Identification of the proteoglycan binding site in apolipoprotein B48

An initial event in atherosclerosis is the retention of lipoproteins within the intima of the vessel wall. Previously we identified Site B (residues 3359-3369) in apolipoprotein (apo) B100 as the proteoglycan binding sequence in low density lipoproteins (LDLs) and showed that the atherogenicity of apoB-containing lipoproteins is linked to their affinity for artery wall proteoglycans. However, both apoB100- and apoB48-containing lipoproteins are equally atherogenic even though Site B lies in the carboxyl-terminal half of apoB100 and is absent in apoB48. If binding to proteoglycans is a key step in atherogenesis, apoB48-containing lipoproteins must bind to proteoglycans via other proteoglycan binding sites in the amino-terminal 48% of apoB. In vitro studies have identified five clusters of basic amino acids in delipidated apoB48 that bind negatively charged glycosaminoglycans. To determine which of these sites is functional on LDL particles, we analyzed the proteoglycan binding activity of recombinant human LDLs from transgenic mice or rat hepatoma cells. Substitution of neutral amino acids for the basic amino acids in Site B-Ib (residues 84-94) abolished the proteoglycan binding activity of recombinant apoB53. Carboxyl-truncated apoB80 bound biglycan with higher affinity than apoB100 and apoB48. ApoB80 in which Site B was mutated had the same affinity for proteoglycans as apoB48. These data support the hypothesis that the carboxyl terminus of apoB100 "masks" Site B-Ib, the amino-terminal proteoglycan binding site, and that this site is exposed in carboxyl-truncated forms of apoB. The presence of a proteoglycan binding site in the amino-terminal region of apoB may explain why apoB48- and apoB100-containing lipoproteins are equally atherogenic.     

LDL particle core enrichment in cholesteryl oleate increases proteoglycan binding and promotes atherosclerosis

Several studies in humans and animals suggest that LDL particle core enrichment in cholesteryl oleate (CO) is associated with increased atherosclerosis. Diet enrichment with MUFAs enhances LDL CO content. Steroyl O-acyltransferase 2 (SOAT2) is the enzyme that catalyzes the synthesis of much of the CO found in LDL, and gene deletion of SOAT2 minimizes CO in LDL and protects against atherosclerosis. The purpose of this study was to test the hypothesis that the increased atherosclerosis associated with LDL core enrichment in CO results from an increased affinity of the LDL particle for arterial proteoglycans. ApoB-100-only Ldlr^−/− mice with and without Soat2 gene deletions were fed diets enriched in either cis-MUFA or n-3 PUFA, and LDL particles were isolated. LDL:proteogylcan binding was measured using surface plasmon resonance. Particles with higher CO content consistently bound with higher affinity to human biglycan and the amount of binding was shown to be proportional to the extent of atherosclerosis of the LDL donor mice. The data strongly support the thesis that atherosclerosis was induced through enhanced proteoglycan binding of LDL resulting from LDL core CO enrichment.     

Heparin binding triggers human VLDL remodeling by circulating lipoprotein lipase: Relevance to VLDL functionality in health and disease

Hydrolysis of VLDL triacylglycerol (TG) by lipoprotein lipase (LpL) is a major step in energy metabolism and VLDL-to-LDL maturation. Most functional LpL is anchored to the vascular endothelium, yet a small amount circulates on TG-rich lipoproteins. As circulating LpL has low catalytic activity, its role in VLDL remodeling is unclear. We use pre-heparin plasma and heparin-sepharose affinity chromatography to isolate VLDL fractions from normolipidemic, hypertriglyceridemic, or type-2 diabetic subjects. LpL is detected only in the heparin-bound fraction. Transient binding to heparin activates this VLDL-associated LpL, which hydrolyses TG, leading to gradual VLDL remodeling into IDL/LDL and HDL-size particles. The products and the timeframe of this remodeling closely resemble VLDL-to-LDL maturation in vivo. Importantly, the VLDL fraction that does not bind heparin is not remodeled. This relatively inert LpL-free VLDL is rich in TG and apoC-III, poor in apoE and apoC-II, shows impaired functionality as a substrate for the exogenous LpL or CETP, and likely has prolonged residence time in blood, which is expected to promote atherogenesis. This non-bound VLDL fraction increases in hypertriglyceridemia and in type-2 diabetes but decreases upon diabetes treatment that restores the glycemic control. In stark contrast, heparin binding by LDL increases in type-2 diabetes triggering pro-atherogenic LDL modifications. Therefore, the effects of heparin binding are associated negatively with atherogenesis for VLDL but positively for LDL. Collectively, the results reveal that binding to glycosaminoglycans initiates VLDL remodeling by circulating LpL, and suggest heparin binding as a marker of VLDL functionality and a readout for treatment of metabolic disorders.     

