Correlation of antiphospholipid antibody recognition with the structure of synthetic oxidized phospholipids. Importance of Schiff base formation and aldol condensation.

The oxidation of low density lipoproteins (LDL) has been correlated with atherogenesis through a variety of pathways. The process involves nonspecific fragmentation, oxidative breakdown, and modification of the lipids and protein of LDL. The process yields a variety of bioactive products, including aldehyde-containing phospholipids, which can cross-react with primary amines (i.e. peptides or phospholipid head groups) to yield Schiff base products. We also demonstrate that such oxidized phospholipid products may further react through a post-oxidation chemical pathway involving aldol condensation. EO6, an IgM monoclonal autoantibody to oxidized phospholipids, blocks the uptake of oxidized LDL (OxLDL) by macrophages. Because the epitope(s) of EO6 also blocks the uptake of OxLDL, a series of oxidized phospholipids, their peptide complexes, and their aldol condensates have been synthesized and characterized, and their antigenicity has been determined. This study defines structural motifs of oxidized phospholipids responsible for antigenicity for EO6. Certain monomeric phospholipids containing short chain fatty acids were antigenic whether oxidized or not in the sn-2 position. However, oxidized phospholipids containing sn-1 long chain fatty acids were not antigenic unless the sn-2 oxidized fatty acid contained an aldehyde that first reacted with a peptide yielding a Schiff base or the sn-2 oxidized fatty acid underwent an aldol type self-condensation. Our data indicate that the phosphorylcholine head group is essential for antigenicity, but its availability depends on the oxidized phospholipid conformation. We suggest that upon oxidation, similar reactions occur in phospholipids on the surface of LDL, generating ligands for macrophage recognition. Synthetic imine adducts of oxidized phospholipids of this type are capable of blocking the uptake of OxLDL.     

The binding of oxidized low density lipoprotein to mouse CD36 is mediated in part by oxidized phospholipids that are associated with both the lipid and protein moieties of the lipoprotein

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.     

Autoantibodies to OxLDL fail to alter the clearance of injected OxLDL in apolipoprotein E-deficient mice

This study tests the hypothesis that autoantibodies to oxidation epitopes on oxidized LDL (OxLDL) promote the clearance of OxLDL from the plasma. Human LDL (hLDL) was injected into immune-competent apolipoprotein E-deficient (apoE(-/-)) mice and immune-deficient apoE(-/-)/recombination-activating gene-deficient mice that lack mature T and B cells and thus antibodies. There was a progressive decrease in human apoB-100 in the plasma in all mice, but the rate of clearance was not greater in the immune-competent mice than in the immune-deficient mice. Interestingly, oxidized phospholipid (OxPL) epitopes as detected by the EO6 antibody on the hLDL increased over time, suggesting de novo oxidation of the LDL or transfer of OxPL to the particles. Because the native LDL was not extensively modified, we also examined the clearance of copper OxLDL. Although the extensively OxLDL was cleared faster than the native LDL, there was no difference in the rate of clearance as a function of immune status. There appeared to be some transfer of OxPL to the endogenous murine LDL. Together, these results suggest that oxidation-specific antibodies do not participate to any great extent in the clearance of OxLDL from plasma. However, it is possible that such antibodies may bind to oxidation epitopes and modulate lesion formation within the vessel wall.     

A modification of apolipoprotein B accounts for most of the induction of macrophage growth by oxidized low density lipoprotein

