Oxidation-specific epitopes are important targets of innate immunity.

During the oxidation of LDL, a central pathophysiological component of atherogenesis, a wide variety of chemical and physical changes occur leading to the generation of oxidation-specific neoepitopes. These epitopes are not only immunogenic, leading to adaptive humoral responses, but are also a prominent target of multiple arcs of innate immunity. The pattern recognition receptors (PRRs) of innate immunity are germ line encoded, conserved by natural selection, and bind to pathogen-associated molecular patterns (PAMPs) common on multiple structures. However, it is not intuitive as to why they should recognize oxidation-specific neoepitopes. Yet it is clear that multiple macrophage scavenger receptors, which are classic PRRs, recognize oxidation-specific epitopes, such as those found on oxidized LDL (OxLDL). Other innate proteins, such as C-reactive protein, also bind to OxLDL. Natural antibodies (NAbs), the humoral arc of innate immunity, provide a nonredundant role in the first line of defence against pathogens, but are also believed to provide important homeostatic house-keeping functions against self-antigens. Our work demonstrates that oxidation-specific epitopes, as found on OxLDL, are a major target of NAbs. In this review, we will discuss the specific example of the prototypic NAb T15/E06, which is increased in atherosclerotic mice and mediates atheroprotection, and discuss the potential role of NAbs in atherogenesis, and in inflammation in general. We also review data that oxidation-specific epitopes are generated whenever cells undergo programmed cell death, forming a common set of PAMPs recognized by oxidation-specific PRRs on macrophages, NAbs and innate proteins. We present the hypothesis that oxidation-specific epitopes on apoptotic cells exerted evolutionary pressure for the conservation of these PRRs and also serve to maintain the expansion of a substantial proportion of NAbs directed to these stress-induced self-antigens.

Leukocyte transglutaminase 2 expression limits atherosclerotic lesion size.

OBJECTIVE: Transglutaminase 2 (TG2), a broadly expressed regulator of protein cross-linking, wound healing, and tissue fibrosis, mediates apoptotic cell ingestion and transforming growth factor-beta release by macrophages and thereby can limit leukocyte-mediated inflammation. In atherosclerosis, oxidative stress and accumulation of unesterified cholesterol stimulate atherosclerotic lesion cell apoptosis. Cell death in advanced atherosclerotic lesions promotes lesion expansion and vulnerable plaques prone to rupture. Hence, we tested the hypothesis that leukocyte TG2 expression limits atherosclerosis. METHODS AND RESULTS: We transplanted TG2-/- or TG2+/+ bone marrow into lethally irradiated low-density lipoprotein receptor (LDLR)-/- mice and evaluated diet-induced atherosclerosis after 16 weeks. We subsequently studied cultured TG2-/- and congenic TG2+/+ mouse macrophages for selected atherogenesis regulatory functions. Atherosclerotic aortic valve lesions in LDLR-/- recipients of TG2-/- bone marrow were larger and more subintimal lesional macrophage penetration than in TG2+/+ marrow recipients. Lesion intimal TG2 expression appeared robust in TG2+/+ but not TG2-/- marrow recipients. Cultured TG2-/- macrophages demonstrated diminished phagocytosis of apoptotic leukocytes, unaltered endocytosis, and degradation of oxidized LDL but decreased retinoic acid induction of the reverse cholesterol transport and apoptotic cell uptake mediator ABCA1. CONCLUSIONS: We conclude that macrophage TG2 expression promotes both apoptotic cell clearance and ABCA1 expression in vitro and limits atherosclerotic lesion size in vivo.

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.

Increased levels of high-density lipoprotein cholesterol are ineffective in inhibiting the development of immune responses to oxidized low-density lipoprotein and atherosclerosis in transgenic rabbits expressing human apolipoprotein (apo) A-I with severe hypercholesterolaemia.