High affinity binding between lipoprotein lipase and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity, and is modulated by glycosaminoglycans

Lipoprotein lipase (LPL) physically associates with lipoproteins and hydrolyzes triglycerides. To characterize the binding of LPL to lipoproteins, we studied the binding of low density lipoproteins (LDL), apolipoprotein (apo) B17, and various apoB-FLAG (DYKDDDDK octapeptide) chimeras to purified LPL. LDL bound to LPL with high affinity (K(d) values of 10(-12) m) similar to that observed for the binding of LDL to its receptors and 1D1, a monoclonal antibody to LDL, and was greater than its affinity for microsomal triglyceride transfer protein. LDL-LPL binding was sensitive to both salt and detergents, indicating the involvement of both hydrophobic and hydrophilic interactions. In contrast, the N-terminal 17% of apoB interacted with LPL mainly via ionic interactions. Binding of various apoB fusion peptides suggested that LPL bound to apoB at multiple sites within apoB17. Tetrahydrolipstatin, a potent enzyme activity inhibitor, had no effect on apoB-LPL binding, indicating that the enzyme activity was not required for apoB binding. LDL-LPL binding was inhibited by monoclonal antibodies that recognize amino acids 380-410 in the C-terminal region of LPL, a region also shown to interact with heparin and LDL receptor-related protein. The LDL-LPL binding was also inhibited by glycosaminoglycans (GAGs); heparin inhibited the interactions by approximately 50% and removal of trace amounts of heparin from LPL preparations increased LDL binding. Thus, we conclude that the high affinity binding between LPL and lipoproteins involves multiple ionic and hydrophobic interactions, does not require enzyme activity and is modulated by GAGs. It is proposed that LPL contains a surface exposed positively charged amino acid cluster that may be important for various physiological interactions of LPL with different biologically important molecules. Moreover, we postulate that by binding to this cluster, GAGs modulate the association between LDL and LPL and the in vivo metabolism of LPL.     

Early-glycation of apolipoprotein E: effect on its binding to LDL receptor,scavenger receptor A and heparan sulfates

Glycation is responsible for disruption of lipoprotein functions leading to the development of atherosclerosis in diabetes. The effects of apolipoprotein E (apoE) glycation were investigated with respect to its interaction with receptors. The interaction of apoE with the low density lipoprotein receptor (LDL-R) and scavenger receptor A (SR-A) was measured by competition experiments performed using, respectively, on a human fibroblast cell line ¹²⁵I-LDL, and on a murine macrophage cell line (J774) ¹²⁵I-acetylated LDL, and unlabeled apoE/phospholipid complexes. Glycated apoE binding to heparin and heparan sulfates (HS) was assessed by surface plasmon resonance (SPR) technology. Site-directed mutagenesis was then performed on Lys-75, the major glycation site of the protein. The prepared mutant protein proved to be useful as a tool to study the role of Lys-75 in apoE glycation. The findings showed that, although glycation has no effect on apoE binding either to the LDL-R or to SR-A, it impairs its binding to immobilized heparin and HS. The glycation of Lys-75 was found to be proceed rapidly and contributed significantly to total protein glycation. We propose that, in the case of diabetes, glycation may lead to the atherogenicity of apoE-containing lipoproteins disturbing their uptake via the HS proteoglycan pathway.     

