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Apoptosis is recognized as important for normal cellular homeostasis in multicellular organisms. Although there have been great advances in our knowledge of the molecular events regulating apoptosis, much less is known about the receptors on phagocytes responsible for apoptotic cell recognition and phagocytosis or the ligands on apoptotic cells mediating such recognition. The observations that apoptotic cells are under increased oxidative stress and that oxidized low-density lipoprotein (OxLDL) competes with apoptotic cells for macrophage binding suggested the hypothesis that both OxLDL and apoptotic cells share oxidatively modified moieties on their surfaces that serve as ligands for macrophage recognition. To test this hypothesis, we used murine monoclonal autoantibodies that bind to oxidation-specific epitopes on OxLDL. In particular, antibodies EO6 and EO3 recognize oxidized phospholipids, including 1-palmitoyl 2-(5-oxovaleroyl) phosphatidylcholine (POVPC), and antibodies EO12 and EO14 recognize malondialdehyde-lysine, as in malondialdehyde-LDL. Using FACS analysis, we demonstrated that each of these EO antibodies bound to apoptotic cells but not to normal cells, whereas control IgM antibodies did not. Confocal microscopy demonstrated cell-surface expression of the oxidation-specific epitopes on apoptotic cells. Furthermore, each of these antibodies inhibited the phagocytosis of apoptotic cells by elicited peritoneal macrophages, as did OxLDL. In addition, an adduct of POVPC with BSA also effectively prevented phagocytosis. These data demonstrate that apoptotic cells express oxidation-specific epitopes-including oxidized phospholipids-on their cell surface, and that these serve as ligands for recognition and phagocytosis by elicited macrophages.

It has been shown previously that the binding of oxidized low-density lipoprotein (OxLDL) to resident mouse peritoneal macrophages can be inhibited (up to 70%) by the apoprotein B (apoB) isolated from OxLDL, suggesting that macrophage recognition of OxLDL is primarily dependent on its modified protein moiety. However, recent experiments have demonstrated that the lipids isolated from OxLDL and reconstituted into a microemulsion can also strongly inhibit uptake of OxLDL (up to 80%). The present studies show that lipid microemulsions prepared from OxLDL bind to thioglycollate-elicited macrophages at 4 degrees C in a saturable fashion and inhibit the binding of intact OxLDL and also of the apoB from OxLDL. Reciprocally, the binding of the OxLDL-lipid microemulsions was strongly inhibited by intact OxLDL. A conjugate of synthetic 1-palmitoyl 2(5-oxovaleroyl) phosphatidylcholine (an oxidation product of 1-palmitoyl 2-arachidonoyl phosphatidylcholine) with serum albumin, shown previously to inhibit macrophage binding of intact OxLDL, also inhibited the binding of both the apoprotein and the lipid microemulsions prepared from OxLDL. Finally, a monoclonal antibody against oxidized phospholipids, one that inhibits binding of intact OxLDL to macrophages, also inhibited the binding of both the resolubilized apoB and the lipid microemulsions prepared from OxLDL. These studies support the conclusions that: (i) at least some of the macrophage receptors for oxidized LDL can recognize both the lipid and the protein moieties; and (ii) oxidized phospholipids, in the lipid phase of the lipoprotein and/or covalently linked to the apoB of OxLDL, likely play a role in that recognition.

Background: Atherosclerotic lesions in intracranial arteries occur later and are less extensive than in extracranial arteries. To investigate potential mechanisms responsible for this difference, in particular the atherogenic response to hypercholesterolemia and LDL oxidation, we compared the extent of fatty streak formation and the composition of these very early lesions in intracranial arteries of human fetuses from normocholesterolemic and hypercholesterolemic mothers with those in extracranial arteries., Methods and Results: Lesions were quantified by computer-assisted image analysis of 30 oil red O-stained sections, each from the middle cerebral, basilar, and common carotid arteries and the abdominal aorta of human fetuses (spontaneous abortions and premature newborns who died within 12 hours of birth; both of fetal age 6.2+/-1.3 months) from 43 hypercholesterolemic mothers and 34 normocholesterolemic mothers. Macrophages, apolipoprotein B, and 2 epitopes of oxidized LDL in lesions were determined immunocytochemically. Activities of superoxide dismutase, catalase, and glutathione peroxidase in the arterial wall were also determined. Lesion numbers and sizes were dramatically greater in the abdominal aorta (area of the largest lesion per section: 66.5+/-10.9 x10(3) microm2) and the carotid (11. 6+/-5.3 x10(3) microm2) than in the basilar and middle cerebral artery (0.4+/-0.1 and 0.8+/-0.2 x10(3) microm2, respectively; P<0. 0001). Hypercholesterolemia resulted in a significant increase of lesion size in extracranial arteries but only a marginal increase in intracranial arteries. In analogy, hypercholesterolemia induced a much greater increase in the intimal presence of macrophages, apolipoprotein B, and oxidized LDL (oxidation-specific epitopes) in extracranial than in intracranial arteries. Immunocytochemistry did not indicate that lesions of intracranial arteries contain relatively less oxidized LDL than similar-size lesions of extracranial arteries. Activities of Mn-superoxide dismutase but not of Zn-superoxide dismutase, catalase, or glutathione peroxidase were significantly higher in both intracranial arteries., Conclusions: Exposure to hypercholesterolemia during fetal development results in extensive formation of fatty streaks in extracranial but not intracranial arteries. The fact that such a difference in lesion formation occurs in the absence of many other atherogenic risk factors found later in life suggests that differences in the atherogenic response to hypercholesterolemia are an important contributor to the slower onset of the disease in intracranial vessels in adults. Fetal arteries may allow elucidation of the mechanisms responsible, for example, better protection of intracranial arteries against free radical-mediated atherogenic processes.

We recently cloned monoclonal IgM autoantibodies which bind to epitopes of oxidized low-density lipoprotein (OxLDL) from apoE-deficient mice (EO- autoantibodies). We now demonstrate that those EO- autoantibodies that were originally selected for binding to copper-oxidized low-density lipoproteins (CuOx-LDL), also bound both to the oxidized protein and to the oxidized lipid moieties of CuOx-LDL. The same EO- autoantibodies showed specific binding to products of oxidized 1-palmitoyl-2-arachidonoyl-phosphatidylcholine (OxPAPC) and to the specific oxidized phospholipid, 1-palmitoyl-2-(5-oxovaleroyl)-phosphatidyl-choline (POVPC), whereas oxidation of fatty acids (linoleic or arachidonic acid) or cholesteryl esters (cholesteryl-oleate or cholesteryl-linoleate) did not yield any binding activity. Those EO- autoantibodies that bound to oxidized phospholipids (e.g., EO6) inhibited the binding and degradation of CuOx-LDL by mouse peritoneal macrophages up to 91%, whereas other IgM EO- autoantibodies, selected for binding to malondialdehyde (MDA)-LDL, had no influence on binding of either CuOx-LDL or MDA-LDL by macrophages. F(ab')2 fragments of EO6 were equally effective as the intact EO6 in preventing the binding of CuOx-LDL by macrophages. The molar ratios of IgM to LDL needed to maximally inhibit the binding varied from approximately 8 to 25 with different CuOx-LDL preparations. Finally, a POVPC-bovine serum albumin (BSA) adduct also inhibited CuOx-LDL uptake by macrophages. These data suggest that oxidized phospholipid epitopes, present either as lipids or as lipid-protein adducts, represent one class of ligands involved in the recognition of OxLDL by macrophages, and that apoE-deficient mice have IgM autoantibodies that can bind to these neoepitopes and inhibit OxLDL uptake.