High levels of high-density lipoprotein (HDL) cholesterol have been reported to protect against the development of atherosclerosis in humans by increasing reverse cholesterol transport and inhibiting the oxidation of low-density lipoprotein (LDL) due to the paraoxonase content of HDL. The purpose of the present study was to assess if there are any relationships between in vivo increases in serum levels of immunological LDL oxidation markers [autoantibodies against oxidized LDL, autoantibodies against malondialdehyde-modified LDL, LDL immune complexes and anti-cardiolipin autoantibodies], paraoxonase activity and the development of atherosclerosis in control rabbits and in transgenic rabbits expressing human apolipoprotein (apo) A-I. A total of 13 apo A-I transgenic rabbits and 18 non-transgenic littermates were fed on a cholesterol-rich diet (0.4%, w/w) for 14 weeks, and were monitored at weeks 0, 2, 6, 10 and 14. Aortic atherosclerotic lesions were measured at the end of this period. Human apo A-I transgenic rabbits with high HDL cholesterol levels were not protected against the development of atherosclerosis when they were fed on a cholesterol-rich diet which induced dramatic hypercholesterolaemia. Immunological markers of LDL oxidation increased and serum paraoxonase activity decreased similarly in control and transgenic rabbits. In conclusion, the present study demonstrates that high HDL cholesterol levels are ineffective in inhibiting increases in immunological markers of LDL oxidation and the development of atherosclerosis in a mammal with severe hypercholesterolaemia.

Scavenger receptors, oxidized LDL, and atherosclerosis.

Oxidized LDL (OxLDL) competes with oxidatively damaged and apoptotic cells for binding to mouse peritoneal macrophages, implying the presence of one or more common domains. However, the nature of the ligands involved has not been determined. Studies in this laboratory over the last several years provide evidence that oxidized phospholipids, present in OxLDL and also in the membrane of apoptotic cells, represent one such ligand. These oxidized phospholipids, either in the lipid phase of OxLDL or becoming attached covalently to apoprotein B during LDL oxidation, have been shown to play a major role in the binding of OxLDL to CD36 and to SR-B1 expressed in transfected cells. The lipid and protein moieties compete with each other to some extent, indicating that they are binding to at least one common site. A monoclonal antibody selected because of its reactivity with OxLDL proved to be an antibody against oxidized phospholipids (but not native phospholipids). This antibody (EO6) blocked the uptake of OxLDL by CD36 and by SR-B1 in transfected cells by as much as 80%; it also inhibited macrophage phagocytosis of apoptotic cells by about 40%. Thus, the persistence of receptors for OxLDL during evolution is probably accounted for by their role in recognition of ligands on the surfaces of oxidatively damaged or apoptotic cells. This has important implications in biology generally and specifically in atherogenesis, because apoptosis is a prominent feature of late lesions.

Oxidized LDL reduces monocyte CCR2 expression through pathways involving peroxisome proliferator-activated receptor gamma.

The CCR2-mediated recruitment of monocytes into the vessel wall plays an important role in all stages of atherosclerosis. In recent studies, we have shown that lipoproteins can modulate CCR2 expression and have identified native LDL as a positive regulator. In contrast, oxidized LDL (OxLDL), which is mainly formed in the aortic intima, reduces CCR2 expression, promotes monocyte retention, and may cause pathological accumulation of monocytes in the vessel wall. We now provide evidence that OxLDL reduces monocyte CCR2 expression by activating intracellular signaling pathways that may involve peroxisome proliferator-activated receptor gamma (PPARgamma). Receptor-mediated uptake of the lipoprotein particle was required and allows for delivery of the exogenous ligand to the nuclear receptor. The suppression of CCR2 expression by OxLDL was mediated by lipid components of OxLDL, such as the oxidized linoleic acid metabolites 9-HODE and 13-HODE, known activators of PPARgamma. Modified apoB had no such effect. Consistent with a participation of the PPARgamma signaling pathway, BRL49653 reduced CCR2 expression in freshly isolated human monocytes ex vivo and in circulating mouse monocytes in vivo. These results implicate PPARgamma in the inhibition of CCR2 gene expression by oxidized lipids, which may help retain monocytes at sites of inflammation, such as the atherosclerotic lesion.