Binding of low density lipoproteins to lipoprotein lipase is dependent on lipids but not on apolipoprotein B

Lipoprotein lipase (LPL) efficiently mediates the binding of lipoprotein particles to lipoprotein receptors and to proteoglycans at cell surfaces and in the extracellular matrix. It has been proposed that LPL increases the retention of atherogenic lipoproteins in the vessel wall and mediates the uptake of lipoproteins in cells, thereby promoting lipid accumulation and plaque formation. We investigated the interaction between LPL and low density lipoproteins (LDLs) with special reference to the protein-protein interaction between LPL and apolipoprotein B (apoB). Chemical modification of lysines and arginines in apoB or mutation of its main proteoglycan binding site did not abolish the interaction of LDL with LPL as shown by surface plasmon resonance (SPR) and by experiments with THP-I macrophages. Recombinant LDL with either apoB100 or apoB48 bound with similar affinity. In contrast, partial delipidation of LDL markedly decreased binding to LPL. In cell culture experiments, phosphatidylcholine-containing liposomes competed efficiently with LDL for binding to LPL. Each LDL particle bound several (up to 15) LPL dimers as determined by SPR and by experiments with THP-I macrophages. A recombinant NH(2)-terminal fragment of apoB (apoB17) bound with low affinity to LPL as shown by SPR, but this interaction was completely abolished by partial delipidation of apoB17. We conclude that the LPL-apoB interaction is not significant in bridging LDL to cell surfaces and matrix components; the main interaction is between LPL and the LDL lipids.     

Statin-exposed vascular smooth muscle cells secrete proteoglycans with decreased binding affinity for LDL

Retention of LDL in the artery intima is mediated by extracellular matrix proteoglycans and plays an important role in the initiation of atherosclerosis. Compared with quiescent cells, proliferating smooth muscle cells secrete proteoglycans with elongated glycosaminoglycan side chains, which have an increased binding affinity to LDL. Because 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors (statins) decrease smooth muscle cell proliferation, we hypothesized that statin exposure would decrease both the size and LDL binding affinity of vascular proteoglycans. Monkey aortic smooth muscle cells grown in culture were exposed to simvastatin (10 and 100 microM) and cerivastatin (0.1 and 1 microM), and newly secreted proteoglycans were quantified and characterized. Both simvastatin and cerivastatin caused a concentration-dependent reduction in cell growth and reduced 35SO4 incorporation into secreted proteoglycans, on both an absolute and a per cell basis. Interestingly, statin exposure increased the apparent molecular weight and hydrodynamic size of secreted proteoglycans. However, proteoglycans secreted from statin-exposed cells demonstrated a reduction in binding affinity to LDL. Thus, statins may induce atheroprotective changes in vascular proteoglycans and lower LDL retention in the vessel wall. These findings suggest a mechanism whereby statins may benefit atherosclerosis in a manner unrelated to serum LDL lowering.     

Oxidized phospholipids, linked to apolipoprotein B of oxidized LDL, are ligands for macrophage scavenger receptors

Previous studies have shown that macrophage receptors for oxidized LDL (OxLDL) recognize both the lipid and protein moieties, and that a monoclonal antibody against OxLDL, EO6, also recognizes both species. The present studies show directly that during LDL oxidation phospholipids become covalently attached to apolipoprotein B (apoB). After exhaustive extraction of lipids, apoB of native LDL contained 4 +/- 3 moles of phosphorus/mole protein. In contrast, apoB of OxLDL contained approximately 75 moles of phosphorus/mole protein. Saponification of this apoB released phosphorus, choline, and saturated fatty acids in a molar ratio of 1.0:0.98:0.84. When LDL was reductively methylated prior to oxidation, the amount of phospholipid covalently bound was reduced by about 80%, indicating that the phospholipids attach at lysine epsilon amino groups. Progressive decreases in the phospholipid associated with apoB of OxLDL decreased the ability of the protein to compete for binding to macrophage scavenger receptors and decreased its reactivity with antibody EO6.We postulate that some oxidized phospholipids containing fatty acid aldehydes at the sn-2 position bind to lysine residues of apoB while others remain unreacted within the lipid phase. This would account for the interchangeability of lipid and apolipoprotein of OxLDL with respect to receptor binding and antibody recognition.     