It has recently been shown that macrophage proliferation occurs during the progression of atherosclerotic lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this include the interaction of oxidized LDL with integral plasma membrane proteins coupled to signaling pathways, the release of growth factors and autocrine activation of growth factor receptors, or the potentiation of mitogenic signal transduction by a component of oxidized LDL after internalization. The present study was undertaken to further elucidate the mechanisms involved in the growth-stimulating effect of oxidized LDL in macrophages. Only extensively oxidized LDL caused significant growth stimulation, whereas mildly oxidized LDL, native LDL, and acetyl LDL were ineffective. LDL that had been methylated before oxidation (to block lysine derivatization by oxidation products and thereby prevent the formation of a scavenger receptor ligand) did not promote growth, even though extensive lipid peroxidation had occurred. The growth stimulation could not be attributed to lysophosphatidylcholine (lyso-PC) because incubation of oxidized LDL with fatty acid-free bovine serum albumin resulted in a 97% decrease in lyso-PC content but only a 20% decrease in mitogenic activity. Similarly, treatment of acetyl LDL with phospholipase A2 converted more than 90% of the initial content of phosphatidylcholine (PC) to lyso-PC, but the phospholipase A2-treated acetyl LDL was nearly 10-fold less potent than oxidized LDL at stimulating growth. Platelet-activating factor receptor antagonists partly inhibited growth stimulation by oxidized LDL, but platelet-activating factor itself did not induce growth. Digestion of oxidized LDL with phospholipase A2 resulted in the hydrolysis of PC and oxidized PC but did not attenuate growth induction. Native LDL, treated with autoxidized arachidonic acid under conditions that caused extensive modification of lysine residues by lipid peroxidation products but did not result in oxidation of LDL lipids, was equal to oxidized LDL in potency at stimulating macrophage growth. Albumin modified by arachidonic acid peroxidation products also stimulated growth, demonstrating that LDL lipids are not essential for this effect. These findings suggest that oxidatively modified apolipoprotein B is the main growth-stimulating component of oxidized LDL, but that oxidized phospholipids may play a secondary role.     

Different apolipoprotein B breakdown patterns in models of oxidized low density lipoprotein.

Low density lipoprotein (LDL) oxidation is characterized by alterations in biological properties and structure of the lipoprotein particles, including breakdown and modification of apolipoprotein B (apoB). We compared apoB breakdown patterns in different models of minimally and extensively oxidized LDL using Western blotting techniques and several monoclonal and polyclonal antibodies. It was found that copper and endothelial cell-mediated oxidation produced a relatively similar apoB banding pattern with progressive fragmentation of apoB during LDL oxidation, whereas malondialdehyde (MDA)- and hydroxynonenal (HNE) -modified LDL produced an aggregated apoB. It is conceivable that apoB fragments present in copper and endothelial cell oxidized LDL lead to the exposure on the lipoprotein surface of different protein epitopes than in aggregated MDA-LDL and HNE-LDL. Although all models of extensively oxidized LDL led to increased lipid uptake in macrophages, mild degrees of oxidation interfered with LDL uptake in fibroblasts and extensively oxidized LDL impaired degradation of native LDL in fibroblasts. We suggest that in order to improve interpretation and comparison of results, data obtained with various models of oxidized LDL should be compared to the simpliest and most reproducible models of 3 h and 18 h copper-oxidized LDL (apoB breakdown) and MDA-LDL (apoB aggregation) since different models of oxidized LDL have significant differences in apoB breakdown and aggregation patterns which may affect immunological and biological properties of oxidized LDL.     

Scavenger receptor class B type I as a receptor for oxidized low density lipoprotein

Scavenger receptor class B type I (SR-BI) has been established as the primary mediator of the selective transfer of lipids from HDL to mammalian cells. In addition to its role in cholesterol metabolism, SR-BI has been shown to bind apoptotic cells and thus could in theory also function as a scavenger receptor. We now show that SR-BI binds oxidized LDL (OxLDL) with high affinity (K(d) of 4.0 +/- 0.5 microg/ml) and mediates internalization and degradation to an extent comparable to that of other scavenger receptors, when normalized to binding activity. The best competitors for OxLDL binding to SR-BI were oxidized lipoproteins, whereas native or acetylated lipoproteins only competed for a small fraction of OxLDL binding. Both the isolated lipids and the isolated protein from OxLDL bound with high affinity to SR-BI and showed partial reciprocal competition. Monoclonal antibody EO6, an antibody against oxidized phospholipids, and 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine (POVPC) both competed effectively with intact OxLDL and with isolated lipids from OxLDL for SR-BI binding.Together, these results demonstrate a potential function of SR-BI, in addition to its role in selective uptake of lipids, to mediate internalization of OxLDL by macrophages and suggest a central role for oxidized phospholipids in this process.     