Absence of relationship between plasma Lp(a), Lp-AI, anti-oxidized LDL autoantibodies, LDL immune complexes concentrations and restenosis after percutaneous transluminal coronary angioplasty.

To determine the relation between the concentration of Lp(a), LpAI, immunological markers of LDL oxidation (antioxidized-LDL autoantibodies (LDL-AB), LDL immune complexes (LDL-IC)) and restenosis after percutaneous transluminal coronary angioplasty (PTCA) in a Caucasian population (France), we studied 77 consecutive patients who successfully underwent PTCA. All were evaluated by follow-up angiography at an average of 6 months after PTCA and were divided into two groups: existence of restenosis (32 patients, group (G+)) and absence of restenosis (45 patients, negative group (G-)). The prevalence of diabetes mellitus was higher in the restenosis positive group than in the negative group (28% versus 2% respectively, P=0.001). Before and after adjustment in diabetes mellitus frequency there was no difference in the usual lipid parameters (total cholesterol, LDL-cholesterol, HDL-cholesterol, triglycerides, phospholipids, apolipoprotein AI, apolipoprotein B) between the two groups of patients nor in the other parameters (Before adjustment: Lp(a): 0.306+/-0.352 g/l (G+) vs. 0.263+/-0.270 g/l (G-); LpAI: 0.414+/-0.126 g/l (G+) vs. 0.390+/-0.092 g/l (G-); LDL-AB: arbitrary unit (AU) 3.75+/-1.91 (G+) vs. 3.67+/-1.24 (G-); LDL-IC: (AU) 0.93+/-0.82 (G+) vs. 0.86+/-0.44 (G-)). Spearman correlation coefficients did not report any correlation between late loss, loss index, gain and the above mentioned plasma parameters. In conclusion, usual tested plasma lipids, Lp(a), LpAI and in vivo markers of LDL oxidation (LDL-AB and LDL-IC) are not risk factors for restenosis after PTCA in this French population.

Paraoxonase activity is dramatically decreased in patients positive for anticardiolipin antibodies.

It has been reported that paraoxonase 1 (PON1) activity inhibits low-density lipoprotein (LDL) oxidation and modulates the risk of coronary heart disease. This study shows that autoantibodies (IgG) directed against modified LDL were increased in 71 patients positive for anticardiolipin antibodies. In a representative subgroup of these patients (n = 36) PON1 activity was dramatically decreased and the prevalence of the RR genotype of this enzyme tended to be increased in patients who had developed arterial thrombosis. This study suggests that PON1 abnormalities play a role in the antiphospholipid syndrome.

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.

Paraoxonase activity is reduced by a pro-atherosclerotic diet in rabbits.

Serum paraoxonase (PON1) is believed to protect against the development of atherosclerosis because of its ability to retard the oxidation of low-density lipoprotein (LDL) by hydrolysing LDL-associated phospholipid and cholesteryl-ester hydroperoxides. We have examined the relationship between PON1 and atherosclerosis development in transgenic rabbits overexpressing human apolipoprotein (apo) A-I and nontransgenic littermates fed a pro-atherogenic diet. PON1 activity was higher in transgenic (4006.1 +/- 716.7 nmol/min/ml) compared to control (3078.5 +/- 623.3 nmol/min/ml) rabbits (P < 0.01) while high-density lipoprotein (HDL) cholesterol was 1.84 +/- 0.54 mmol/L in transgenic rabbits and 0.57 +/- 0.21 mmol/L in control rabbits (P = 0.0001). After feeding rabbits a high-cholesterol diet for 14 weeks HDL-cholesterol fell by 70% in both transgenic and control rabbits (P < 0.001 compared to week 0) PON1 activity fell by 50% in both groups of rabbits (P < 0. 01 compared to week 0). The amount of thoracic aortic surface area covered by lesions was 29 +/- 16% in the control group and 26 +/- 15% in the transgenic group (P = NS). A pro-atherosclerotic diet reduces PON1 which may exaggerate the effects of the diet on the development of atherosclerosis.

Paraoxonase polymorphism (Gln192Arg) as a determinant of the response of human coronary arteries to serotonin.

