, was a more potent inhibitor of SR-BI than an activator of peroxisome proliferator-activated receptor alpha, a target of its active fenofibric acid (FFA) derivative. Nevertheless, FFA, four other fibrates (clofibrate, gemfibrozil, ciprofibrate, and bezafibrate), and niacin had little, if any, effect on SR-BI, suggesting that they do not directly target SR-BI in vivo. However, similarities of HDL376 treatment and SR-BI gene knockout on HDL metabolism in vivo (increased HDL-cholesterol and HDL particle sizes) and structure-activity relationship analysis suggest that SR-BI may be a target of HDL376 in vivo. HDL376 and other inhibitors may help elucidate SR-BI function in diverse mammalian models and determine the therapeutic potential of SR-BI-directed pharmaceuticals.     

Effect of cigarette smoking on high density lipoprotein phospholipids

The effect of acute inhalation of cigarette smoke on high density lipoprotein (HDL) phospholipid composition in White Carneau pigeons was examined. Four treatments included: 1) Shelf Control birds fed a chow diet and retained in their cages; 2) Sham pigeons fed a cholesterol-saturated fat diet and exposed to fresh air by a smoking machine; 3) Low nicotine-low carbon monoxide (LoLo) animals also fed the cholesterol diet and exposed to low concentrations of these cigarette smoke products; and 4) High nicotine-high carbon monoxide (HiHi) birds fed the cholesterol diet and subjected to high concentrations of these inhalants. The cholesterol-fat diet caused an increase in the concentration of most HDL phospholipid classes. Exposure to the HiHi regimen resulted in an increase in the HDL cholesterol/phospholipid ratio and a reduction in the concentration of HDL phosphatidyl ethanolamine, phosphatidyl serine/inositol, sphingomyelin and lysophosphatidyl choline. Cigarette smoking may thus attenuate HDL's anti-atherogenic properties by altering surface phospholipid components.     

Functional LCAT deficiency in human apolipoprotein A-I transgenic, SR-BI knockout mice

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-ITg SR-BI-/-) mice. Compared with hA-ITg mice, hA-ITg SR-BI-/- mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13-18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-ITg SR-BI-/- mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-ITg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-ITg SR-BI-/- mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.     

CETP does not affect triglyceride production or clearance in APOE*3-Leiden mice

The cholesteryl ester transfer protein (CETP) facilitates the bidirectional transfer of cholesteryl esters and triglycerides (TG) between HDL and (V)LDL. By shifting cholesterol in plasma from HDL to (V)LDL in exchange for VLDL-TG, CETP aggravates atherosclerosis in hyperlipidemic APOE*3-Leiden (E3L) mice. The aim of this study was to investigate the role of CETP in TG metabolism and high-fat diet-induced obesity by using E3L mice with and without the expression of the human CETP gene. On chow, plasma lipid levels were comparable between both male and female E3L and E3L.CETP mice. Further mechanistic studies were performed using male mice. CETP expression increased the level of TG in HDL. CETP did not affect the postprandial plasma TG response or the hepatic VLDL-TG and VLDL-apolipoprotein B production rate. Moreover, CETP did not affect the plasma TG clearance rate or organ-specific TG uptake after infusion of VLDL-like emulsion particles. In line with the absence of an effect of CETP on tissue-specific TG uptake, CETP also did not affect weight gain in response to a high-fat diet. In conclusion, the CETP-induced increase of TG in the HDL fraction of E3L mice is not associated with changes in the production of TG or with tissue-specific clearance of TG from the plasma.     

