Neil J. Hime

Specific dietary polyphenols attenuate atherosclerosis in apolipoprotein E-knockout mice by alleviating inflammation and endothelial dysfunction.

Objective—Animal and clinical studies have suggested that polyphenols in fruits, red wine, and tea may delay the development of atherosclerosis through their antioxidant and anti-inflammatory properties. We investigated whether individual dietary polyphenols representing different polyphenolic classes, namely quercetin (flavonol), (−)-epicatechin (flavan-3-ol), theaflavin (dimeric catechin), sesamin (lignan), or chlorogenic acid (phenolic acid), reduce atherosclerotic lesion formation in the apolipoprotein E (ApoE)−/− gene–knockout mouse. Methods and Results—Quercetin and theaflavin (64-mg/kg body mass daily) significantly attenuated atherosclerotic lesion size in the aortic sinus and thoracic aorta (P<0.05 versus ApoE−/− control mice). Quercetin significantly reduced aortic F2-isoprostane, vascular superoxide, vascular leukotriene B4, and plasma-sP-selectin concentrations; and augmented vascular endothelial NO synthase activity, heme oxygenase-1 protein, and urinary nitrate excretion (P<0.05 versus control ApoE−/− mice). Theaflavin showed similar, although less extensive, significant effects. Although (−)-epicatechin significantly reduced F2-isoprostane, superoxide, and endothelin-1 production (P<0.05 versus control ApoE−/− mice), it had no significant effect on lesion size. Sesamin and chlorogenic acid treatments exerted no significant effects. Quercetin, but not (−)-epicatechin, significantly increased the expression of heme oxygenase-1 protein in lesions versus ApoE−/− controls. Conclusion—Specific dietary polyphenols, in particular quercetin and theaflavin, may attenuate atherosclerosis in ApoE−/− gene–knockout mice by alleviating inflammation, improving NO bioavailability, and inducing heme oxygenase-1. These data suggest that the cardiovascular protection associated with diets rich in fruits, vegetables, and some beverages may in part be the result of flavonoids, such as quercetin.

What Is So Special About Apolipoprotein AI in Reverse Cholesterol Transport

An initial step in reverse cholesterol transport is the movement of unesterified cholesterol from peripheral cells to high-density lipoproteins (HDLs). This transfer usually occurs in extracellular spaces, such as the subendothelial space of a vessel wall, and is promoted by the interaction of lipid-free or lipid-poor apolipoprotein (apo)AI with ATP binding cassette A1 cellular transporters on macrophages (M&PHgr;). Because HDL does not interact with M&PHgr; ATP binding cassette A1 and apoAI is not synthesized by macrophages, this apoAI must be generated from spherical HDL. In this brief review, we propose that spherical apoAI is derived from HDL by remodeling events that are accomplished by proteins secreted by cholesteryl ester–loaded foam cells, including the lipid transfer proteins, phospholipid transfer protein, and cholesteryl ester transfer protein, and the triglyceride hydrolases hepatic lipase and lipoprotein lipase.

Evidence That Cholesteryl Ester Transfer Protein-mediated Reductions in Reconstituted High Density Lipoprotein Size Involve Particle Fusion

It is well established that cholesteryl ester transfer protein (CETP) changes the size of high density lipoproteins (HDL) during incubation in vitro It has been suggested that HDL·CETP·HDL ternary complex formation is involved in these changes. The present results, which are consistent with CETP changing the size of spherical reconstituted HDL (rHDL) by a mechanism involving fusion, support the ternary complex hypothesis. When rHDL containing a core of cholesteryl esters and either three molecules of apolipoprotein (apo) A-I/particle, (A-I)rHDL, or six molecules of apoA-II/particle, (A-II)rHDL, were incubated individually with CETP, their respective diameters decreased from 9.4 to 7.8 nm and from 9.8 to 8.8 nm. The small (A-I)rHDL and (A-II)rHDL contained, respectively, two molecules of apoA-I/particle and four molecules of apoA-II/particle. As all of the rHDL lipids and apolipoproteins were quantitatively recovered at the end of the incubations, it was apparent that there was a 50% increase in the number of particles. This increase in the number of particles can be explained as follows: (i) sequential binding of two rHDL to CETP to generate a ternary complex, (ii) fusion of the rHDL in the ternary complex, and (iii) rearrangement of the fusion product into three small particles. Various spectroscopic techniques were used to show that the small rHDL were structurally distinct from the original rHDL. These results provide the first evidence that CETP mediates the fusion of spherical rHDL.