Capillary electrochromatography and quartz crystal microbalance, valuable techniques in the study of heparin-lipoprotein interactions

Atherosclerosis is initiated when lipoproteins bind to proteoglycans (PGs) in arterial walls. The binding is mediated by apolipoprotein apoB-100 and/or apoE, both of which have binding affinity toward heparin. We developed covalently bound heparin coatings for APTES-modified silica capillaries and SiO(2) chips and carried out capillary electrochromatography (CEC) and quartz crystal microbalance (QCM) studies on the interactions of heparin with selected peptide fragments of apoB-100 and apoE and, for CEC, also with low- and high-density lipoproteins (LDL and HDL), the latter with and without apoE. The peptides are known to mediate interactions of HDL and LDL with arterial PGs. Interactions and affinities were expressed in CEC as retention factors and reduced mobilities and in continuous flow QCM techniques as affinity constants. Both techniques showed heparin interactions to be stronger with apoB-100 peptide than with apoE peptide fragment, and they confirmed that the sulfate groups in heparin play an especially important role in interactions with apoB-100 peptide fragments. In addition, CEC confirmed the importance of sulfate groups of heparin in interactions between heparin and LDL and between heparin and apoE-containing HDL. CEC and QCM acted as excellent platforms to mimic these biologically important interactions, with small sample and reagent consumption.     

ApoC-III content of apoB-containing lipoproteins is associated with binding to the vascular proteoglycan biglycan

Retention of apolipoprotein (apo)B and apoE-containing lipoproteins by extracellular vascular proteoglycans is critical in atherogenesis. Moreover, high circulating apoC-III levels are associated with increased atherosclerosis risk. To test whether apoC-III content of apoB-containing lipoproteins affects their ability to bind to the vascular proteoglycan biglycan, we evaluated the impact of apoC-III on the interaction of [(35)S]SO(4)-biglycan derived from cultured arterial smooth muscle cells with lipoproteins obtained from individuals across a spectrum of lipid concentrations. The extent of biglycan binding correlated positively with apoC-III levels within VLDL (r = 0.78, P < 0.01), IDL (r = 0.67, P < 0.01), and LDL (r = 0.52, P < 0.05). Moreover, the biglycan binding of VLDL, IDL, and LDL was reduced after depletion of apoC-III-containing lipoprotein particles in plasma by anti-apoC-III immunoaffinity chromatography. Since apoC-III does not bind biglycan directly, enhanced biglycan binding may result from a conformational change associated with increased apo C-III content by which apoB and/or apoE become more accessible to proteoglycans. This may be an intrinsic property of lipoproteins, since exogenous apoC-III enrichment of LDL and VLDL did not increase binding. ApoC-III content may thus be a marker for lipoproteins characterized as having an increased ability to bind proteoglycans.     

High binding affinity of electronegative LDL to human aortic proteoglycans depends on its aggregation level

Electronegative LDL [LDL(-)] is an atherogenic subfraction of plasma LDL that has increased apolipoprotein E (apoE) and apoC-III content, high density, and increased susceptibility to aggregation. These characteristics suggest that LDL(-) could bind to proteoglycans (PGs); therefore, our aim was to evaluate its affinity to PGs. Binding of LDL(-) and native LDL [LDL(+)] to human aortic PGs was determined by precipitation of LDL-glycosaminoglycan complexes, LDL incubation in PG-coated microtiter wells, and affinity chromatography on PG column. All methods showed that LDL(-) had higher binding affinity to PGs than did LDL(+). PG capacity to bind LDL(-) was increased approximately 4-fold compared with LDL(+) in precipitation and microtiter assays. Chromatography on PG column showed LDL(-) to consist of two subpopulations, one with higher and one with lower PG binding affinity than LDL(+). Unexpectedly, the lower PG affinity subpopulation had increased apoE and apoC-III content. In contrast, the high PG affinity subpopulation presented phospholipase C (PLC)-like activity and increased aggregation. These results suggest that PLC-like activity could alter LDL lipid composition, thereby promoting particle aggregation and binding to PGs. This propensity of a subpopulation of LDL(-) to bind to PGs could facilitate its retention in the extracellular matrix of arterial intima and contribute to atherosclerosis progression.     