Separation of the principal HDL subclasses by iodixanol ultracentrifugation

HDL subclasses detection, in cardiovascular risk, has been limited due to the time-consuming nature of current techniques. We have developed a time-saving and reliable separation of the principal HDL subclasses employing iodixanol density gradient ultracentrifugation (IxDGUC) combined with digital photography. HDL subclasses were separated in 2.5 h from prestained plasma on a three-step iodixanol gradient. HDL subclass profiles were generated by digital photography and gel scan software. Plasma samples (n = 46) were used to optimize the gradient for the resolution of HDL heterogeneity and to compare profiles generated by IxDGUC with gradient gel electrophoresis (GGE); further characterization from participants (n = 548) with a range of lipid profiles was also performed. HDL subclass profiles generated by IxDGUC were comparable to those separated by GGE as indicated by a significant association between areas under the curve for both HDL₂ and HDL₃ (HDL₂, r = 0.896, P < 0.01; HDL₃, r = 0.894, P < 0.01). The method was highly reproducible, with intra- and interassay coefficient of variation percentage < 5 for percentage area under the curve HDL₂ and HDL₃, and < 1% for peak Rf and peak density. The method provides time-saving and cost-effective detection and preparation of the principal HDL subclasses.     

Estrogen increases hepatic lipase levels in inbred strains of mice: A possible mechanism for estrogen-dependent lowering of high density lipoprotein

We have shown mouse to be an useful animal model for studies on the estrogen-mediated synthesis and secretion of lipoproteins since, unlike in rats, low density lipoprotein receptors are not upregulated in mice [3]. This results into the elevation of plasma levels of apolipoprotein (apo) B and apoE, and lowering of apoA-I-containing particles. The mechanisms of apoB and apoE elevation by estrogen have been elucidated [6], but the mechanism of lowering of plasma levels of HDL is still not known. Among other factors, apoA-I, cholesterol ester transfer protein (CETP), scavenger receptor B1 (SR-B1), and hepatic lipase are potential candidates that modulate plasma levels of HDL. Since estrogen treatment increased hepatic apoA-I mRNA and apoA-I synthesis, and mouse express undetectable levels of CETP, we tested the hypothesis that estradiol-mediated lowering of HDL in mice may occur through modulation of hepatic lipase (HL). Four mouse strains (C57L, C57BL, BALB, C3H) were administered supraphysiological doses of estradiol, and plasma levels of HDL as well as HL mRNA were quantitated. In all 4 strains estradiol decreased plasma levels of HDL by 30%, and increased HL mRNA 2–3 fold. In a separate experiment groups of male C57BL mouse were castrated or sham-operated, and low and high doses of estradiol administered. We found 1.4–2.5 fold elevation of HL mRNA with concomitant lowering of HDL levels. Ten other mouse strains examined also showed estradiol-induced elevation of HL mRNA, but the extent of elevation was found to be strain-specific. Based on these studies, we conclude that hepatic lipase is an important determinant of plasma levels of HDL and that HL mRNA is modulated by estrogen which in turn may participate in the lowering of plasma levels of HDL.     

Remodeling of apolipoprotein E-containing spherical reconstituted high density lipoproteins by phospholipid transfer protein

Phospholipid transfer protein (PLTP) transfers phospholipids between HDL and other lipoproteins in plasma. It also remodels spherical, apolipoprotein A-I (apoA-I)-containing HDL into large and small particles in a process involving the dissociation of lipid-free/lipid-poor apoA-I. ApoE is another apolipoprotein that is mostly associated with large, spherical HDL that do not contain apoA-I. Three isoforms of apoE have been identified in human plasma: apoE2, apoE3, and apoE4. This study investigates the remodeling of spherical apoE-containing HDL by PLTP and the ability of PLTP to transfer phospholipids between apoE-containing HDL and phospholipid vesicles. Spherical reconstituted high density lipoproteins (rHDL) containing apoA-I [(A-I)rHDL], apoE2 [(E2)rHDL], apoE3 [(E3)rHDL], or apoE4 [(E4)rHDL] as the sole apolipoprotein were prepared by incubating discoidal rHDL with low density lipoproteins and lecithin:cholesterol acyltransferase. PLTP remodeled the spherical, apoE-containing rHDL into large and small particles without the dissociation of apoE. The PLTP-mediated remodeling of apoE-containing rHDL was more extensive than that of (A-I)rHDL. PLTP transferred phospholipids from small unilamellar vesicles to apoE-containing rHDL in an isoform-dependent manner, but at a rate slower than that for spherical (A-I)rHDL. It is concluded that apoE enhances the capacity of PLTP to remodel HDL but reduces the ability of HDL to participate in PLTP-mediated phospholipid transfers.