Background-Oxidation of LDL plays a role in endothelial dysfunction. Paraoxonase, an enzyme present on HDL, protects LDL against oxidation. Paraoxonase activity is genetically determined in part, and 3 genotypes have been described with variable enzymatic activity. We hypothesized that the paraoxonase polymorphism might influence endothelial function. Methods and Results-Twenty-seven patients with clinical manifestations of coronary artery disease underwent provocative testing by intracoronary administration of serotonin. None of the coronary arteries studied had significant (>50%) stenosis. Ten patients had the QQ genotype and 17 had the QR genotype. At proximal segments, the mean percentage reduction in lumen diameter in response to serotonin was greater in QQ patients than in QR patients (10(-5) mol/L: P<0.05; 10(-4) mol/L: P<0.006). Similarly, at distal segments, constriction in response to serotonin was greater in QQ patients than in QR patients (10(-6) mol/L: P<0. 03; 10(-5) mol/L: P<0.07). Conclusions-These results suggest a higher synthesis or release of endothelium-derived relaxing factors to counteract the vasoconstrictor effect of serotonin in patients with the R allele. These findings provide evidence that the paraoxonase polymorphism may play a role in the regulation of coronary vasomotor tone.

Alpha-tocopherol but not beta-tocopherol inhibits thrombin-induced PKC activation and endothelin secretion in endothelial cells.

INTRODUCTION: Production of endothelin by endothelial cells depends on protein-kinase C (PKC) stimulation which has been reported to be inhibited by alpha-tocopherol (alpha-Toch) but not by beta-tocopherol (beta-Toch). The goal of this study was to determine whether alpha-Toch and beta-Toch inhibit endothelin secretion by endothelial cells. METHODS: and results In a first set of experiments, cultured bovine aortic endothelial cells (BAEC) were incubated for 48h with 100 micromol/l alpha-Toch or vehicle (0.1% ethanol), then cells were stimulated for 4 h or 20 h with thrombin. After stimulating bovine aortic endothelial cells with thrombin for 4 h, alpha-Toch inhibited PKC activity by 63% and endothelin secretion by 44%, whereas after 20 h of incubation with thrombin, alpha-Toch decreased the peptide secretion by 51%. In a second set of experiments, BAEC were incubated with increased concentrations (from 0 to 100 micromol/l) of alpha-Toch or beta-Toch, PKC activity and endothelin secretion were measured after thrombin stimulation as previously reported. In these experiments, alpha-Toch strongly inhibited thrombin-induced PKC activity and endothelin secretion in a dose-dependent manner, whereas beta-Toch was more than 10-fold less active than alpha-Toch in inhibiting these stimulations. Tocopherols (alpha-Toch + beta-Toch) produced a proportional correlation on both PKC stimulation and endothelin secretion by inhibiting the effect of thrombin. CONCLUSION: These data suggest that alpha-Toch strongly inhibits thrombin-induced endothelin secretion in vitro at least partly through PKC inhibition.

Endothelial derived vasorelaxation is impaired in human APO A-I transgenic rabbits.

Endothelium-derived relaxing factor (nitric oxide: NO) may provide an endogenous defence against atherosclerosis which impairs endothelium-dependent vascular relaxation. Atherosclerosis development is inhibited in cholesterol fed human apo A-I transgenic rabbits (Duverger, N., Circulation, 1996, 94, 713-717). We investigated if endothelium-dependent vascular relaxation is modified in human apo A-I transgenic rabbits by testing in vitro endothelium-dependent receptor-dependent vascular relaxation to acetylcholine and endothelium-dependent receptor-independent vascular relaxation to A23187 of abdominal aorta, precontracted with phenylephrine, in human apo A-I transgenic rabbits (n=4) versus non transgenic littermates (n=4). Endothelium-independent vascular relaxation was investigated with sodium nitroprusside. Vascular precontraction to phenylephrine was significantly increased in human apo A-I transgenic rabbits (p<0.05) while endothelium-independent vascular relaxation to nitroprusside was similar between human apo A-I transgenic rabbits and control rabbits. Endothelium-dependent receptor-dependent and receptor-independent vascular relaxations were reduced in human apo A-I transgenic rabbits (p<0.05). Maximum endothelium-dependent receptor-dependent vascular relaxation was negatively correlated with HDL-cholesterol and total apo A-I (rabbit+ human) plasma levels (r=0.87 and 0.86, p=0.01, respectively) but not with atherogenic plasma lipid (VLDL-cholesterol, LDL-cholesterol, VLDL+LDL cholesterol, triglycerides, apolipoprotein B) levels. These results suggest that the transgenesis of human apo A-I in rabbits impairs signal transduction of endothelial NO synthesis.