Background: Oxidatively modified low-density lipoprotein (LDL) is present in atherosclerotic but not normal arteries and plays a crucial role in the pathogenesis and adverse consequences of atherosclerotic lesions. We previously generated a series of monoclonal antibodies (MoAb) against oxidation-specific neo-epitopes formed during the oxidative modification of LDL. MDA2, a prototype MoAb, recognizes malondialdehyde-lysine epitopes (eg, in malondi-aldehyde-modified LDL) within atherosclerotic lesions. We describe the in vivo characteristics of MDA2 and initial noninvasive imaging studies of atherosclerosis in rabbits., Methods: To assess the in vivo specificity of MDA2 for atherosclerotic lesions, iodine 125-MDA2 was intravenously injected into 7 LDL-receptor deficient Watanabe heritable hyperlipidemic (WHHL) and 2 normal New Zealand white (NZW) rabbits, and the aortic plaque uptake was evaluated 24 hours later. 125I-Halb, an isotype-matched irrelevant MoAb that binds to human albumin, was injected into 5 WHHL and 2 NZW rabbits as a control. Aortic autoradiography was performed, and the mean uptake of MoAbs was measured as the percent injected dose per gram aortic tissue. Gamma camera imaging was then carried out in 7 WHHL rabbits and 2 NZW rabbits with 99mTc-MDA2. Imaging was carried out at 10 minutes and at 12 or 24 hours. Malondialdehyde-LDL was then injected to clear the blood pool signal, and final images were obtained 2 hours later., Results: Mean uptake of 125I-MDA2 in the entire aorta was 17.4-fold higher in WHHL than in NZW aortas (P < .001), and 2.8-fold higher than 125I-Halb in WHHL aortas. 125I-MDA2 also had higher specificity for lesioned areas than 125I-Halb (plaque/normal ratio 6.3 vs 2.9, P < .001). Autoradiograph of aortas of 125I-MDA2-injected WHHL rabbits revealed uptake in lipid-stained lesions with absence of signal in adjacent normal arterial tissue. Immunostaining of WHHL lesions, which accumulated MDA2 as noted on autoradiography, revealed that uptake was highest in areas with abundant foam cells and in lipid-rich necrotic core areas. Autoradiograph of aortas from NZW rabbits injected with 125I-MDA2 did not yield any visible signal. Planar gamma camera in vivo scintigraphy revealed a visible signal in 4/7 WHHL rabbits, which was confirmed by aortic Sudan staining., Conclusion: Radiolabeled MDA2 shows excellent in vivo uptake and specificity for atherosclerotic lesions containing abundant oxidation-specific epitopes. The in vivo imaging studies suggest that noninvasive imaging of oxidation-rich atherosclerotic lesions with radiolabeled MDA2 may be feasible in human beings with optimization of the imaging methods.

We and others previously showed that immunization of rabbits with different forms of oxidized low density lipoprotein (LDL) significantly reduced atherogenesis. We now investigated the effect of continued immunization on atherosclerosis in LDL receptor-deficient (LDLR-/-) mice to determine whether a similar reduction of atherosclerosis occurred in murine models and whether this was due to humoral immune responses, ie, formation of high titers of antibodies to oxidation-specific epitopes. Three groups of LDLR-/- mice were repeatedly immunized with homologous malondialdehyde-modified LDL (MDA-LDL), native LDL, or phosphate-buffered saline (PBS) for 7 weeks. Extensive hypercholesterolemia and accelerated atherogenesis were then induced by feeding a cholesterol-rich diet for 17 weeks, during which immunizations were continued. Binding of immunoglobulin (Ig) M and IgG antibodies, as well as IgG1 and IgG2a isotypes, to several epitopes of oxidized LDL were followed throughout the study. After 24 weeks of intervention, atherosclerosis in the aortic origin was significantly reduced by 46.3% and 36.9% in mice immunized with MDA-LDL and native LDL, respectively, compared with PBS (133 558 and 157 141 versus 248 867 microm2 per section, respectively). However, the humoral immune response to oxidative neoepitopes in the MDA-LDL group was very different from that of the LDL or PBS group. IgG antibody binding to MDA-LDL and other epitopes of oxidized LDL, such as oxidized phospholipid (cardiolipin), oxidized cholesterol, or oxidized cholesteryl linoleate, but not native LDL, increased markedly in mice immunized with MDA-LDL, but not in mice immunized with native LDL or PBS. In the MDA-LDL group, both T helper cell (Th)2-dependent IgG1 antibody and Th1-dependent IgG2a antibody binding to oxidative neoepitopes increased significantly over time. The fact that mice immunized with both MDA-LDL and native LDL had a significant reduction in atherosclerosis, whereas only the MDA-LDL group developed very high titers of antibodies to oxidation-specific epitopes, suggests that the antiatherogenic effect of immunization is not primarily dependent on very high titers of antibodies to oxidation-specific epitopes but is more likely to result from the activation of cellular immune responses.

Lipid oxidation has been shown to be a prominent feature of atherosclerosis. The presence of isoprostanes and other lipid oxidation products has been clearly demonstrated in lesions. Furthermore, antibodies to oxidized phospholipids have been identified in animal and human models. Recent evidence suggests that oxidized phospholipids may activate all vascular cell types. The identification and isolation of particular oxidative products has provided some important information about the receptors for these active lipids.

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that has been demonstrated to regulate fat cell development and glucose homeostasis. PPARgamma is also expressed in a subset of macrophages and negatively regulates the expression of several proinflammatory genes in response to natural and synthetic ligands. We here demonstrate that PPARgamma is expressed in macrophage foam cells of human atherosclerotic lesions, in a pattern that is highly correlated with that of oxidation-specific epitopes. Oxidized low density lipoprotein (oxLDL) and macrophage colony-stimulating factor, which are known to be present in atherosclerotic lesions, stimulated PPARgamma expression in primary macrophages and monocytic cell lines. PPARgamma mRNA expression was also induced in primary macrophages and THP-1 monocytic leukemia cells by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Inhibition of protein kinase C blocked the induction of PPARgamma expression by TPA, but not by oxLDL, suggesting that more than one signaling pathway regulates PPARgamma expression in macrophages. TPA induced the expression of PPARgamma in RAW 264.7 macrophages by increasing transcription from the PPARgamma1 and PPARgamma3 promoters. In concert, these observations provide insights into the regulation of PPARgamma expression in activated macrophages and raise the possibility that PPARgamma ligands may influence the progression of atherosclerosis.