Lysosomal accumulation of oxidized phosphatidylcholine-apolipoprotein B complex in macrophages: intracellular fate of oxidized low density lipoprotein

Oxidized phosphatidylcholine (OxPC) formed in oxidized low density lipoprotein (OxLDL) is thought to be involved in the development of atherosclerosis. OxPC has been found in foam cells in atherosclerotic lesions and suggested to be the epitope for OxLDL recognition by macrophages. OxPC is present as a complex with apolipoprotein B (apoB) in OxLDL, since some OxPC can bind with proteins. In the current study, the intracellular fate of OxPC-apoB complexes after internalization of OxLDL by macrophages was investigated. Murine macrophage cell line J774.1 was incubated with either OxLDL or acetylated LDL for 24 h, then the cells were further incubated for up to 24 h in new medium without lipoprotein. Modified apoB in the cells was quantitated by sandwich ELISA using monoclonal antibodies against OxPC and apoB. Intracellular OxLDL decreased rapidly for the first 4 h to approx. 20% of that before medium change, with the apparent metabolism of OxPC-apoB complex ceasing. OxPC-apoB complexes that remained in the cells after 24 h chasing increased as the period of OxLDL loading in macrophages prolongs. Acetylated LDL in the cells decreased quickly and disappeared after 4 h of chasing. Subcellular fractionation using sucrose density gradient ultracentrifugation of macrophages, which had already accumulated OxPC-apoB complexes by 24 h of incubation with OxLDL and further 24 h chasing, showed that the complex was co-localized with endosomal and lysosomal markers. Immunohistochemical double staining studies demonstrated that OxPC and apoB co-localize in foam cells in early atherosclerotic lesions obtained from human coronary artery. These results suggest that OxPC-apoB complexes originating from OxLDL accumulate in foam cells in human atherosclerotic lesions as well as in macrophages in vitro.     

Modification of human serum low density lipoprotein by oxidation--characterization and pathophysiological implications

Plasma low density lipoprotein (LDL) can undergo free radical oxidation either catalyzed by divalent cations, such as Cu2+ or Fe2+ or promoted by incubation with cultured cells such as endothelial cells, smooth muscle cells and monocytes. The content of vitamin E, beta-carotene and unsaturated fatty acids is decreased in oxidized LDL. A breakdown of apolipoprotein-B (apoB), hydrolysis of the phospholipids, an increase of thiobarbituric acid reactive substances and the generation of aldehydes also occur. Changes in the ratio of lipid to protein, the electrophoretic mobility and the fluorescent properties have also been reported to accompany oxidation of this lipoprotein. The functional changes of oxidized LDL include its recognition by the scavenger receptor on macrophages, its cytotoxicity especially to proliferating cells, its chemotactic properties with respect to monocyte-macrophages and its regulation of platelet-derived growth factor-like protein (PDGFc) production by endothelial cells. In this article we summarize some of the contributions to this topic and present speculations relating oxidized LDL to pathological conditions such as atherosclerosis.     

Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products

Modification of low density lipoproteins (LDL) by oxidation has been shown to permit recognition by the acetyl-LDL receptor of macrophages. The extensive oxidation of LDL that is required before interaction occurs with this receptor produces major alterations in both the lipid and protein components of LDL. Several chemical modifications of LDL also lead to recognition by this receptor; all of these involve derivatization of lysine residues of apolipoprotein B by adducts that neutralize the positively charged epsilon-amino group. The present studies show that oxidation also results in derivatization of LDL lysine residues. Analysis of amino acid composition indicated that 32% of lysine residues were modified after oxidation of LDL by exposure to 5 microM CuSO4 for 20 h. About one-half of the derivatized lysines were labile under the conditions of acid hydrolysis. Fluorescence of LDL protein was greatly increased by oxidation, with excitation maximum at 350 nm and emission maximum at 433 nm. When LDL containing phosphatidylcholine with isotopically labeled arachidonic acid in the sn-2 position was oxidized, there was a 5-fold increase in radioactivity bound to protein compared to nonoxidized LDL or oxidized LDL labeled with 2-[1-14C]palmitoyl phosphatidylcholine. Prior methylation of LDL prevented the rapid uptake and degradation by macrophages that normally accompanies oxidation. These findings suggest that oxidation of LDL is accompanied by derivatization of lysine epsilon-amino groups by lipid products and that these adducts may be important in the interaction of oxidized LDL with the acetyl-LDL receptor.     

