Identification of autoantibodies in human plasma recognizing an apoB-100 LDL receptor binding site peptide

The aim of this study was to test the hypothesis that autoantibodies recognize amino acid sequences in the LDL receptor binding region of apolipoprotein B-100 (apoB-100). Autoantibodies against an unmodified or malondialdehyde (MDA)-modified LDL receptor binding site peptide were determined by ELISA in baseline plasma samples of 78 cases with coronary events and 149 matched controls recruited from the prospective Malm? Diet Cancer Study. IgG and IgM recognizing this peptide were detected in all subjects but did not differ between cases and controls. Inverse associations were observed between IgG against the native binding site and plasma oxidized LDL (r = -0.21, P < 0.005), but there were no significant associations with total or LDL cholesterol levels. In univariate analyses, inverse associations were found between baseline carotid intima-media thickness and IgG against the MDA-modified binding site (r = -0.14, P < 0.05), but this association was lost when controlling for other major cardiovascular risk factors. Specificity studies demonstrated that the binding of autoantibodies to these sequences could be inhibited by oxidized but not by native LDL. Autoantibodies recognizing the LDL receptor binding site in apoB-100 are frequently expressed. Their association with plasma oxidized LDL suggests that they have been generated in response to breakdown products of LDL oxidation, but their influence on cholesterol metabolism and the development of atherosclerosis appears limited.     

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.     

Plant sterol or stanol esters retard lesion formation in LDL receptor-deficient mice independent of changes in serum plant sterols

Statins do not always decrease coronary heart disease mortality, which was speculated based on increased serum plant sterols observed during statin treatment. To evaluate plant sterol atherogenicity, we fed low density lipoprotein-receptor deficient (LDLr(+/-)) mice for 35 weeks with Western diets (control) alone or enriched with atorvastatin or atorvastatin plus plant sterols or stanols. Atorvastatin decreased serum cholesterol by 22% and lesion area by 57%. Adding plant sterols or stanols to atorvastatin decreased serum cholesterol by 39% and 41%. Cholesterol-standardized serum plant sterol concentrations increased by 4- to 11-fold during sterol plus atorvastatin treatment versus stanol plus atorvastatin treatment. However, lesion size decreased similarly in the sterol plus atorvastatin (-99% vs. control) and the stanol plus atorvastatin (-98%) groups, with comparable serum cholesterol levels, suggesting that increased plant sterol concentrations are not atherogenic. Our second study confirms this conclusion. Compared with lesions after a 33 week atherogenic period, lesion size further increased in controls (+97%) during 12 more weeks on the diet, whereas 12 weeks with the addition of plant sterols or stanols decreased lesion size (66% and 64%). These findings indicate that in LDLr(+/-) mice 1) increased cholesterol-standardized serum plant sterol concentrations are not atherogenic, 2) adding plant sterols/stanols to atorvastatin further inhibits lesion formation, and 3) plant sterols/stanols inhibit the progression or even induce the regression of existing lesions.     

LDL phospholipid hydrolysis produces modified electronegative particles with an unfolded apoB-100 protein

Electronegative low density lipoprotein (LDL(-)) formation that structurally resembles LDL(-) isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A(2) (PLA(2)). PLA(2) treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL(-) formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL(-) subfraction from plasma and PLA(2)-treated LDL (PLA(2)-LDL) to amyloid oligomer-specific antibody was observed. Higher beta-strand structural content and unfolding proportionate to the loss of alpha-helical structure of apolipoprotein B-100 (apoB-100) of LDL(-) isolated from both native and PLA(2)-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA(2)-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL(-). In contrast, PLA(2)-LDL was more resistant to copper-mediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids. The observed similarities between PLA(2)-LDL(-)-derived LDL(-) and plasma LDL(-) implicate a role for secretory PLA(2) in producing modified LDL(-) that is facilitated by unfolding of apoB-100.     