Apolipoprotein E mediates binding of normal very low density lipoprotein to heparin but is not required for high affinity receptor binding

The relationship between the cholesteryl ester content of normal human very low density lipoprotein (VLDL) and its ability to bind to apolipoprotein E (apoE), heparin, and the low density lipoprotein (LDL) receptor have been compared. Plasma VLDL were separated by heparin affinity chromatography into two fractions: one with apoE and one without. Both fractions had the same cholesteryl ester content relative to apolipoprotein B (apoB). LDL, on the other hand, had a greater cholesteryl ester content. VLDL were modified by lipolysis to express the ability to bind apoE (Ishikawa, Y., Fielding, C. J., and Fielding, P. E. (1988) J. Biol. Chem. 263, 2744-2749). Lipolyzed VLDL with or without apoE were compared for their ability to bind to heparin or the up-regulated fibroblast LDL receptor. Lipolyzed VLDL bound with the same affinity to the receptor whether or not the particles contained apoE. ApoB, not apoE, appears then to be the important ligand for normal VLDL. On the other hand, modified VLDL without apoE, even though binding to the LDL receptor, did not bind to heparin. These data suggest that apoE mediates heparin binding in normal VLDL, that apoB mediates receptor binding, and that the cholesteryl ester content of VLDL is not a factor in the induction of the ability to bind apoE.     

Glycation of apolipoprotein E impairs its binding to heparin: identification of the major glycation site.

The increased glycation of plasma apolipoproteins represents a possible major factor for lipid disturbances and accelerated atherogenesis in diabetic patients. The glycation of apolipoprotein E (apoE), a key lipid-transport protein in plasma, was studied both in vivo and in vitro. ApoE was shown to be glycated in plasma very low density lipoproteins of both normal subjects and hyperglycemic, diabetic patients. However, diabetic patients with hyperglycemia showed a 2-3-fold increased level of apoE glycation. ApoE from diabetic plasma showed decreased binding to heparin compared to normal plasma apoE. The rate of Amadori product formation in apoE in vitro was similar to that for albumin and apolipoproteins A-I and A-II. The glycation of apoE in vitro significantly decreased its ability to bind to heparin, a critical process in the sequestration and uptake of apoE-containing lipoproteins by cells. Diethylenetriaminepentaacetic acid, a transition metal chelator, had no effect on the loss of apoE heparin-binding activity, suggesting that glycation rather than glycoxidation is responsible for this effect. In contrast, glycation had no effect on the interaction of apoE with amyloid beta-peptide. ApoE glycation was demonstrated to be isoform-specific. ApoE(2) showed a higher glycation rate and the following order was observed: apoE(2)>apoE(4)>apoE(3). The major glycated site of apoE was found to be Lys-75. These findings suggest that apoE is glycated in an isoform-specific manner and that the glycation, in turn, significantly decreases apoE heparin-binding activity. We propose that apoE glycation impairs lipoprotein-cell interactions, which are mediated via heparan sulfate proteoglycans and may result in the enhancement of lipid abnormalities in hyperglycemic, diabetic patients.     

Oxidized LDL bind to nonproteoglycan components of smooth muscle extracellular matrices

Arterial wall lipid retention is believed to be due primarily to ionic interactions between lipoproteins and proteoglycans. Thus, oxidized low density lipoproteins (LDL), with decreased positive charge relative to native LDL, should have decreased interaction with negatively charged proteoglycans. However, oxidized LDL does accumulate within arterial lesions. Therefore, this study investigated the binding of native and oxidized LDL to a complex smooth muscle extracellular matrix and the role of ionic charge interactions in their binding. LDL was modified with 2,2-azo-bis(2-amidinopropane)-2HCl, hypochlorite, soybean lipoxygenase, and phospholipase or copper sulfate. The extracellular matrix had 15- to 45-fold greater binding capacity for the different forms of oxidized LDL than for native LDL. However, the affinity of binding for all forms of oxidized LDL was high (K(a) = approximately 10(-9) M) and was similar to that for native LDL. Preincubation of the lipoproteins with chondroitin sulfate decreased the binding of native LDL, but had no effect on the binding of oxidized LDL. Digestion of matrices with chondroitin ABC lyase and heparinase decreased the binding of native LDL, but increased the binding of oxidized LDL; matrix digestion with pronase or trypsin markedly reduced the binding of both native and oxidized LDL.Thus, the binding of native LDL involves matrix proteoglycans, whereas the binding of oxidized LDL involves a nonproteoglycan component(s) of the matrix. The markedly enhanced retention of oxidized LDL compared with native LDL may play an important role in the progression of atherosclerosis.     

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.     