Oxidized LDL (oxLDL) is present in atherosclerotic lesions and is believed to play a key role in atherogenesis. Mainly on the basis of cell culture studies, oxLDL has been shown to produce many biological effects that influence the atherosclerotic process. To study LDL oxidation in vivo, we have established a model in which Sprague-Dawley rats are given a single injection of unmodified human LDL (> or = 4 mg/kg body weight). Within 6 hours, an accumulation of apolipoprotein B and epitopes present on oxLDL are detected in the arterial endothelium and media. The presence of oxLDL is associated with activation of the transcription factor nuclear factor-kappaB in the endothelium as well as endothelial expression of intercellular adhesion molecule-1. Injection of LDL enriched with the antioxidant probucol resulted in arterial accumulation of apolipoprotein B, but the expression of oxLDL-specific epitopes was reduced at 24 hours. Thus, this simple model has the potential to analyze the mechanisms behind and biological effects of LDL oxidation in vivo.

Transgenic mice, named GFAP-IL6, that express interleukin-6 in astrocytes in the central nervous system (CNS) have a constitutive blood-brain barrier (BBB) defect and develop a progressive neurodegenerative disease. Based on ultrastructural observations showing electron-dense pigment in the brain of the GFAP-IL6 mice, we hypothesized that iron metabolism was altered in the brains of these animals. Enhanced histochemical methods revealed abnormal iron deposition in the cerebellum from 1 month of age that worsened with progression of the disease. Immunohistochemical analysis of iron-binding proteins (IBP) showed increased ferritin immunoreactivity and a decreased signal from the transferrin receptor in symptomatic animals. Atomic absorption spectroscopy revealed a 40% increase of total iron concentration in the cerebellum at the symptomatic stage. In order to obtain evidence that accumulation of this oxidizing metal was toxic, we looked for the presence of oxidative damage. Using the MAL-2 antibody, extensive lipid peroxidation (LP) was detected in the neocortex and the cerebellum in symptomatic animals. Ultrastructural analysis indicated lipofuscin deposition at the sites of neuro-axonal degeneration and abnormal iron deposition. These results suggest that the IL6-induced BBB defect precipitates iron accumulation in the GFAP-IL6 mouse brain and that subsequent IBP regulation mediates protective responses. As these defenses become overwhelmed, the iron overload seems to promote LP, which may contribute to the neurodegeneration that ensues. This transgenic mouse model of IL6-mediated neurodegeneration provides a unique opportunity to examine several aspects of iron metabolism in the brain, including its entry at the site of the BBB, its distribution through the IBP, and its mechanisms of toxicity.

Several lines of evidence suggest that the cellular enzyme 15 lipoxygenase (15-LO) may be important in promoting the oxidation of lipoproteins in vivo. In previous studies we have shown that fibroblasts transfected with 15-LO "seed" LDL with lipoperoxides such that subsequent oxidation readily generates an LDL that is taken up by macrophages through scavenger receptors. We now demonstrate that LDL incubated with 15-LO cells is "minimally modified" and has bioactive properties. Characterization of LDL incubated with 15-LO cells reveals that lipid peroxidation is modest, with low levels of TBARS generated (12.6 +/- 4.7 nmole MDA per mg protein) and small amounts of 18:2 lost as a result of oxidation (7%, compared with extensive loss [82%] with copper oxidation). The 15-LO-conditioned LDL showed mildly increased electrophoretic mobility on agarose gels, and on polyacrylamide gels it showed only mild protein degradation compared with copper-oxidized LDL. Additionally 15-LO-conditioned LDL competed very well for the LDL receptor of fibroblasts but did not compete for macrophage uptake of 125I-acetylated LDL. Importantly, compared with LDL incubated on beta-galactosidase (lac Z)-transfected control cells, LDL incubated on 15-LO cells stimulated monocyte chemotaxis (15-LO-LDL, 6.9 +/- 1.2 monocytes per field versus lac Z-LDL, 0 +/- 0.9 monocytes per field) and when added to endothelial cells enhanced adhesion (15-LO-LDL, 31.1 +/- 5.0 monocytes per field versus lac Z-LDL, 0 +/- 2.0 monocytes per field). Preincubation of 15-LO cells with 15-LO inhibitors significantly inhibited the generation of bioactive LDL. Lipid extracts of LDL conditioned on 15-LO cells showed chemotactic activity not related to lysophosphatidylcholine levels. Preincubation of target endothelial cells with several different platelet-activating factor receptor antagonists prevented stimulation of monocyte adhesion by 15-LO-conditioned LDL. When probucol- or vitamin E-enriched LDL was incubated with 15-LO cells it was less oxidized and less bioactive, which suggests that these cells seed LDL with LOOH, which then requires further propagation of lipid peroxidation to yield bioactivity. These studies demonstrate that fibroblasts expressing 15-LO reliably produce a bioactive "minimally modified" LDL, which may explain in part how cellular 15-LO activity may generate atherogenic LDL in vivo.

Circulating autoantibodies to phospholipids (aPLs), such as cardiolipin (CL), are found in patients with antiphospholipid antibody syndrome (APS). We recently demonstrated that many aPLs bound to CL only after it had been oxidized (OxCL), but not to a reduced CL analogue that could not undergo oxidation. We now show that the neoepitopes recognized by some aPLs consist of adducts formed between breakdown products of oxidized phospholipid and associated proteins, such as beta2 glycoprotein 1 (beta2GP1). Addition of human beta2GP1, polylysine, native low-density lipoprotein, or apolipoprotein AI to OxCL-coated wells increased the anticardiolipin antibody (aCL) binding from APS sera that first had been diluted so that no aCL binding to OxCL could be detected. No increase in aCL binding was observed when these proteins were added to wells coated with reduced CL. The ability of beta2GP1, polylysine, or low-density lipoprotein to be a "cofactor" for aCL binding to OxCL was greatly reduced when the proteins were methylated. Incubation of beta2GP1 with oxidized 1-palmitoyl-2-linoleyl-[1-14C]-phosphatidylcholine (PC), but not with dipalmitoyl-[1-14C]-PC, led to formation of covalent adducts with beta2GP1 recognized by APS sera. These data suggest that the reactive groups of OxCL, such as aldehydes generated during the decomposition of oxidized polyunsaturated fatty acids, form covalent adducts with beta2GP1 (and other proteins) and that these are epitopes for aCLs. Knowledge that the epitopes recognized by many aPLs are adducts of oxidized phospholipid and associated proteins, including beta2GP1, may give new insights into the pathogenic events underlying the clinical manifestations of APS.