Oxidized high-density lipoproteins modulate endothelin secretion by adult bovine aortic endothelial cells.

BACKGROUND: High-density lipoprotein (HDL) cholesterol levels are well established as an inverse risk factor for atherosclerosis. This fact is probably related to the ability of HDL to induce cholesterol efflux from the vascular cell. It is also possible that HDL affects the production of different mediators implicated in the development of atheroslerosis. Endothelin is a vasconstricting mitogenic peptide involved in the development of atherosclerosis. We studied whether native HDL, oxidized HDL and tetranitromethane HDL modulate the endothelin secretion of cultured adult bovine aortic endothelial cells. METHODS: We determined the effect of native HDL and modified HDLs (oxidized HDL and tetranitromethane HDL) on the secretion of endothelin by cultured adult bovine aortic endothelial cells. An endothelin radioimmunoassay system was used to quantify levels of immunoreactive endothelin in the cultured media. RESULTS: Native HDL, tetranitromethane HDL and oxidized HDL produced a highly significant stimulation of endothelin secretion (maximum 294% of control), even at low concentrations (10 and 20 micrograms/ml). Oxidized HDL2 and oxidized HDL3 produced a biphasic effect, with maximum secretion occurring with 100 micrograms/ml oxidized HDL3 (294% of control) and 50 micrograms/ml oxidized HDL2 (252% of control). The secretion of the peptide decreased with higher concentrations of oxidized HDL2 and oxidized HDL3. CONCLUSION: Because modified HDLs (oxidized HDL and tetranitromethane HDL) do not bind to the 'HDL receptor' to stimulate endothelin secretion, we propose that the stimulation of secretion is mediated by unspecific binding of the lipoprotein to the cell membrane. Nevertheless, oxidized HDL and tetranitromethane HDL may stimulate endothelin secretion via the scavenger-receptor pathway. Our results suggest that HDL and modified HDL participate in the regulation of vascular tone.

Detection of autoantibodies against oxidized low-density lipoproteins and of IgG-bound low density lipoproteins in patients with coronary artery disease.

The role of oxidized low-density lipoprotein (ox-LDL) in the pathogenesis of atherosclerosis has been the object of intense investigation. It has been proposed that, due to the antigenic properties of ox-LDL, the anti-ox-LDL antibody titre could represent a useful index of in vivo LDL oxidation. On the other hand, LDL immune complexes (LDL-IC) have been demonstrated in patients with coronary disease and could play an atherogenic role. The goal of our study was to investigate anti-malondialdehyde (MDA)-LDL autoantibodies and LDL-IC in a cohort of patients with coronary artery disease. Seventy control subjects and 70 coronary angiographically documented patients were compared; in addition 32 healthy male non-smokers were compared with 32 healthy male smokers (> 10 cigarettes/day). All patients were matched for age and cholesterolemia. Enzyme-linked immunosorbent assay was used to measure anti-MDA-LDL autoantibodies and LDL-IC. Titres of anti-MDA-LDL autoantibodies were not larger in patients with documented coronary artery stenosis and in smokers than they were in controls and non-smokers. The titre of LDL-IC was not higher in patients with coronary artery stenosis than in controls. The results thus indicate that in populations matched for age and cholesterolemia the titres of anti-MDA-LDL autoantibodies and the titre of LDL-IC are not increased in patients suffering from coronary artery stenosis. Furthermore, cigarette smoking does not induce higher titres of anti-MDA-LDL autoantibodies in healthy patients.