Regulation of macrophage apoE secretion and sterol efflux by the LDL receptor

Factors that regulate apolipoprotein E (apoE) secretion by macrophages will have important effects on vessel wall lipid flux and atherosclerosis. Macrophages express the LDL receptor, which binds apoE with high affinity and could thereby affect the net secretion of apoE from macrophages. In these studies, we demonstrate that treatment of J774 macrophages transfected to constitutively express a human apoE3 cDNA with simvastatin, to increase LDL receptor activity, reduces the secretion of apoE. To further examine the relationship between LDL receptor expression and apoE secretion from macrophages, mouse peritoneal macrophages (MPMs) were isolated from mice with constitutively high expression of human LDL receptor to increase overall LDL receptor expression by 2- to 3-fold. Cells with increased LDL receptor expression also showed reduced apoE secretion compared with MPMs with basal LDL receptor expression. The effect of changes in LDL receptor expression on apoE secretion was isoform-specific, with greater reduction of apoE4 compared with apoE3 secretion and no reduction of apoE2 secretion, paralleling the known affinity of each isoform for LDL receptor binding. The effect of the LDL receptor on apoE secretion for each isoform was further reflected in LDL receptor-dependent changes in apoE-mediated cholesterol efflux. These results establish a regulatory interaction between two branches of macrophage sterol homeostatic pathways that could facilitate a rapid response to changes in macrophage sterol content relative to need.     

Enhancement by LDL of transfer of L-4F and oxidized lipids to HDL in C57BL/6J mice and human plasma

The apoA-I mimetic peptide L-4F [(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2) synthesized from all L-amino acids] has shown potential for the treatment of a variety of diseases. Here, we demonstrate that LDL promotes association between L-4F and HDL. A 2- to 3-fold greater association of L-4F with human HDL was observed in the presence of human LDL as compared with HDL by itself. This association further increased when LDL was supplemented with the oxidized lipid 15S-hydroxy-5Z, 8Z, 11Z, 13E-eicosatetraenoic acid (15HETE). Additionally, L-4F significantly (P = 0.02) promoted the transfer of 15HETE from LDL to HDL. The transfer of L-4F from LDL to HDL was demonstrated both in vitro and in C57BL/6J mice. L-4F, injected into C57BL/6J mice, associated rapidly with HDL and was then cleared quickly from the circulation. Similarly, L-4F loaded onto human HDL and injected into C57BL/6J mice was cleared quickly with T(1/2) = 23.6 min. This was accompanied by a decline in human apoA-I with little or no effect on the mouse apoA-I. Based on these results, we propose that i) LDL promotes the association of L-4F with HDL and ii) in the presence of L-4F, oxidized lipids in LDL are rapidly transferred to HDL allowing these oxidized lipids to be acted upon by HDL-associated enzymes and/or cleared from the circulation.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions

The atherogenicity theory for triglyceride-rich lipoproteins (TRLs; VLDL + intermediate density lipoprotein) generally cites the action of apolipoprotein C-III (apoC-III), a component of some TRLs, to retard their metabolism in plasma. We studied the kinetics of multiple TRL and LDL subfractions according to the content of apoC-III and apoE in 11 hypertriglyceridemic and normolipidemic persons. The liver secretes mainly two types of apoB lipoproteins: TRL with apoC-III and LDL without apoC-III. Approximately 45% of TRLs with apoC-III are secreted together with apoE. Contrary to expectation, TRLs with apoC-III but not apoE have fast catabolism, losing some or all of their apoC-III and becoming LDL. In contrast, apoE directs TRL flux toward rapid clearance, limiting LDL formation. Direct clearance of TRL with apoC-III is suppressed among particles also containing apoE. TRLs without apoC-III or apoE are a minor, slow-metabolizing precursor of LDL with little direct removal. Increased VLDL apoC-III levels are correlated with increased VLDL production rather than with slow particle turnover. Finally, hypertriglyceridemic subjects have significantly greater production of apoC-III-containing VLDL and global prolongation in residence time of all particle types. ApoE may be the key determinant of the metabolic fate of atherogenic apoC-III-containing TRLs in plasma, channeling them toward removal from the circulation and reducing the formation of LDLs, both those with apoC-III and the main type without apoC-III.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.     

Influence of age and sex on levels of anti-oxidized LDL antibodies and anti-LDL immune complexes in the general population

Most studies of antibodies to oxidized LDL have been undertaken in patients with different diseases and cardiovascular risk factors. However, very few studies have researched the distribution and determining factors of antibodies to oxidized LDL in the general population. A total of 1,354 persons (817 females and 537 males) aged 5-65 years were included in this study. They were selected randomly from the population census of Málaga, in southern Spain. The females had lower levels of total cholesterol and triglycerides and higher levels of HDL-cholesterol and a very significant increase (P < 0.0001) in levels of anti-oxidized LDL [low density lipoprotein modified by malondialdehyde (MDA-LDL)] antibodies but no difference in levels of immune complexes consisting of LDL and IgG antibodies (anti-LDL immune complex). Younger persons (16-35 years) had higher levels of anti-oxidized LDL (MDA-LDL) antibodies than persons older than 35 years (P = 0.05). Levels of immune complexes were significantly higher (P = 0.05) in persons aged 5-15 years than in persons older than 40 years. A very weak association was found between levels of anti-oxidized LDL (MDA-LDL) antibodies and anti-LDL immune complexes. The higher prevalence of anti-oxidized LDL (MDA-LDL) antibodies in females and young persons is in agreement with studies that found an inverse association between atherosclerosis and the level of these antibodies.     