Genetic hemochromatosis (GH) is associated with excess iron deposition in hepatocytes, which results in progressive hepatic injury. The pathogenesis of hepatic injury in GH is poorly understood. In this study, we found enhanced oxidative stress in patients with GH, as evidenced by hepatic malondialdehyde (MDA)-protein adducts and by increased oxidatively modified serum proteins. MDA-lysine epitopes and oxidatively modified serum proteins, as well as immunoglobulin G autoantibodies against MDA-lysine epitopes, were increased in untreated GH patients and to a lesser extent in GH heterozygotes compared with normal individuals. These markers of ongoing oxidative stress decreased with phlebotomy treatment in GH patients. In addition, TGF-beta1 colocalized with hepatic iron and MDA protein adducts in hepatocytes and sinusoidal cells of hepatic acinar zone 1 and normalized after iron removal. Our data suggest that iron overload increases both lipid peroxidation and TGF-beta1 expression, which together could promote hepatic injury and fibrogenesis.

Proteinuria in passive Heymann nephritis is primarily caused by reactive oxygen species that are produced by glomerular cells. Reactive oxygen species apparently exert their damaging effects on the glomerular filter by lipid peroxidation and subsequent adduct formation on matrix proteins of glomerular basement membranes. This raised the question as to the source of polyunsaturated fatty acids required as substrates for lipid peroxidation. Here we have localized by immunocytochemistry rat apolipoprotein E and apolipoprotein B within subepithelial immune deposits. Moreover, apolipoprotein B extracted from isolated glomeruli of proteinuric passive Heymann nephritis rats shows degradation and lipid peroxidation adduct formation, similar to apoproteins of oxidized lipoproteins in atherosclerotic lesions. These data provide evidence that lipoproteins accumulate within immune deposits and suggest that their lipids generate lipid-peroxidation-derived reactive compounds.

Apolipoprotein (apo) E-deficient mice develop atherosclerotic lesions that contain epitopes formed during the oxidative modification of lipoproteins, and they demonstrate high titers of circulating autoantibodies against such epitopes, suggesting that this murine strain may provide a model to investigate the atherogenic mechanisms of oxidized lipoproteins (Palinski et al, Arterioscler Thromb. 1994; 14:605-616). To test the hypothesis that lipoprotein oxidation contributes to lesion formation in apoE-deficient mice, we studied the effect of the antioxidant N,N'-diphenyl 1,4-phenylenediamine (DPPD) in mice fed a high-fat diet containing 0.15% cholesterol. Animals were divided into two subgroups matched for sex and plasma cholesterol levels, and DPPD (0.5% wt/wt) was added to the diet of one subgroup. Throughout the 6 months of intervention, DPPD treatment had no significant effect on plasma cholesterol. Plasma levels of DPPD at the end of the experiment were 33.1 mumol/L. As judged by resistance to loss of polyunsaturated fatty acids, lipoproteins (d < 1.019 g/mL) from DPPD-treated animals showed greater resistance to copper-induced oxidation than lipoproteins from control animals. In addition, there was a greater than twofold prolongation of the lag time in the formation of conjugated dienes in the LDL and IDL fractions of DPPD-treated mice. Atherosclerosis was significantly reduced, by 36% in the DPPD-treated mice (14.0 +/- 4.53% of aortic surface area versus 21.9 +/- 11.6%; n = 32; P < .02). These results are consistent with the hypothesis that lipoprotein oxidation contributes to atherogenesis in apoE-deficient mice. However, further studies with other antioxidants are needed to validate this hypothesis.

Increasing evidence indicates that immune processes modulate atherogenesis. Oxidized LDL (Ox-LDL) is immunogenic, and autoantibodies recognizing epitopes of Ox-LDL have been described in plasma and in atherosclerotic lesions of several species. To determine whether the titer of such autoantibodies correlates with the extent of atherosclerosis, we followed the development of antibodies against malondialdehyde-lysine, an epitope of Ox-LDL, in two groups of LDL receptor-deficient mice for 6 months. One group was fed an atherogenic diet (21% fat and 0.15% cholesterol) that resulted in marked hypercholesterolemia and extensive aortic atherosclerosis; the other group was fed regular rodent chow (4% fat) that did not alter plasma cholesterol levels and induced minimal atherosclerosis. Autoantibody titers significantly increased over time in the group on the atherogenic diet, whereas they remained constant in the chow-fed group. When data from both groups were pooled, a significant correlation was found between the autoantibody titers and the extent of atherosclerosis (r = .61, P < .01). Autoantibody titers also correlated with plasma cholesterol levels (r = .48, P < .05). These results suggest that the rise in autoantibody titers to an epitope of Ox-LDL in this murine model is partially determined by the extent of atherosclerosis but could also be influenced by the degree of hypercholesterolemia or other factors that may influence lipid peroxidation.

Atherosclerosis is known to be accelerated in diabetic patients, but the mechanisms of this acceleration are poorly understood. Nonenzymatic glycosylation of long-lived proteins results in the formation of advanced glycosylation end products (AGEs), which are extensively cross-linked and could contribute to atherogenesis. Oxidative modification of LDL is also an important process in atherogenesis. In vitro evidence suggests that hyperglycemia may enhance lipid peroxidation, and conversely, that increased lipid peroxidation may enhance AGE formation. If such interactions occur in vivo, we hypothesized that AGE should be found in atherosclerotic lesions of euglycemic LDL receptor-deficient rabbits in areas rich in lipids and oxidized lipoproteins. To demonstrate the presence of AGEs, we developed antisera against a specific "model" compound of AGE, 2-furoyl-4(5)-(2-furanyl)-1H-imidazole (FFI) by using FFI-hexanoic acid (FFI-HA)-protein adducts as the antigen and against AGEs in general by using AGE-albumin as the antigen. Antisera generated with FFI-HA-protein adducts recognized FFI-HA alone as well as FFI-protein adducts. Native proteins or proteins conjugated with aldehydes formed during lipid peroxidation in vitro were not recognized by these antisera. Immunocytochemistry with both FFI-specific and AGE-specific antisera revealed the presence of these epitopes in atherosclerotic lesions of euglycemic LDL receptor-deficient rabbits but not in normal aortic tissues. AGE epitopes within atherosclerotic lesions were predominantly found in similar locations as epitopes generated during modification of the lipoproteins by oxidation, consistent with the hypothesized interactions between oxidation and glycosylation. Indirect evidence in support of the in vivo presence of FFI-like structures was also obtained by the observation that both diabetic and euglycemic human subjects contained autoantibodies that recognize FFI-protein adducts. Taken together, these data provide immunological evidence for the in vivo presence of FFI-like structures and other AGE-protein adducts in atherosclerotic lesions, even in euglycemic conditions.