The Influence of Apolipoproteins on the Hepatic Lipase-mediated Hydrolysis of High Density Lipoprotein Phospholipid and Triacylglycerol

This study describes the influence of apolipoproteins on the hepatic lipase (HL)-mediated hydrolysis of phospholipids and triacylglycerol in high density lipoproteins (HDL). HL-mediated hydrolysis was assessed in well characterized, homogeneous preparations of spherical reconstituted high density lipoproteins (rHDL). The rHDL were comparable in size and lipid composition and contained either apoA-I ((A-I)rHDL) or apoA-II ((A-II)rHDL) as their sole apolipoprotein constituent. Preparations of rHDL containing only cholesteryl esters (CE) in their core, (A-I/CE)rHDL and (A-II/CE)rHDL, were used to assess phospholipid hydrolysis. Preparations of rHDL that contained triacylglycerol as their predominant core lipid, (A-I/TG)rHDL and (A-II/TG)rHDL, were used to assess both triacylglycerol and phospholipid hydrolysis. The rHDL contained trace amounts of either radiolabeled phospholipid or radiolabeled triacylglycerol. Hydrolysis was measured as the release of radiolabeled nonesterified fatty acids (NEFA) from the rHDL. Kinetic analysis showed that HL had a greater affinity for the phospholipids in (A-II/CE)rHDL (K m (app) = 0.2 mm) than in (A-I/CE)rHDL (K m (app) = 3.1 mm). This was also evident when hydrolysis was measured directly by quantitating NEFA mass. HL also had a greater affinity for the phospholipids and triacylglycerol in (A-II/TG)rHDL than in (A-I/TG)rHDL. TheV max for phospholipid hydrolysis was, by contrast, greater for (A-I/CE)rHDL than for (A-II/CE)rHDL: 309.3versus 49.1 nmol of NEFA formed/ml of HL/h. ComparableV max values were obtained for the hydrolysis of the phospholipids in (A-II/TG)rHDL and (A-I/TG)rHDL. In the case of triacylglycerol hydrolysis, the respective V maxvalues for (A-I/TG)rHDL and (A-II/TG)rHDL were 1154.8 and 240.2 nmol of NEFA formed/ml of HL/h. These results show that apolipoproteins have a major influence on the kinetics of HL-mediated phospholipid and triacylglycerol hydrolysis in rHDL.

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.

Anti-atherosclerotic and anti-diabetic properties of probucol and related compounds

Abstract Probucol is a diphenolic compound with anti-oxidant and anti-inflammatory properties that reduces atherosclerosis and restenosis. Unfortunately, adverse effects on blood lipoproteins and cardiac electrophysiology have curtailed its use as a drug. Compounds related to probucol that have improved efficacy without the adverse effects offer promise as novel therapies of cardiovascular disease. Recent results suggest that these compounds may be used for the prevention of type 2 diabetes, a disease that is increasing in prevalence and importance world-wide. In this review, the molecular mechanisms underlying the beneficial activities of probucol and related compounds are described.

Bone marrow-derived HL mitigates bone marrow-derived CETP-mediated decreases in HDL in mice globally deficient in HL and the LDLr

The objective of this study was to determine the combined effects of HL and cholesteryl ester transfer protein (CETP), derived exclusively from bone marrow (BM), on plasma lipids and atherosclerosis in high-fat-fed, atherosclerosis-prone mice. We transferred BM expressing these proteins into male and female double-knockout HL-deficient, LDL receptor-deficient mice (HL−/−LDLr−/−). Four BM chimeras were generated, where BM-derived cells expressed 1) HL but not CETP, 2) CETP and HL, 3) CETP but not HL, or 4) neither CETP nor HL. After high-fat feeding, plasma HDL-cholesterol (HDL-C) was decreased in mice with BM expressing CETP but not HL (17 ± 4 and 19 ± 3 mg/dl, female and male mice, respectively) compared with mice with BM expressing neither CETP nor HL (87 ± 3 and 95 ± 4 mg/dl, female and male mice, respectively, P < 0.001 for both sexes). In female mice, the presence of BM-derived HL mitigated this CETP-mediated decrease in HDL-C. BM-derived CETP decreased the cholesterol component of HDL particles and increased plasma cholesterol. BM-derived HL mitigated these effects of CETP. Atherosclerosis was not significantly different between BM chimeras. These results suggest that BM-derived HL mitigates the HDL-lowering, HDL-modulating, and cholesterol-raising effects of BM-derived CETP and warrant further studies to characterize the functional properties of these protein interactions.