Potent modification of low density lipoprotein by group X secretory phospholipase A2 is linked to macrophage foam cell formation

The deposition of cholesterol ester within foam cells of the artery wall is fundamental to the pathogenesis of atherosclerosis. Modifications of low density lipoprotein (LDL), such as oxidation, are prerequisite events for the formation of foam cells. We demonstrate here that group X secretory phospholipase A2 (sPLA2-X) may be involved in this process. sPLA2-X was found to induce potent hydrolysis of phosphatidylcholine in LDL leading to the production of large amounts of unsaturated fatty acids and lysophosphatidylcholine (lyso-PC), which contrasted with little, if any, lipolytic modification of LDL by the classic types of group IB and IIA secretory PLA2s. Treatment with sPLA2-X caused an increase in the negative charge of LDL with little modification of apolipoprotein B (apoB) in contrast to the excessive aggregation and fragmentation of apoB in oxidized LDL. The sPLA2-X-modified LDL was efficiently incorporated into macrophages to induce the accumulation of cellular cholesterol ester and the formation of non-membrane-bound lipid droplets in the cytoplasm, whereas the extensive accumulation of multilayered structures was found in the cytoplasm in oxidized LDL-treated macrophages. Immunohistochemical analysis revealed marked expression of sPLA2-X in foam cell lesions in the arterial intima of high fat-fed apolipoprotein E-deficient mice. These findings suggest that modification of LDL by sPLA2-X in the arterial vessels is one of the mechanisms responsible for the generation of atherogenic lipoprotein particles as well as the production of various lipid mediators, including unsaturated fatty acids and lyso-PC.     

The effects of branched chain fatty acid incorporation into Neurospora crassa membranes

Phytanic acid (3,7,11,15-tetramethylhexadecanoic acid), an unusual branched chain fatty acid thought to disrupt the hydrophobic regions of membranes, can be incorporated into the lipids of growing Neurospora cultures. The phytanic acid must be supplied in a water soluble form, esterified to a Tween detergent (Tween-Phytanic). This fatty acid and its oxidation product, pristanic acid, were found in both the phospholipid and neutral lipid fractions of Neurospora. In phospholipids of the wild-type strain, phytanic acid was present to the extent of 4 to 5 moles percent of the fatty acids and pristanic acid, about 41 moles percent. The neutral lipids contained 42 and 4 moles percent of phytanic and pristanic acids respectively. By employing a fatty acid-requiring mutant strain (cel^?), the phytanic acid level was raised to a maximum of 16 moles percent in the phospholipids and to 63 moles percent in the neutral lipids. Under this condition, the level of pristanic acid was reduced to about 6 moles percent in phospholipids and 1 mole percent in the neutral lipids. The phytanic acid levels could not be further elevated by increased supplementation with phytanic acid or by a change in the growth temperature. In strains with a high phytanic acid content, the complete fatty acid distribution of the phospholipids and neutral lipids was determined. In the neutral lipids, phytanic acid appeared to replace the 18 carbon fatty acids, particularly linoleic acid. The presence of phytanic acid in the phospholipids was confirmed by mass spectrometry, and by the isolation of a phospholipid fraction containing this fatty acid via silicic acid column chromatography. Most of the phytanic acid in phospholipids appeared to be in phosphatidylethanolamine, and 2 lines of evidence suggest that it was esterified to both positions of this molecule. In the fatty acid-requiring mutant strain (cel^?), the replacement by phytanic acid of 10 to 15% of the fatty acids in the phospholipid produced an aberrant morphological change in the growth pattern of Neurospora and caused this organism to be osmotically more fragile than the wild-type strain. The lack of noticeable effect of the high levels of pristanic acid in the phospholipids suggests that it is not just the presence of the methyl groups in a branched chain fatty acid which leads to the altered membrane function in this organism.     

Bioactive products of phospholipid oxidation: isolation, identification, measurement and activities

There is considerable evidence to suggest that oxidation of LDL plays an important role in atherogenesis. Polyunsaturated fatty acids, a major oxidative target, are present as phospholipids in the outer core of the lipoprotein particle. Studies from several laboratories have shown an increase in the levels of phospholipid oxidation products in atherosclerotic lesions and of antibodies to oxidized phospholipids in mice and humans with lesions. Significantly, phospholipid oxidation products have been demonstrated (in vitro) to selectively activate processes in vascular wall cells that may contribute to atherogenesis. This review discusses activities, methods for isolation, identification and measurement of bioactive phospholipids. Past studies suggest that defined and relatively simple current technologies allow identification of bioactive phospholipid oxidation products and measurement of their levels in tissue.     