We previously demonstrated that aminoguanidine (AMGN) was able to prevent oxidative modification of LDL. Initially, we thought that this occurred solely because AMGN trapped reactive breakdown products of lipid peroxidation and prevented apoB modification, similar to AMGN's proposed ability to trap reactive glucose intermediates and prevent advanced glycosylation end-product formation. We now demonstrate that AMGN also displays dose-dependent pro-oxidant and antioxidant activity toward LDL. Moderate doses of AMGN (0.05 to 1.0 mmol/L) prevented lipid peroxidation in LDL exposed to copper. AMGN prevented the loss of polyunsaturated fatty acids and delayed or prevented conjugated-diene formation, both of which are sensitive indicators of lipid peroxidation. The same doses of AMGN also prevented apoB modification, a step distal to lipid peroxidation, as evidenced by the ability to (1) prevent fluorescence at 420 nm, (2) block enhanced electrophoretic mobility, and (3) prevent changes leading to enhanced macrophage uptake. Thus, AMGN inhibits LDL modification both by inhibiting lipid peroxidation as well as by trapping reactive breakdown products of lipid peroxidation. It was also demonstrated that for every LDL, there was also a very low dose of AMGN (about 0.01 mmol/L) that actually promoted lipid oxidation and subsequent protein modification. This activity of AMGN could be enhanced by increasing the content of lipid hydroperoxide in the LDL, eg, by aging or radioiodinating the LDL. Conversely, the pro-oxidant activity could be reduced by pretreatment of LDL with ebselen or vitamin E. We propose a mechanism by which AMGN effects pro-oxidant activity toward LDL at very low concentrations and antioxidant activity at higher concentrations and discuss the practical implications of these observations.

Background: The primary mechanisms of ethanol-induced tissue damage have been suggested to include aldehyde-derived protein modifications resulting from ethanol metabolism and lipid peroxidation. Conjugation of reactive aldehydes to a variety of target proteins and cellular constituents have been recently reported. This research was undertaken in order to examine the presence of covalent chemical addition products (adducts) of proteins and acetaldehyde, the first metabolite of ethanol, and those with malondialdehyde, a product of lipid peroxidation, as formed in vivo., Experimental Design: Specific antibodies recognizing acetaldehyde- and malondialdehyde-modified epitopes in proteins were used in immunoperoxidase and double immunofluorescence stainings of liver specimens obtained from ethanol-fed rats and micropigs and from human alcoholics., Results: The centrilobular region of the liver contained the protein modifications both in alcohol-consuming humans and in animals fed ethanol before any apparent histologic damage. With inflammation and fibrosis, such protein modifications were more widespread, and the positive staining for the malondialdehyde-derived modification became more dominant. The presence of the adducts colocalized with the areas of fatty infiltration, focal necrosis and fibrosis. In addition, the erythrocytes of alcohol consumers were found to contain such modifications., Conclusions: The studies support the view that covalent damage to proteins and cellular constituents induced by aldehyde-derived modifications in vivo may play a role in the sequence of events leading to liver disease in alcohol consumers. Species and dietary differences may be important in the relative contribution of lipid peroxidation to alcohol-induced tissue damage.

Apolipoprotein (apo) E-deficient transgenic mice develop marked hyperlipidemia and progressive atherosclerotic lesions. To explore whether oxidative modification of lipoproteins is involved in atherogenesis in this murine model, we performed extensive immunocytochemical studies. Atherosclerotic lesions ranging from early fatty streaks to very advanced plaques were examined from the aortic valve region and the thoracic and abdominal aorta. Using guinea pig antisera against malondialdehyde (MDA)-lysine and 4-hydroxynonenal-lysine, two epitopes generated during the oxidative modification of low-density lipoprotein (LDL), we demonstrated the presence of these "oxidation-specific epitopes" in atherosclerotic lesions. In early lesions, oxidation-specific epitopes were found predominantly in macrophage-rich areas, whereas diffuse extracellular staining predominated in necrotic areas of advanced lesions. We have previously shown that autoantibodies against MDA-lysine are present in the circulation of humans and rabbits and that the immunoglobulin fraction extracted from their lesions contains autoantibodies against several "oxidation-specific" epitopes. Sera from apoE-deficient mice also contained circulating autoantibodies to MDA-lysine, and both early and advanced lesions were rich in murine immunoglobulins. Titers of serum autoantibodies were significantly higher in apoE-deficient mice than in C57BL/6 mice. Autoantibodies in murine plasma recognized MDA-lysine epitopes in atherosclerotic lesions of rabbits, and the immunostaining was competitively inhibited by excess human MDA-LDL. Similar findings were obtained by competitive radioimmunoassay. Finally, a morphometric technique was developed and tested in these mice that allows a quantitative assessment of aortic atherosclerosis. These findings suggest that in apoE-deficient mice, lipoprotein oxidation is involved in atherogenesis and that these transgenic mice constitute an appropriate model with which to study the antiatherogenic effect of antioxidant intervention.

Oxidised low-density lipoprotein (Ox-LDL) has been associated with arterial foam-cell formation, and autoantibodies to Ox-LDL are present in human serum. Lipid peroxidation is enhanced in pre-eclampsia. We assessed whether the titre of IgG autoantibody to an epitope of Ox-LDL, malondialdehyde-conjugated low-density lipoprotein (MDA-LDL), was increased in the sera of pre-eclamptic patients. 16 such patients had significantly higher mean titres of autoantibodies to MDA-LDL than healthy pregnant women (p = 0.028). In a multiple regression model, pre-eclamptic patients still had a significantly higher mean titre (p = 0.048). Enhanced lipid peroxidation may be involved in the foam-cell formation of decidua and in the pathogenesis of pre-eclampsia.

Atherosclerotic lesions contain relatively large quantities of IgG. We have previously shown that both human and rabbit sera contain autoantibodies against epitopes of oxidized (Ox) low-density lipoprotein (LDL) and that LDL isolated from atherosclerotic lesions contains small amounts of tightly bound IgG. However, it is not known whether IgG isolated from atherosclerotic lesions recognizes epitopes present in native LDL or Ox-LDL. IgG was isolated from Watanabe heritable hyperlipidemic (WHHL) rabbit atherosclerotic lesions by sequential salt extractions, purified by fast protein liquid chromatography on protein G, and used in a solid-phase radioimmunoassay. IgG and immune complexes were also isolated from the saline extracts of human lesions by adsorption onto latex beads coated with anti-human IgG antibodies or protein A. IgG isolated from rabbit lesions showed significant titers against malondialdehyde (MDA)-modified LDL and LDL oxidized by copper ions for 4 and 18 hours but not against native LDL. On Western blots, lesion IgG stained MDA-LDL and fragments of Ox-LDL. Western blots of immune complexes isolated from human lesions revealed the presence in the isolated complexes of both apoprotein B and apoprotein B fragments, which reacted with antibodies to MDA-lysine. Furthermore, rabbit lesion IgG immunostained epitopes of Ox-LDL present in human atherosclerotic lesions. Immunostains obtained with rabbit lesion IgG were similar to those obtained with a monoclonal antibody specific for MDA-lysine. The results show that human and rabbit atherosclerotic lesions contain IgG that recognizes epitopes characteristic of Ox-LDL. These data suggest that immunologic processes may be an important component of the atherogenic process.