Impact of myeloperoxidase-LDL interactions on enzyme activity and subsequent posttranslational oxidative modifications of apoB-100

Oxidation of LDL by the myeloperoxidase (MPO)-H₂O₂-chloride system is a key event in the development of atherosclerosis. The present study aimed at investigating the interaction of MPO with native and modified LDL and at revealing posttranslational modifications on apoB-100 (the unique apolipoprotein of LDL) in vitro and in vivo. Using amperometry, we demonstrate that MPO activity increases up to 90% when it is adsorbed at the surface of LDL. This phenomenon is apparently reflected by local structural changes in MPO observed by circular dichroism. Using MS, we further analyzed in vitro modifications of apoB-100 by hypochlorous acid (HOCl) generated by the MPO-H₂O₂-chloride system or added as a reagent. A total of 97 peptides containing modified residues could be identified. Furthermore, differences were observed between LDL oxidized by reagent HOCl or HOCl generated by the MPO-H₂O₂-chloride system. Finally, LDL was isolated from patients with high cardiovascular risk to confirm that our in vitro findings are also relevant in vivo. We show that several HOCl-mediated modifications of apoB-100 identified in vitro were also present on LDL isolated from patients who have increased levels of plasma MPO and MPO-modified LDL. In conclusion, these data emphasize the specificity of MPO to oxidize LDL.     

Apolipoprotein A-I mimetic peptide 4F blocks sphingomyelinase-induced LDL aggregation

Lipolytic modification of LDL particles by SMase generates LDL aggregates with a strong affinity for human arterial proteoglycans and may so enhance LDL retention in the arterial wall. Here, we evaluated the effects of apoA-I mimetic peptide 4F on structural and functional properties of the SMase-modified LDL particles. LDL particles with and without 4F were incubated with SMase, after which their aggregation, structure, and proteoglycan binding were analyzed. At a molar ratio of L-4F to apoB-100 of 2.5 to 20:1, 4F dose-dependently inhibited SMase-induced LDL aggregation. At a molar ratio of 20:1, SMase-induced aggregation was fully blocked. Binding of 4F to LDL particles inhibited SMase-induced hydrolysis of LDL by 10% and prevented SMase-induced LDL aggregation. In addition, the binding of the SMase-modified LDL particles to human aortic proteoglycans was dose-dependently inhibited by pretreating LDL with 4F. The 4F stabilized apoB-100 conformation and inhibited SMase-induced conformational changes of apoB-100. Molecular dynamic simulations showed that upon binding to protein-free LDL surface, 4F locally alters membrane order and fluidity and induces structural changes to the lipid layer. Collectively, 4F stabilizes LDL particles by preventing the SMase-induced conformational changes in apoB-100 and so blocks SMase-induced LDL aggregation and the resulting increase in LDL retention.     

Low density lipoprotein (LDL), atherosclerosis and antioxidants

A crucial and causative role in the pathogenesis of atherosclerosis is believed to be the oxidative modification of low density lipoprotein (LDL). The oxidation of LDL involves released free radical driven lipid peroxidation. Several lines of evidence support the role of oxidized LDL in atherogenesis. Epidemiologic studies have demonstrated an association between an increased intake of dietary antioxidant vitamins, such as vitamin E and vitamin C and reduced morbidity and mortality from coronary artery diseases. It is thus hypothesized that dietary antioxidants may help prevent the development and progression of atherosclerosis. The oxidation of LDL has been shown to be reduced by antioxidants, and, in animal models, improved antioxidants may offer possibilities for the prevention of atherosclerosis. The results of several on going long randomized intervention trials will provide valuahle information on the efficacy and safety of improved antioxidants in the prevention of atherosclerosis. This review a evaluates current literature involving antioxidants and vascular disease, with a particular focus on the potential mechanisms.     