Phospholipids in oxidized LDL not adducted to apoB are recognized by the CD36 scavenger receptor

Previous studies have shown that oxidation of low-density lipoprotein (oxLDL) results in its recognition by scavenger receptors on macrophages. Whereas blockage of lysyl residues on apoB-100 of oxLDL by lipid peroxidation products appears to be critical for recognition by the scavenger receptor class A (SR-A), modification of the lipid moiety has been suggested to be responsible for recognition by the scavenger class B receptor, CD36. We studied the recognition by scavenger receptors of oxidized LDL in which lysyl residues are blocked prior to oxidation through methylation [ox(m)LDL]. This permits us to minimize any contribution of modified apoB-100 to the recognition of oxLDL, but does not disrupt the native configuration of lipids in the particle. We found that ox(m)LDL was recognized by receptors on mouse peritoneal macrophages (MPM) almost as well as oxLDL. Ox(m)LDL was recognized by CD36-transfected cells but not by SR-A-transfected cells. Oxidized phospholipids (oxPC) transferred from oxLDL or directly from oxPC to LDL, conveyed recognition by CD36-transfected cells, confirming that CD36 recognized unbound oxidized phospholipids in ox(m)LDL. Collectively, these results suggest that oxPC not adducted to apoB within the intact oxLDL particle are recognized by the macrophage scavenger receptor CD36, that these lipids are not recognized by SR-A, and that they can transfer from oxidized to unoxidized LDL and induce CD36 recognition.     

Composition and structure of lipopolysaccharide-human plasma low density lipoprotein complex. Analytical ultracentrifugation, 31P-NMR, ESR and fluorescence spectroscopy studies

Complexes of Salmonella typhimurium lipopolysaccharide toxin (LPS) with low density lipoproteins (LDL) prepared in vitro have been analyzed. LPS-LDL complexes were found to comprise approx. 0.24 mg LPS/mg LDL protein. The major protein of complexes was apolipoprotein apoB-100 (greater than or equal to 90-95%). Incorporation of LPS molecules into LDL was accompanied by small changes in lipid composition, i.e. the phosphatidylcholine content was diminished by approx. 11% and the free fatty acid concentration was raised 2-fold. Analytical ultracentrifugation showed that insertion of LPS into LDL results in the increase of a portion of particles with higher density (lower flotation coefficient) compared to initial LDL. As was evidenced by ESR, in LPS-LDL complexes, the phospholipid hydrocarbon chains are more ordered than in LDL. 31P-NMR spectra indicated that in LPS-LDL complexes the mobility of phospholipid polar headgroups is restricted in comparison with LDL. Application of the shift reagent (Pr3+) revealed that phospholipid molecules form a monolayer structure on the surface of complexes. Upon binding of LPS to LDL, a maximum of the apoB intrinsic fluorescence was slightly red-shifted (1-2 nm) which may testify that the localization of apoB remains nearly unchanged. For LPS-LDL complexes, the accessibility of apoB fluorophores to quenchers (I-, Cs+, acrylamide) did not dramatically differ from that of LDL. It is concluded that rather large amounts of LPS (about 9-10 molecules) can accommodate in one LDL particle without severely perturbing its original composition and structure. Moreover, in the LPS-LDL complexes, oligosaccharide chains of LPS screen notably neither phospholipid polar headgroups nor, what is very important, apoB. LPS-LDL complexes are suggested to be able in vivo to bind to cellular apoB/E receptors, possible LPS receptors and scavenger-receptors of macrophages (monocytes).     

Expression of vascular endothelial growth factor during the development of cardiac hypertrophy in spontaneously hypertensive rats

We have recently demonstrated that lipids, particularly cholesterol, covalently bound to apolipoprotein B (apoB) are a stable marker of low density lipoprotein (LDL) oxidation (Tertov et al. 1995). The present study is an attempt to assess the relationship between the degree of LDL oxidation, evaluated by the content of apoB-bound cholesterol and the ability of LDL to induce cholesterol accumulation in cultured human aortic intimal smooth muscle cells, i.e. LDL atherogenicity. Native LDL was oxidized in vitro by copper ions, 2,2-azobis-(2-aminopropane hydrochloride), or sodium hypochlorite. Minimum degree of LDL in vitro oxidation necessary to convert LDL into atherogenic one was accompanied by an increase of apoB-bound cholesterol to the level much higher than that usually observed in freshly isolated atherogenic LDL from human blood. Moreover, elimination of LDL aggregates from in vitro oxidized LDL preparations by gel filtration led to loss of its atherogenic properties. Thus, the ability to induce cholesterol accumulation in cells, i.e. the atherogenicity of in vitro oxidized LDL is a result of LDL aggregation but not oxidation. We also studied the relationship between LDL atherogenicity and apoB-bound cholesterol content in LDL freshly isolated from healthy subjects and normo- and hypercholesterolemic patients with coronary atherosclerosis. The ability of human LDL to induce cholesterol accumulation in aortic smooth muscle cells did not correlate with the degree of in vivo LDL oxidation (r = 0.12, n = 90). It is concluded that LDL atherogenicity does not depend on the degree of lipid peroxidation in LDL particle.     