It is now apparent that low density lipoprotein (LDL) is very susceptible to lipid peroxidation and that the resulting oxidized LDL has altered biological properties. Radiation, particularly of longer duration and lower intensities, initiates lipid peroxidation, yet radioiodination with 125I and 131I is a frequently used method to label LDL for biological studies. To test the possibility that this procedure alters the biological properties of LDL, native LDL was radioiodinated with 125I/131I using ICl to average specific activities of approximately 300 and approximately 100 cpm/ng protein, respectively. Lipid peroxidation was monitored by TBARS and conjugated diene formation. Biological properties were monitored by fibroblast and macrophage uptake of LDL as well as by rate of plasma clearance (FCR) in guinea pigs. 131I-labeled LDL showed enhanced indices of lipid peroxidation compared to 125I-labeled LDL and both were greater than native LDL. The FCR of 131I-labeled LDL was greater than that of 125I-labeled LDL (by 20-40%) and both increased progressively (by > 250%) when measured at 2, 6, and 13 days after iodination. The radioiodinated LDL samples were also more susceptible to pro-oxidant conditions. Thus, after exposure to Cu2+, 131I-labeled LDL showed greatly enhanced lipid peroxidation, decreased uptake by fibroblasts, increased uptake by macrophages and greatly accelerated FCR in guinea pigs. Exposure of LDL to 131I-labeled albumin produced similar changes. Protecting LDL with antioxidants such as BHT and ascorbate immediately after radioiodination generally ameliorated the adverse effects.

Previous studies have demonstrated the presence of apoprotein (apo) E protein and message in arterial lesions. To determine the source of the synthesized apoE, we performed simultaneous in situ hybridization and immunocytochemistry on human and rabbit atherosclerotic tissue. Studies of serial sections of aortic atherosclerotic lesions from humans and hypercholesterolemic New Zealand White rabbits and Watanabe heritable hyperlipidemic rabbits revealed a similar pattern of macrophage-specific apoE expression in the rabbit and human lesions. In early lesions of rabbit atherosclerotic tissue, in which many macrophages were present, there was abundant expression of apoE mRNA. Northern blot analyses of total mRNA obtained from arterial macrophage-derived foam cells, freshly isolated from ballooned, cholesterol-fed New Zealand White rabbits, demonstrated positive hybridization with an apoE-specific riboprobe. Western blot analyses of conditioned media from the isolated foam cells placed in culture for up to 24 hours demonstrated the presence of secreted apoE. These studies demonstrated that in atherosclerotic lesions, arterial wall macrophages synthesize and secrete apoE and probably account for most of the apoE synthesized in the atherosclerotic artery.

Experimental and epidemiological evidence supports the hypothesis that oxidation of low density lipoprotein (LDL) appears to be important in mediating the atherogenicity of LDL. To test this hypothesis in humans, it will be necessary to perform intervention studies in large populations. We performed two studies to assess the effectiveness of supplementation with beta-carotene and vitamin E, used alone and in combination with each other, and with vitamin C, to protect LDL from oxidation. In phase 1, after a placebo period, eight subjects were given beta-carotene (60 mg/day) for 3 months, then beta-carotene plus vitamin E (1,600 mg/day) for another 3 months, and then beta-carotene plus vitamin E plus vitamin C (2 g/day) for 3 months. During phase 2, beta-carotene and vitamin C were discontinued, and subjects took only vitamin E for 5 months. During each period, LDL samples were isolated, and measurements of susceptibility to oxidation were performed. beta-Carotene levels in LDL increased nearly 20-fold, but LDL susceptibility to oxidation did not change. Addition of vitamin E increased LDL vitamin E levels nearly 2.5-fold, and this decreased LDL oxidation 30-40%. During the vitamin C supplementation period, plasma levels of beta-carotene and vitamin E rose, but only beta-carotene increased in LDL. However, the susceptibility of LDL to oxidation in this period was not decreased further. During phase 2, when subjects took only vitamin E, LDL susceptibility to oxidation was decreased by 50% as measured by thiobarbituric acid-reactive substances, conjugated dienes, and lipid peroxide formation as well as by macrophage degradation. Thus, long-term supplementation with large doses of vitamin E alone, but not beta-carotene, conferred increased protection to LDL in in vitro assays of oxidation. These data should be useful in planning therapeutic strategies to test the antioxidant hypothesis in humans.

It has been suggested that alpha-tocopherol, a safe and effective antioxidant, be used in clinical trials to evaluate the ability of antioxidant therapy to inhibit atherosclerosis. Recent reports, however, have raised the possibility that there may be greater enrichment of plasma low density lipoprotein (LDL) in alpha-tocopherol resulting from the use of the naturally occurring RRR-alpha-tocopherol isomer compared with the other isomers present in the synthetic racemic form of alpha-tocopherol. Therefore, we fed equal dosages (1,600 mg/day) of the two forms of vitamin E to 16 men and women for 8 weeks and compared the effects of this supplementation on the susceptibility of isolated lipoproteins to oxidation. Neither form of vitamin E had appreciable effects on lipid or lipoprotein levels. alpha-Tocopherol levels in LDL increased at a similar rate in both groups and were nearly twofold higher than baseline levels by the end of the study. The susceptibility of LDL to oxidation was measured by formation of conjugated dienes, lipid peroxides, and thiobarbituric acid-reactive substances, as well as by macrophage degradation of LDL exposed to oxidizing conditions in vitro. The susceptibility of LDL to oxidation was decreased in both vitamin E groups compared with the baseline value, and this reduction occurred to a similar extent in both vitamin E-supplemented groups. alpha-Tocopherol levels in LDL also strongly correlated with all measures of LDL oxidation. This study demonstrates that, at this dosage, supplementation with either the natural or synthetic form of alpha-tocopherol provided equal antioxidant protection to LDL.

Self-aggregates of low density lipoprotein (LDL) are taken up and degraded more rapidly by macrophages than is native LDL. That enhanced uptake is attributable in part to phagocytosis via the LDL receptor pathway. However, arterial macrophages appear to express little LDL receptor activity. The present studies demonstrate an alternative mechanism by which LDL aggregates could contribute to foam cell formation. This could occur by the formation of large immune complexes that are taken up by macrophages via the Fc receptor. When immune complexes were formed with native, soluble LDL and MB47, a monoclonal antibody specific to the apoprotein B domain recognized by the LDL receptor, the subsequent uptake and degradation of the LDL by macrophages were inhibited 50-80% compared with native LDL alone. In contrast, when aggregated LDL was bound to MB47 at a similar molar ratio, the subsequent degradation of the insoluble immune complexes was two- to fivefold greater than that of aggregated LDL alone. The enhanced uptake was abolished when Fab or F(ab')2 fragments of MB47 were substituted for the intact antibody, indicating that the increased uptake was via the Fc receptor pathway. Furthermore, the uptake of the immune complexes of aggregated LDL was reduced by competition for the Fc receptor with heat-aggregated immunoglobulin. There was also an increase in the rate of cellular cholesterol esterification and an increase in macrophage cholesteryl ester mass. Since aggregates of LDL as well as autoantibodies against modified LDL have been demonstrated in atherosclerotic lesions, it is possible that immune complexes of aggregates of modified LDL may be generated in the intima.(ABSTRACT TRUNCATED AT 250 WORDS)