Lipolytic and ligand-binding functions of hepatic lipase protect against atherosclerosis in LDL receptor-deficient mice

To elucidate the separate contributions of the lipolytic versus ligand-binding functions of hepatic lipase (HL) to lipoprotein metabolism and atherosclerosis, and to investigate the role of the low density lipoprotein receptor (LDLr) in these processes, we compared mice expressing catalytically active HL (HL-WT) with mice expressing inactive HL (HL-S145G) in a background lacking endogenous HL and the LDLr (LDLr-KOxHL-KO). HL-WT and HL-S145G reduced (P < 0.05 for all) cholesterol (55% vs. 20%), non-HDL-cholesterol (63% vs. 22%), and apolipoprotein B (apoB; 34% vs. 16%) by enhancing the catabolism of autologous (125)I-apoB-intermediate density lipoprotein (IDL)/LDL (fractional catabolic rate in day(-1): 6.07 +/- 0.25, LDLr-KOxHL-WT; 4.76 +/- 0.30, LDLr-KOxHL-S145G; 3.70 +/- 0.13, LDLr-KOxHL-KO); HL-WT had a greater impact on the concentration, composition, particle size, and catabolism of apoB-containing lipoproteins (apoB-Lps) and HDL. Importantly, consistent with the changes in apoB-Lps, atherosclerosis in LDLr-KOxHL-KO mice fed a regular chow diet (RCD) was reduced by both HL-WT and HL-S145G (by 71% and 51% in cross-sectional analysis, and by 85% and 67% in en face analysis; P < 0.05 for all). These data identify physiologically relevant but distinct roles for the lipolytic versus ligand-binding functions of HL in apoB-Lp metabolism and atherosclerosis and demonstrate that their differential effects on these processes are mediated by changes in catabolism via non-LDLr pathways. These changes, evident even in the presence of apoE, establish an antiatherogenic role of the ligand-binding function of HL in LDLr-deficient mice.     

Lipoprotein lipase-facilitated uptake of LDL is mediated by the LDL receptor

LPL mediates the uptake of lipoproteins into different cell types independent of its catalytic activity. The mechanism of this process and its physiological relevance are not clear. Taking into account the importance of the endothelial barrier for lipoprotein uptake, in vitro studies with primary aortic endothelial cells from wild-type and low density lipoprotein receptor (LDLR)-deficient (LDLR(-/-)) mice were performed. Addition of LPL almost doubled the uptake of LDL into wild-type cells. However, there was virtually no LPL-mediated change of LDL uptake into LDLR(-/-) cells. Upregulation of LDLR by lipoprotein-deficient serum/lovastatin in wild-type cells resulted in a 7-fold increase of LPL-mediated LDL uptake. Uptake of chylomicron remnants was not affected by LDLR expression. In proteoglycan-deficient cells, LPL did not increase the uptake of lipoproteins. The physiological relevance of this pathway was studied in mice that were both LDLR(-/-) and transgenic for catalytically inactive LPL in muscle. In the presence of LDLR, inactive LPL reduced LDL cholesterol significantly (13-24%). In the absence of LDLR, LDL cholesterol was not affected by transgenic LPL. Metabolic studies showed that in the presence of LDLR, LPL increased the muscular uptake of LDL by 77%. In the absence of LDLR, transgenic LPL did not augment LDL uptake. Chylomicron uptake was not affected by the LDLR genotype. We conclude that LPL-mediated cellular uptake of LDL, but not of chylomicrons, is dependent on the presence of both LDLR and proteoglycans.     

Aggregated LDL and lipid dispersions induce lysosomal cholesteryl ester accumulation in macrophage foam cells

Macrophage foam cells in atherosclerotic lesions accumulate substantial cholesterol stores within large, swollen lysosomes. Previous studies with mildly oxidized low density lipoprotein (OxLDL)-treated THP-1 macrophages suggest an initial buildup of free cholesterol (FC), followed by an inhibition of lysosomal cholesteryl ester (CE) hydrolysis and a subsequent lysosomal accumulation of unhydrolyzed lipoprotein CE. We examined whether other potential sources of cholesterol found within atherosclerotic lesions could also induce similar lysosomal accumulation. Biochemical analysis combined with microscopic analysis showed that treatment of THP-1 macrophages with aggregated low density lipoprotein (AggLDL) or CE-rich lipid dispersions (DISP) produced a similar lysosomal accumulation of both FC and CE. Co-treatment with an ACAT inhibitor, CP113,818, confirmed that the CE accumulation was primarily the result of the inhibition of lysosomal CE hydrolysis. The rate of unhydrolyzed CE buildup was more rapid with DISP than with AggLDL. However, with both treatments, FC appeared to accumulate in lysosomes before the inhibition in hydrolysis and CE accumulation, a sequence shared with mildly OxLDL. Thus, lysosomal accumulation of FC and CE can be attributable to more general mechanisms than just the inhibition of hydrolysis by oxidized lipids.