Actin polymerization in macrophages in response to oxidized LDL and apoptotic cells: role of 12/15-lipoxygenase and phosphoinositide 3-kinase

Formation of filamentous F-actin drives many cellular processes, including phagocytosis and cell spreading. We have recently reported that mouse macrophage 12/15-lipoxygenase (12/15-LO) activity promotes F-actin formation in filopodia during phagocytosis of apoptotic cells. Oxidized low-density lipoprotein (OxLDL) also stimulates robust F-actin formation and spreading of macrophages. However, unlike apoptotic cells, OxLDL did not cause specific translocation of 12/15-LO to the cell membrane, neither in macrophages nor in GFP-15LO-transfected COS-7 cells. Moreover, inhibition of 12/15-LO activity in macrophages by a specific inhibitor or by 12/15-LO gene disruption did not affect OxLDL-induced actin polymerization. Among LDL modifications modeling OxLDL, LDL modified by incubation with 15LO-overexpressing fibroblasts was as active in eliciting F-actin response as was OxLDL. This LDL modification is well known to produce minimally modified LDL (mmLDL), which is bioactive and carries lipid oxidation products similar to those produced by 12/15-LO catalysis. MmLDL activated phosphoinositide 3-kinase (PI3K), and PI3K inhibitors abolished mmLDL-induced macrophage spreading. We hypothesize that OxLDL and mmLDL may contribute oxidized lipids to the macrophage cell membrane and thereby mimic intracellular 12/15-LO activity, which leads to uncontrolled actin polymerization and dramatic cytoskeletal changes in macrophages.     

Lipid and lipoprotein profile in women with polycystic ovary syndrome

Polycystic ovary syndrome (PCOS) is a common endocrine disorder characterized by obesity-related risk factors for cardiovascular disease. The objective of our study was to determine values of key lipid and lipoprotein fractions in PCOS, and their possible relation to insulin resistance. A total of 75 women with PCOS (aged 23.1 +/- 5.1 years, BMI 24.9 +/- 4.7 kg/m(2)), and 56 age- and BMI-matched controls were investigated. In all subjects, basal glucose, cholesterol (total, HDL, and LDL), oxidized LDL (OxLDL), triglycerides, apolipoprotein (apo)A1, apoB, and apoE, nonesterified fatty acids, insulin, testosterone, sex hormone-binding globulin, homeostasis model assessment (HOMA) index, and free androgen index were determined in the follicular phase of the cycle. PCOS patients compared with controls had increased indices of insulin resistance, basal insulin (p < 0.001), and HOMA index (p < 0.001), and worsened insulin resistance-related dyslipidemia with decreased HDL cholesterol (p < 0.01), elevated triglycerides (p = 0.010), and pronounced LDL oxidation (p < 0.001). In conclusion, characteristic dyslipidemia of insulin resistance and unfavorable proatherogenic lipoprotein ratios were present only in women with PCOS and not in controls. Elevated OxLDL and the relation of apoE and nonesterified fatty acids with insulin resistance suggest that women with PCOS are at increased risk for premature atherosclerosis.     

An immunochemical marker of low density lipoprotein oxidation

Using monoclonal antibodies against apolipoprotein B (apoB) we studied changes in apoB immunoreactivity during copper ion-mediated oxidation of human low density lipoprotein (LDL). The radioimmunoassay experiments demonstrated the decrease of immunoreactivity of three different epitopes of apoB located in different parts of the protein; at the same time the immunoreactivity of another epitope, previously mapped to the C-terminal 20 amino acids of apoB increased markedly during the first 6 h of LDL oxidation and diminished gradually upon prolonged incubation with copper ions. The fate of LDL during oxidation was also monitored using electrophoretic techniques combined with immunodetection. These experiments showed a rapid fragmentation and disappearance of immunoreactive apoB. They also indicated that the diminishing LDL immunoreactivity detectable during oxidation is associated with apoB fragments still attached to the lipid core. The changes in apoB immunoreactivity during Cu2+ treatment of LDL are similar to those observed upon LDL aging. Therefore, it appears that the enhancement of immunoreactivity of the C-terminus of apoB is a general phenomenon associated with various kinds of oxidative modifications of LDL.     