Oxidative modification of LDL renders it immunogenic and autoantibodies to epitopes of oxidised LDL, such as malondialdehyde (MDA)-lysine, are found in serum and recognise material in atheromatous tissue. However, there has been no prospective study to assess the importance of oxidised LDL among patients with vascular disease. We compared the titre of autoantibodies to MDA-modified LDL and native LDL in baseline serum samples of 30 eastern Finnish men with accelerated two-year progression of carotid atherosclerosis and 30 age-matched controls without progression. Neither group had specific antibody binding to native LDL. A titre was defined as a ratio of antibody binding to MDA-LDL/binding to native LDL. Cases had a significantly higher titre to MDA-LDL (2.67 vs 2.06, p = 0.003). Cases also had a greater proportion of smokers (37% vs 3%), higher LDL cholesterol (4.2 mmol/l vs 3.6 mmol/l), and higher serum copper concentration (1.14 mg/l vs 1.04 mg/l). Even after adjusting for these variables and the severity of baseline atherosclerosis, the difference in antibody titre remained significant in a multifactorial logistic model (p = 0.031). Thus, the titre of autoantibodies to MDA-LDL was an independent predictor of the progression of carotid atherosclerosis in these Finnish men. Our data provide further support for a role of oxidatively modified LDL in atherogenesis.

We have used gene transfer vectors derived from a replication-defective mutant of herpes simplex virus type 1 (HSV-1) expressing the hepatitis B virus surface antigen (HBsAg), Escherichia coli beta-galactosidase (beta-gal), or canine factor IX (cFIX) from the immediate early promoter of human cytomegalovirus (hCMV) to infect mouse liver by direct injection or through the portal vein. By either route, high levels of transgene expression were demonstrated by the detection of immunoreactive HBsAg or cFIX in the circulation and by histochemical detection of beta-gal activity in situ. The results were striking in that the serum level of cFIX reached 10% of the normal murine levels. Although the level of transgene expression from the hCMV promoter was transient, a significant number of persistent vectors could be rescued from the livers of recipient mice up to 2 months after inoculation. Replacement of the hCMV promoter with the HSV-1 latency-associated transcript (LAT) promoter resulted in reduced but prolonged expression of both HBsAg and cFIX. The very high level of factor IX expression suggests that clinically useful gene transfer may eventually be feasible through direct vector delivery to the liver.

To determine whether probucol's ability to confer antioxidant protection to low density lipoprotein (LDL) could be dissociated from its ability to lower high density lipoprotein (HDL) cholesterol, 17 hypercholesterolemic patients were treated with either a standard dose, 1 g/day (4 tablets), or a low dose, 250 mg/day (1 tablet), of probucol for a 6-month period. Effects of therapy on lipoprotein levels and on susceptibility of LDL to in vitro oxidation were measured at frequent intervals. Probucol levels in plasma LDL rose less rapidly in the 1-tablet group but were nearly 50% of levels in the 4-tablet group after 6 months. HDL cholesterol and apolipoprotein A-1 decreased 17.6% and 27.9%, respectively, in the 1-tablet group compared with 28.0% and 38.3%, respectively, in the 4-tablet group (p = 0.07 and p = 0.06). In the 4-tablet group, LDL was protected from copper and endothelial cell-mediated oxidation after 2 months of therapy. In the 1-tablet group, equal degrees of protection occurred, but only after 6 months of therapy. In the whole study group, the decrease in LDL susceptibility to copper or endothelial cell-mediated oxidative modification was correlated with the content of probucol in LDL (r = 0.73, r = 0.65, p less than 0.005). Additionally, the decrease in HDL cholesterol level was correlated with the increase in protection to LDL from oxidative modification (r = 0.67 for copper, r = 0.58 for endothelial cells, p less than 0.05 for both) and also with the content of probucol in LDL (r = 0.6, p = 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Primary cultures of rat hepatocytes embedded and grown to high density in a wafer of reticulated polyurethane have been infected in vitro with a retroviral vector expressing the hepatitis B virus surface antigen (HBsAg). Wafers containing infected cells and implanted directly between the liver lobes of recipient rats became vascularized and firmly attached to the liver parenchyma. Stable levels of HBsAg were detected in the blood circulation for at least 4 months after grafting. Hepatocytes recovered from the recipient rats up to 4 months after implantation were recultured under selective conditions and found to produce high levels of HBsAg.

In this study, the authors demonstrate the expression of mRNA and the presence of protein for macrophage colony-stimulating factor (MCSF) in atherosclerotic lesions from humans and rabbits. In situ hybridization of serial sections of human fatty streaks demonstrated expression of MCSF mRNA by cells dispersed throughout the lesions. Immunocytochemical staining with a panel of MCSF-specific antibodies showed extensive cell-associated staining of all of the cell types in the lesions. Immunocytochemical studies of atherosclerotic lesions from Watanabe heritable hyperlipidemic (WHHL) and cholesterol-fed rabbits demonstrated a similar cell-associated pattern of staining. There was no MCSF-specific staining of aortas from normal rabbits or of cultured aortic smooth muscle cells from either humans or rabbits. Macrophage-derived foam cells (MFC) were isolated from the aortas of ballooned, cholesterol-fed rabbits. A Northern blot demonstrated that RNA isolated from the MFC hybridized with a human cDNA probe for MCSF. RNA from alveolar macrophages isolated simultaneously from the same rabbits did not hybridize with the MCSF probe. Conditioned media from an 18- to 24-hour incubation of the MFC contained colony-stimulating activity as demonstrated in a mouse bone marrow culture assay. Most of this colony-stimulating activity was neutralized by preincubating the conditioned media with an MCSF-specific antibody.

Oxidized low-density lipoprotein (LDL) is more atherogenic than native LDL. The initial step in the oxidation is the peroxidation of polyunsaturated fatty acids. Thus, decreasing the concentration of polyunsaturated fatty acids should reduce the susceptibility of LDL to oxidation. Therefore, we tested the possibility that diets enriched in oleate might result in LDL that is less susceptible to oxidative modification. LDL isolated from subjects consuming an oleate-enriched diet, compared with LDL from subjects on a linoleate-enriched diet, contained significantly more oleate (28.7% vs 11.5%) and less linoleate (31.9% vs 50.9%). Generation of conjugated dienes was significantly lower in the LDL from the oleate group. Most important, after incubation with endothelial cells, LDL from the oleate group underwent less degradation by macrophages. These studies demonstrate the feasibility of altering the diet in a way that will not raise LDL cholesterol concentrations and yet will decrease the susceptibility of LDL to oxidative modification.