The molecular mechanism for the genetic disorder familial defective apolipoprotein B100

Familial defective apolipoprotein B100 (FDB) is a genetic disorder in which low density lipoproteins (LDL) bind defectively to the LDL receptor, resulting in hypercholesterolemia and premature atherosclerosis. FDB is caused by a mutation (R3500Q) that changes the conformation of apolipoprotein (apo) B100 near the receptor-binding site. We previously showed that arginine, not simply a positive charge, at residue 3500 is essential for normal receptor binding and that the carboxyl terminus of apoB100 is necessary for mutations affecting arginine 3500 to disrupt LDL receptor binding. Thus, normal receptor binding involves an interaction between arginine 3500 and tryptophan 4369 in the carboxyl tail of apoB100. W4369Y LDL and R3500Q LDL isolated from transgenic mice had identically defective LDL binding and a higher affinity for the monoclonal antibody MB47, which has an epitope flanking residue 3500. We conclude that arginine 3500 interacts with tryptophan 4369 and facilitates the conformation of apoB100 required for normal receptor binding of LDL. From our findings, we developed a model that explains how the carboxyl terminus of apoB100 interacts with the backbone of apoB100 that enwraps the LDL particle. Our model also explains how all known ligand-defective mutations in apoB100, including a newly discovered R3480W mutation in apoB100, cause defective receptor binding.     

Paradoxical protective effect of aminoguanidine toward low-density lipoprotein oxidation: inhibition of apolipoprotein B fragmentation without preventing its carbonylation. Mechanism of action of aminoguanidine

Oxidative modification of low-density lipoproteins (LDLs) is an important feature in the initiation and progression of atherosclerosis. Aminoguanidine (AMG), classically described as an inhibitor of advanced glycation end products, turned out to be also efficient in animal models as an antioxidant against lipid peroxidation. The originality of the present study was based on the simultaneous assessment of the oxidation of LDL lipid and protein moieties in order to characterize the molecular sites of AMG protection. Oxidation of the LDL lipid moiety was monitored by measuring conjugated dienes (CD) and hydroperoxide molecular species from cholesteryl esters (CEOOH) and phosphatidylcholines (PCOOH). LDL protein oxidative modifications were assessed by evaluating apoB carbonylation and fragmentation. The LDL oxidation was mediated by water gamma radiolysis, which has the advantage of being quantitative and highly selective with regard to the free radicals produced. Here, we reported that AMG resulted in a protection of LDLs against lipid peroxidation (both in the lag phase and in the propagation phase) and against apoB fragmentation in a concentration-dependent manner, due to the scavenging effect of AMG toward lipid peroxyl radicals. Paradoxically, AMG was poorly efficient against apoB carbonylation that began during the lag phase. We hypothesize that, even in the presence of AMG, a nonnegligible proportion of (*)OH radicals remained able to initiate oxidation of the LDL protein moiety, leading to apoB carbonylation.     

Molecular action of vitamin E in lipoprotein oxidation: implications for atherosclerosis

The oxidation theory of atherosclerosis proposes that the oxidative modification of low-density lipoproteins (LDL) plays a central role in the disease. Although a direct causative role of LDL oxidation for atherogenesis has not been established, oxidized lipoproteins are detected in atherosclerotic lesions, and in vitro oxidized LDL exhibits putative pro-atherogenic activities. alpha-Tocopherol (alpha-TOH; vitamin E), the major lipid-soluble antioxidant present in lipoproteins, is thought to be antiatherogenic. However, results of vitamin E interventions on atherosclerosis in experimental animals and cardiovascular disease in humans have been inconclusive. Also, recent mechanistic studies demonstrate that the role of alpha-TOH during the early stages of lipoprotein lipid peroxidation is complex and that the vitamin does not act as a chain-breaking antioxidant. In the absence of co-antioxidants, compounds capable of reducing the alpha-TOH radical and exporting the radical from the lipoprotein particle, alpha-TOH exhibits anti- or pro-oxidant activity for lipoprotein lipids depending on the degree of radical flux and reactivity of the oxidant. The model of tocopherol-mediated peroxidation (TMP) explains the complex molecular action of alpha-TOH during lipoprotein lipid peroxidation and antioxidation. This article outlines the salient features of TMP, comments on whether TMP is relevant for in vivo lipoprotein lipid oxidation, and discusses how co-antioxidants may be required to attenuate lipoprotein lipid oxidation in vivo and perhaps atherosclerosis.