Covalent binding of oxidized cholesteryl esters to protein: implications for oxidative modification of low density lipoprotein and atherosclerosis

It has been proposed that plasma low density lipoproteins (LDL) undergo oxidative modification before they can produce foam cells in atherosclerosis. The oxidation of LDL generates a variety of reactive aldehydic products, which covalently bind to the LDL apolipoprotein B-100 (apoB). In the present study, to investigate the mechanisms contributing to the modification of LDL, we analyzed oxidized cholesteryl esters generated during the autoxidation of LDL and characterized their covalent binding to the lysine residues of LDL apoB. In addition, we raised a monoclonal antibody specific to a lysine-bound oxidized cholesteryl ester and determined its production in human atherosclerotic lesions. The peroxidation of LDL with Cu2+ produced 9-oxononanoylcholesterol (9-ONC) and 5-oxovaleroylcholesterol as the major oxidized cholesteryl esters. We observed that the levels of 9-ONC and 5-oxovaleroylcholesterol peaked at 12 h and significantly decreased thereafter. The reduction of the core aldehyde levels was accompanied by (i) the formation of free 7-ketocholesterol and 7-ketocholesteryl ester core aldehydes and (ii) an increase in the amounts of apoB-bound cholesterol and 7-ketocholesterol, suggesting that the cholesteryl ester core aldehydes were further converted to their 7-ketocholesterol- and apoB-bound derivatives. To detect the protein-bound 9-ONC, we raised the monoclonal antibody 2A81, directed against 9-ONC-modified protein, and found that it extensively recognized protein-bound cholesteryl ester core aldehydes. Agarose gel electrophoresis followed by immunoblot analysis of the oxidized LDL clearly demonstrated the formation of antigenic structures. Furthermore, immunohistochemical analysis of the atherosclerotic lesions from the human aorta showed that immunoreactive materials with mAb 2A81 were indeed present in the lesions, in which the intense immunoreactivity was mainly located in the macrophage-derived foam cells and the thickening neointima of the arterial walls. The results of this study suggest that the binding of cholesteryl ester core aldehydes to LDL might represent the process common to the oxidative modification of lipoproteins.     

The influence of the triglyceride content of low density lipoprotein on the interaction of apolipoprotein B-100 with cells

To study the effect of triglyceride content of low density lipoprotein (LDL) on its physicochemical and biological properties, we have depleted the triglyceride by incubation with hepatic lipase (HL-LDL) and raised the triglyceride by incubation of HL-LDL with very low density lipoprotein and lipoprotein-deficient serum. HL-LDL was taken up by human monocyte-derived macrophages and by human skin fibroblasts at an increased rate compared to untreated LDL. Incubation of the various LDL preparations revealed that cellular LDL degradation as well as LDL-mediated cholesterol esterification were inversely related to the triglyceride content of the LDL preparation. Modification of the triglyceride content of LDL also was associated with changes in the free fatty acid content, but the interaction of the LDL with cells was unaffected by the level of this component. The triglyceride content of LDL was found to be reciprocally related to the number of free lysine amino groups of LDL apolipoprotein B (apoB) which could be labeled with trinitrobenzenesulfonic acid. 13C-Nuclear magnetic resonance (NMR) spectra of native LDL and HL-LDL samples containing [13CH3]2 lysine residues formed by reductive methylation (11-13% modification) showed that the arrangement of apoB lysines is perturbed by the exposure to hepatic lipase. The ratio of labeled lysines with pK 8.9 to those with pK 10.5 exposed on the surface of LDL particles was decreased by about 40% by lipase treatment. These effects are apparently due to changes in local apoB conformation because circular dichroism spectra revealed that the average secondary structure of the entire apoB molecule is the same in native LDL and HL-LDL. The triglyceride content of LDL reciprocally affected its binding to a monoclonal antibody which recognizes epitopes around the LDL receptor binding domain of apoB. The above evidence indicates that modulation of the core triglyceride and possibly also surface phospholipid content of LDL can alter the conformation of apoB on the surface of the particle, thereby influencing the interaction with cell surface LDL receptors.