We have examined the apolipoprotein content of the lipoproteins of several marine mammals by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their apolipoprotein (apo) B-100, apoB-48, and apoA-I migrated to virtually the same position as the comparable human apolipoproteins. In cetaceans (bottlenose dolphins and killer whales), the molecular mass of the apoE was identical to that of human apoE (35 kDa). In contrast, in the lipoproteins of pinnipeds (harbor seals, sea lions, and walrus) there was no protein comparable in size to human apoE; however, there were two proteins in the 40- to 44-kDa range. The protein with the higher apparent molecular weight (44 kDa) was apoA-IV, as determined by NH2-terminal amino acid sequencing. Sequencing of the NH2-terminal 15 amino acids of the lower molecular weight protein (40-42 kDa) revealed no obvious homology with human apoE. However, a human apoE-specific monoclonal antibody, 1D7, bound to the 40- to 42-kDa protein, allowing us to identify that protein as apoE. Sequencing of sea lion apoE cDNA clones demonstrated that sea lion apoE is 311 amino acids in length, 12 residues longer than human apoE. All 12 additional residues are located in the NH2-terminal 31 amino acids, a region that has extremely low homology with the NH2-terminal portion of human apoE. The remainder of the sea lion apoE sequence is 74% homologous to human apoE. The sea lion apoE cDNA was expressed in Chinese hamster ovary (CHO) cells as well as CHO ldlD cells, a cell line that is deficient in O-glycosylation of proteins. The size of the sea lion apoE secreted by these two cell lines, compared with the apoE in sea lion plasma, indicated that the predominant form of apoE in sea lion plasma must be posttranslationally modified. Thus, our studies have demonstrated that the higher apparent molecular weight of pinniped (sea lion) apoE is due to a longer polypeptide chain as well as posttranslational modification of the protein.

Oxidatively modified low density lipoprotein (LDL) exhibits several potentially atherogenic properties, and inhibition of LDL oxidation in rabbits decreases the rate of the development of atherosclerotic lesions. In vitro studies have suggested that cellular lipoxygenases may be involved in LDL oxidation, and we have shown previously that 15-lipoxygenase and oxidized LDL are present in rabbit atherosclerotic lesions. We now report that epitopes of oxidized LDL are also found in macrophage-rich areas of human fatty streaks as well as in more advanced human atherosclerotic lesions. Using in situ hybridization and immunostaining techniques, we also report that 15-lipoxygenase mRNA and protein colocalize to the same macrophage-rich areas. Moreover, these same lesions express abundant mRNA for the acetyl LDL receptor but no detectable mRNA for the LDL receptor. We suggest that atherogenesis in human arteries may be linked to macrophage-induced oxidative modification of LDL mediated by 15-lipoxygenase, leading to subsequent enhanced macrophage uptake, partly by way of the acetyl LDL receptor.

Pure macrophage-derived foam cells (MFC) were isolated from the aortas of rabbits made atherosclerotic by balloon deendothelialization followed by diet-induced hypercholesterolemia. The MFC were isolated under sterile conditions using an enzymatic digestion procedure and discontinuous density gradient centrifugation. The purity of the MFC preparations was verified immunocytochemically with the macrophage specific monoclonal antibody RAM-11. MFC plated in medium containing 0.5% FCS for 24 h contained approximately 600 micrograms cholesterol per mg cell protein, 80% of which was esterified cholesterol. The MFC specifically degraded low density lipoprotein (LDL), acetyl-LDL, copper oxidized LDL, and beta-very low density lipoprotein (beta-VLDL) at rates comparable to mouse peritoneal macrophages (MPM) in 5-h assays. MFC within sections of the atherosclerotic lesions from the ballooned rabbits as well as the MFC isolated from the same lesions in the presence of antioxidants, exhibited positive immunoreactivity with polyclonal guinea pig antisera and mouse monoclonal antibodies directed against malondialdehyde-LDL, and 4-hydroxynonal-LDL. The MFC also exhibited the capacity to induce the oxidation of LDL at rates comparable to those exhibited by MPM and rabbit aortic endothelial cells. These data provide direct evidence that arterial wall macrophages express modified LDL receptors in vivo, contain epitopes found in oxidized-LDL and are capable of oxidizing LDL even when maximally loaded with cholesterol.

The frontiers in atherosclerosis research are moving from lipoprotein metabolism and control of hyperlipidemia to the cellular events in the artery wall. Emerging hypotheses, including the oxidative modification hypothesis, are already suggesting new approaches that could complement and be additive to control of hypercholesterolemia in the prevention of atherosclerosis.

In hepatic iron overload, iron-catalyzed lipid peroxidation has been implicated in the mechanisms of hepatocellular injury. Lipid peroxidation may produce reactive aldehydes such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), which may form aldehyde-protein adducts. We investigated whether lipid peroxidation occurred in rats fed a diet containing 3% carbonyl iron for 5-13 wk, and if this resulted in the formation of MDA- and 4-HNE- protein adducts. Chronic iron feeding resulted in hepatic iron overload (greater than 10-fold) and concomitantly induced a 2-fold increase in hepatic lipid peroxidation. Using an antiserum specific for MDA-lysine protein adducts, we demonstrated by immunohistochemistry the presence of aldehyde-protein adducts in the cytosol of periportal hepatocytes, which co-localized with iron. In addition, MDA- and 4-HNE-lysine adducts were found in plasma proteins of animals with iron overload. Only MDA adducts were detected in albumin, while other plasma proteins including a approximately 120-kD protein had both MDA and 4-HNE adducts. In this animal model of hepatic iron overload, injury occurs primarily in periportal hepatocytes, where MDA-lysine protein adducts and excess iron co-localized.

We have reported the use of a retroviral vector to introduce the low density lipoprotein (LDL) receptor gene into receptor-deficient Watanabe heritable hyperlipidemic (WHHL) rabbit fibroblasts (Miyanohara, A., M. F. Sharkey, D. Steinberg, J. L. Witztum, and T. Friedmann. 1988. Proc. Natl. Acad. Sci. USA. 85: 6538-6542). Because the cDNA for the LDL receptor did not contain the 5' sterol regulatory element that confers sterol-mediated inhibition of LDL receptor transcription, we did not anticipate that LDL receptor activity transduced by this vector would be sterol-responsive. However, we now demonstrate sterol-mediated down-regulation of receptor protein in the infected cells by a mechanism that appears to be mediated at a post-transcriptional level. Down-regulation of LDL receptor activity occurred when infected WHHL cells were preincubated with either LDL or cholesterol plus 25-hydroxycholesterol. Identically organized vectors bearing cDNAs encoding irrelevant genes such as firefly luciferase or bacterial beta-galactosidase exhibited no sterol regulation of reporter gene activity. Insulin receptor activity in WHHL fibroblasts and in WHHL fibroblasts infected with the LDL receptor retroviral vector was also unchanged by sterol. Transfection of LDL receptor-deficient Chinese hamster ovary (CHO) cells with a nonretroviral vector containing the same LDL receptor cDNA also resulted in sterol responsiveness of the transduced LDL receptor. These experiments suggest that the effect of sterol was specific for the LDL receptor transcript. Transgene LDL receptor mRNA levels from the infected cells were unaffected by sterol, indicating that the sterol-mediated reduction in LDL receptor activity did not result from alterations in steady state mRNA levels. These data suggest the existence of post-transcriptional level mechanisms that are responsible for sterol regulation of expression of the transduced LDL receptor gene.