Influence of sphingomyelin on the structure and function of reconstituted high density lipoproteins

The effect of sphingomyelin (SPM) on the structure and function of discoidal and spherical reconstituted high density lipoproteins (rHDL) has been studied. Three preparations of discoidal rHDL with 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC)/SPM/unesterified cholesterol (UC)/apolipoprotein (apo)A-I molar ratios of 99.6/0. 0/10.2/1.0, 86.0/13.6/10.8/1.0, and 72.5/26.3/11.4/1.0 were prepared by cholate dialysis. SPM did not affect discoidal rHDL size or surface charge. Esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT) was inhibited in the SPM-containing discoidal rHDL. When the discoidal rHDL of POPC/SPM/UC/apoA-I molar ratio 99.6/0.0/10.2/1.0 were incubated with low density lipoproteins (LDL) and LCAT, SPM transferred spontaneously from the LDL to the rHDL (t1/2 = 0.8 h) and spherical particles with a POPC/SPM/UC/CE/apoA-I molar ratio of 24.6/4.9/3. 6/24.9/1.0 were formed. Depleting the spherical rHDL of SPM head groups by incubation with sphingomyelinase increased the negative charge on the surface, but did not change their size. Cholesteryl ester transfer protein (CETP)-mediated transfers of cholesteryl esters and triglyceride between spherical rHDL and Intralipid were not affected by SPM head group depletion. The effect of SPM on rHDL structure was assessed spectroscopically. SPM increased POPC acyl chain and head group packing in the discoidal rHDL. When the spherical rHDL were depleted of SPM head groups, POPC acyl chain packing order decreased, but head group packing order was not affected. SPM inhibited the lipid-water interfacial hydration of discoidal rHDL. This parameter was not affected when the spherical rHDL were depleted of SPM head groups. The SPM molecule and the SPM head group, respectively, inhibited the unfolding of apoA-I in discoidal and spherical rHDL. It is concluded that (i) SPM influences the structure of discoidal and spherical rHDL, (ii) SPM inhibits the LCAT reaction in discoidal rHDL, and (iii) the SPM head group does not affect CETP-mediated lipid transfers into or out of spherical rHDL.

Influence of cholesteryl ester transfer protein on the composition, size and structure of spherical, reconstituted high density lipoproteins

The effect of cholesteryl ester transfer protein (CETP) on the size, composition, and structure of spherical, reconstituted HDL (rHDL) which contain apolipoprotein (apo) A-I as their sole apolipoprotein has been studied. Spherical rHDL were incubated with CETP and Intralipid for up to 24 h. During this time CETP promoted transfers of cholesteryl esters (CE) and triglyceride (TG) between rHDL and Intralipid. As a result, the rHDL became depleted of CE and enriched in TG. However, as the loss of CE from the rHDL was greater than the gain of TG, the concentration of core lipids in the rHDL decreased. The decrease in the concentration of rHDL core lipids, which was evident throughout the incubation, was accompanied by a reduction in rHDL diameter from 9.2 to 8.0 nm, the dissociation of apoA-I from rHDL and a decrease in the number of apoA-I molecules, from three/particle in the 9.2-nm rHDL, to two/particle in the 8.0-nm rHDL. Spectroscopic studies showed that the lipid-water interface and phospholipid packing of the 8.0-nm rHDL were, respectively, more polar and less ordered than those of the 9.2-nm rHDL. Quenching studies with KI revealed that the number of exposed apoA-I Trp residues in the 9.2- and 8.0-nm rHDL was two and three, respectively. Circular dichroism established that the 9.2- and 8.0-nm rHDL had identical apoA-I alpha-helical contents. The 9.2- and 8.0-nm rHDL also had identical surface charges as determined by agarose gel electrophoresis. Denaturation studies with guanidine hydrochloride demonstrated that apoA-I is more stable in 8.0-nm rHDL than in 9.2-nm rHDL. It is concluded that CETP converts rHDL to small, TG-enriched, apoA-I-depleted particles with increased lipid-water interfacial hydration and less ordered phospholipid packing. These changes are associated with enhanced stability and minor changes to the conformation of the apoA-I which remains associated with the rHDL.